Since algae were eluted off the resin by 5% sulfuric acid in methanol, a reagent that catalyzes the transesterification of esterified fatty acid to FAMEs, tests were carried out to measure conversion of lipids in the elu — ate to FAME. To quantify FAME and other lipids, normal-phase HPLC was used in conjunction with an evaporative light scattering detector and mass spectrometry (HPLC-ELSD/MS;) [24]. The advantage here is that one can rapidly measure the amount of FAME generated, its fatty acid composition, and the amount of residual triacylglycerol starting material present in the reaction product as well as in crude total lipid extracts. In the reaction product, the presence of residual TAG is an indicator that the transesterification reaction did not reach completion. Preliminary tests carried out by extracting the sulfuric acid methanol eluate with hexane to obtain the products at various times after elution showed that 12 h at room temperature was sufficient to consume all the TAG. What remains in the extract are mainly saturated hydrocarbons, which have been characterized in more detail elsewhere [24], and FAME.

As a reference, algal lipids were converted to FAME by a two step pro­cedure that entailed treatment of dry algal pellets with base to hydrolyze fatty acid esters followed by re-esterification of free fatty acids in sulfuric acid and methanol [26]. The results showed 21.7%, 20.9%, and 35.2% of dry weights were recovered as FAME for healthy Neochloris, stressed Neochloris, and KAS 603 respectively [Figure 2(a)]. Acid-catalyzed trans­esterification of resin-bound algae resulted in 13.6%, 6.9%, and 37.6% of dry weight recovered as FAME for healthy Neochloris, stressed Neochloris, and KAS 603 respectively. For comparison, FAME synthesis yields are shown alongside total lipid extract amounts [Figure 2(a)]. Crude lipid ex­tract constituted 31.4% of total dry weight for healthy Neochloris, 35.4% for stressed Neochloris, and 41.3% for KAS 603. HPLC analysis of the crude lipid extract showed that TAG constituted 0.4%, 8.6%, and 11.7% of dry weight for healthy Neochloris, stressed Neochloris, and KAS 603, respectively.

For KAS 603 comparable yields of FAME were obtained using either method and the total FAME was close to the weight of total lipid. For

Neochloris, both methods generated substantially less FAME than total lipid and the resin-bound algae yielded only 60% of the FAME generated from the dried pellet. Clearly, substantially more FAME can be generated by direct transesterification than can be accounted for by TAG alone. For healthy Neochloris, there was hardly any TAG present in the extracts yet 15%-20% of the DCW could be recovered as FAME. For KAS 603, 10% of the DCW was present as TAG but nearly 40% of the DCW was recov­ered as FAME. These data suggest that much of the FAME is derived from polar lipids such as glycolipids and phospholipids.

One of the surprising results of this study is the finding that Neochloris accumulates high amounts of TAG when subjected to nitrogen depriva­tion [23], yet with only a modest increase in total lipid. While TAG in­creased nearly ten-fold, total lipid still constituted approximately 20% of dry weight, and FAME yield from dried biomass was comparable between healthy and stressed Neochloris. The most notable difference between FAME generated from healthy and stressed Neochloris was found in the fatty acid composition. This can be seen by analyzing positive mode APCI mass spectra of the various FAME reactions. Mass signatures were deter­mined based on the fragmentation behavior of FAME standards. Fatty acyl groups in FAME were identified and quantified by positive mode APCI — MS. Figures 2(b) and 2(c) indicate a trend towards a higher degree of fatty acid saturation with increased TAG content. For example, healthy Neochlo­ris, having little TAG content, yielded more C18:3 and C21:4 species and less C16:0, C18:0, and C20:0 than the other two algal groups. In contrast, stressed Neochloris and KAS 603, having a higher TAG content, yielded more C16:0, C18:0, and C20:0, and less C18:3 and C21:4 than healthy Neochloris. This shift in TAG fatty acid composition from unsaturated to saturated species with nitrogen starvation has been reported previously [21].