## 3 Results and Discussion
### Microalgal Growth Experiments  
Comparative heterotrophic growth of ALP2 microalgae on 10 g L\({}^{-1}\) glucose or acetate-buffered hydrolysate corresponding to the same glucose concentration is shown (Figure 8). The hydrolysate culture resulted in a final \(DCW\) of g L\({}^{-1}\) and % higher \(DCW\) than that achieved on glucose alone. Similarly superior growth on hydrolysate was previously witnessed when _Chlorella pyrenoidosa_ was heterotrophically cultivated using hydrolysate from lignocellulosic rice straw waste pretreated using trifluoroacetate at 95 \({}^{\circ}\)C and ethanol at 210 \({}^{\circ}\)C [6]. Food waste effluent 5% (\(\
u/v\)) dilutions at both initial pH 8 and 9 resulted in higher final absorbance readings for ALP2 cultures compared to those involving flush dairy manure  
Figure 8: Comparative heterotrophic growth of ALP2 on hydrolysate and 10 g L\({}^{-1}\) glucose. effluent (data not shown). Flow cytometry also demonstrated segregation of green ALP2 microalgae from light-blocking particulate matter of the dairy manure effluent that was absent in food waste effluent (Figure 9). Food waste effluent was further evaluated instead because of its greater optical clarity and superior growth performance. The combined effect of NaHCO\({}_{3}\) concentration, food waste effluent dilution, and periodic pH adjustment with acid on ALP2 growth and lipid accumulation is presented in Table 6. Consistent with induction of nitrogen-depletion stress, cultures with minimal 5% (\(v/v\)) effluent levels exhibited the lowest final \(DCW\) but generally resulted in the highest lipid content compared to 10%, 15%, and 20% (\(v/v\)). High salt and NaHCO\({}_{3}\) alkaline levels prolonged an exclamatory lag-phase but did not limit the ability of ALP2 to reach high biomass when pH was not daily adjusted. ALP2 initially grew the fastest and reached an extremely high pH and stationary phase in cultures containing the least amount of NaHCO\({}_{3}\) where buffering was minimal for all effluent levels. For instance, a pH of 11.84 was reached at 8.4 g L\({}^{-1}\) NaHCO\({}_{3}\) and 5% (\(v/v\)) effluent. Such a stationary phase may be attributed to ammonia inhibition occurring at high pH. Daily pH 10 adjustment with HCl acid for the 8.4 g L\({}^{-1}\) NaHCO\({}_{3}\) and 20% effluent condition resulted in a significant 100.89% increase in the \(OD_{680}\) after 120 h, higher total fat content, and a more saturated fatty acid profile, compared to no pH adjustment (data not shown). For similar cultures, the normalized BODIPY fluorescence, indicative of relative neutral lipid content, was 4.97, 3.04, 15.54, and 8.43 for 5% AD effluent + 8.4 g L\({}^{-1}\) NaHCO\({}_{3}\), 20% AD effluent + 8.4 g L\({}^{-1}\) NaHCO\({}_{3}\), 5% AD effluent + 33.6 g L\({}^{-1}\) NaHCO\({}_{3}\), 20% AD effluent + 33.6 g L\({}^{-1}\) NaHCO\({}_{3}\), respectively (Figure 10). Figure 9: Flow cytometric forward scatter vs. side scatter plot (**a**) and PE Cy 5.5 Fluorescence vs. Side Scatter Plot (**b**) of gated anaerobically digested manure particles and ALP2 microalgal populations. At 8.4 g L\({}^{-1}\) NaHCO3, the 5% vs. 22% AD effluent lipid content ratio was 1.64, based on flow cytometry and BODIPY, compared to 1.39 using FAME GC-FID. The total fat content and fatty acid profile of the ALP2 culture at 8.4 g L\({}^{-1}\) NaHCO3 and 20% effluent with acid addition is shown in Table 7. The four prominent fatty acids were palmitic (C16:0), oleic (C18:1n9), linoleic (C18:2n6), and linoleic (C18:3n3). Interestingly, oleic, linoleic, and linolenic composed 50% of the total fatty acids. To the best of our knowledge, the only oil with a higher concentration of omega-3 linolenic fatty acid is flaxseed oil. With such a favorable omega-6/omega-3 ratio, the results on the residues of this microalgae after and neutral TAG lipid extraction suggest these residues could be used as substitutes of omega-6/omega-3.