Ays)BEPA content (pg cell1)two mM 9 mM 18 mM1.two 1.0 0.8 0.6 0.4 0.two 0.0 02 mM 9 mM 18 mMCulture time (Days)CDHA production (mg L1)five four 3 two 1 0 0DHA content material (pg cell1)2 mM 9 mM 18 mMD0.5 0.four 0.3 0.two 0.1 0.2 mM 9 mM 18 mMCulture time (days)Culture time (days)Following complete nitrate depletion, the maximum cellular lipid content material (depending on total fatty acids, Figure 1F) was greater in cultures supplemented with 18 mM bicarbonate (6.9 1.0 pg cell1), compared to cultures with 9 mM bicarbonate addition (three.8 0.five pg cell1). Despite the precipitation of salts observed, this result suggests greater inorganic carbon availability with improved bicarbonate supply for de novo synthesis of organic carbon compounds. Interestingly, lipid accumulation was hugely repressed at low carbon provide (2 mM bicarbonate), displaying practically no adjustments over time, even below nutrient depletion.H-Lys(Aloc)-OH Price As observed in earlier research for quite a few chlorophyte species [39,43], cellular lipid accumulation in P. lutheri also correlated with elevated pH in the time of nitrate depletion. In spite of the proven ability of bicarbonate addition to improve cellular lipid content material in P. lutheri, carbon supply seems to become a limiting aspect for lipid accumulation. Indeed, ToledosCervantes et al. (2013) also reported that lipid accumulation in Scenedesmus obtusiusculus considerably elevated at 5 CO2, but not when the species was batchcultivated at 0.04 COMar. Drugs 2013,(air without having CO2enrichment) [44]. In P. lutheri, Tonon et al. (2002) showed a decrease within the total fatty acid content material per cell upon entry into the stationary phase [10], which might be explained by limited carbon availability utilizing aeration by shaking the flasks at 150 rpm (without the need of further carbon supply). Furthermore, as suggested in some prior research [39,44,45], lipid accumulation is actually a transient metabolic approach reaching a maximum shortly following nutrient limitation.Formula of (S)-BI-DIME Cellular lipid content in P.PMID:33734886 lutheri reached a maximum following nine and 14 days, using 18 and 9 mM bicarbonate, respectively, and subsequent decline was observed, indicating that lipid accumulation ceased and/or cellular lipids had been being utilized or degraded. Interestingly, lower in cellular lipid content corresponded to a reduction in pH in the culture media immediately after nine days. Through batch culture, harvesting time constitutes, as a result, a critical parameter to get the highest cellular lipid content and reach optimum productivity. Lastly, maximum volumetric lipid production (based on total fatty acids, Figure 1E) was observed just after 14 days, utilizing 98 mM bicarbonate addition ( 705 mg L1). 2.two. Combined Effect of Nitrogen Limitation and Bicarbonate Addition on Total Fatty Acid Composition and n3 LCPUFA Production It can be properly established that lipid metabolism and fatty acid profiles are extremely dependent on nutrient availability [168]. Nitrate limitation or starvation are recognized crucial aspects controlling oil accumulation and connected adjustments in fatty acid composition [22,27,54,55]. In P. lutheri, independent on the bicarbonate concentrations, the principle alterations in total fatty acid (TFA) profiles resulting from nitrate limitation were accounted for by relative increases in SFA and MUFA levels, especially 16:0 and 16:1 n7, concomitant with a decrease in the proportion of PUFA (i.e., 18:4 n3, EPA and DHA). 16:0 and 16:1 n7 fatty acids enhanced from 18.5 9.7 and 17.9 4.6 to 26.two 9.9 and 29.3 0.three of TFA, respectively; whilst EPA and DHA decreased from 16.1 eight.7 and 8.six .6 to 9.4 1.