## 4 Discussion
To increase the cultivation time of NCYC1146 will be addressed in the future to determine if this strain can produce astaxanthin in greater cultivation times than in this study. The _Thraustochytrids_ have demonstrated that the DHA and carotenoid production are related across acetyl-coenzyme A (CoA) and the availability of NADPH, both used in the synthesis of these substances [14]. Therefore, the carotenoid total production and DHA as secondary metabolites of the NCYC4007 and NCYC1146 strains, respectively, would indicate that the use of raw glycerol could provide a good supply of carbon for the synthesis of said substances. In this way, DHA is synthesized from malonyl-CoA coming from Acetyl-CoA, which, at the same time, is a key precursor for the synthesis of acetoacetyl-CoA used for the subsequent biosynthesis of carotenoids and squalene in _Thraustochytrids_[14]. In this way, the simultaneous synthesis of both DHA and carotenoids in these marine _Rbloodotrula_, using raw glycerol as a substrate, suggests that this microorganism should possess the analog biosynthetic pathways described by _Thraustochytrids_. Subsequent studies should clarify whether both strains also have the ability to synthesize squalene, a substance that shares the biosynthetic pathways described above and that also has commercial value, as well as DHA and carotenoids. The carotenoid concentration with raw glycerol reached by strain NCYC 4007 is greater than observed by [PERSON] et al. [9] for the same strain but using glucose as a carbon source. The NCYC1146 strain also synthesizes the DHA (7% ODW of DHA at the end of the study; Figure 4b), substance of which in preliminary studies just reached 0.3% ODW when the strain was cultured with glucose. The synthesis of a secondary metabolite was observed in the cultures of the NCYC4007 (carotenoids) and NCYC1146 strains (DHA) when raw glycerol was used as a substrate, which suggests that an interconnection should exist between the biosynthesis of these two substances in these marine _Rbloodotrula_, as observed in _Thraustochytrium_ sp. AMCQS5-5 [12] and _Thraustochytrids_ species [14]. To the best of our knowledge, the glucose accumulation in the culture medium when raw glycerol was used as a carbon source has not been reported in oleaginous microorganisms. Nevertheless, the appearance when raw glycerol was used could be that the excess glycerol promotes a gluconeogenesis and subsequently eliminates the synthesized glucose from the cell. To determine glucose in the culture medium could be a useful parameter to determine the optimum raw glycerol concentration that could be used. It has been observed that an excess of glycerol can produce an inhibition of DHA synthesis in _Schizochytrium limacinun_[38] and _Schizochytrium limacinun_ SR21 [30] when used in percentages greater than 8.5% and 9%, respectively, values inferior to those used in this study (10%). In addition, excess glycerol has demonstrated the inhibition of \(\beta\)-carotene synthesis in _Blakesela trispora_[39] and, furthermore, could restrict xanthine production, which uses \(\beta\)-carotene as a precursor [14]. Another interesting result to consider is the sudden increase in the intracellular phosphorus concentration from 96 h in NCYC4007, which is not as noticeable in NCYC1146 (Figure 4c,d). In NCYC4007, the intracellular phosphorus concentrations present a significant and strong positive correlation (semi-log; R\({}^{2}\) = 0.8217; \(p\) = 0.001195) with the DHA concentrations; what is suggested is that this latter could be synthesized as a phospholipid rather than as a triacylglycerol. If the DHA synthesized by NCYC4007 were in the phospholipid form, this would have a greater oxidative stability, as well as intestinal bioavailability, if used for human and animal nutrition [40].