## 4 Discussion
Similarly, the results obtained in the first trial revealed that MT1 supplemented with TM1 outperformed MB10 and was further used for the whole optimization process. After these significant improvements in the formulation of macronutrients and trace metals, the next step was to optimize the carbon source and its concentration. In the present study, _Aurantiochytrium_ sp. was grown using glucose and glycerol independently and in combination as the C-source. Glucose is the most used substrate in heterotrophic cultivation, since it displays, generally, higher growth rates and biomass yields in most heterotrophic microorganisms, including thraustochytrids [25]. Nevertheless, alternative carbon sources, such as glycerol, must be pursued particularly from waste streams, with the goal of reducing production costs [25]. Glycerol is a carbon source of excellence that has received attention in the biotechnology field due to its abundance as a cheap by-product of biodiesel industries [25; 35; 41]. Moreover, glycerol used as a feedstock in heterotrophic growth maintains the food-grade status of the produced biomass and sub-products [4; 41]. Thraustochytrids, such as _Aurantiochytrium_, have been reported as being able to utilize glycerol as a carbon source, obtaining comparable and even higher biomass and DHA yields [35; 41]. Nevertheless, when 100 g/L of glycerol was tested, growth was inhibited. In fact, inhibition at glycerol concentrations above 50 g/L was already reported for _Aurantiochytrium limacinum_[42]. The same philosophy was used to evaluate the impact of different N sources in the growth of _Aurantiochytrium_ sp. In the first N optimization trial, three traditionally used N sources present in MT1 were evaluated independently, namely yeast extract, ammonium sulphate and glutamate. Results showed that both yeast extract and glutamate displayed similar growth results to the whole MT1 medium. These results unveiled the larger contribution of glutamate for growth and the possibility of using it as sole N source. This is a highly interesting finding because that eliminates the need of adding yeast extract, reducing medium costs. Accordingly, glutamate is often used as N source in thraustochytrid-based DHA production, since it accelerates growth and promotes DHA accumulation [2; 7; 11]. Interestingly, [PERSON] et al. [21] attained slightly higher biomass, lipid and DHA content with glutamate, rather than with yeast extract. In a similar experiment, [PERSON] et al. [10] reported glutamate as being able to promote growth up to higher biomass concentration; however, a right balance should be found, as N depletion is a known trigger of lipid accumulation, and excess N might be detrimental to DHA production [2; 4; 7]. Regarding alternative and more sustainable N sources, corn steep powder displayed a greater potential for the improved growth of _Aurantiochytrium_ sp., when compared to cheese whey and MNBIO. High fructose corn syrup or corn steep liquor/powder (CSP) is a co-product of corn wet milling process, a well-reported agro-waste often used as a sugar substitute, due to its low cost and high solubility [43]. Apart from its elevated fermentable sugar content, it contains between 35%-45% of protein, being both an N and C source. Interestingly, [PERSON] et al. [44] obtained a _Schizochytrium limacinum_ biomass yield of 15 g/L DW using yeast extract and CSP, but a higher lipid content and twice as much DHA yield (1.7 g/L) was achieved with CSP, rather than with yeast extract. In this assay, the cultures using CSP as N source reached around 12 g/L DW. [PERSON] et al. [34] also recommended 2-10 g/L CSP in fed-batch mode, in order to obtain high-density cultures and high DHA productivities. On the other hand, [PERSON] et al. [32] reported the lowest _Aurantiochytrium_ sp. productivity and maximum biomass concentration with CSP, when compared to yeast extract, peptone and tryptone. Therefore, CSP is an interesting N source for thraustochytrid-based DHA fermentation, but its utilization and effectiveness seem to be strain-dependent.