## 1 Introduction  
Microalgae are a diverse group of organisms recognized for their capacity to produce biomolecules of interest for industrial applications (energy, food, nutrition, or pharma) [1, 2, 3, 4]. Although most of them are cultivated under phototropic conditions and their products are already on the market, the industry is now focusing on heterotrophic cultivation due to the similar characteristics to traditional fermentation process. Heterotrophic microalgae contain mainly proteins and lipids, Omega-3 polyunsaturatedated fatty acids (Omega-3 PUFA), in particular long chain PUFA docosahexaenoicenoic acid, DHA (22:6 n3) and eicosapentadecenoic acid, EPA (20:5 n3). They are essential compounds for nutrition and human health that the human body only produces in small quantities and therefore must be acquired through the diet. Traditional sources of these compounds are marine origin, fish and crustaceans in our diet since prehistory [5, 6, 7]. Nevertheless,the progressive population increase and the knowledge about their effect in nutrition and health promote access limitations to traditional sources of Omega-3 PUFA [8]. To fill the gap between the demand and supply of Omega-3 compounds in the different industrial sectors, alternative sources for Omega-3 were investigated in recent years. In this context, heterotrophic microalgae represent an interesting alternative as they are also primary producers of Omega-3 [9; 10; 11; 12]. The capacity of certain species of microalgae to produce high amounts of Omega-3 PUFA, in particular DHA under heterotrophic conditions, turn them into a very attractive source for the production of these compounds for industrial application. However, there are still challenges in the establishment of economic feasible production processes from microalgae [13; 14; 15]. The main drawback is the high cost associated with the use of glucose; the main carbon source used for the growth of microalgae in heterotrophic cultures. Techno-economic analysis carried out by experts indicated that the higher costs at industrial scale were mainly associated with the stage of cultivation, considering that glucose can constitute up to 80% of the cost of the medium. Therefore, it becomes a major disadvantage, from an economic point of view, to produce high added-value compounds by heterotrophic microalgae [16; 17]. However, the high potential of heterotrophic cultivation leads the industry to search for more sustainable routes to produce Omega-3 PUFA though the identification of alternative carbon sources. Great efforts are being taken by the scientific community to investigate alternatives to the use of glucose, as is reflected in the increase in publications in this field in the last ten years. In our previous reports, a compilation of the state of the art on the use of alternative carbon sources was published [18]. Forest biomass, glycerol or pre-treated whey permeates were discussed as substitutes of glucose. Cane molasses and sugarcane waste was also investigated for this purpose, and it was concluded that the presence of toxic substances may cause inhibition and can molasses might be used in combination with other carbon sources [19; 20; 21; 22; 23; 24]. In recent years, the use of volatile fatty acids (VFA) as an alternative carbon source has been studied [15; 25; 26]. VFAs are short-chain fatty acids (two to six carbon atoms) that can provide microalgae a carbon chain ready to be used for further elongation, towards long-chain unsaturated Omega-3. However, current production routes using petrochemical feedstocks to obtain VFAs are considered as non-sustainable and therefore, more environmentally friendly pathways are being investigated.