Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid (PUFA) that belongs to the ω-3 group. In recent years, DHA has attracted much attention because of its recognized beneficial effect on human health. At present, fish oil is the major source of DHA, but it may be produced by microorganisms with additional benefits. Marine microorganisms may contain large amounts of DHA and are considered a potential source of this important fatty acid. Some of these organisms can be grown heterotrophically on organic substrates without light, offering the possibility of greatly increasing microalgal cell concentration under controlled and monitored conditions, resulting in a very high quality product. Among the heterotrophic marine dinoflagellates, Crypthecodinium cohnii has been identified as a prolific producer of DHA. The organism is extraordinary in that it produces no other PUFAs than DHA in its cell lipid in any significant amount, which makes the DHA purification process very attractive, particularly for pharmaceutical and nutraceutical applications. This paper reviews recent advances in the biotechnological production of DHA by C. cohnii.

Energy crises, global warming, and climatic changes call for technological and commercial advances in manufacturing high-quality transportation fuels from unconventional feedstocks. Microalgae is one of the most promising sources of biofuels due to the high yields attained per unit area and because it does not displace food crops. Neochloris oleabundans (Neo) microalga is an important promising microbial source of single-cell oil (SCO). Different experimental growth and lipid production conditions were evaluated and compared by using optical density (540 nm), dry-weight determination, and flow cytometry (FC). Best Neo average biomass productivity was obtained at 30 degrees C under conditions of nitrogen-sufficiency and CO(2) supplementation (N+/30 degrees C/CO(2)), with an average doubling time of 1.4 days. The second and third highest productivities occurred with N-sufficient cultures without CO(2) supplementation at 26 degrees C (N+/26 degrees C) and at 30 degrees C (N+/30 degrees C), with doubling times of 1.7 and 2.2 days, respectively. Microbial lipid production was monitored by flow cytometry using Nile red (NR), a lipophilic fluorochrome that possesses several advantageous characteristics for in situ screening near real time (at line). Results showed maximum lipid content (56%) after 6 days of nitrogen depletion under nitrogen starvation without CO(2) supplementation (N-/30 degrees C), followed by N-/30 degrees C/CO(2) and N-/26 degrees C conditions with 52% lipid content, after 5 and 6 days of N starvation, respectively. The adequate fatty acid profile and iodine value of Neo lipids reinforced this microalga as a good source of SCO, in particular for use as biodiesel.

Carotenoids are one of the most common classes of pigments that occur in nature. Due to their biological properties, they are widely used in phytomedicine and in the chemical, pharmaceutical, cosmetic, food and feed industries. Accordingly, their global market is continuously growing, and it is expected to reach about US$1.4 billion in 2018. Carotenoids can be easily produced by chemical synthesis, although their biotechnological production is rapidly becoming an appealing alternative to the chemical route, partly due to consumer concerns against synthetic pigments. Among the yeasts, and apart from the pigmented species Phaffia rhodozyma (and its teleomorph Xanthophyllomyces dendrorhous), a handful of species of the genera Rhodosporidium, Rhodotorula, Sporobolomyces and Sporidiobolus are well known carotenoid producers. These are known as 'red yeasts', and their ability to synthesize mixtures of carotenoids from low-cost carbon sources has been broadly studied recently. Here, in agreement with the renewed interest in microbial carotenoids, the recent literature is reviewed regarding the taxonomy of the genera Rhodosporidium, Rhodotorula, Sporobolomyces and Sporidiobolus, the stress factors that influence their carotenogenesis, and the most advanced analytical tools for evaluation of carotenoid production.Moreover, a synopsis of the molecular and ''-omic'' tools available for elucidation of the metabolic pathways of the microbial carotenoids is reported.

Microalgae biomass can be a feasible source of ω‐3 fatty acids due to its stable and reliable composition. In the present study, the Crypthecodinium cohnii growth and docosahexaenoic acid (DHA, 22:6ω3) production in a 100 L glucose‐fed batch fermentation was evaluated. The lipid compounds were extracted by supercritical carbon dioxide (SC‐CO2) from C. cohnii CCMP 316 biomas, was and their fatty acid composition was analysed. Supercritical fluid extraction runs were performed at temperatures of 313 and 323 K and pressures of 20.0, 25.0 and 30.0 MPa. The optimum extraction conditions were found to be 30.0 MPa and 323 K. Under those conditions, almost 50% of the total oil contained in the raw material was extracted after 3 h and the DHA composition attained 72% w/w of total fatty acids. The high DHA percentage of total fatty acids obtained by SC‐CO2 suggested that this extraction method may be suitable for the production of C. cohnii value added products directed towards pharmaceutical purposes. Furthermore, the fatty acid composition of the remaining lipid fraction from the residual biomass with lower content in polyunsaturated fatty acids could be adequate for further uses as feedstock for biodiesel, contributing to the economy of the overall process suggesting an integrated biorefinery approach.

In this work, carob pulp syrup was used as carbon source in C. cohnii fermentations for docosahexaenoic acid production. In preliminary experiments different carob pulp dilutions supplemented with sea salt were tested. The highest biomass productivity (4 mg/lh) and specific growth rate (0.04/h) were observed at the highest carob pulp dilution (1:10.5 (v/v), corresponding to 8.8 g/l glucose). Ammonium chloride and yeast extract were tested as nitrogen sources using different carob pulp syrup dilutions, supplemented with sea salt as growth medium. The best results were observed for yeast extract as nitrogen source. A C. cohnii fed-batch fermentation was carried out using diluted carob pulp syrup (1:10.5 v/v) supplemented with yeast extract and sea salt. The biomass productivity was 420 mg/lh, and the specific growth rate 0.05/h. Under these conditions the DHA concentration and DHA production volumetric rate attained 1.9 g/l and 18.5 mg/lh respectively after 100.4 h. The easy, clean and safe handling of carob pulp syrup makes this feedstock a promising carbon source for large-scale DHA production from C. cohnii. In this way, this carob industry by-product could be usefully disposed of through microbial production of a high value fermentation product.

Microbial oils have been considered a renewable feedstock for bioenergy not competing with food crops for arable land, freshwater and biodiverse natural landscapes. Microalgal oils may also have other purposes (niche markets) besides biofuels production such as pharmaceutical, nutraceutical, cosmetic and food industries. The polyunsaturated fatty acids (PUFAs) obtained from oleaginous microalgae show benefits over other PUFAs sources such as fish oils, being odorless, and non-dependent on fish stocks. Heterotrophic microalgae can use low-cost substrates such as organic wastes/residues containing carbon, simultaneously producing PUFAs together with other lipids that can be further converted into bioenergy, for combined heat and power (CHP), or liquid biofuels, to be integrated in the transportation system. This review analyses the different strategies that have been recently used to cultivate and further process heterotrophic microalgae for lipids, with emphasis on omega-3 rich compounds. It also highlights the importance of studying an integrated process approach based on the use of low-cost substrates associated to the microalgal biomass biorefinery, identifying the best sustainability methodology to be applied to the whole integrated system.

h i g h l i g h t sCarop pulp syrup (CPS) and sugarcane molasses (SCM) were used as carbon sources. CPS at 75 g L À1 induced the highest fatty acid and carotenoid productivities. Flow cytometry detected differences between the cell membrane grown on CPS and SCM. R. toruloides growth on CPS induced lower ratio of permeabilised cells than on SCM. a r t i c l e i n f o b s t r a c tThe present work studied low-cost carbon sources for carotenoid and lipid production using the yeast Rhodosporidum toruloides NCYC 921. Carob pulp syrup and sugarcane molasses at different concentrations were used as low-cost carbon sources in R. toruloides batch cultivations. Carob pulp syrup containing a total sugar concentration of 75 g L À1 induced the highest total fatty acid productivity (1.90 g L À1 h À1 ) and the highest carotenoid productivity (9.79 lg L À1 h À1 ). Flow cytometric analysis revealed that most of the yeast cells (>60%) grown on carob pulp syrup displayed intact polarised membranes, conversely to the cells grown on sugarcane molasses, wherein a large proportion (>45%) displayed permeabilised cytoplasmic membranes.

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