Liset Flores

Thraustochytrids attract biotechnological interest because of their ability to synthesize carotenoids and lipids rich in long-chain omega-3 poly-unsaturated fatty acids: specifically, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). These fatty acids can be found among the different lipid classes. In this work, the effects of initial glucose concentration on the growth and the content of lipids and carotenoids in Thraustochytrium sp. RT2316-16 were determined. The fatty acid composition of the neutral and phospholipid fractions was compared. Results obtained in shake flasks showed that increasing the initial glucose concentration from 5 to 20 g L⁻¹ did not affect the growth rate during the first 24 h but increased the maximum biomass concentration 1.4-fold (from 6.3 to 8.6 g L⁻¹) and the carotenoid concentration 1.9-fold while reducing the percentages of EPA and DHA in total fatty acids. DHA was one of the major fatty acids in the phospholipids (37–42% of total fatty acids). The extraction of total lipids from the dry biomass with supercritical CO₂ (SCC) had a low yield (59 mg (g DW)⁻¹; 34% of the extraction yield attained with the Bligh and Dyer method). However, when ethanol was used as the co-solvent (SCC-E), the extraction of phospholipids (343 ± 17 mg (g extract)⁻¹) and carotenoids was improved. The composition of the fatty acids in the lipid extracts depended on the lipid extraction method (SCC, SCC-E, Soxhlet with hexane, or the Bligh and Dyer method).

The biomass and lipid production responses of the psychrophilic marine thraustochytrid Thraustochytrium sp. RT2316-16 were assessed in chemically defined media comprising glucose, up to 17 amino acids and up to 9 B-vitamins and mineral salts. Compared to the control medium with all amino acids and B-vitamins (biomass concentration: 7.1 ± 0.1 g L⁻¹; total lipid content: 30.4 ± 0.5% of the DW), the growth of RT2316-16 was reduced by more than 50% in the medium that lacked cyanocobalamin or pyridoxamine. The total lipid content of the biomass grown in the absence of vitamins was 63% lower than in the biomass produced in the control medium. The composition of the B-vitamin mixture modulated the fatty acid composition, an effect that may have been related to the availability of dissolved oxygen. In bioreactor culture with the dissolved oxygen level controlled to ≥10% of air saturation, the microorganism consumed all 17 amino acids; 8 of the amino acids were fully consumed within a 0–33 h period, in which the specific growth rate was 0.065 h⁻¹. Under these culture conditions, the sum of eicosapentaenoic acid and docosahexaenoic acid in the total fatty acid content rose from 15% (at time 0) to 54% (after 95 h). A medium that contained the 9 amino acids that were not preferentially consumed favored the accumulation of total lipids, but reduced biomass growth.

Interest in probiotics has not diminished, and techniques to protect them from the environment in which they are found are constantly being innovated. Spray-drying is the most studied and industrially used technique to encapsulate probiotics. Recently, a new process has been developed in which particle formation, alginate cross-linking, and drying are carried out in a single step. In this study, Bifidobacterium infantis, Bifidobacterium longum, Lactobacillus plantarum, and Lactobacillus rhamnosus were microencapsulated by spray-drying using a cross-linked alginate matrix supplemented with chia seed mucilage (CM) or flaxseed mucilage (FM) as the coating material. All formulations evaluated, supplemented with 0.4% (w/v) of CM or FM, including the control formulation showed high survival rates, varying between 87% and 97%. The viability of microencapsulated probiotics was affected by storage temperature. At 4 °C, viability decreased slightly, and after 90 days, the viable probiotic count ranged from 7 to 11 Log CFU/g of dry powder. Meanwhile, viability did not exceed 4 Log CFU/g of dry powder at 37 °C. Probiotic microencapsulation in cross-linked alginate matrices and chia or flaxseed mucilage by spray-drying is presented as a promising alternative for their protection, potentially improving the long-term stability and efficacy of the probiotic product.

This work analyzes the production of total lipids and the content of CoQ₉ and CoQ₁₀ in the biomass of Thraustochytrium sp. RT2316-16 grown in media containing a single amino acid at a concentration of 1 g L⁻¹ as the sole nitrogen source; glucose (5 g L⁻¹) was used as the carbon source. Biomass concentration and the content of total lipids and CoQ were determined as a function of the incubation time; ten amino acids were evaluated. The final concentration of the total biomass was found to be between 2.2 ± 0.1 (aspartate) and 3.9 ± 0.1 g L⁻¹ (glutamate). The biomass grown in media containing glutamate, serine or phenylalanine reached a content of total lipids higher than 20% of the cell dry weight (DW) after 72, 60 and 72 h of incubation, respectively. The highest contents of CoQ₉ (39.0 ± 0.7 µg g⁻¹ DW) and CoQ₁₀ (167.4 ± 3.4 mg g⁻¹ DW) in the biomass of the thraustochytrid were obtained when glutamate and cysteine were used as the nitrogen source, respectively. Fatty acid oxidation, which decreased the total lipid content during the first 12 h of incubation, and the oxidation of hydrogen sulfide when cysteine was the nitrogen source, might be related to the content of CoQ₁₀ in the biomass of the thraustochytrid.

Coenzyme Q (CoQ; ubiquinone) is an essential component of the respiratory chain. It is also a potent antioxidant that prevents oxidative damage to DNA, biological membranes, and lipoproteins. CoQ comprises a six-carbon ring with polar substituents that interact with electron acceptors and donors, and a hydrophobic polyisoprenoid chain that allows for its localization in cellular membranes. Human CoQ has 10 isoprenoid units (CoQ₁₀) within the polyisoprenoid chain. Few microorganisms produce CoQ₁₀. This work shows that Thraustochytrium sp. RT2316-16 produces CoQ₁₀ and CoQ₉. The CoQ₁₀ content in RT2316-16 depended strongly on the composition of the growth medium and the age of the culture, whereas the CoQ₉ content was less variable probably because it served a different function in the cell. Adding p-hydroxybenzoic acid to the culture media positively influenced the CoQ₁₀ content of the cell. The absence of some B vitamins and p-aminobenzoic acid in the culture medium negatively affected the growth of RT2316-16, but reduced the decline in CoQ₁₀ that otherwise occurred during growth. The highest content of CoQ₉ and CoQ₁₀ in the biomass were 855 μg g⁻¹ and 10 mg g⁻¹, respectively. The results presented here suggest that the thraustochytrid RT2316-16 can be a potential vehicle for producing CoQ₁₀. Metabolic signals that trigger the synthesis of CoQ₁₀ in RT2316-16 need to be determined for optimizing culture conditions.

Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and carotenoids are needed as human dietary supplements and are essential components in commercial feeds for the production of aquacultured seafood. Microorganisms such as thraustochytrids are potential natural sources of these compounds. This research reports on the lipid and carotenoid production capacity of thraustochytrids that were isolated from coastal waters of Antarctica. Of the 22 isolates, 21 produced lipids containing EPA+DHA, and the amount of these fatty acids exceeded 20% of the total fatty acids in 12 isolates. Ten isolates were shown to produce carotenoids (27.4–63.9 μg/g dry biomass). The isolate RT2316-16, identified as Thraustochytrium sp., was the best producer of biomass (7.2 g/L in five days) rich in carotenoids (63.9 μg/g) and, therefore, became the focus of this investigation. The main carotenoids in RT2316-16 were β-carotene and canthaxanthin. The content of EPA+DHA in the total lipids (34 ± 3% w/w in dry biomass) depended on the stage of growth of RT2316-16. Lipid and carotenoid content of the biomass and its concentration could be enhanced by modifying the composition of the culture medium. The estimated genome size of RT2316-16 was 44 Mb. Of the 5656 genes predicted from the genome, 4559 were annotated. These included genes of most of the enzymes in the elongation and desaturation pathway of synthesis of ω-3 polyunsaturated fatty acids. Carotenoid precursors in RT2316-16 were synthesized through the mevalonate pathway. A β-carotene synthase gene, with a different domain organization compared to the gene in other thraustochytrids, explained the carotenoid profile of RT2316-16.

Seaweed processing generates liquid fraction residual that could be used as a low-cost nutrient source for microbial production of metabolites. The Rhodotorula strain is able to produce antimicrobial compounds known as sophorolipids. Our aim was to evaluate sophorolipid production, with antibacterial activity, by marine Rhodotorula rubra using liquid fraction residual (LFR) from the brown seaweed Macrocystis pyrifera as the nutrient source. LFR having a composition of 32% w/w carbohydrate, 1% w/w lipids, 15% w/w protein and 52% w/w ash. The best culture condition for sophorolipid production was LFR 40% v/v, without yeast extract, artificial seawater 80% v/v at 15 °C by 3 growth days, with the antibacterial activity of 24.4 ± 3.1 % on Escherichia coli and 21.1 ± 3.8 % on Staphylococcus aureus. It was possible to identify mono-acetylated acidic and methyl ester acidic sophorolipid. These compounds possess potential as pathogen controllers for application in the food industry.

Oblongichytrium RT2316-13 synthesizes lipids rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The content of these fatty acids in the total lipids depended on growth temperature. Sequencing technology was used in this work to examine the thraustochytrid’s response to a decrease in growth temperature from 15 °C to 5 °C. Around 4% (2944) of the genes were differentially expressed (DE) and only a few of the DE genes (533 upregulated; 206 downregulated) had significant matches to those in the SwissProt database. Most of the annotated DE genes were related to cell membrane composition (fatty acids, sterols, phosphatidylinositol), the membrane enzymes linked to cell energetics, and membrane structure (cytoskeletal proteins and enzymes). In RT2316-13, the synthesis of long-chain polyunsaturated fatty acids occurred through ω3- and ω6-pathways. Enzymes of the alternative pathways (Δ8-desaturase and Δ9-elongase) were also expressed. The upregulation of the genes coding for a Δ5-desaturase and a Δ5-elongase involved in the synthesis of EPA and DHA, explained the enrichment of total lipid with these two long-chain fatty acids at the low temperature. This molecular response has the potential to be used for producing microbial lipids with a fatty acids profile similar to that of fish oils.