Antarctic thraustochytrids: Producers of long‐chain omega‐3 polyunsaturated fatty acids

Abstract Thraustochytrids have been isolated from different aquatic systems; however, few studies have reported their occurrence in Antarctica. In this study, 13 strains close to strains belonging to the genera Oblongichytrium, Thraustochytrium, and Aurantiochytrium were isolated from seawater samples collected near the Antarctic Base Professor Julio Escudero (S 62°12′57′ E 58°57′35″). Docosahexaenoic acid (DHA) was found in the total lipids of all the isolates; DHA content of the biomass (dry weight) varied between 3.3 and 33 mg/g under the growth conditions for isolation. Five of the Antarctic thraustochytrids were able to accumulate lipids at levels higher than 20% w/w. Two strains, RT2316‐7 and RT2316‐13, were selected to test the effect of the incubation temperature (at 5°C for 14 days and at 15°C for 5 days). Incubation temperature had little effect on the lipid content and biomass yield; however, its effect on the fatty acid composition was significant (p < .05). The low incubation temperature favored the accumulation of eicosapentaenoic acid (EPA), palmitic acid and stearic acid in the total lipids of RT2316‐7. Percentage of EPA, DHA and the omega‐6 fatty acid dihomo‐γ‐linolenic acid of total fatty acids of RT2316‐13 was higher at the low incubation temperature. RT2316‐13 accumulated the highest lipid content (30.0 ± 0.5%) with a carbon to nitrogen mass ratio equal to 16.9. On the contrary, lipid accumulation in RT2316‐7 occurred at high concentration of the nitrogen sources (monosodium glutamate or yeast extract). The capability to accumulate lipids with a fatty acid profile that can be tuned through cultivation temperature make the Antarctic thraustochytrid RT2316‐13 a candidate for the production of lipids with different uses.


| INTRODUC TI ON
Docosahexaenoic acid (DHA, C22:6n3) is one of the long-chain most unsaturated omega-3 fatty acid, which is normally found in marine oily fish. DHA is also the main omega-3 fatty acid in the human brain where it is synthesized from the essential fatty acid α-linolenic acid (ALA; 18:3n3). (Weiser, Butt, & Mohajeri, 2016) However, the rate of conversion of ALA into DHA would not provide the required levels for optimal neurological functions, and its intake is suggested by health organizations.
Marine thraustochytrids are known because of their ability to synthesize DHA which is found distributed in cell membrane lipids and neutral lipids (triacylglycerols). The role of DHA in the cells of thraustochytrids is not fully understood but should contribute to the survival under the prevailing conditions of their habitats. One of those conditions is salinity since most of DHA producer strains requires sea salts, albeit at different levels. Few studies have evaluated the effect of salt concentration on fatty acid desaturation in thraustochytrids. (Burja, Radianingtyas, Windust, & Barrow, 2006;Kang, Jeh, Seo, Chun, & Hur, 2007) Enhanced tolerance to salt stress has been demonstrated in Saccharomyces cereviciae (Rodríguez-Vargas, Sánchez-García, Martínez-Rivas, Prieto, & Randez-Gil, 2007) and Synechococcus sp. (Allakhverdiev, Kinoshita, Inaba, Suzuki, & Murata, 2001) genetically engineered to increase the unsaturation of their fatty acids. The activation of the Na + /H + antiport system via enhanced fluidity of membranes is a plausible explanation. (Allakhverdiev et al., 2001).
The ability of microorganisms to adjust fluidity of cell membrane is essential to maintain important metabolic functions such as permeability, substrate uptake and the electron transport chain. (Denich, Beaudette, Lee, & Trevors, 2003) Unsaturation of fatty acids is a very effective mechanism for cell adaptation to low temperatures, because it has a higher effect on lipid fluidity. Changes in the unsaturation of fatty acids can be realized by de novo synthesis or desaturation of already existing fatty acids. (Russell, 1984) Low temperature would be a selection pressure for the evolution of microorganisms able to produce PUFA. (Nichols, Nichols, & McMeekin, 1995) In fact, sea ice surrounding Antarctica is one habitat where many PUFA producer bacteria have been isolated. (Nichols, 2003) The analysis of a large dataset of fatty acid profile from marine and terrestrial organisms led to the conclusion that compared to terrestrial organisms eicosapentaeonic acid (EPA, C20:5n3) and DHA are more abundant in marine organisms. (Colombo, Wacker, Parrish, Kainz, & Arts, 2017) In addition, the content of PUFA in polar and temperate marine organisms is higher than in organisms from the tropics.
To date, the main source of EPA and DHA is fish oil. Fatty acid composition of fish oil is very dependent on the fish (Sargent, 1997), and both the season (Bandarra, Batista, Nunes, Empis, & Christie, 1997) and latitude (Colombo et al., 2017) of the capture. Fish oil contains relative high concentrations of marine pollutants, especially persistent organic pollutants. (Sun et al., 2018) Therefore, a refining process is necessary to produce fish oil for human consumption.
The biotechnological production of DHA using heterotrophic microorganisms offers various advantages; (a) it is a sustainable process that could be based on cheap and renewable carbon and nitrogen sources, (b) under controlled growth conditions lipid yield and quality can be reproduced, and (c) it is free of pollutants.
Considering the biotechnological potential of thraustochytrids for the production of DHA, the aim of this study was to isolate novel strains from seawater and marine sediment samples collected in the Chilean Antarctic peninsula, to identify the isolates at a molecular level, and to evaluate the biomass and DHA production. The effect of incubation temperature on the biomass and lipid production, and the fatty acid composition of the total lipids was evaluated for two of the Antarctic thraustochytrids.

| Isolation of thraustochytrid strains
Samples of water and sediments were collected in sterile bottles from the coast near the Antarctic Base Professor Julio Escudero (S 62°12′57″ E 58°57′35″) in December 2016. The samples were supplemented with sterile pine pollen (ca. 15 mg/200 ml) and kept at 5°C. (Gaertner, 1968) After 15 days, pollen grains with attached microorganisms were collected by filtration (0.45 μm) and dispersed on solid medium. The composition of the solid medium was as follows: glucose (Merck) 1 g/L, yeast extract (BBL ™ , Becton, Dickinson and Co.) 6 g/L, and agar (Merck) 15 g/L, in artificial seawater 50% v/v. (Shene et al., 2013) Streptomycin sulfate and penicillin G (0.3 g/L of each) (Sigma) were used to avoid proliferation of bacteria. Incubation of the plates was carried out at 5 and 20°C until the colonies were visible. Individual colonies from plates incubated at 5 and 20°C were subcultured on solid medium at the same temperature until pure isolates were obtained (microscopic inspection). The isolated microorganisms were grown in a liquid medium (glucose 2 g/L, yeast extract 6 g/L, monosodium glutamate (Merck) 0.6 g/L, in ASW 50% v/v) at the temperature at which they were isolated. Stock cultures, prepared by transferring 0.5 ml of the grown culture into a microtube containing sterile glycerol (0.5 ml), were maintained at −80°C.

| Microscopic analysis
Cell morphology was observed using an optical microscope (Olympus CX22LED) with 40× magnification. Lipid distribution in cells stained with BODIPY ® 581/591 (Thermo Fisher Scientific, Molecular Probes ™ ) (300 μl of the cell culture incubated with 10 μl of the fluorophore) was observed using an Olympus Fluoview (FV) 1,000 confocal laser microscopy (Olympus). The images were obtained with a 100× magnification (immersion oil) using the software included in the equipment (FV10-ASW 2.0).

| DNA extraction and identification of isolates
The UltraClean ™ Kit (Mo Bio Laboratories, Inc.) was used for DNA isolation. DNA was quantified in an Infinity R 200 Pro microplate reader (Tecan). The sequence of the 18S rRNA gene was amplified using the conditions for PCR described by Mo et al. with some modi-fications. (Mo, Douek, & Rinkevich, 2002)

| Biomass growth and lipid production
The inoculum to the culture experiments was prepared in Erlenmeyer flasks (250 ml) containing 100 ml of the sterile medium (glucose 20 g/L, yeast extract 6 g/L, monosodium glutamate 0.6 g/L, in 50% v/v ASW) to which 5 ml of the grown culture, described in 2.

| Effect of the medium composition on the biomass and lipid production
The effect of the initial glucose concentration (0, 10, 20, 30, and 40 g/L), yeast extract concentration (0, 3, 6, and 9 g/L), and monosodium glutamate concentration (0.0, 0.3, 0.6, and 0.9 g/L) on the biomass concentration and lipid content of RT2316-7 and RT2316-13 after 5 d incubation at 15°C was determined. The effect of each factor (concentration of glucose, yeast extract, and monosodium glutamate) was evaluated by changing one factor at the time. Experiments were carried out in duplicate as described in 2.4.

| Lipid content and fatty acid composition
Lipids in the freeze-dried biomass (50 mg) were extracted (1 hr, 215 g) with 9.5 ml of a solvent system chloroform/methanol/phosphate buffer (50 mM, pH 7.4) in the volume ratio of 2.5:5.0:2.0. (Bligh & Dyer, 1959) The mixture was transferred to a conical separating funnel containing 2.5 ml of chloroform; after mixing, 2.5 ml of phos-

| Statistical analysis
One-way ANOVA was used for data analysis. Duncan multiple range test was used for mean comparison at a level of 5%.

| Isolation and identification of Antarctic thraustochytrids
Thirteen thraustochytrid strains were isolated and molecularly identified through 18S rRNA sequencing (Table A1). A phylogenetic tree constructed with the sequences is shown in Figure 1; for comparison purposes, the phylogenetic tree also included the sequence of a Thraustochytrium striatum previously isolated. (Shene et al., 2019) The isolates formed four groups of closest relatives and the more distantly related was RT2316-9. Microscopic observation revealed that some strains grew forming cell aggregates while in other cells were found isolated ( Figure A1). Another morphological characteristic that allowed to differentiate the isolates was the presence of zoospores after transferring a grown culture to fresh medium; this was observed in strains RT2316-5, RT2316-10, RT2316-11, RT2316-12, and RT2316-13. Under the growth conditions used, strains RT2316-5, RT2316-10, RT2316-7, and RT2316-8 exhibited an orange pigmentation.

| Biomass concentration and lipid content of the biomass of isolated strains
To evaluate the production of biomass and lipids, the Antarctic thraustochytrids were cultivated in a glucose-yeast extract-monosodium glutamate medium. The incubation temperature was that at which isolation was made (5 or 20°C). The strains RT2316-12 and RT2316-13, incubated at 5°C, reached biomass concentrations higher than 5 g/L ( Figure 2a). Among the strains isolated and cultivated at 20°C, RT2316-11 and RT2316-4 reached biomass concentrations higher than 2.5 g/L. The lowest biomass concentration (<0.5 g/L) corresponded to RT2316-10 ( Figure 2a).
The content of DHA in the dry biomass varied between 3.3 (in RT2316-1) and 33 mg/g (in RT2316-13) ( Figure 2c). Fatty acid composition of the total lipids extracted from the biomass of the Antarctic thraustochytrids is shown in Figure 2d. The lipids of all the strains contained EPA and DHA; DHA percentage ranged from 25.5% (in RT2316-12) to 74.5% (in RT2316-3) while EPA percentage varied between 1.2% (in RT2316-5) and 20.5% (in RT2316-13). The highest percentage of palmitic acid and stearic acid was found in the lipids of RT2316-2 and RT2316-1, respectively.

| Effect of incubation temperature on biomass, lipids, and DHA production by two selected strains
The strains RT2316-13 and RT2316-7 were selected to study the effect of incubation temperature on biomass, lipids, and DHA pro-  At the low temperature, the growth curve of RT2316-7 exhibited two phases ( Figure 3a). The specific growth rate decreased from 1.7 × 10 −2 hr −1 (48-192 hr) to 2.0 × 10 −3 hr −1 (192-336 hr). Only 25.9% of the initial glucose was consumed during the 14 days incubation, and the final biomass concentration was 1.8 g/L ( Figure 3a).
As the incubation time increased, EPA and DHA percentage of total fatty acids of cells cultivated at the low and high temperature F I G U R E 2 Biomass concentration (a), lipid content of the biomass (b), fatty acid composition of the total lipids (c), and DHA content of the biomass of the Antarctic thraustochytrids (d). Gray and white bars in a, b, and c correspond to the strains cultivated at 5 and 20°C, respectively. The strains are (1) RT2316-13, (2) RT2316-12, (3) RT2316-11, (4) RT2316-4, (5) RT2316-6, (6) RT2316-8, (7) RT2316-7, (8) RT2316-9, (9) RT2316-3, The main fatty acids in the total fatty acids of RT2316-7 cultivated at 5°C were palmitic acid (13.8 ± 2.5-18.8 ± 1.1% w/w), stearic acid (8.5 ± 0.0-15.8 ± 0.4% w/w), EPA (16.1 ± 1.2-27.2 ± 1.1% w/w), and DHA (25.9 ± 2.0-39.6 ± 5.0% w/w) ( Table 2). The omega-3 PUFA represented more than 40% of the total fatty acids of RT2316-7 cultivated at the low temperature whereas the content of omega-6 PUFA was lower than 14%. At the high incubation temperature, DGLA percentage of total fatty acids was significantly higher (on average 5.7-fold) than in total fatty acids of RT2316-7 cultivated at 5°C. DHA percentage of the total fatty acids was not significantly affected (p > .05) by the incubation temperature. On the contrary, the high incubation temperature decreased (on average 30%) the EPA percentage of the total fatty acids of RT2316-7. The SFA percentage of total fatty acids of RT2316-7 cultivated at 15°C was lower (9.5%-18.1% w/w) than in RT2316-7 cultivated at 5°C (22.7%-34.9% w/w). On average, total fatty acids of RT2316-7 cultivated at 15°C had a PUFA/SFA ratio higher than the total fatty acids of RT2316-7 cultivated at 5°C. Table A2 shows the effect of glucose concentration, yeast extract concentration, and monosodium glutamate concentration on the biomass concentration and lipid content of RT2316-7 and RT2316-13 cultivated at 15°C for 5 d. Biomass concentration of RT2316-13 was significantly higher (p < .05) when the initial glucose concentration was 30 g/L. The increase of glucose concentration from 0 to 30 g/L increased 9.9-and 1.6-fold the biomass concentration and the lipid content of the biomass, respectively. The initial glucose concentration had a small effect on the biomass concentration of RT2316-7;

| Effect of medium composition on the production of biomass and lipids
the highest biomass concentration was obtained with an initial concentration of glucose equal to 20 g/L. The increase of glucose concentration from 0 to 20 g/L increased 1.4-fold both the biomass concentration and lipid content of RT2316-7. Yeast extract is a complex mixture of nitrogen compounds (amino nitrogen 6.9% w/w, total nitrogen 11.4% w/w) (BD biosciences, 2015) which also provides minerals and some carbohydrates. The increase of yeast extracts concentration from 0 to 9 g/L allowed increases of 5.7-and 8.5-fold in the biomass concentrations of RT2316-13 and RT2316-7, respectively. The lipid content of decreased significantly (p < .05) when the initial yeast extract concentration increased; the lowest lipid content of RT2316-13 (18.4 ± 1.0% w/w) was obtained with the highest concentration of yeast extract (9 g/L). The observed increase in lipid content of RT2316-7 was not significant (p > .05). The effect of F I G U R E 3 Growth curve (□) of the Antarctic thraustochytrids RT2316-7 (a and b) and RT2316-13 (c and d) cultured at 5°C (a and c) and 15°C (b and d).
Concentration of glucose (•) and the total lipid content of the biomass (×) are also shown TA B L E 1 Effect of incubation time (days) and incubation temperature (5 and 15°C) on the fatty acid composition of the total lipids produced by the Antarctic thraustochytrid RT2316-13. Incubation was made for 14 d at 5°C and for 5 d at 15°C  TA B L E 2 Effect of the incubation time (days) and incubation temperature (5 and 15°C) on the fatty acid composition of the total lipids produced by the Antarctic thraustochytrid RT2316-7. Incubation was made for 14 d at 5°C and for 5 d at 15°C  monosodium glutamate concentration, at levels tested, was significant (p < .05) on the lipid content of both strains; the lipid content of RT2316-7 increased 1.4-fold when the monosodium glutamate concentration increased from 0 to 0.9 g/L whereas for RT2316-13, the highest lipid content of was obtained with a concentration equal to 0.3 g/L.

| D ISCUSS I ON
Thraustochytrids are eukaryotic protists, with abundant occurrence in diverse aquatic ecosystems. It has been reported that thraustochytrids are the most common fungus-like organisms in the sea, with densities up to 73,000 cells/L in marine sediments. (Raghukumar, 1992) In Antarctic waters, lower densities (5-100 cells/L) have been reported. (Bahnweg & Sparrow, 1974) Riemann and Schaumann reported a dense occurrence of thraustochytrid-like cells in the lower section of a fast ice core drilled close to the southern shelf ice margin of the Weddell Sea in the Antarctic. (Riemann & Schaumann, 1993) To our knowledge, only three reports have described and characterized strains belonging to the genera Thraustochytrium and Aplanochytrium in Antarctic samples. (Bahnweg & Sparrow, 1974;Caamaño et al., 2017;Moro, Negrisolo, Callegaro, & Andreoli, 2003) In this work, strains close to members of the genera Oblongichytrium, Thraustochytrium, and Aurantiochytrium, were isolated; in addition, 3 strains close to members of Thraustochytriidae family were isolated. All the isolates were able to produce lipids containing DHA.
Nevertheless, significant differences in biomass concentration were observed when these were cultivated in the glucose-yeast extractmonosodium glutamate medium not optimized for the production of lipids. Most of the Antarctic thraustochytrids were able to reach biomass concentrations higher than British isolates (ca. 0.78 g/L) and with higher lipid content. (Marchan et al., 2017) Among the Antarctic thraustochytrids, five strains (RT2316-10, RT2316-4, RT2316-11, RT2316-12, and RT2316-13) were able to accumulate lipids at levels higher than 20% w/w suggesting that these would be oleaginous strains.
Of the two Antarctic thraustochytrids selected to characterize the lipid production and the lipid composition as a function of incubation time at different temperatures, RT2316-7 is particularly attractive because of the high DHA percentage of the total fatty acids. Nevertheless, because of the scarce glucose uptake, the design of a medium suitable for biomass and lipid production will be necessary. Compared to RT2316-7, the strain RT2316-13 is characterized by a higher glucose affinity, a higher specific growth rate, and a higher content of lipids, which compensate the lower DHA percentage of the total fatty acids. The fatty acid composition of RT2316-7 and RT2316-13 might suggest that EPA and DHA are synthesized through a pathway different from that used for the synthesis of other PUFA. In thraustochytrids, long-chain PUFA can be synthesized via the aerobic desaturase/elongase pathway or the anaerobic polyketide synthase (PKS-like PUFA synthase). (Metz, 2001) The former comprises a series of alternating desaturation and elongation steps acting on SFA produced in fatty acid synthase pathway. (Matsuda et al., 2012) The PKS-like PUFA synthase is a multiple enzyme system in which reiterative cycles of condensation, reduction, dehydration, and isomerization steps, introduce the double bounds as the fatty acid chain is elongated. Compared to the aerobic desaturase/elongase pathway, the PKS-like PUFA synthase pathway would produce relatively pure end-products.
It was noted that the incubation temperature and time have dramatic effects on the fatty acid composition of RT2316-13. In a batch culture, the incubation time involves all the changes in the composition of the medium that occur as biomass grows. On the other hand, incubation temperature determines the rate of enzymatic reactions and the specific growth rate. At 15°C, the specific growth rate of RT2316-13 was 5.4-fold higher than the specific growth rate at 5°C. At the low temperature, EPA and DHA were the main fatty acids in the total fatty acids of the initially harvested biomass whereas at the high temperature the percentage of omega-6 PUFAs (linoleic acid and DGLA) was higher. Since the two omega-6 PUFAs were also found in the total fatty acids of the biomass cultivated at 5°C, it could be assumed that these are precursors of EPA. For this, a Δ5 fatty acid desaturase acting on DGLA would produce arachidonic acid (found at low levels, of total fatty acids) at 15°C and was not observed at 30°C. These results might suggest that the effect of temperature on the activity Δ6 desaturase, responsible for linoleic acid (C18:2n6) desaturation into γ-linolenic acid (C18:3n6) a precursor of DGLA, from different sources is the same. Although the initial objective of this study was to isolate novel thraustochytrids for the production of DHA, DGLA has also attracted the interest because of its medical applications. (Kawashima et al., 2000) DGLA percentage of the total fatty acids of RT2316-13 is similar to that reported in a mutant M. alpina (39.8 -43.1% w/w) in which the low Δ5 activity decreases the DGLA desaturation to arachidonic acid. (Kawashima et al., 2000).
The strain RT2316-7 accumulates lipids with a high content of EPA and DHA that together represent between 44.5% and 64.6% of the total fatty acids. The DHA percentage (27.9-49.4%) of the total fatty acids of RT2316-7 is two-to fourfold higher than of sardine oil (11%). (Morais et al., 2001) Therefore, this Antarctic thraustochytrid is a promising candidate for the biotechnological production of lipids rich in long-chain omega-3 PUFA. The composition of the total fatty acids of RT2316-7 suggests that DHA is not a product of elongation and desaturation activities on SFA. Incubation temperature (5 or 15°C) has no effect on the DHA percentage of the total fatty acids of RT2316-7. However, the high temperature promotes the elongation and desaturation of palmitic acid into DGLA.
The capability of microorganisms to grow at low temperatures is attributed to the ability to desaturate fatty acids because the presence of PUFA is relevant to maintain the correct phase of membrane lipids. In agreement with this, the ratio PUFA/SFA of the total fatty acids of RT2316-13 was, on average, higher in cells cultivated at 5°C than in cells cultivated at 15°C. This pattern would not be exhibited by all microorganisms as it is the case of RT2316-7. A possible explanation would be that not all PUFA are needed to maintain membrane fluidity at a low incubation temperature. On the other hand, the different lipid content of both strains (ca. 10% in RT2316-7 and 30% in RT2316-13) suggests that the extracted lipids from RT2316-7 correspond to membrane lipids whereas those extracted from RT2316-13 are both storage and membrane lipids.
Although optimizing culture conditions for the production of DHA was not an objective of the present study, the effect of the concentration of the 3 components of the growth medium on the biomass and lipid production by the two selected strains was analyzed. A high carbon to nitrogen mass ratio is usually effective for increasing the lipid content of oleaginous microorganisms. (Converti, Casazza, Ortiz, Perego, & Borghi, 2009) This effect was observed in RT2316-13 which accumulated the highest lipid content (30.0 ± 0.5%) with a carbon to nitrogen mass ratio equal to 16.9. On the contrary, lipid accumulation in RT2316-7 occurred at a high concentration of monosodium glutamate or yeast extract. This is not surprising if it is considered that because of the low glucose uptake an alternative carbon source is consumed by this strain. Some amino acids can be consumed to be converted into pyruvate or intermediates of the citric acid cycle (glucogenic amino acids) while others can be channeled into acetyl-CoA (ketogenic amino acids), the building block for the synthesis of fatty acids.
Thraustochytrids, similar to strains belonging to different genera able to produce lipids containing DHA, were isolated from water and sediment samples collected at the coast near the Antarctic Base Professor Julio Escudero (S 62°12′57″E 58°57′35″). Interestingly, one of the strains for which the isolation methodology was carried out at 5°C was able to produce the highest biomass concentration with a high lipid content. A low incubation temperature (5°C) increased the percentage of EPA and DGLA of the total fatty acids of RT2316-7 and RT2316-13, respectively. A more detailed analysis of the fatty acid composition of membrane lipids (phospholipids, glycolipids, and phosphoglycerides) could show if these changes are the responses of an adaptation mechanism that allow these two strains to grow at low temperatures.

This research was supported by Institute Antarctic Chilean project
Inach RT 23-16, postdoctoral project Fondecyt N°3170610, the Centre for Biotechnology and Bioengineering (CeBiB) FB-0001, and Direction of Investigation, Universidad de La Frontera (GAP funding and undergraduate program).

CO N FLI C T O F I NTE R E S T S
None declared. A. wrote the manuscript.

E TH I C A L A PPROVA L
None required.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data are provided in full in the results section and the Appendix of this paper apart from the partial sequences of the small subunit ribosomal RNA gene which are available at the GenBank (https ://www.ncbi.nlm.nih.gov/) under the assigned accession numbers given in Table A1.