Screening and Identification of Coastal Chilean Thraustochytrids for Arachidonic Acid Production: Biotechnological Potential of Ulkenia visurgensis Lng2-Strain

Thraustochytrids are unicellular heterotrophic marine protists that have been described as producing a high content of polyunsaturated fatty acids (PUFAs). Among them, arachidonic acid (ARA) stands out as a precursor of several mediators of pivotal importance for the immune system. However, the biotechnological potential of thraustochytrids for ARA production has not been developed. The objective of this study is to isolate and identify native strains from different Chilean coastal environments and evaluate in vitro the effect of culture parameters such as C/N ratio (19 and 33) and temperature (15 °C and 23 °C) on biomass production and arachidonic acid content. A total of nine strains were identified and classified into four genera of the Thraustochitridae family. The Lng2 strain with 99% identity belongs to the species Ulkenia visurgenis and was the most prominent one for ARA production. Temperature had an effect on the PUFA profile but not on the ARA content nor on the biomass yield. Additionally, the C/N ratio has been identified as a key parameter. The ARA productivity increased by 92% (from 0.6 to 8.3 ARA mg/g-DW) and its total biomass by 62.7% (from 1.9 to 5.1 g/L) at a high C/N ratio (33) as compared to the control.

PUFAs are necessary as nutrients for human and animal health, growth, and development [11,12]. Two families represent these PUFAs: n-6 (u ω-6) and n-3 (u ω-3), which are biosynthesized from linoleic acid and linolenic acid, respectively; humans cannot synthesize Considering the biotechnological potential of thraustochytridiae in the production o ARA, studies that undertake the search for ARA-producing strains are needed. Accord ingly, the objective of this work was to isolate and identify thraustochytrids from differen Chilean estuarine and coastal environments, evaluating in vitro their ARA productio and studying the effect of culture parameters such as C/N ratio and temperature on bio mass production and ARA content.

Isolation and Culture Conditions
Marine samples were collected from five different Chilean coastal sites and habitats Samples were obtained from the water column (15-20 cm depth) or sediment. A samplin sites map was constructed using the Google Maps platform (Figure 1). For thrausto chytrids isolation, the pollen technique was performed [48]. The presence of thrausto chytrids adhered to pine pollen was verified by optical microscopy. Subsequently, sam ples were filtered (nylon filter, 20 μm) and cultivated on solid culture medium 790 (yeas extract 1 g/L, peptone 1 g/L, glucose 5 g/L, and agar 15 g/L) prepared in diluted artificia seawater (NaCl 27.12 g/L, MgCl2×6H2O 5.23 g/L, MgSO4×7H2O 6.77 g/L, CaCl2×H2O 0.1 g/L, KCl 0.725 g/L, and NaHCO3 0.202 g/L) and a final concentration of 0.3 g/L of strepto mycin and penicillin G (Sigma Aldrich Co., Steinheim, Germany). Incubation was per formed at 22 °C for 5 to 7 days. Selected colonies were subcultured in liquid 790.
The obtained sequences were compared with those available in the Genbank database (National Center for Biotechnology Information, USA: NCBI Home page http://www.ncbi accessed on 10 October 2021). The search for the most related homologous sequences was performed using the BLAST program available on the NCBI web server.
Taxonomic evaluation, DNA alignment, and interpretation of sequences were performed by using Geneious Prime software [52]. BLAST was performed against the GenBank database. Representative sequences alignment was done by using MUSCLE [53]. Jmodeltest 2.1.10 [54] software was used for the alignment best-fit model of nucleotide substitution selection. Phylogenetic reconstruction was done by using Bayesian inference (BI) method in MrBayes 3.2.6 [55] geneious plugin.

Screening for High ARA-Producing Strains and Total Biomass Assessment
Pure strains obtained in 2.2 were cultivated in triplicate, in 100 mL 790-liquid medium using 250 mL flasks, at 20 • C and 120 rpm for 7 days. To determine the final biomass resulting from each cultivated strain, the biomass was washed thrice, centrifuged at 1798 rcf for 5 min, and finally, the biomass was lyophilized, and dry weight (DW) was determined gravimetrically.
For the extraction of total lipids, Folch's method [56] was applied. Each sample was homogenized in a chloroform/methanol (2:1, v/v) mixture, then centrifuged at 1614 rcf in a refrigerated centrifuge (Ependorf, model 4303), to separate the water-soluble and organic phases. The organic phase was then separated, and the chloroform was evaporated using a mini-evaporator. The amount of total lipids present in each sample was expressed as a percentage of its dry weight. Fatty acids methyl esters (FAME) were prepared from the extracted lipids according to Morrison and Smith (1964) [57].
Fatty acids were separated in a gas chromatograph (Hewlett Packard 6890 series II Plus, Wilmington, NC, USA) using a 30 m × 0.25 mm × 0.20 µm capillary column SP TM 2380 (Supelco, Bellefonte, PA, USA). Helium was used as a carrier gas. FAME was analyzed by comparison with a well characterized standard such as SUPELCO TM 37 component FAME Mix (Sigma-Aldrich, St. Louis, MO, USA). Fatty acids were expressed as percentage of total identified FAME. The productivity of ARA, EPA, and DHA were expressed in mg/dry biomass.

Temperature Effect on Growth Curve and Glucose Consumption
The highest ARA-producing strains detected in 2.3, i.e., ARA mg /biomass g-DW were cultivated at different temperatures (15 • C and 23 • C) for 5.5 days at 120 rpm. Biomass was estimated in triplicate, initially every 3 h and then every 12 h, by cell DW as follows: Cell cultures were harvested, centrifuged (1798 rcf for 5 min), washed (three times) with distilled water, and subsequently lyophilized for gravimetric determination. Glucose consumption was estimated spectrophotometrically at the same time intervals as biomass, using a commercial SPINREACT Kit (Glucose-LQ).

C/N Ratio Effect on Productivity de ARA
To evaluate the effect of C/N ratio on ARA production, batch fermentations were performed using the selected Ulkenia visurgensis (Leng2 strain).
An accurate determination of C and N content present in the reagents used was performed by elemental analysis Thermo Scientific (FlashSmart™ Elemental Analyzer) at Universidad Católica de Temuco (Table 1). While preparing the culture media, the C/N ratio was modified by altering the content of the main carbon source (glucose from 10 to 40 g/L) and the content of Peptone (from 2 to 4 g/L) and yeast extract (from 1 to 2 g/L).
The C/N ratio of each medium, control (15), C/N 19 (1), and C/N 33 (2), was calculated considering the amount of carbon and nitrogen provided by the reagents: glucose, peptone, and yeast extract. The experiments were performed in triplicate and cultured for 5 days with orbital agitation (120 rpm) at 23 • C.
The C/N ratio of each medium, listed in Table 1, was calculated by the amount of carbon and nitrogen provided by glucose, peptone, and yeast extract. The experiments were performed in triplicate and cultured for 5 days with orbital agitation (120 rpm) at 23 • C.

Statistical Analysis
Statistical analyses (ANOVA and Student's t-test) were performed using the MATLAB program (R2019b, The MathWorks Inc., Natick, MA, USA; 2018) to compare biomass and ARA yield obtained in the native strains and the treatments with C/N ratio in the Lng2 strain, respectively. A significance level of 95% (p < 0.05) was used to determine significant differences.
Under microscopic observation, the strain Lng2 (U. visurgensis) presents a spherical shape with mature sporangia containing zoospores ( Figure 2). The comparison of the 18S rRNA gene shows that the Lng2 strain exhibits a common ancestor, with two strains; Ulkenia aff visurgensis BAFCult 3541 (HQ228980) and Ulkenia aff visurgensis BAFCult 3529 (HQ228958), with a bootstrap value of 0.99 and 0.94, respectively ( Figure 3). The 18S rRNA partial sequence of strain Lng2 are available in GenBank (accession number: OM228767) ( Figure 3). Under microscopic observation, the strain Lng2 (U. visurgensis) presents a spherical shape with mature sporangia containing zoospores (   The productivity of ARA, EPA, and DHA in mg/biomass g-DW and their respective percentages (related to their total lipids) was determined in all the native strains isolates (Table 3). ARA was detected in the nine identified strains with percentages of total lipids from a minimum of 2.59% to a maximum of 9.31% in the Pch3 and LNO strains, respectively. The maximum cellular biomass (DW) corresponds to the Lng3 strain with 1.12 g-DW/L. However, its percentage of ARA (2.97%) is low, limiting its biotechnological potential. On the other hand, the Lng2 strain isolated from an estuarine zone, with 5.44% of ARA has the highest productivity (5.81 ARA mg/ biomass g DW); although its biomass is low (0.45 g/L), its DHA percentage levels (44.78%), EPA (16.26%), and total lipids (10.68%) are remarkably high compared to the other strains. Based on Lng2 (U. visurgensis) high PUFAs productivity including ARA, the culture parameters were assayed for that strain by evaluating the effect of temperature and the C/N ratio on the productivity of ARA and cell biomass.   Figure 4 shows the total biomass growth kinetics of Lng2 strain during a 5.5 d (132 h) culture at two temperatures (15 and 23 • C) and its respective glucose consumption. Lng2 grew faster at 23 • C than at 15 • C. The exponential growth 3-5 d (72-120 h) and the stationary phase (from 5 d onwards) occurred at the same intervals for both temperatures. At 23 • C, the maximum biomass was obtained at 1.723 g-DW/L at 120 h. Additionally, in relation to glucose, it was observed that the remaining substrate is 1.04 g glucose/L at 23 • C (132 h). However, at 15 • C and similar culture hours, the value is three times higher than in the higher temperature (3.44 g glucose/L), which is an interesting trait for industrial scaling prospects.
OR PEER REVIEW 9 of 16 The bars indicate ± standard error (n = 3).
The effect of temperature (15 °C and 23 °C) on the fatty acid profile and yield of ARA was evaluated during the exponential phase (from 3 d) and at the beginning of the stationary phase (5 d) ( Table 4). The maximum biomass is 1.46 g-DW/L (exponential phase) and the minimum is 1.09 g-DW/L (stationary phase), both at 23 °C. The maximum yield of ARA (mg/biomass g DW) is at 3.87 (15°C) and the minimum is at 1.88 (23 °C), both in the exponential phase.
Lower temperature does affect the profile of fatty acids, mainly PUFAs. At 15 °C, the total percentage of ARA in the biomass is 47.1% higher than the culture at 23 °C (3.99 and 1.88%, respectively) during the exponential phase (Table 4). In addition, PUFAs represent 44% of the total fatty acids at the same temperature and cultivated period. The effect of temperature (15 • C and 23 • C) on the fatty acid profile and yield of ARA was evaluated during the exponential phase (from 3 d) and at the beginning of the stationary phase (5 d) ( Table 4). The maximum biomass is 1.46 g-DW/L (exponential phase) and the minimum is 1.09 g-DW/L (stationary phase), both at 23 • C. The maximum yield of ARA (mg/biomass g DW) is at 3.87 (15 • C) and the minimum is at 1.88 (23 • C), both in the exponential phase.  1 Indicator of Fatty acids expressed as percentage of total identified FAME. 2 The productivity of ARA was expressed in mg/dry biomass.
Lower temperature does affect the profile of fatty acids, mainly PUFAs. At 15 • C, the total percentage of ARA in the biomass is 47.1% higher than the culture at 23 • C (3.99 and 1.88%, respectively) during the exponential phase (Table 4). In addition, PUFAs represent 44% of the total fatty acids at the same temperature and cultivated period. Figure 5 describes the important effect that the C/N ratio has on cell biomass and ARA productivity. At a C/N ratio of 19, the biomass increases from 1.9 g-DW /L (control) to 4.4 g-DW/L (i.e., a 56.8% increase). However, this value is even higher at a C/N ratio of 33 with final biomass of 5.3 g-DW/L, i.e., 64.1% more than the control. Additionally, the highest productivity of ARA also corresponds to this last C/N ratio of 33 with 8.3 ARA mg/biomass g-DW, which is the highest ARA yield found in this study. Figure 6 shows the distribution of fatty acids with respect to the carbon and nitrogen ratio present in the culture medium. Saturated fatty acids predominate in the C/N ratios of 19 and 33 with percentages of 50.3% and 55.6%, respectively. Additionally, PUFAs are the second most important group of total lipids, their values are 44.1% for the control strain and 43.1% at C/N ratio of 19 and 32.2 for the C/N ratio of 33. The C/N ratio has an important effect on the total lipids (%). This value increase from 1.5% to 27.5% and 27.6% in a C/N ratio of 19 and 33, respectively, was achieved, i.e., 94.6% more than the control. Although an increase of 1.7-fold of the C/N ratio does not affect the proportion of total lipids, it triggers a high ARA yield ( Figure 5B). The bars indicate ± standard error (n = 3). * Indicates significant differences between control and C/N treatments (p < 0.05).

Effect of Culture Conditions on Cell Biomass and ARA Yields
Among all the identified native strains, non-significant differences between their total biomass yields were found. The same trend was observed for the ARA yields among these native strains, where non-significant differences were found (ANOVA, p > 0.05) ( Table 3).
Temperature had a non-significant effect on the LNG2 strain cultures (Table 4. ANOVA, p > 0.05), whereas the two C/N ratios assayed had a significant effect as compared to the control, on the LNG2 strain cultures ( Figure 5 Student's test, p < 0.05). important effect on the total lipids (%). This value increase from 1.5% to 27.5% and 27.6% in a C/N ratio of 19 and 33, respectively, was achieved, i.e., 94.6% more than the control. Although an increase of 1.7-fold of the C/N ratio does not affect the proportion of total lipids, it triggers a high ARA yield ( Figure 5B).

Effect of Culture Conditions on Cell Biomass and ARA Yields
Among all the identified native strains, non-significant differences between their total biomass yields were found. The same trend was observed for the ARA yields among these native strains, where non-significant differences were found (ANOVA, p > 0.05) ( Table 3).
Temperature had a non-significant effect on the LNG2 strain cultures (Table 4. ANOVA, p > 0.05), whereas the two C/N ratios assayed had a significant effect as compared to the control, on the LNG2 strain cultures ( Figure 5 Student's test, p < 0.05).

Morphological Characteristics and Genetic Identification
The literature describes a wide geographical distribution of thraustochytrids and highlights their versatility to adapt to various marine environments with particular characteristics of temperature, light, and suspended decomposing material [1,3,38]. In this study, samples of thraustochytrids were isolated from coastal environments along Chile. The genus Ulkenia sp. was the most frequently identified. Thraustochytrids have been reported in mangroves [58,59] whose intertidal zones are exposed to freshwater courses. Interestingly, three genera of thraustochytrids (Thraustochytrium sp., U. visurgensis, and Botryochytrium sp.) were identified in the Lenga estuary, whose salinity fluctuates due to

Morphological Characteristics and Genetic Identification
The literature describes a wide geographical distribution of thraustochytrids and highlights their versatility to adapt to various marine environments with particular characteristics of temperature, light, and suspended decomposing material [1,3,38]. In this study, samples of thraustochytrids were isolated from coastal environments along Chile. The genus Ulkenia sp. was the most frequently identified. Thraustochytrids have been reported in mangroves [58,59] whose intertidal zones are exposed to freshwater courses. Interestingly, three genera of thraustochytrids (Thraustochytrium sp., U. visurgensis, and Botryochytrium sp.) were identified in the Lenga estuary, whose salinity fluctuates due to freshwater inflows from a river. Additionally, to our knowledge, this research reports the genus Botryochytrium sp. for the first time in Chile [6,7,39,60].

Biomass and Yield in Strain Natives
The Lng2 native strain of the genus U. visugensis has a 5.44% ARA content out of the total lipids and its yield is 5.81 ARA mg/ biomass g-DW (Table 2). These values are significant as compared to the ones reported of strains isolated from an estuary and cultivated in an optimized medium (composed of 1 g peptone, 1 g yeast extract, 20 g glucose, 1 L seawater, 25 • C) where the yields for S. mangrovei strains I AO-1 and IXm-6, Schizochitryum sp. strain BSn-1 and Thraustrochytrium sp. strain Ira-8 are 0.9, 0.4, 0.7, and 5.5 ARA mg/biomass g-DW, respectively [61]. The latter reported yields are lower than the ones obtained from the native Lng2 strain cultivated in a non-optimized medium in this research.

Evaluation of the Effect of Temperature on Strain Lng2 (U.visurgensis)
Temperature is one of the most important environmental factors affecting growth rate. Low temperatures inhibit cell development but stimulate the synthesis of fatty acids, mainly PUFAs [28,45]. For example, the biomass in Aurantiochytrium sp. strain mh0186 grown at 10 • C (0.6 g-DW/L) was considerably lower than at 15 • C (6.7 g-DW/L). However, the percentage of DHA increased from 59% to 75% (total lipids basis) when the temperature dropped from 15 • C to 10 • C [62]. A proteomics study indicates that cold stress inhibits the supply of cellular energy by glycolysis and the Krebs cycle, which could explain the low biomass yields [63]. However, in this study, a greater temperature difference (23 • C and 15 • C) did not significantly affect the cell biomass yield of Lng2 strain (always over 1 g-DW/L) ( Table 4). In general, this yield fits in the usual range reported for batch cultures in thraustochytrids (from 0.5 to 2.3 g-DW/L) [6]. A feasible explanation for the non-significant effect of temperature on biomass yields found in the Lng2 strain could be related to the estuarine origin of that strain ( Table 2). As an estuary is a dynamic ecosystem, its marine population is constantly subjected to physicochemical changes [64]. These adaptability traits [65,66] could account for the behavior of the Lng2 strain (isolated from an estuary) whose biomass yield is not affected by the assayed temperatures. Therefore, similar biomass yields are obtained regardless of whether it is cultivated at 15 • C or 23 • C (Table 4). On the other hand, in the Lng2 strain, low temperatures stimulate the synthesis of PUFAS and increase its ARA content as well, with differences of 23% (from 15 • C to 23 • C) and 53% (from 15 • C to 23 • C), respectively, at the exponential phase. These results are in accordance with previous reports [62,67,68]. These changes are associated with membrane integrity and functional maintenance. ARA and DHA are incorporated into the membrane to preserve its fluidity and permeability [11,45,46]. On the other hand, the yield of ARA is similar at both culture temperatures assayed ( Table 4). The plasticity of the strain to adapt to low temperatures without slowing down its cell biomass yield hints at the potential of the Lng2 strain for both ARA and PUFAs production in industrial-scale fermentation.

Evaluation the Reason C/N in the Biomass and Yield of ARA in Strain Lng2 (U. visurgensis)
Nutrients such as carbon and nitrogen are key in the cellular development of thraustochytrids. Our results confirm that the concentration of glucose (from 5 g-DW/L to 40 g-DW/L) as a carbon source directly affects the increase in its total biomass (i.e., 62.7% with respect to the control). Previous studies [7] with the strain T. areum ATCC 34304 obtained an increase of 13.8% when glucose was added to the culture medium (from 5 to 20 g-DW/L). Accordingly, the yield of ARA had a significant increase at a higher concentration of glucose in culture reaching up to 8.3 ARA mg/biomass g-DW, i.e., 92.8% with respect to the control in just 3 days. Other studies on the microalga Porphyridium purpureum obtained yields of 10.87 and 10.61 ARA mg/biomass g-DW after 18 days of culture, by adding NaCO3 and 5-ALA, respectively, hence obtaining an increase of 11% and 16.1% with respect to the control conditions [69].
The results obtained in this study indicate that the C/N ratio has a significant effect on ARA yield and final biomass. However, excess carbon leads to the synthesis of saturated lipids and to an inhibition of the synthesis of membrane-associated PUFAs [70]. Similar results were observed in this research at a high C/N ratio (33) as the percentage of saturated fatty acids was 9.5% higher than the control and 25.6% lower in PUFAs content. However, there was an increase in total lipids of 94.5% with respect to control conditions ( Figure 6). It is necessary in future studies to accurately determine the N concentration required by the Lng2 strain in order to optimize ARA synthesis.
Thraustochytrids are fast-growing heterotrophic organisms that can use alternative and low-cost carbon sources for cultivation, for example, lignocellulosic biomass [47] and waste from pig industries [71]. This nutritional plasticity allows efficient use of resources and decreases the costs associated with production, and desired characteristics in a strain with commercial potential [72][73][74][75]. The increase in ARA productivity in the Lng2 U. visurgensis strain mediated by nutritional variation is a key factor to be considered for future studies by optimizing the culture medium. In all, the results found in this study highlight the Lng2 strain as a potential candidate for ARA production.

Conclusions
A total of nine strains collected in diverse coastal Chilean environments were isolated and successfully maintained in laboratory conditions. They were classified into four genera corresponding to the thraustochitridae family; Thraustochytrium sp., U. visurgensis, Botryochytrium sp., and Ulkenia sp.
The Lng2 strain was identified as U. visurgensis with a 99.43% identity. Their sequences were included in the GenBank database (number access: OM228767.1). An initial screening singled out the Lng2 strain (U. visurgensis) as the most promising one for ARA production. Results derived from the temperature effect (15 • C and 23 • C) indicate that this factor affects the PUFA profile but not the ARA content nor the biomass yield.
The proportion of nutrients (carbon and nitrogen) is a key parameter for the Lng2 strain. The productivity of ARA increased by 92% (from 0.6 to 8.3 ARA mg/g-DW) and its total biomass by 62.7% (from 1.9 to 5.1 g/L) at a high C/N ratio (33) as compared to control. This research provides evidence of the potential of Lng2 strain for ARA production.