Production of squalene and fatty acids byThraustochytrium sp. RT2316-16: Effects of dissolved oxygen and the medium composition

doi:10.21203/rs.3.rs-6614112/v1

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Production of squalene and fatty acids byThraustochytrium sp. RT2316-16: Effects of dissolved oxygen and the medium composition

https://doi.org/10.21203/rs.3.rs-6614112/v1

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published 16 Sep, 2025

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The psychrophilic marine microbe Thraustochytrium sp. RT2316-16 produced carotenoids, lipids containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and the triterpenoid squalene. The composition of the culture medium and the dissolved oxygen (DO) concentration affected both the squalene content of the biomass and the composition of the fatty acids. Accumulation of total lipids and squalene in the biomass was favored if a high mass ratio of glucose (or glycerol) relative to organic nitrogen was combined with a DO concentration of 20% of air saturation. The highest concentration of squalene in the medium containing glucose was 1485 ± 63 mg L^–1, whereas it was 906 ± 45 mg L^–1 in the medium containing glycerol. Relatively high concentrations of EPA and DHA were obtained under conditions that promoted the growth of the lipid-free biomass in a medium with glucose. A fed-batch operation was found to be suitable for enhancing the concentrations of EPA and DHA.

Thraustochytrids

Thraustochytrium sp.

Microbial squalene

Microbial fatty acids

Eicosapentaenoic acid

Docosahexaenoic acid

This study focused on the production of squalene and fatty acids by the marine psychrophilic thraustochytrid Thraustochytrium sp. RT2316-16. Squalene (SQ, C₃₀H₅₀) is a triterpenoid with important applications in cosmetics and pharmaceuticals. Consumption of squalene lowers the risk of heart disease (Aguilera et al., 2005), lowers blood cholesterol (Lou-Bonafonte et al., 2018), and imparts other health benefits (Brown et al., 2019; Güneş, 2013; Ronco and De Stéfani, 2013). Similarly, consumption of polyunsaturated fatty acids such as eicosapentaenoic acid (EPA, 20:5(n-3), C₂₀H₃₀O₂) and docosahexaenoic acid (DHA, 22:6(n-3), C₂₂H₃₂O₂) provides numerous health benefits (Kapoor et al., 2021).

Squalene is a metabolic intermediate of the steroid biosynthesis pathways of all animals and plants. In eukaryotes the precursors of squalene (i.e., isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP)), are synthesized from acetyl-CoA in the mevalonate pathway. IPP is used to produce farnesyl diphosphate (farnesyl-PP), a precursor of dolichols, carotenoids, prenylated proteins, ubiquinone and sterols. Squalene synthase, a membrane-associated enzyme of the endoplasmic reticulum, catalyzes the production of squalene in a two-step reaction starting from two units of farnesyl-PP (Tansey and Shechter, 2000). The accumulation of squalene in the cells is determined by the activity of squalene epoxidase (SQE), also known as squalene monooxygenase. SQE is a flavin adenosine dinucleotide (FAD)-containing epoxidase that uses nicotinamide adenine dinucleotide phosphate (NADPH) and molecular oxygen to oxidize squalene to 2,3-oxidosqualene. As SQE is inhibited by the antifungal compound terbinafine, its accumulation in the cell is enhanced in the presence of terbinafine (Fan et al., 2010). Microbial production of squalene is further reviewed elsewhere (Paramasivan and Mutturi, 2022; Shalu et al., 2024; Xu et al., 2016). Production of fatty acids by thraustochytrids has also been reviewed (Chi et al., 2022; Marchan et al., 2018; Menzorov et al., 2024; Morabito et al., 2019; Raghukumar, 2008).

Thraustochytrids are obligate aerobic heterotrophs found in many marine coastal waters (Lyu et al., 2020; Marchan et al., 2018; Raghukumar and Damare, 2011). Among microorganisms, thraustochytrids are promising producers of squalene. For example, a squalene content as high as 198 mg g^–1 (1.29 g L^–1) has been reported in species such as Aurantiochytrium sp. 18W-13a (Kaya et al., 2011). In addition, thraustochytrids are well-known producers of lipids that are especially rich in DHA. The total lipid content of some species approaches 50% of cell dry mass. Saturated fatty acids in thraustochytrids are synthesized through the classical fatty acid synthase (FAS) pathway whereas the polyunsaturated fatty acids such as DHA are synthesized through polyketide-like PUFA synthase (the anaerobic pathway), although some species may use elongases and desaturases (the aerobic pathway) (Morabito et al., 2019).

The accumulation of lipids in thraustochytrids is affected by the culture conditions, including the nutritional composition of the culture medium (concentrations and types of carbon and nitrogen sources), the culture age, and the temperature among other factors (Sohedein et al., 2020). For example, in Thraustochytrium sp. the squalene content could be elevated by raising the concentration of NaCl from nil to 5 g L^–1 (Zhang et al., 2021). This phenomenon was ascribed to enhanced oxygen consumption by the cells grown with NaCl resulting in boosted production of the adenosine triphosphate (ATP) needed in reactions of the mevalonate pathway.

The present study assessed the effects of dissolved oxygen concentration in the culture medium on the production of squalene by Thraustochytrium sp. RT2316-16, a psychrophilic marine protist that produces carotenoids, lipids containing EPA and DHA (Leyton et al., 2022), and the co-enzyme Q₁₀ (Flores et al., 2023; Flores and Shene, 2024). As in other thraustochytrids, composition of the culture medium affects the growth rate and the biomass composition of RT2316-16. Typically, the media used for growing RT2316-16 contain yeast extract as a source of organic nitrogen and glucose or glycerol as sources of carbon. In this work, RT2361-16 was grown in media with different carbon and nitrogen sources, under different controlled concentrations of dissolved oxygen, to assess the effects on biomass growth and production of total lipids, squalene and fatty acids. As an alternative to the relatively expensive yeast extract, a hydrolyzed lupine (Lupinus albus) extract was evaluated as a source of organic nitrogen in comparison with yeast extract.

2.1. Microorganism and culture conditions

All experiments used a pure culture of Thraustochytrium sp. RT2316-16 (Leyton et al., 2021) and were conducted aseptically. An initial inoculum was prepared by transferring a loopful (5 µL) of a stock culture (maintained at 4°C) to 100 mL of the M1 medium in a 250 mL Erlenmeyer flask. The medium M1 had the following composition: glucose (Merck, Darmstadt, Germany) 20 g L^–1, yeast extract (Merck) 6 g L^–1, and monosodium glutamate (Merck) 0.6 g L^–1. The medium was made using artificial seawater (see Shene et al. (2013) for composition) diluted with distilled water in a volume ratio of 1:1. In addition, the medium contained a vitamin solution V-I (3.6 mL L^–1), a vitamin solution V-II (3.6 mL L^–1), and a solution of mineral salts (24 mL L^–1). The compositions of V-I, V-II and the mineral salts solutions are specified in Section S1 (Supplemental Material). The vitamins and salt solutions were filter sterilized (0.2 µm sterile membrane filter) prior to use. The inoculated medium was incubated (15 ± 1°C) in an orbital shaker (150 rpm) for 4 days. An aliquot (10 mL) of the resulting culture was used to inoculate 300 mL (500 mL Erlenmeyer flask) of a medium of the same composition that was to be used in the subsequent bioreactor culture. The incubation conditions were as specified above.

The media evaluated in bioreactor cultures were the following: (1) the medium M1 of the above specified composition; (2) the medium M2 (glucose 5 g L^–1, yeast extract 12 g L^–1, monosodium glutamate 1.2 g L^–1); (3) the medium M3 (glycerol (Merck, Darmstadt, Germany) 20 g L^–1, yeast extract 6 g L^–1, monosodium glutamate 0.6 g L^–1); and (4) the medium M4 (lupine extract (see Section 2.2) to replace yeast extract and monosodium glutamate, and glucose 20 g L^–1).

The bioreactor batch cultures were carried out in a 6 L Minifors 2 stirred vessel (Infors HT, Switzerland) with a working volume of 3.8 L. The inoculum volume was 300 mL. The dissolved oxygen (DO) concentration was controlled at the specified values using a cascade control protocol involving initial control with the stirrer speed (200–500 rpm) and subsequent control with the aeration rate (1–2 L min^–1). Cultures were terminated once the DO concentration began to uncontrollably rise to above the setpoint value indicating a lack of oxygen consumption by the culture. The incubation temperature was 15 ± 1°C. Samples (40 mL) were taken aseptically twice a day. The biomass was recovered by centrifugation (2057×g, 4°C, 10 min) and stored at −18°C for analysis. The culture supernatant was analyzed for the concentrations of the residual carbon and nitrogen sources.

A fed-batch experiment was also carried out. This began as a batch culture using the medium M1. The first feeding (250 mL) used a concentrated solution containing glucose (140 g L^–1), yeast extract (84 g L^–1) and monosodium glutamate (8.4 g L^–1). The second feeding (250 mL) comprised of only glucose (140 g L^–1).

2.2. Preparation of the hydrolyzed lupine extract

Lupine flour was purchased from Avelup S.A. (Temuco, Chile). The flour was mixed with NaOH (0.05 M) to obtain a flour concentration of 50 g L⁻¹. A bacterial (Bacillus sp.) protease solution (Sigma Aldrich, USA) was added (700 µL protease per g of lupine flour). After brief mixing, this slurry was incubated (2 h, 50°C) in an orbital shaker (150 rpm). The suspended solids were removed by filtration (Whatman 40 paper filter; pore size of 8 µm). The filtrate (organic nitrogen (Or-N) concentration = 35 g L^–1) was stored at − 20°C until use.

2.2. Analyses

2.2.1. Concentration of total biomass, lipid-free biomass and total lipids

The dry mass concentration of biomass (x, g L⁻¹) was determined gravimetrically. The culture sample (10 mL) was centrifuged (2057×g, 10 min) and the cell pellet was dried to constant weight at 60°C. The concentration of lipid-free biomass (X_LF, g L⁻¹) was calculated using the following equation:

$$\:{X}_{LF}=x\left(1-\frac{l}{100}\right)$$

In the above equation, $\:l$ was the mass percentage of total lipids in the dry biomass. The data on the total lipids in the dry biomass are provided in Tables S2–S5 (see Supplementary Material) for the various experiments. The concentration of total lipids (TL, g L⁻¹) was calculated using the following equation:

$$\:TL=\frac{x\:l}{100}$$

2.2.2. Total lipids in biomass and the fatty acid composition of the lipids

The total lipids in the biomass were extracted using a published method (Bligh and Dyer, 1959). A 50 mg portion of the dried biomass was extracted (1 h, 150 rpm) with 9.5 mL of a solvent mixture of chloroform/methanol/phosphate buffer (50 mM, pH 7.4) in the volume ratio of 5:10:4. The slurry was transferred to a separating funnel containing 2.5 mL of chloroform. After mixing, 2.5 mL of phosphate buffer was added, the contents were mixed briefly and allowed to separate. The chloroform layer was recovered, and the solvent was evaporated at room temperature in a fume hood. The recovered residue (total lipids) was weighed. The extracted lipids were methylated, and the composition of the fatty acid methyl esters was determined by gas chromatography (Leyton et al., 2021).

2.2.3. Squalene content of biomass

The squalene content was measured in the total lipids as previously reported (Budge and Barry, 2019). Serial dilutions of a standard solution of squalene (0 to 32.9 mg mL^–1; Sigma-Aldrich, St. Louis, MO, USA) were prepared in chloroform. The calibration curve was linear (R² = 0.993) in the above specified concentration range.

2.2.4. Concentrations of glucose and glycerol

Residual glucose concentration in the cell-free culture medium was measured using the 3,5-dinitrosalicylic acid (DNS) method (Miller 1959). The residual concentration of glycerol was measured by HPLC (Alliance Waters e2695 separation module; Waters, Mildford, MA, USA) as previously described (Leyton et al., 2022).

2.2.5. Concentration of organic nitrogen

Concentration of organic nitrogen (amino acids) (Or-N) was determined using the o-phthalaldehyde (OPA) method (Nielsen et al., 2006). An aliquot (1 mL) of the reagent solution was mixed with 100 µL of the sample and incubated for 1 to 2 min at 25°C. Afterwards, the spectrophotometric absorbance was measured at 340 nm. A calibration curve made using standard solutions of yeast extract (0–1 g L^–1) was used to quantify the Or-N in the sample.

3.1. Growth and lipid production in batch cultures

The effects of dissolved oxygen (DO) concentration (% of air saturation level) on growth of lipid-free biomass of RT2316-16, and the production of total lipids and fatty acids were evaluated at two levels of DO in three different culture media: the medium M1 combined a high concentration of glucose (20 g L^–1) with low concentrations of the organic nitrogen (Or-N; yeast extract 6 g L^–1 and monosodium glutamate 0.6 g L^–1); the medium M2 combined a low glucose concentration (5 g L^–1) with a high concentration of Or-N (yeast extract 12 g L^–1 and monosodium glutamate 1.2 g L^–1); and the medium M3. The latter was identical to the medium M1, except that it used glycerol instead of glucose. A fourth medium was also evaluated. In this medium an extract of hydrolyzed lupine served as a source of Or-N instead of yeast extract and sodium glutamate, and the DO concentration was 20% of air saturation.

Time profiles of the concentration of the lipid-free biomass, the total lipids, the nutrients, and the key fatty acids (palmitic acid, stearic acid, oleic acid, EPA, and DHA) for the culture in medium M1 at two dissolved oxygen concentrations (DO = 5% and 20%) are shown in Fig. 1. The growth of lipid-free biomass in medium M1 with the DO set at 20% followed a diauxic-type pattern (Fig. 1a): during 0–55 h and 101–149 h, the growth was faster than in the intervening period. The rate of consumption of Or-N declined after 77 h, and at termination (150 h) Or-N was not fully consumed (Or-N = 0.7 g L^–1; Fig. 1a). In the medium M1 at a DO level of 5%, relatively rapid growth of the lipid-free biomass continued to nearly 100 h and a diauxic pattern was not observed (Fig. 1b). The peak concentration of the lipid-free biomass was 5.8 ± 0.4 g L^–1 at 94 h (Fig. 1b), nearly 50% higher than the peak concentration at a DO saturation level of 20% (Fig. 1a). The estimated growth rate during in the period 0–94 h was 0.057 g (L h)⁻¹. At the lower oxygen level (DO = 5%), glucose was consumed more rapidly than at the higher oxygen level, and by 119 h had been fully consumed (Fig. 1b). In contrast, at the 20% DO level, the glucose consumption rate appeared to go through multiple distinct phases and by the end of the culture (150 h) a substantial amount (1 g L^–1) of glucose remained (Fig. 1a).

The DO concentration also affected the total lipids content of the biomass grown in the medium M1 (Table S1; see Supplemental Material) and the production profile of total lipids (Fig. 1a, b). In the low oxygen regimen (DO = 5%), the concentration of total lipids did not increase after around 70 h, and the average final concentration was 1.2 ± 0.5 g L^–1 (Fig. 1b). In contrast, in the high oxygen regimen (DO = 20%) the production profile of total lipids had a pattern similar to the growth profile of the lipid-free biomass (Fig. 1a): the concentration of total lipids increased from 0.1 to 1.3 ± 0.1 g L^–1 between 0 and 93 h and, then, in a second stage (101–149 h), the concentration increased again to finally reach 2.9 ± 0.2 g L^–1 (Fig. 1a). This final total lipid concentration was ~ 53% greater than the result under the low oxygen regimen (Fig. 1b).

The fatty acids in the biomass of RT2316-16 included saturated fatty acids (myristic acid, C14:0; palmitic acid C16:0; and stearic acid C18:0), monounsaturated fatty acids (palmitoleic acid, C16:1; and oleic acid, C18:1) and polyunsaturated fatty acids (PUFA) (linoleic acid, C18:2^D9,12; g-linolenic acid, C18:3^D6,9,12; dihomo-γ-linolenic acid, C20:3^D8,11,14; eicosatrienoic acid, C20:3^D11,14,17; arachidonic acid, C20:4^D5,8,11,14; EPA; and DHA). The fatty acids C16:0, C18:0, C18:1, EPA and DHA amounted to 71–91% of the total fatty acids in high oxygen (20% DO) culture (Fig. 1c). During the first growth phase of the lipid-free biomass (Fig. 1a), the highest concentrations of C16:0, EPA and DHA were 177 ± 15, 224 ± 12 and 550 ± 25 mg L^–1, respectively, whereas the concentrations of C18:0 and C18:1 were less than 50 mg L^–1 each (Fig. 1c). In the second growth phase of the lipid-free biomass (Fig. 1a), the concentrations of C16:0 and C18:1 began to increase to finally reach 525 ± 33 mg L^–1 for C16:0 and 214 ± 20 mg L^–1 for C18:1 (Fig. 1c). The average concentrations of EPA and DHA in this period (101–150 h) were 61 ± 9 mg L^–1 and 129 ± 10 mg L^–1, respectively. The increase in the concentration of C16:0 was in direct proportion to the increase in the total lipids content of the biomass (Table S1; see Supplemental Material) that took place during the second phase of growth of the lipid-free biomass around 77 h (Fig. 1a). Around the same time, i.e. 77 h, the rate of consumption of Or-N decreased (Fig. 1a).

C16:0 was the main fatty acid (22–56% of total fatty acids) in the biomass grown using the medium M1 with the DO level controlled at 5% of air saturation (Fig. 1d). The concentration of C16:0 began to increase once the growth of lipid-free biomass had ceased (Fig. 1b). The C16:0 concentration reached 430 ± 32 mg L^–1 after 129 h (Fig. 1d). After glucose and organic nitrogen (Or-N) were depleted (Fig. 1b), the C16:0 concentration decreased substantially (Fig. 1d). The concentrations of C18:0 and C18:1 followed a pattern similar to that of C16:0 (Fig. 1d). The concentrations of EPA and DHA showed a small increase before the concentration of C16:0 increased abruptly (Fig. 1d) although the peak concentrations (31 mg L^–1 for EPA; 88 mg L^–1 for DHA) were smaller than the peak concentration obtained with the higher DO level (Fig. 1c).

The RT2316-16 culture profiles in the medium M2 with the two settings of the DO concentrations (DO = 10% and 20%) are shown in Fig. 2. The combination of the medium M2 and a DO level of 20% increased the average growth rate of the lipid-free biomass (averaged growth rate during 12–120 h was 0.057 g (L h)^–1) resulting in a final concentration of the lipid-free biomass (5.9 ± 0.4 g L^–1; Fig. 2a) that was 52% higher than the concentration attained with the medium M1 with the same DO level (Fig. 1a). At the lower DO concentration of 10%, the maximum concentration of the lipid-free biomass was lower (4.3 ± 0.3 g L^–1; Fig. 2b), and after 72 h the lipid-free biomass did not grow as the glucose had been consumed.

As was observed in the medium M1 (Fig. 1), the higher oxygen level (DO = 20%) promoted accumulation of total lipids in the biomass (Table S2; see Supplemental Material). Thus, the final concentration of total lipids (1.0 ± 0.1 g L^–1) (Fig. 2a) was 2-fold greater than the concentration at the 10% DO level (Fig. 2b). In all cultures in Fig. 2, the final concentration of the Or-N was the same (1.1 ± 0.1 g L^–1). As the initial concentration of glucose in the medium M2 was low, it was exhausted before Or-N was fully consumed (Fig. 2a, b). At the high oxygen level (DO = 20%), the residual Or-N allowed growth of the lipid-free biomass after glucose had been consumed (Fig. 2a).

DHA was the main fatty acid (30–50% of the total fatty acids) in the biomass grown using the medium M2 (Fig. 2c, d). The concentration of DHA was influenced by the DO level of the culture: the highest DHA concentration (145 ± 9 mg L^–1) occurred near the end (145 h) of the culture if the DO level was 20% (Fig. 2c), but at the lower DO level of 10%, the highest DHA concentration (67 ± 7 mg L^–1) occurred at the end of the growth phase of the lipid-free biomass (Fig. 2d). The final concentration of EPA in the medium M2 with the DO set at 20% was 73 ± 5 mg L^–1, whereas it was much lower (10 ± 2 mg L^–1) at the DO setting of 10%.

In the high oxygen (DO = 20%) culture, the concentration profiles of EPA and DHA generally paralleled the concentration profile of the lipid-free biomass (Fig. 2a). The decrease in concentrations of C16:0, C18:0 and C18:1 was a consequence of the decreased total lipids in the biomass after 60 h (Table S2; see Supplemental Material). At 60 h, the rate of consumption of Or-N was also sharply reduced compared to the earlier period (Fig. 2a).

The RT2316-16 culture profiles in the medium M3 with glycerol as the carbon source and DO levels of 10% and 20% are shown in Fig. 3. With the DO level set at 20%, the concentration of lipid-free biomass increased at an average rate of 0.079 g (L h)^–1 during the first 46 h (Fig. 3a). The highest concentration of the lipid-free biomass was 4.7 ± 0.3 g L^–1 at ~ 70 h (Fig. 3a). By the time glycerol had depleted to a low level (119 h), the concentration of the lipid-free biomass had declined to 3.9 ± 0.3 g L^–1 (Fig. 3a). During rapid growth of the lipid-free biomass, the Or-N was consumed rapidly and 87% of the original Or-N had been consumed by 22 h (Fig. 3a). Around 22 h, the total lipids in the biomass began to increase, reaching 40.3 ± 2.0% w w^–1 in the dry biomass after 104 h (Table S3; see Supplemental Material). In these cultures, the highest concentration of total lipids was 2.7 ± 0.2 g L^–1. At the lower oxygen concentration (DO = 10%) in the medium M3, the highest concentration of the lipid-free biomass (4.4 ± 0.3 g L^–1 at 61 h; Fig. 3b) was not significantly different (p > 0.05) than the concentration in the high-oxygen culture (Fig. 3a). However, the rate of increase of the lipid-free biomass concentration on the low-oxygen culture (DO = 10%) was ~ 20% higher than in the high-oxygen culture. The total lipids concentration increased to eventually reach 2.7 ± 0.2 g L^–1 at the end of the culture (Fig. 3b).

In high-oxygen environment (DO = 20%), the fatty acid concentration did not exceed 65 mg L^–1 during the first 64 h (Fig. 3c). During this period the total lipids content of the dry biomass was less than 17% w w^–1 (Table S3; see Supplemental Material). After 64 h, the concentration of fatty acids increased, reaching 1153 mg L^–1 at 104 h, mainly due to an increase on concentration of C16:0 (17–44% of the total fatty acids) (Fig. 3c). The C16:0 concentration peaked after ~ 70 h once the growth of the lipid-free biomass had ceased (Fig. 3a, c). This pattern was like the trend observed for the total lipids in the biomass (Table S3; see Supplemental Material). The average concentration of EPA between 55 h and 129 h was 52 mg L^–1, whereas that of DHA during the same period was 83 mg L^–1 (Fig. 3c). At the lower oxygen level (DO = 10%), the patterns of changes in concentrations of fatty acids (Fig. 3d) were similar to those seen in the high-oxygen environment. Thus, the C16:0 concentration began to increase (Fig. 3d) once the rate of consumption of Or-N slowed at 36 h (Fig. 3b). The lower oxygen level elevated the concentrations of stearic acid and oleic acid: in the low-oxygen conditions (61–132 h) stearic acid constituted ~ 17% of total fatty acids and oleic acid constituted ~ 31% (Fig. 3d). In contrast, in the high oxygen environment (55–129 h) stearic acid amounted to ~ 5% of total fatty acids and oleic acid constituted ~ 20% of the total fatty acids (Fig. 3c).

3.2. Fed-batch culture

In fed-batch culture with the DO level controlled at 20% of air saturation and the preceding batch stage initiated with the M1 medium, the first feeding (250 mL of concentrated glucose and Or-N (yeast extract and monosodium glutamate)) occurred at 92 h and the second feeding (250 mL, glucose only) occurred at 163 h. The data are shown in Fig. 4.

In the batch phase (i.e., before the first feeding), the growth was slow (Fig. 4a) as had been observed earlier in batch cultures with the medium M1 in combination with a DO concentration of 20% of air saturation (Fig. 1a). The first feeding increased the concentrations of the nutrients (Or-N and glucose), resulting in resumed growth from a near stationary phase just prior to the feeding (Fig. 4a). In the period between the two feedings (i.e., 92–163 h; Fig. 4a) the concentration of the lipid-free biomass increased nearly 2.3-fold from 3.0 ± 0.2 g L^–1. The second feeding resulted in a further rapid increase in the concentration of the lipid-free biomass, notwithstanding the initial drop in concentration (at 163 h) due to the dilution associated with the feeding (Fig. 4a). The peak concentration of the lipid-free biomass (10.8 ± 0.6 g L^–1) was reached at 187 h (Fig. 4a). The concentration of the total lipids increased from 0.2 ± 0.1g L^–1 to 0.5 ± 0.1 g L^–1 near the end of the initial batch phase, and then further to 1.5 ± 0.1 g L^–1 near the end of the first fed-batch phase, and to 2.1 ± 0.2 g L^–1 at the end of the second fed-batch phase (Fig. 4a). The main fatty acid in the biomass was DHA (Fig. 4b). The concentration of DHA increased sharply after the second feeding to ultimately reach 245 ± 11 mg L^–1 (Fig. 4b). The time profile of the concentration of EPA was similar to that of DHA, and the final concentration of EPA was 107 ± 8 mg L^–1 (Fig. 4b). The other fatty acids (C16:0, C18:0, C18:1) also increased in concentration after the first feeding (Fig. 4b).

3.3. Effect of the lupine extract as organic N source

The hydrolyzed lupine extract was evaluated as a source of organic nitrogen (Or-N) instead of the more expensive yeast extract and monosodium glutamate. The DO level was controlled at 20% of air saturation. The resulting culture profiles are shown in Fig. 5.

The lupine extract Or-N was consumed rapidly: a > 50% consumption within the first 24 h (Fig. 5a). Afterwards, the average residual concentration of Or-N was 0.7 g L^–1 (Fig. 5a). Glucose was consumed relatively slowly during 7–54 h, and after 71 h it had declined to around 0.6 g L^–1 (Fig. 5a). The lipid-free biomass grew relatively rapidly until around 50 h but continued to grow more slowly until the end of the culture (Fig. 5a). The final concentration of the lipid-free biomass was 5.0 ± 0.9 g L^–1 (Fig. 5a). This final concentration was 30% greater than the concentration obtained with the medium M1 and the same DO level (Fig. 1a). In the lupine extract medium, the average growth rate (0–47 h) of the lipid-free biomass was 0.089 g (L h)^–1. The concentration of total lipids continued to increase after cessation of growth of the lipid-free biomass (Fig. 5a). The final concentration of total lipids was 1.7 ± 0.1 g L^–1 (Fig. 5a). The main fatty acid in the biomass was C16:0 (Fig. 5b). The final concentration of C16:0 was 280 ± 12 mg L^–1 at 167 h (Fig. 5b). The concentrations of C18:1, EPA and DHA increased until 33 h, the time at which the growth of the lipid-free biomass ceased (Fig. 5a). The concentrations of C18:1 and DHA declined once glucose was exhausted and while the C16:0 concentration still increased.

3.4. Effects of dissolved oxygen concentration and the composition of the growth medium on squalene in the biomass

The squalene content in the biomass and the squalene concentration in the whole culture broth were determined for the various culture conditions discussed in the earlier sections (Figs. 1–5). The data are shown in Fig. 6. The high DO level (DO = 20%) positively affected the squalene content of the biomass grown in the medium M1 (Fig. 6a). During the first growth phase of the lipid-free biomass (Fig. 1a), the squalene content in the biomass increased but once the stationary phase commenced, there was a decline in the squalene content of the biomass (Fig. 6a). When the lipid-free biomass resumed growth (Fig. 1a), its squalene content began to increase (Fig. 6a). At the end of the culture, the squalene content of the biomass peaked at 218 ± 11 mg g^–1 (Fig. 6a). In this same culture, the final concentration of squalene was 1485 ± 63 mg L^–1 (Fig. 6a). In the medium M1, with the DO set at 5%, the squalene content of the biomass began a slow increase after 57 h paralleling the growth of the lipid-free biomass and finally reached 118 ± 6 mg g^–1 once the lipid-free biomass had ceased to grow (Fig. 1b). In this culture, the average squalene concentration towards the end was 454 ± 90 mg L^–1 (Fig. 6a).

The biomass grown in the medium M2 had a low content of squalene (14–57 mg g^–1) (Fig. 6b). After 120 h, the squalene concentration was 95 ± 8 mg L^–1 in the high-oxygen environment (DO = 20%), but it was much lower (67 ± 4 mg L^–1) in the low-oxygen (DO = 10%) condition (Fig. 6b).

The dissolved oxygen level significantly affected (p < 0.05) the squalene content of the biomass grown using glycerol (Fig. 6c). Initially, the squalene content of the biomass decreased: under low oxygen (DO = 10%) the squalene content declined from 128 ± 6 mg g^–1 to 7.2 ± 1 mg g^–1 in the first 12 h whereas a similar decline took place over a longer period (the first 34 h) in the high-oxygen (DO = 20%) environment. Subsequently, the squalene content rose to 68 ± 4 mg g^–1 in the low-oxygen environment, and to 145 ± 7 mg g^–1 in the high-oxygen environment. Irrespective of the dissolved oxygen level in the culture, the increase in the biomass squalene content occurred during a period of increase in the concentration of the lipid-free biomass. At both oxygen levels, the squalene content in the biomass initially decreased and then rose towards the end of the culture (Fig. 6c). The highest squalene concentration (906 ± 45 mg L^–1 at 129 h) occurred in the high-oxygen (DO = 20%) culture (Fig. 6c). This concentration was 2.8-fold greater than the maximum concentration in the low-oxygen culture (Fig. 6c).

The biomass grown using the lupine extract had a relatively low squalene content, and the squalene content decreased from 65 ± 3 mg g^–1 at 31 h to 22 ± 1 mg g^–1 at 172 h (Fig. 6d). In the fed-batch culture, the squalene content of the biomass increased a little during the slow growth phase (i.e., from around 50 h before the first feeding) (Fig. 6e). None of the two feedings promoted accumulation of squalene in the biomass. On average the squalene concentration in the culture was 185 ± 71 mg L^–1 (Fig. 6e).

Some thraustochytrids accumulate lipids and other compounds of commercial interest, including the long-chain omega-3 polyunsaturated fatty acids such as EPA and DHA, and the triterpenoid hydrocarbon squalene. Some of these products are currently sourced from oils of wild-caught marine fish, a mode of production that is considered ecologically unsustainable (Chi et al., 2022). For example, most of the squalene is obtained from shark liver oil. Potentially sustainable alternative sources are microbial oils such as thraustochytrid lipids. Commercial production of thraustochytrid-derived DHA has proven successful (Chi et al., 2022), suggesting that other microbial oils have the potential to be commercialized. Hence the rationale for the present study on production of marine microbial lipids.

The quantity and composition of the lipids in microbial biomass is highly sensitive to the nutrients provided in the culture medium and the other culture conditions. In Thraustochytrium sp. RT2316-16, the focus of the present study, the culture conditions were previously shown to affect the production of EPA, DHA, carotenoids, and phospholipids in shake flasks (Leyton et al., 2022; Valdebenito et al., 2023). Shake flask culture does not typically allow any level of control of the concentration of dissolved oxygen (DO) in the medium. Therefore, the present work was carried out in a stirred tank bioreactor at various controlled concentrations of dissolved oxygen to elucidate the impact of oxygen concentration on the production of total lipids, fatty acids and squalene. As the concentration of metabolically active microbial biomass increased with growth, the volumetric oxygen consumption rate in the culture increased. To compensate for the increased consumption of oxygen, the oxygen transfer rate in the bioreactor was automatically increased to ensure that the measured dissolved oxygen concentration remained at the controlled value. A cascaded control combining increased agitation speed of the mixing impeller and the air flow rate was used to achieve effective control of the dissolved oxygen concentration.

The growth kinetics of the lipid-free biomass and the production of total lipids were found to be strongly influenced both by the DO concentration and the nutritional composition of the culture medium. For growth in the medium M1, a relatively high dissolved oxygen concentration (DO = 20% of air saturation) promoted accumulation of total lipids in the biomass (Table S1; see Supplemental Material) compared to lower oxygen levels. Although a lower DO (10% of air saturation) satisfactorily supported the growth of the lipid-free biomass, the total lipids in the biomass were reduced.

In oleaginous microorganisms, the accumulation of triacylglycerol lipids typically occurs after cell growth has ceased due to exhaustion of some key nutrient (e.g., nitrogen, phosphorus) other than the carbon source. Once a key non-carbon nutrient is missing, the available organic carbon can no longer support cell growth but continues to be used to build up reserves of lipids that do not contain the missing nutrient. For example, triacylglycerols are comprised of only carbon, oxygen and hydrogen. Similarly, squalene is comprised of only carbon and hydrogen. The cessation of growth of the lipid-free biomass in the medium M1 with the DO controlled at 20% was not due to a depletion of glucose or organic nitrogen (Or-N) although the uptake rates of both glucose and Or-N were affected once growth ceased (Fig. 1a). The observed cessation of growth may have been due to possible production of some growth-inhibitory metabolite, or generation of reactive oxygen species in a high-oxygen environment, and/or the depletion of some essential component in the Or-N fraction of the nitrogen source (yeast extract). The arrest of the lipid-free biomass growth was transient, and the growth resumed after a short period (Fig. 1a). A transient arrest of growth, or a diauxic growth (or diauxie) pattern, is consistent with complete consumption of a readily metabolizable nutrient such as a component of Or-N, followed by an adaptation period of little or no growth during which the microorganism builds the enzyme machinery necessary for metabolizing the other available nitrogen-containing components within the Or-N mixture.

Compared to the medium M1, the medium M2 had a higher concentration of Or-N but less glucose. In M2, the effect of the DO concentration on production of the lipid-free biomass was opposite to that seen in the medium M1, i.e. a higher concentration of dissolved oxygen (DO = 20%) promoted the growth of lipid-free biomass and the phenomenon of transient arrested growth did not occur. This concurred with the earlier inference that depletion of some component of Or-N was responsible for the temporarily arrested growth in the medium M1 that was low in Or-N compared to the medium M2. The relatively lower concentration of the lipid-free biomass in the medium M1 was an apparent effect of a limited supply of some essential component of Or-N, possibly an amino acid of the yeast extract. A higher concentration of Or-N alleviated this limitation in the medium M2. Amino acids contain both nitrogen and carbon and can be directly used for growth.

Regardless of the DO concentration and the initial concentrations of glucose and Or-N, the rate of consumption of Or-N showed sharp variations at certain times during the batch culture. This was likely related to preferential consumption of some components of the yeast extract, with the others being consumed after the preferred component was no longer available. These changes in the nitrogen components being metabolized may have promoted an increased accumulation of total lipids in the biomass (Fig. 1a) and may also have affected the composition of the fatty acids in the lipids.

In earlier studies in shake flasks without control of the dissolved oxygen concentration, the concentration of the lipid-free biomass in medium M1 was 4.3 g L^–1 and the concentration of the total lipids was 1.7 g L^–1 (Valdebenito et al., 2023). Also in shake flasks, the concentration of the lipid-free biomass in medium M2 was 3.7 g L^–1 and the concentration of the total lipids was 0.4 g L^–1 (Valdebenito et al., 2023). These concentrations were comparable to those obtained in the present work with a low dissolved oxygen concentration (DO = 5%) in medium M1 (i.e., lipid-free biomass concentration = 4.9 g L^–1, and total lipids = 1.2 g L^–1). In the less oxygenated environment (DO = 10%), in medium M2 in the present work, the lipid-free biomass concentration was 4.3 g L^–1, and the total lipids concentration was and 0.5 g L^–1. This comparison suggests that an accurate evaluation of the potential of thraustochytrids as producers of lipids requires controlled oxygen cultures; the conventional shake flask culture is unsatisfactory. As thraustochytrids are obligate aerobes, the oxygen concentration in a culture can reasonably be expected to critically influence their metabolism, as observed in the present work.

The data showed that the concentrations of EPA and DHA in cultures with a constant concentration of dissolved oxygen followed similar time-dependent variations. This may suggest that DHA was produced via elongation–desaturation pathway in which EPA is an intermediary; however, the elongation–desaturation pathway lacks certain omega-3 fatty acids (e.g., a-linoleic acid, C18:3^D9,12,15; stearidonic acid, C18:4 ^D6,9,12,15; and eicosatetraenoic acid, C20:4 ^D8,11,14,17) which were not found in RT2316-16. Therefore, in RT2316-16 EPA and DHA were most likely synthesized from the omega-6 fatty acid arachidonic acid (C20:4 ^D5,8,11,14) as has been reported in some unrelated photosynthetic marine microorganisms (Guihéneuf et al., 2013). In RT2316-16 cultures, the highest concentrations of EPA and DHA occurred simultaneously during growth of the lipid-free biomass while Or-N was being consumed. This suggested a possible contribution of amino acid metabolism to the synthesis of polyunsaturated fatty acids, or a requirement of these fatty acids by actively growing cells. In fact, long-chain polyunsaturated fatty acids such as EPA and DHA are known to be essential for maintaining the required fluidity of the membranes of cold-water marine microbes (Yoshida et al., 2016), suggesting an essential requirement of these fatty acids for the cells to build the various internal and external membranes during multiplication.

Although a low Or-N concentration (< 1 g L⁻¹) could stop growth of the lipid-free biomass (Fig. 1b, Fig. 3b), the conditions that triggered the accumulation of total lipids in the biomass seemed to depend on the DO concentration. The results did show an accumulation of the total lipids in the biomass after the Or-N had declined to less than 1 g L⁻¹, and these lipids were rich in the fatty acids C16:0 and C18:1 (Fig. 1d; Fig. 3c, d; Fig. 5b). Glycerol proved to be a good carbon source for producing lipids rich in saturated fatty acids (C16:0 and C18:0). The high rates of production of these fatty acids (Fig. 3c, d) resulted in their accumulation in triacylglycerols as they could not undergo elongation and desaturation sufficiently rapidly. Hence, the concentrations of EPA and DHA increased relatively slowly. In a plausible scenario, the metabolism of glycerol may have promoted a more rapid consumption of the Or-N, as the latter could supply both carbon and nitrogen. A more rapid consumption of Or-N was apparent in the oxygen-rich (DO = 20%) environment in glycerol-based medium (Fig. 3). Once the Or-N had been depleted, glycerol was metabolized for producing C16:0.

When lupine extract was the source of Or-N, most of the Or-N was consumed within 24 h, and the subsequent consumption of glucose resulted in production of C16:0 (Fig. 5). Although this pattern was comparable to when yeast extract was used to provide Or-N, the glucose–lupine and glycerol–yeast extract cultures had some differences: the concentration of C16:0 was lower, and it was produced more slowly in the glucose–lupine culture.

Squalene is an intermediate metabolite in the sterol synthesis pathway. The products of this pathway influence properties of cell membranes such as fluidity and permeability. Thraustochytrids are known to produce sterols (Bi et al., 2023; Ishibashi et al., 2023; Menzorov et al., 2024), although there is no specific information on production of sterols by Thraustochytrium sp. RT2316-16. In related thraustochytrids such as Schizochytrium (now Aurantiochytrium; Chi et al., 2022) sp. S31, sterols such as cholesterol, stigmasterol, lanosterol, and cycloartenol are produced (Bi et al., 2023). The biosynthesis of sterols and fatty acids in some thraustochytrids appears to be co-regulated. For example, genetic modifications for increased production of DHA have reduced production of squalene and sterols in Schizochytrium (now Aurantiochytrium; Chi et al., 2022) sp. HX-308 (Ren et al., 2015). Conversely, modifications to the mevalonate pathway for increased production of squalene and carotenoids have reduced the amount of DHA in the biomass of in Schizochytrium sp. HX-308 (Huang et al., 2021).

In the absence of nutrient limitations (including oxygen limitation), squalene is unlikely to accumulate in the biomass as it does not participate in other metabolic pathways. Nonetheless, an imbalance between the rate of synthesis of squalene and its consumption through oxidation by squalene monooxygenase or squalene epoxidase (Bi et al., 2023) could result in accumulation of squalene. The rates of enzymatic reactions depend both on the concentrations of the enzymes and the concentrations of the substrates, farnesyl pyrophosphate and squalene in this case. Farnesyl pyrophosphate is produced in the mevalonate pathway in which 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) is the rate limiting enzyme in Aurantiochytrium sp. 18W-13a (Yang et al., 2022). The HMGR activity in the strain 18W-13a strongly correlated with the squalene content. If a similar scenario applied to RT2316-16, anything that increased the flux through the mevalonate pathway (e.g., an excess of acetyl-CoA, the starting metabolite of the pathway), would promote accumulation of squalene. Availability excess acetyl-CoA is also necessary for accumulation of lipids. In RT2316-16 the conditions that promoted lipid accumulation were the medium M1 with a high oxygen level (DO = 20%) and the medium M3 with the DO levels of ≥10%.

Dissolved oxygen level and the composition of the culture medium substantially influenced the squalene content in the biomass (Fig. 6). The conditions that promoted the accumulation of total lipids (i.e., the medium M1 with either glucose, or glycerol, and a DO level of 20%), also promoted the accumulation of squalene. The highest observed concentrations of total lipids and squalene were 1485 ± 63 mg L^–1 and 906 ± 45 mg L^–1 (Fig. 6a), respectively. The effect of the dissolved oxygen concentration on the squalene content of the biomass of RT2316-16 was different compared to data reported for other thraustochytrids. For example, in the thraustochytrid ACEM 6063, possibly Schizochytrium (or Aurantiochytrium) sp.), a low level of dissolved oxygen (DO = 5%) promoted squalene accumulation to around 2 mg g^–1 in the biomass (Lewis et al., 2001). Squalene accumulation under low oxygen was hypothesized to be associated with oxygen inhibition of certain downstream enzymes that consumed squalene to generate the other metabolites needed for the synthesis of sterols (Lewis et al., 2001).

When RT2316-16 was cultured in a medium that combined a high concentration of Or-N and a low concentration of glucose (i.e., the medium M2), the biomass was low in squalene and total lipids, irrespective of the dissolved oxygen concentration used, but the available nutrients did not limit the growth of the lipid-free biomass at the higher oxygen level (DO = 20%; Fig. 2a). The relatively high levels of EPA and DHA compared to the other fatty acids (C16:0, C18:0, C18:1cis; Fig. 2c) and the low squalene content of the biomass (Fig. 6b), suggested a combination of the medium M2 and a high dissolved oxygen level (DO = 20%) if the objective was to produce polyunsaturated fatty acids rather than squalene, as often wanted in thraustochytrid cultures (Ren et al., 2015).

The fed-batch culture (Fig. 4) with the tested feeding strategies did not prove useful for producing squalene-rich biomass, possibly because there was no nutrient limitation as was evidenced by a continuous increase in the concentration of the lipid-free biomass during the entire culture. The relatively low lipid content of the biomass produced under these conditions was also consistent with an absence of a growth limitation.

In studies with a different thraustochytrid (Schizochytrium sp. S31; Schütte et al., 2024) squalene production was compared among batch, fed-batch and continuous culture operations. Continuous culture implemented with a relatively high dilution rate (dilution rate = 0.025 h^− 1) resulted in biomass with the highest squalene content (39.7 ± 1.3 mg g^–1). In a steady-state continuous culture, the specific growth rate of the biomass is identical to the dilution rate (Chisti, 2010), meaning that the growth rate was high. In such high dilution rate cultures, the steady state concentrations of all nutrients in the culture vessel tend to be high, implying an absence of nutritional stress. Although the most squalene-rich biomass was produced in continuous culture, the highest concentration of squalene was produced in a fed-batch operation with pulsed feeding of glucose (Schütte et al., 2024). This suggests that a more comprehensive future evaluation of feeding strategies in fed-batch operations may be worthwhile for enhancing the productivity of squalene in Thraustochytrium sp. RT2316-16.

The composition of the culture medium and the concentration of dissolved oxygen (DO) affected both the squalene content of the biomass and the composition of the fatty acids in the total lipids. A medium relatively rich in glucose compared to Or-N (the medium M1) in combination with a DO level of 20%, limited the growth of lipid-free biomass and favored the accumulation of both total lipids and squalene in the biomass. Replacing glucose with an equal mass concentration of glycerol in the medium M1 enhanced squalene accumulation in the biomass.

The specific nitrogen compounds present in the organic nitrogen source affected the composition of the lipids produced. The microorganism consumed lupine extract Or-N more rapidly compared to yeast extract, promoting the synthesis of palmitic acid and oleic acid to the detriment of EPA and DHA.

Relatively high concentrations of the long-chain omega-3 fatty acids EPA and DHA could be obtained under conditions that promoted growth of the lipid-free biomass if the Or-N and glucose concentrations were kept above a minimum threshold, for example, by using a fed-batch operation. In a 149-h batch operation, the maximum content of squalene in the biomass (218 mg g⁻¹) occurred under the following conditions: a DO concentration of 20%, 15°C, and the medium M1.

Ethics statement for the use of human and animal subjects (may require consent to participate and consent to publish for human subjects)

Not applicable.

Consent for publication

Not applicable.

Competing interests

Authors declare no conflicts of interest.

Author’s contributions

Paris Paredes: Conceptualization, Methodology, Investigation, Writing - Original draft. Liset Flores: Methodology, Writing - Review & Editing, Visualization. Mariela Bustamante: Methodology, Writing - Review & Editing. Yusuf Chisti: Writing - Reviewing and Editing. Juan A. Asenjo: Funding, Writing - Reviewing and Editing. Carolina Shene: Conceptualization, Methodology, Investigation, Writing - Reviewing and Editing.

Funding

This research was funded by: ANID doctoral grant 21200659; the Centre for Biotechnology and Bioengineering (CeBiB) CEBIB AFB240001; and Fondecyt project 1230547.

Availability of data and materials

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgement

Not applicable.

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Production of squalene and fatty acids byThraustochytrium sp. RT2316-16: Effects of dissolved oxygen and the medium composition

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Microorganism and culture conditions

2.2. Preparation of the hydrolyzed lupine extract

2.2. Analyses

2.2.1. Concentration of total biomass, lipid-free biomass and total lipids

2.2.2. Total lipids in biomass and the fatty acid composition of the lipids

2.2.3. Squalene content of biomass

2.2.4. Concentrations of glucose and glycerol

2.2.5. Concentration of organic nitrogen

3. Results

3.1. Growth and lipid production in batch cultures

3.2. Fed-batch culture

3.3. Effect of the lupine extract as organic N source

4. Discussion

5. Conclusions

Declarations

References

Supplementary Files

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