تولید دوکوزاهگزانوئیک‌اسید توسط سویة بومی آئورانتیوکیتریوم در تخمیر خوراک‌دهی‌شده

Introduction

Docosahexaenoic acid (DHA) is an essential polyunsaturated fatty acid (PUFA) with a wide range of applications in the food and pharmaceutical industries (1). DHA contains 6 double bonds or unsaturated C=C linkages in its long chain backbone structure (2) and prevents the development of a wide variety of diseases such as cardiovascular failures and several types of cancer and tumor (3-7). Also, DHA is essential for the normal growth of infants’ central nervous system and visual sight and hence is secreted in human breast milk (8). Fish oil is the world’s traditional source of DHA. However using fish-derived oil is not achieved in the food industry yet, because of its fishy taste and smell, and high sensitivity to oxidation during food processing (9). Another drawback of using fish oil in infant formula is the presence of eicosapentaenoic acid (EPA), which is not recommended in milk formulation (10). Therefore, alternative and sustainable sources of DHA are under research and investigation. Aurantiochytrium is a marine thraustochytrid with the ability to grow on the culture media containing various carbon sources. This strain is known for the production of a high amount of DHA (3). Production of this microbial oil is feasible to scale up through biotechnological processes (11). Cultivation of Aurantiochytrium at high cell densities and on a large scale requires proper oxygen transferring between culture broth and gas phase. Also, it has been shown that the production of biomass and DHA is mainly affected by the ratio of carbon to nitrogen (C/N) and dissolved oxygen (DO) (2, 12-15). Also, the effect of a wide range of raw materials (16-18), physicochemical parameters (13, 19, 20), and fermentation conditions were studied on the production of DHA by microbial strains (14, 21-24). Furthermore, batch, fed-batch, and continuous cultures were investigated (12, 25, 26). Barclay (1992) studied the production of DHA in 14 L bioreactor at 48 hours using glucose, low salinity, and chloride level, and ammonium sulfate as the sole nitrogen source (27). The results showed that 65-70 g.L^-1 biomass and 8 g.L^-1 DHA were produced with the productivity of 0.1 g.L^-1.h^-1 (28). Also, fed˗batch cultivation was used with low dissolved oxygen and ammonium as nitrogen sources and results showed 170-210 g.L^-1 biomass with 409 mg.L^-1.h^-1 DHA productivity (29). In this research, a strain of Aurantiochytrium was used to produce oil rich in DHA in a fed-batch cultivation fermentor. Growth and oil production at a small scale (250 mL flasks) were studied in previous studies (30). The aims of the present study were to investigate the production of biomass, oil, and DHA in a bench-scale fermentor. So, experiments were conducted in a 3L fermentor in order to assess the effects of medium composition and various C/N ratios on biomass, oil, and DHA production. Glycerol and glucose were studied separately as the sole carbon source. Finally, the findings of this research paved the way for the large-scale production of oil rich in DHA as a sustainable alternative for fish oil.

Materials and Methods

Fed-batch Cultivation in the Fermentor: Aurantiochytrium sp. qe-4 (KR091914.1) had been isolated and identified earlier from the mangrove forests of Qeshm in the south of Iran (N2642 E5540) (30). The strain was cultured in the medium containing 30 g.L^-1 glycerol or glucose, 5 g.L^-1 yeast extract (YE), 3 g.L^-1 peptones, 0.3 g.L^-1 penicillin/streptomycin, and 60% v/v natural seawater (30). A 3 L Bench-Top fermentor FS-01-A was used for fed-batch cultivation of Aurantiochytrium strain. Before inoculation, saturation was controlled by agitation speed (250-300 rpm) and sparging of 0.5-1.5 volume of air per volume of medium per minute (vvm) at 28 °C and pH 7±0.2 (acid and base: 2 N NaOH and 2 N HCl). Two media (I and II) were used to investigate the production of DHA by Aurantiochytrium in 2 L fermentation medium in the fermentor. The medium I contained 30 g.L^-1 glycerol, 10 g.L^-1 YE, 10 g.L^-1 peptones, (C/N: 1.5), 100% natural seawater. Fresh seed culture was used as inoculum (10% v/v). Also, medium II contained 40 g.L^-1 glucose, 6 g.L^-1 YE; 20 g.L^-1 monosodium glutamate (C/N: 2), 100% natural seawater, and a 10% v/v seed culture. Cell suspensions were taken at time intervals to measure cell dry weight (CDW), residual glycerol and glucose, oil, and DHA concentration. Medium Ι and ΙΙ were prepared only with glycerol and glucose as the sole carbon source.

Controlling of Parameters during Fermentation: The aeration in the medium I was controlled at 1 vvm and 0.5 vvm at the growth phase (0-168 h) and lipid production phase (168-240 h), respectively. Moreover, aeration in culture with glucose as the sole carbon source was controlled at 1.5 vvm and 1 vvm at the growth phase (0-190 h) and lipid accumulation phase (190- 337 h), respectively. In both cultures, the agitation speed, pH, and temperature were kept at 300 rpm, 7, and 28 °C, respectively. Fermentation data were monitored through equipment readouts. During the fermentation, the formation of foam was notobserved, so no surfactant and antifoam were used.

Determination of Cell Growth and Biomass: CDW was measured by the sampling of 10 mL suspension culture and centrifuged at 5000 rpm for 10 min. Then, the removed supernatant and pellets were dried at 110 °C for 48 h (31).

Glycerol Measurement: The concentration of glycerol was determined in the culture supernatants in time intervals. Briefly, two reagents including reagent I (periodate reagent: 18 mg.mL^-1 sodium periodate dissolved in 10% v/v acetic acid/distilled water (DW)) and reagent II (acetylacetone reagent: 1% v/v acetylacetone in isopropyl alcohol) were prepared and used. The calibration curve (glycerol 50-200 mg.L^-1) was determined in a 96-well microplate. Forty μL of the culture supernatant was added into each well and then 40 μL of the reagent I was added, mixed, and incubated for 10 min. Then, reagent II (125 μL) was added to the wells and mixed and incubated for 25 min. The absorption was assessed at 410 nm by a microplate reader. Residual glycerol was determined in the culture supernatant according to the standard curve (32).

Glucose Measurement: The dinitrosalicylic acid (DNS) method was used for the determination of glucose. Briefly, 1 mL sugar solution was prepared in DW and mixed with 4 mL of the DNS reagent. Solutions were incubated in a boiling water bath for 5 min and then transferred to the ice for rapid cooling. Then, tubes were brought to room temperature and absorbance was determined at 540 nm using spectrophotometer CARY 50 (33).

Lipid Extraction: One-hundred mg of CDW were weighted in screw cap tubes and then 2 mL DW was added. Samples were ultrasonicated for 5 min and chloroform (2.5 mL) and methanol (5 mL) were added. Ultrasonication and homogenization were done for 5 min and 2.5 mL chloroform and 2.5 mL of DW were added and shaked for 30 s. Then, suspensions were centrifuged (4000 rpm for 15 min) to separate both organic and inorganic phases. The bottom layer (organic phase) was separated and the solvent was evaporated. Finally, extracted oils were determined gravimetrically (30).

Measurement of DHA: The DHA content was analyzed by the gas chromatography (GC) method. Three mL of methanolic sulfuric acid 4% (v/v) was added to a certain amount of extracted lipid in screw cap tubes and vortexed for 10 s. Then, tubes were incubated in a water bath (90 min at 80 °C). After that, tubes were cooled and 1 mL of DW was added and vortexed for 10 s. Finally, 1 mL of hexane was used for the extraction of fatty acid methyl esters (FAMEs). The organic layer was recovered and Na₂SO₄ was added to the tubes for the elimination of remained H₂O. Agilent 6890 along with a flame ionization detector (FID) and DB-23 (30 m × 0.32 mm, 0.25 μm; Agilent Technologies) capillary column were used for GC analysis. The carrier gas was nitrogen. Then, 0.5 μL of each FAMEs was injected under splitless injection mode. The temperature of the injector and detector was set at 300 °C. The temperature program of the column was set as 50 °C; 2 min, 10 °C.min^-1 to 180 °C; 5 min, 5 °C.min^-1 to 240 °C; 7 min. C19:0-FAME (Sigma) was used as an internal standard and mixed PUFA-standard (Sigma) was used for determining the retention time of each FAME peak (30). Finally, Biomass yield (Y_x/s), oil yield (Y_p/s), and DHA yield (Y_DHA/s) coefficients were assessed and determined (22).

Results

Aurantiochytrium Growth during fed-batch Fermentation

Medium Ι: Feeding strategies are important in fed-batch culture systems to achieve high productivity of intracellular lipids. As shown in figure 1A, biomass, oil, and glycerol consumption were monitored during the cultivation of Aurantiochytrium. After 114 h of cultivation, 10 g.L^-1 glycerol, 3.3 g.L^-1 YE, 3.3 g.L^-1 peptones (C/N: 1.5) were fed to the fermentor. This feeding was for the growth and multiplication of cells. Glycerol was consumed and reached below 6 g.L^-1 after the first feeding. With the consumption of 26 g.L^-1 glycerol in 186 hours of cultivation, biomass increased to 18 g.L^-1. The concentration of glycerol was kept between 5-25 g.L^-1 during the fermentation process. The second feeding was used for oil accumulation and contained only 17 g.L^-1 glycerol. This feeding was done at 186 h of cultivation. After this feeding, oil increased to 8.8 g.L^-1. and final biomass, total oil, and DHA content reached 30.2 g.L^-1, 8.8 g.L^-1, and 22.08% (of total fatty acids) in medium Ι, respectively. The maximum productivity of DHA was 7.15 mg.L^-1.h^-1.

When glycerol solution was fed to the culture medium at 114 h and 186 h of cultivation, the DO level decreasedwhile the oxygen consumption rate and cell growth increased. The time course of DO concentration during the fermentation is shown in Figure 1B. Analysis of DO level at the end of the process showed that DO less than about 3% of saturation was favored for the production of DHA. A stirrer and intensive mixing are required to reduce viscosity and provide aerobic conditions for DHA production during fermentation. In this fermentation, agitation speed of 300 rpm did not destroy cells structure and was favorable for mixing culture medium.

Medium ΙΙ: A significant increase in biomass was observed between 106-290 h. During this period, biomass increased from 6.4 g.L^-1 to 27.33 g.L^-1. Also, the rate of glucose consumption was high, and glucose was added and fed every 20 h. A certain amount of concentrated glucose solution was added to the culture medium when the residual glucose decreased below 20 g.L^-1. After 340 h, the biomass and total lipid reached 27.6 g.L^-1 and 12.5 g.L^-1, respectively. As shown in Figure 2A, feedings 1 and 2 were contained 20 g.L^-1 glucose. Third feeding  contained 10 g.L^-1 glucose, 20 g.L^-1 MSG and 6 g.L^-1 YE at 105 h of cultivation. When this feeding was added to the culture medium, the DO level  dropped quickly and then agitation increased to 300 rpm (Fig. 2B). This variation in agitation was done by the fermentor because it was set up between 250-300 rpm and agitation can fluctuate in this range to increase or decrease DO during feeding. As shown in Figure 2B, agitation decreased to 250 between 50-100 h of incubation. During this period, several feedings were done and cells were growing. They required more oxygen for their metabolisms, which was provided to them by increasing and decreasing levels of DO and agitation, respectively. These results showed that glucose feeding without a nitrogen source was not suitable for cell growth during the early fermentation period. Table 1 shows various parameters which were obtained from the medium I and II.

Fig. 1- A) Biomass, oil production, and glycerol consuming during fed-batch cultivation of Aurantiochytrium in medium I. B) DO variation in fermentation process at 300 rpm agitation speed

Fig. 2- A) Biomass, oil production, and glucose consumption during fed-batch cultivation of Aurantiochytrium with glucose as a carbon source. B) Profile of DO concentrations in the fed-batch cultivation of Aurantiochytrium

Table 1- Several Parameters Obtained in DHA Production by Aurantiochytrium

TFA: Total Fatty Acids

DHA Analysis: Figures 3A and 3B show a GC chromatogram of methyl esterified fatty acids produced by Aurantiochytrium after 240 of cultivation in medium Ι. Lipids were extracted, esterified, and prepared for analysis. Tables 2 and 3 show the fatty acid composition of the medium I and II. It was observed that the DHA content and productivity reached 1.72 g.L^-1 and 7.15 mg.L^-1.h^-1 in the medium Ι, respectively. DHA content and productivity were 18.63% and 42.71% higher than the results obtained from medium ΙΙ. Overall, these two parameters were improved by using glycerol as the sole carbon source in the fermentation medium.

Fig. 3- A) GC chromatogram of methyl esterified fatty acid produced by Aurantiochytrium after 240 h of cultivation in medium Ι. B) GC chromatogram of methyl esterified fatty acid produced by Aurantiochytrium after 290 of cultivation in medium ΙΙ

Table 2- Fatty Acid Composition (% of Total Fatty Acids) in Time Intervals in Medium Ι

Table 3- Fatty Acid Composition (% of Total Fatty Acids) in Time Intervals in Medium ΙΙ

Determination of Kinetic Parameters: Kinetic parameters obtained from the medium I and II are shown in Table 4. The parameters include biomass yield coefficient (Y_x/s), oil yield coefficient (Y_p/s), and DHA yield coefficient (Y_DHA/s). Y_x/s, Y_p/s, and Y_DHA/s were significantly higher in medium I in comparison to medium ΙΙ.

Table 4- Kinetic Parameters at Different Fermentation Stages

Microscopic Observation of Cells during Fermentation: Aurantiochytrium sp. showed a spherical morphology with a diameter ranging from 4 µm to 20 µm.  Development of lipid body was detected by Nile red staining and fluorescence microscopy. Other pictures were taken by optical microscopy (Axioplan2 Imaging) (Fig. 4). In medium Ι, zoospores were observed during 24-144 h and then transformed into some sporangia through binary cell division. After the addition of feeding at 186 h, cells accumulated intracellular oil bodies (Fig. 4A). As shown in Figure 4B, in medium ΙΙ, cell growth and division stopped at 96h, due to the lack of enough nitrogen source. After the simultaneous addition of MSG and glucose at 120 h, cell growth and division increased. The positive effect of nitrogen with carbon source on cell growth was observed during the fermentation period (128-190 h). After 215 h of cultivation, the cells were saturated with intracellular oil droplets.

Fig. 4- The cell morphology of Aurantiochytrium sp. during fermentation in 3 L fermentor. (A) Medium Ι and magnification of 40 (B) Medium ΙΙ and magnification of 100

Discussion and Conclusion

In the present study, the authors investigated the effects of different carbon sources and DO levels on the cell growth, lipid, and DHA production in Aurantiochytrium sp. at 3 L fermentor. Significant DHA productivity was achieved in the medium containing glycerol compared with the medium with glucose as the sole carbon source (7.15 and 5.01 mg.L^-1.h^-1, respectively). The results of the study showed the cell growth increased to a high amount in the fermentor (30.15 g.L^-1) in comparison to the shake flask (10.32 g.L^-1) in the previous study (30). Janthanomsuk et al. (2015) obtained a high cell density of Aurantiochytrium B072 and high productivity of DHA by the fed-batch pH auxostat fermentation process. They showed that glucose-limited feeding increased the percentage of DHA per total fatty acids up to 37% (w/w) (34). The bioreactor provides more dissolved oxygen for the growing of Aurantiochytrium cells than shake flasks, so vegetative cells consume the most carbon and nitrogen sources for growth and multiplication. Moreover, we used a strategy for the simultaneous feeding of MSG and glucose to improve DHA productivity. Trovao et al. (2020) used two media (I and II) containing monosodium glutamate/glucose and corn steep powder/glycerol, respectively. Their results showed that medium I and II increased DHA content 1.7 and 3 folds, respectively (35). Ren et al. investigated DHA production in a 1500 L bioreactor with glucose and MSG in fed˗batch culture. They achieved 71 g.L^-1 biomass, 35.75 g.L^-1 lipid, and 48.95% DHA (% of total fatty acids) (22).

There are two important factors that effectively increase lipid accumulation in oleaginous microorganisms: 1- the continuous generation of acetyl-CoA, a precursor for fatty acid synthase (FAS); and 2- proper amount of NADPH as the essential reductant in fatty acid biosynthesis (36, 37). ATP: citrate lyase (ACL) enzyme facilitates the formation of acetyl-CoA in the cytosol of oleaginous Aurantiochytrium sp. and malic enzyme (ME) and glucose-6-phosphate dehydrogenase (G6PDH) are responsible for generating NADPH for the biosynthesis of fatty acids (38). Therefore, the limitation of nitrogen sources in the fermentation medium increases the cytosolic level of both acetyl-CoA and NADPH and consequently leads to the accumulation of lipid granules by Aurantiochytrium cells (2).

Furthermore, the addition of MSG can activate acetyl-CoA carboxylase (10) and glucose-6-phosphate dehydrogenase (39), hence it increases enough supplement of acetyl-CoA and NADPH for the biosynthesis of lipid and DHA. Ye et al. (2020) used the fed bath fermentation strategy of mixed carbon sources including glucose and glycerol. The results showed that 52.2 g.L^-1 biomass and 100.7 mg.L^-1.h^-1 DHA were produced. Their results revealed that simultaneous feeding of glucose and glycerol upregulates putative genes in fatty acid synthase (FAS) and polyketide synthase (PKS) pathways (40).

In the lipid accumulation phase, low oxygen amount is needed for DHA production. Chi et al showed that a high level of DO (50% of saturation) during the lipid accumulation phase has a declining effect on lipid content (41). In addition, Kim et al. (2013) showed that the nitrogen source is responsible for the cell growth and multiplication during the early stage of fermentation. Also, their results showed that a carbon source is required during the entire process of cultivation for cell growth and particularly lipid accumulation (42). Jakobsen (2008) showed that a low level of DO in the fermentation medium was favored for the production of DHA (14). Also, Bailey et al. (2003) reported that a high DO level (40%) led to the accumulation of 18.2% (w/w) lipids in CDW. On the other hand, a low DO level (less than 3% of saturation) stimulated the production of DHA up to 24.4% of total fatty acids (29). In conclusion, a low level of oxygen is more effective for lipids accumulation by Aurantiochytrium sp. Also, it is crucial for long-chain PUFAs production via the O₂-independent-PUFA-synthase pathway (43). This work proved and paved the way to use native Aurantiochytrium strain for the production of DHA at the industrial scale.

Acknowledgments

This work is a part of an MSc thesis in chemical engineering. The authors appreciate the Central Lab. of Graduate University of Advanced Technology, Kerman, for fatty acid analysis.