Characteristics and application of aerial microalgae Pilidiocystis sp. FDA-4 with high C18 yield

Abstract

The invention discloses characteristics and application of aerial microalgae Pilidiocystis sp. FDA-4 with high yield of C18, and belongs to the technical field of microalgae biology. The aerial microalgae FDA-4 is preserved in the China Center for Type Culture Collection on July 1, 2025, and the preservation number of the aerial microalgae FDA-4 is CCTCC NO: M 20251502. The strain has excellent low-pH adaptability and organic acid tolerance, can be directly cultured by using lactic acid bacteria waste liquid as the unique carbon source and nutrient medium, the optimal dilution ratio is 100 times, the biomass reaches 0.19 g / L after 12 days of culture, an exogenous nitrogen source does not need to be added and the initial pH does not need to be adjusted, the fatty acid composition is mainly C18 series, and the strain has a wide application prospect. The produced grease can be used for preparing products for reducing the low-density lipoprotein level and preventing cardiovascular diseases; meanwhile, the strain can efficiently convert nutrient substances in the lactic acid bacteria waste liquid, resource utilization of the waste liquid is achieved, and the strain has remarkable application value and industrialization potential.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microalgae biotechnology, and particularly relates to a strain of C18 high-yield aerogenic microalgae Pilidiocystis sp. FDA-4 and its application. BACKGROUND

[0002] As an important biological resource, microalgae can efficiently synthesize a variety of valuable products through photosynthesis, which makes it have important potential in the production of biological products and functional foods. With the increasing demand for efficient and sustainable biological resources, the practical application demand of microalgae is also rising.

[0003] C18 fatty acids, especially C18:1 (oleic acid), C18:2 (linoleic acid) and C18:3 (alpha-linolenic acid), have important applications in many fields. C18:1 is widely used in food and health products, can reduce low-density lipoprotein levels, prevent cardiovascular diseases, and is also one of the key raw materials for biodiesel. C18:2 (linoleic acid) as an omega-6 fatty acid, is essential for regulating the immune system and promoting skin health, and also plays an important role in the plastics, coatings and lubricating oil industries. C18:3 (alpha-linolenic acid) as an omega-3 fatty acid, has anti-inflammatory, cardiovascular protection and promotes brain and vision development effects.

[0004] The existing high-yield C18 fatty acid microalgae species is relatively scarce, and its resource cultivation and application still face many challenges. Aerogenic algae growing on the surface of tree bark need to cope with extreme environmental conditions such as temperature fluctuations, drought and strong light, which requires them to have stronger adaptability and higher metabolic activity and product accumulation capacity. SUMMARY

[0005] The present application provides a high-yield C18 fatty acid aerogenic microalgae Pilidiocystis sp. FDA-4 isolated from the surface of camphor tree bark, and the preservation number of the aerogenic microalgae FDA-4 is CCTCC NO: M 20251502.

[0006] The application further provides a method for preparing C18 fatty acid by using the aerotole microalgae (Pilidiocystis sp.) FDA-4 in claim 1, i.e. culturing the aerotole microalgae FDA-4 by using the diluted Lactobacillus acidophilus waste liquid to reduce the culture cost and realize the resource production of C18 fatty acid. The lactic acid bacteria waste liquid is rich in small molecule organic acids and sugars which are easily absorbed by algae, and is used as a high-quality carbon source in the heterotrophic or mixed culture system. At the same time, the metabolic by-products (such as vitamins and amino acids) in the waste liquid can also promote the growth and metabolic activity of algae. However, due to the complex composition and acidic environment of the lactic acid bacteria waste liquid, conventional algae strains are difficult to grow in this environment. After research and screening, the Pilidiocystis sp. FDA-4 shows good growth potential in the lactic acid bacteria waste liquid, and is suitable for culturing in the lactic acid bacteria waste liquid, thereby realizing the efficient and low-cost resource production of C18 fatty acid.

[0007] Preferably, the dilution multiple of the Lactobacillus acidophilus waste liquid is 80-120, such as 80, 85, 90, 95, 100, 105, 110, 115, 120.

[0008] Preferably, the culture time is at least 6 days, such as 12 days. When the dilution multiple of the lactic acid bacteria waste liquid for culturing the Pilidiocystis sp. FDA-4 is 100 times, the biomass is 0.19 g / L after 12 days of culture.

[0009] The application further provides the application of the above-mentioned aerotole microalgae (Pilidiocystis sp.) FDA-4 in preparing C18 fatty acid, which comprises directly extracting C18 fatty acid in the aerotole microalgae FDA-4.

[0010] Preferably, the aerotole microalgae FDA-4 is further cultured before extraction.

[0011] Preferably, the application of the aerotole microalgae FDA-4 in preparing C18 fatty acid comprises directly extracting C18 fatty acid in the aerotole microalgae FDA-4.

[0012] The application further provides the application of the above-mentioned aerotole microalgae (Pilidiocystis sp.) FDA-4 in the resource treatment of the Lactobacillus acidophilus waste liquid, i.e. culturing the aerotole microalgae FDA-4 by using the diluted Lactobacillus acidophilus waste liquid.

[0013] Compared with the prior art, the application has the following beneficial effects: Compared with existing algae strains, the aerial algae Pilidiocystis sp. FDA-4 from the surface of tree bark exhibits unique environmental adaptability, and can maintain good growth in a culture system with high organic acid and low pH, such as lactic acid bacteria fermentation waste liquid, while other conventional algae are generally inhibited or die, such as Spirulina subsalsa FDA-2 which is used in large scale by the research group. The aerial algae Pilidiocystis sp. FDA-4 can efficiently utilize the soluble organic carbon in the waste liquid, realizing the biological conversion of carbon source and accumulation of oil. The oil produced by the aerial algae Pilidiocystis sp. FDA-4 has a high proportion of C18 as the main component, and the proportion of unsaturated fatty acids reaches as high as 77%. The product has excellent nutritional value and biochemical characteristics, and is suitable for reducing low-density lipoprotein levels and preventing cardiovascular diseases.

[0014] Biological preservation of aerial algae FDA-4: Preservation agency: China Center for Type Culture Collection; Preservation number: CCTCC NO: M 20251502; Preservation date: July 1, 2025; Preservation address: Wuhan, China, Wuhan University; Taxonomic name: Pilidiocystis sp. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The micro-morphology photo of Pilidiocystis sp. FDA-4 in Example 1.

[0016] Figure 2 The phylogenetic tree of Pilidiocystis sp. FDA-4 in Example 1.

[0017] Figure 3 The scanning electron microscope image of Pilidiocystis sp. FDA-4 in Example 1.

[0018] Figure 4 The composition and content of each major fatty acid of Pilidiocystis sp. FDA-4 in Example 2.

[0019] Figure 5 The comparison chart of the proportion of C18 of Pilidiocystis sp. FDA-4 and the proportion of C18 of Spirulina subsalsa FDA-2 in Example 2.

[0020] Figure 6Figure 4. Biomass of Pilidiocystis sp. FDA-4 cultured in different concentrations of lactobacillus spent broth for different days in Example 3.

[0021] Figure 7 Figure 5. Biomass comparison of Pilidiocystis sp. FDA-4 and Spirulina subsalsa FDA-2 cultured in 100-fold dilution of optimal lactobacillus broth for 14 days in Example 3.

[0022] Figure 8 Figure 6. pH of Pilidiocystis sp. FDA-4 cultured in different concentrations of lactobacillus spent broth for different days in Example 4.

[0023] Figure 9 Figure 7. Dissolved oxygen of Pilidiocystis sp. FDA-4 cultured in different concentrations of lactobacillus spent broth for different days in Example 4. DETAILED DESCRIPTION

[0024] Example 1 Collection, observation and identification of Pilidiocystis sp. FDA-4 Pilidiocystis sp. FDA-4 has the accession number CCTCC M 20251502 and is deposited at China Center for Type Culture Collection, and is isolated from the bark of a camphor tree at the coordinates of 31°17'N, 121°30'E.

[0025] (1) Sample collection The sample was collected from the surface of the camphor tree bark in January 2024 and purified.

[0026] (2) Observation of Pilidiocystis sp. FDA-4 The algal cells appear green under a light microscope, and the vegetative cells are fusiform, about 12-20 µm long and 5-8 µm wide. The microstructure is shown in Figure 1 Each stage is mononuclear and contains one protein nucleus. The cell wall is smooth and thin, and the cell is symmetrical on both sides. With cell aging, the cell wall thickens. Its asexual reproduction is performed by forming 2, 4 or 8 similar spores.

[0027] (3) Identification of Pilidiocystis sp. FDA-4 To confirm the taxonomic status of strain FDA-4, its 18S rRNA gene was sequenced, and a phylogenetic tree was constructed based on the sequence using the maximum likelihood method, as shown in Figure 2The results showed that FDA-4 was clustered with Pilidiocystis sequences, confirming its affiliation to Pilidiocystis sp. Scanning electron microscope (SEM) images of Pilidiocystis sp. FDA-4 are shown in FIG. 2. Figure 3 As shown in FIG. 2, the cell morphology was consistent with the characteristics of the genus, and the morphological evidence was confirmed by the results of molecular systematics.

[0028] Example 2 (1) Fatty acid determination of Pilidiocystis sp. FDA-4 Fatty acid methyl esters (FAME) were prepared by acid-catalyzed transesterification, and Pilidiocystis sp. FDA-4 was analyzed by gas chromatography-mass spectrometry (GC-MS).

[0029] Chromatographic conditions: DB-23 capillary column (60 m x 0.25 mm x 0.25 µm); carrier gas helium, constant flow 1.0 mL / min; injection port temperature 250 ℃, split ratio 10:1, injection volume 1 µL.

[0030] During the determination, first prepare the required reagents: mix chloroform and methanol at a volume ratio of 2:1 to prepare a chloroform-methanol solution; prepare a 5% HCl-methanol solution with concentrated hydrochloric acid and methanol; dissolve tridecanoic acid with n-hexane to prepare a 0.2 mg / mL tridecanoic acid solution; similarly, prepare a 0.2 mg / mL pentadecane working solution with n-hexane as the solvent. Then, accurately weigh about 10 mg of freeze-dried algal powder sample into a 1.5 mL GC sample bottle, with two replicates for each sample. Next, use a microsyringe to add 200 µL of chloroform-methanol mixture, 300 µL of 5% HCl-methanol solution, and 25 µL of 0.2 mg / mL tridecanoic acid solution to each sample bottle in turn. After shaking well, place the sample bottles in an 85 ℃ oven for 1 hour.

[0031] After the reaction is complete, remove the sample bottles and cool them to room temperature for 15-60 minutes. Then add 1 mL of n-hexane to each bottle, shake well, and let it stand at room temperature for 1-4 hours. Then take 40 µL of the supernatant and add it to a new GC sample bottle containing 960 µL of n-hexane and 25 µL of 0.2 mg / mL pentadecane, shake well, and prepare for machine detection.

[0032] As shown in FIG. 3, the fatty acid composition of Pilidiocystis sp. FDA-4 was determined. Figure 4As can be seen, the fatty acid composition of Pilidiocystis sp. FDA-4 is mainly composed of C18 series (total proportion of about 77.29%), among which the contents of oleic acid (C18:1, 33.43%) and linoleic acid (C18:2, 30.20%) are the highest, followed by linolenic acid (C18:3, 12.34%) and palmitic acid (C16:0, 19.79%). Overall, the saturated fatty acids account for about 22.24%, the unsaturated fatty acids account for about 77.75%, among which the monounsaturated fatty acids account for 35.21% and the polyunsaturated fatty acids account for 42.54%. The contents of long-chain fatty acids (C20 and above) and short-chain fatty acids (≤C14) are very low, and the C16-C18 fatty acids are mainly present. In comparison, the content of C18 series fatty acids of Spirulina subsalsa FDA-2 is relatively low, about 34.64% (see Table 1), which shows a significant difference in fatty acid composition between the two. Figure 5

[0033] Example 3 Cultivation of Pilidiocystis sp. FDA-4 (1) A certain concentration of Pilidiocystis sp. FDA-4 was inoculated in Lactobacillus acidophilus fermentation waste liquid (after Lactobacillus acidophilus was fermented in MRS medium, the supernatant was obtained by centrifugation), wherein the dilution range of the lactic acid bacteria waste liquid was 10 times to 200 times.

[0034] (2) Pilidiocystis sp. FDA-4 was cultured in lactic acid bacteria waste liquid with different dilution multiples for 0-12 days, the culture temperature was 25°C, the light intensity was 90 µmol / m 2 / s, as shown in Figure 1. Figure 6

[0035] The experimental results show that Pilidiocystis sp. FDA-4 can effectively grow in the lactic acid bacteria waste liquid system, and the growth condition thereof shows a significant difference with the change of the dilution multiple of the waste liquid. When the dilution multiple of the waste liquid is relatively low (10 times, 50 times), the growth of the algal body is relatively inhibited due to the high concentration of organic load and inhibitory substances in the culture liquid; when the dilution multiple is increased to 100 times to 200 times, the inhibitory effect is weakened, and the growth rate of the algal body is significantly improved, among which the growth under the condition of 100 times dilution is the most ideal. Figure 7 ​​The growth of Spirulina subsalsa FDA-2 and Pilidiocystis sp. FDA-4 in lactic acid bacteria wastewater was compared, and it was shown that Pilidiocystis sp. FDA-4 could still maintain good growth in 100-fold diluted lactic acid bacteria wastewater, while Spirulina subsalsa FDA-2 could not maintain normal physiological activity, further verifying the superior adaptability of Pilidiocystis sp. FDA-4 in this culture system.

[0036] The results show that the lactic acid bacteria wastewater contains nutrients (such as organic carbon, nitrogen source and phosphorus source) that can be utilized by algae, and moderate dilution can reduce the inhibitory effect while promoting the normal growth of algae. The research results prove that Pilidiocystis sp. FDA-4 has the ability to directly use lactic acid bacteria wastewater as a carbon source for culture medium, and can realize the resource utilization of lactic acid bacteria wastewater, which has high application value and industrialization potential.

[0037] Example 4 The Pilidiocystis sp. FDA-4 biomass cultured in lactic acid bacteria wastewater for 0-12 days was collected and its pH and dissolved oxygen were measured, as shown in Figure 8 and 9 .

[0038] To evaluate the synergistic effect of oxygen supply conditions and acid-base environment on algal proliferation, the dissolved oxygen and pH of Pilidiocystis sp. FDA-4 in BG11 and lactic acid bacteria wastewater with different dilution ratios were monitored simultaneously. The results showed that the dissolved oxygen of the BG11 group was always close to air saturation (8.3-8.6 mg·L -1 ), and the pH remained in the alkaline range, with the fastest growth of the algae. The initial dissolved oxygen of the lactic acid bacteria wastewater group was significantly lower, and the smaller the dilution ratio, the more obvious the oxygen deficiency. The initial dissolved oxygen of the 100-fold and 200-fold groups was only about 0.7 mg·L -1 , that of the 50-fold group was 2.8 mg·L -1 , and that of the 10-fold group was higher initially but then decreased rapidly, which was consistent with the characteristic of the initial pH being acidic, indicating that high organic load and respiratory oxygen consumption dominated at this stage. As the culture time increased, the pH gradually rose to the weak alkaline range of 8.3-8.9, and the dissolved oxygen concentration also increased and stabilized. The dissolved oxygen concentration of the 100-fold and 200-fold groups stabilized at 7.1-7.8 mg·L -1 , that of the 50-fold and 10-fold groups rose to 6.8 mg·L -1 and 5.2 mg·L -1The pH increase and the change of dissolved oxygen recovery are consistent with the change of algal body from initial inhibition to normal proliferation stage, which indicates that moderate dilution (100-200 times) can simultaneously alleviate the acidity and oxygen deficiency problems, so that the system quickly enters a state favorable to photosynthesis and stable growth of algal body, thereby realizing high-efficiency proliferation and controllable culture of FDA-4 in lactic acid bacterial waste liquid.

[0039] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.

Claims

1. An aerial microalga (Pilidiocystis sp.) FDA-4, characterized in that, The accession number of the aerial microalgae FDA-4 is CCTCC No: M 20251502.

2. A method for preparing C18 fatty acids using the aerial microalgae (Pilidiocystis sp.) FDA-4 as described in claim 1, characterized in that, FDA-4 aerial microalgae were cultured using diluted Lactobacillus acidophilus fermentation waste liquid.

3. The method according to claim 2, characterized in that, The dilution ratio of the Lactobacillus acidophilus fermentation waste liquid is 80-120.

4. The method according to claim 3, characterized in that, The dilution factor of the Lactobacillus acidophilus fermentation waste liquid is 100.

5. The method according to any one of claims 2-4, characterized in that, The culture time is at least 6 days.

6. The application of the aerial microalgae (Pilidiocystis sp.) FDA-4 as described in claim 1 in the preparation of C18 fatty acids, characterized in that, This includes the direct extraction of C18 fatty acids from aerial microalgae FDA-4.

7. The application according to claim 6, characterized in that, Before extraction, aerial microalgae (FDA-4) must be cultured.

8. The application of the aerial microalgae (Pilidiocystis sp.) FDA-4 as described in claim 1 in the resource utilization treatment of Lactobacillus acidophilus fermentation waste liquid, characterized in that, FDA-4 aerial microalgae were cultured using diluted Lactobacillus acidophilus fermentation waste liquid.

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