Isochrysis sp. FQZ-2 capable of heterotrophically growing in low sea salt concentration and application of isochrysis sp. FQZ-2

Abstract

The invention discloses isochrysis sp. FQZ-2 capable of heterotrophically growing in low sea salt concentration and application of the isochrysis sp. FQZ-2. The classification name of the isochrysis galbana is isochrysis galbana FQZ-2, the chemical name of the isochrysis galbana FQZ-2 is isochrysis galbana FQZ-2, the isochrysis galbana is preserved in China Center for Type Culture Collection in Wuhan University, Wuhan, China, the preservation date is June 19, 2024, and the preservation number is CCTCC NO: M 20241307. A water sample is collected from a low-salt-concentration position of a mangrove forest in the Quanzhou bay, and after a series of separation and screening including photoautotrophic enrichment, low-salt domestication and heterotrophic purification, a strain of isochrysis sp. FQZ-2 with heterotrophic ability is obtained. The algal strain can quickly grow and accumulate biomass in a 0-10g / L low sea salt concentration culture medium, and high-added-value products such as EPA (eicosapentaenoic acid), DHA (docosahexaenoic acid) and microalgae protein can be produced on a large scale by using a 10-ton fermentation tank.

Description

Technical Field

[0001] The invention relates to the fields of food and bioenergy, and in particular to a strain of Isochrysis chrysotrichum FQZ-2 capable of heterotrophically growing in low sea salt concentration and application thereof. Background Art

[0002] In the practice of aquaculture, protein-rich feeds are widely used to support the growth and development of aquatic organisms. At present, the main sources of these high-protein feeds for aquaculture usually include high-quality soybean meal and fish meal, which provide the necessary nutritional basis for aquatic animals. As the cornerstone of aquaculture, the healthy growth of fry is crucial to the success of the entire breeding cycle. Therefore, in the early stage of breeding, special attention is paid to providing fry with palatable, nutritious and comprehensive starter feed to ensure the survival rate of fry. Among the many fry feeds, Isochrysis chrysophylla has small cells, no cell walls, rich nutrition and easy digestion, so it is an ideal basic feed for the larvae of marine economic bivalves. In addition, Isochrysis chrysophylla is rich in (n-3) polyunsaturated fatty acids and is an algae species with the potential to produce DHA and EPA. As a starter feed for fry, it can improve the feed intake rate and growth performance of aquatic animals, enhance the immunity of aquatic animals, and improve the nutritional value of aquatic animals.

[0003] With the development of industry and agriculture and the improvement of people's living standards, the amount of aquaculture has increased year by year, and the demand for aquatic feed in the aquaculture industry has also been increasing. However, at present in China, many microalgae producers have not yet carried out large-scale production of Isochrysis chrysophylla. This is mainly because the nutritional mode of Isochrysis chrysophylla is mainly photosynthetic autotrophic. There are many disadvantages in developing two cultivation methods based on this nutritional mode, photobioreactors and outdoor open cultivation. Although photobioreactors can provide a relatively controllable culture environment, they have problems such as large footprint, long production cycle and low algae growth density, resulting in high production costs. Although outdoor open cultivation has lower costs, it is strongly affected by environmental conditions (such as light, temperature, water quality, etc.), and the yield and quality are difficult to guarantee. In addition, there is CO 2 There are a series of challenges such as difficulty in replenishment, high harvesting costs, easy contamination by other organisms and high water quality requirements.

[0004] Compared with photosynthetic autotrophic culture, heterotrophic culture of microalgae has its unique advantages. This culture method allows microalgae to grow directly using external supplemented organic matter in the absence of light. Since it is no longer constrained by light conditions, the growth rate of microalgae is significantly improved, so that a high biomass concentration can be accumulated in a relatively short period of time. In addition, heterotrophic culture is often carried out in a closed fermentation tank. This environment not only facilitates steam sterilization of the entire culture system and effectively reduces the risk of contamination by foreign microorganisms, but also ensures that the temperature, pH value and supply of nutrients during the culture process are under precise control, providing a strong guarantee for the stable growth of microalgae. To this end, obtaining a heterotrophic strain of Isochrysis chrysophylla and using traditional biological fermentation tanks to cultivate the strain can greatly improve the yield and quality of Isochrysis chrysophylla, which has significant social, economic and environmental benefits.

[0005] So far, there are very few Isochrysis strains that have been successfully cultivated heterotrophically. In addition, as a single-cell seaweed, the culture medium of Isochrysis needs to be prepared with seawater or tap water mixed with an equal concentration of sea salt (about 25g / L). High-salinity culture medium will not only cause corrosion problems in fermentation tanks, but also lead to a surge in downstream water treatment costs. Therefore, it is particularly important to select Isochrysis strains from nature that can grow and reproduce rapidly in low-salt or freshwater environments. This type of engineered algae strain helps to reduce the salt concentration during heterotrophic fermentation, thereby reducing the cost of cultivation and subsequent equipment maintenance and water treatment. Summary of the invention

[0006] The invention aims to provide a strain of Isochrysis chrysotrichum FQZ-2 capable of heterotrophically growing in low sea salt concentration and application thereof.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows: A strain of Isochrysis chrysophylla, whose taxonomic name is Isochrysis chrysophylla FQZ-2, and its scientific name is Isochrysis galbana FQZ-2), has been deposited in the China Type Culture Collection, Wuhan University, Wuhan, China. The deposit date is June 19, 2024, and the deposit number is: CCTCC NO: M 20241307.

[0008] The Isochrysis algae FQZ-2 can be applied to heterotrophic fermentation.

[0009] The Isochrysis algae FQZ-2 can be used for fermentation to produce algae oil.

[0010] The Isochrysis algae FQZ-2 can be used for fermentation production of DHA and EPA.

[0011] The present invention has the following beneficial effects: The present invention collects water samples from the low salt concentration (5-15g / L) of the mangroves in Quanzhou Bay, and obtains a heterotrophic Isochrysis FQZ-2 after a series of separation and screening, including photoautotrophic enrichment, low salt acclimation, and heterotrophic purification. The algae strain can rapidly grow and accumulate biomass in a medium with low salt concentrations of 0g / L, 5g / L, and 10g / L, and can be used in a 10-ton fermenter to mass-produce high value-added products such as EPA, DHA, and microalgae protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a plate colony morphology diagram of the algae strain isolated in Example 1 of the present invention.

[0013] Figure 2 This is a morphological picture of the algae strain isolated in Example 1 of the present invention under a 40x objective lens.

[0014] Figure 3 It is the 18SrDNA sequence of the algae strain isolated in Example 1 of the present invention.

[0015] Figure 4 This is the growth curve of strain FQZ-2 in a 10000L fermenter. DETAILED DESCRIPTION Example 1

[0016] Screening and identification of strains 1) Enrichment, screening and domestication of Isochrysis algae that can grow in low sea salt concentrations Collect low-salinity water samples (seawater with a salt concentration of 5-15 g / L) from the Luoyang Bridge in the mangrove forest of Quanzhou Bay, add an appropriate amount of f / 2 nutrient salt to the water sample until the liquid turns yellow-brown, seal it, and culture it at 25°C and 2500 lux light intensity. Shake it by hand 6 times a day to initially obtain a low-salinity mixed culture of microalgae. In the clean bench, drop a drop of the culture into the sterilized f / 2 solid culture medium (sea salt concentration 10 g / L), and use an inoculation needle or inoculation loop (after careful burning and sterilization) to draw the algae liquid parallel to the surface of the agar culture medium. Seal the glass culture dish with a sealing film and culture it at 25°C and 2500 lux light intensity for 10 days. When microalgae grow on the plate, pick a single algae colony and transfer it to a new f / 2 solid plate (sea salt concentration 10 g / L). Repeat the above steps until there is only a single algae colony on the plate.

[0017] The various single algae colonies obtained were inoculated into f / 2 liquid culture medium prepared with fresh water that does not contain sea salt, and cultured at 25°C and 2500lux light intensity for 10 days. The microalgae that cannot adapt to fresh water are inhibited from growing, while the microalgae that can adapt to fresh water can grow, thus obtaining marine microalgae that can grow in fresh water.

[0018] 2) Screening of microalgae capable of heterotrophic cultivation Use fresh water to prepare f / 2 culture medium (without sea salt), and add 10g / L of organic carbon source (glucose, glycerol, sucrose, sodium citrate, sodium acetate, etc.) to it to prepare heterotrophic culture medium suitable for heterotrophic growth of microalgae. Sterilize it by high temperature and high pressure, and use it after it is fully cooled.

[0019] All the pure microalgae culture solutions obtained in step 1) were inoculated into heterotrophic culture medium and cultured at 25°C in the dark for 10 days. Microalgae that can adopt heterotrophic nutrition can grow normally, while microalgae that cannot adopt heterotrophic nutrition will be significantly inhibited in the absence of light. The obtained algae species FQZ-2 that can grow heterotrophically was stored on an f / 2 solid slope with an organic carbon source added.

[0020] 3) Identification of strains The microalgae strain FQZ-2 obtained in step 2) was amplified and the 18SrDNA and ITS sequences were determined. Figure 1 ) and l8S rDNA sequence analysis results ( Figure 3 ), and the strain was identified as Isochrysis chrysotrichum FQZ-2 ( Isochrysis galbana FQZ-2), and sent the preserved strain to the China Type Culture Collection at Wuhan University, Wuhan, China for preservation and registration. The preservation date is June 19, 2024, and the preservation number is: CCTCC NO: M20241307. Example 2

[0021] Application of Isochrysis chrysotrichum FQZ-2 in heterotrophic fermentation 1) The Isochrysis algae FQZ-2 strain on the solid slope was inoculated into a heterotrophic medium without sea salt, and cultured at 25°C and 150 r / min for 5 days to prepare the fermentation FQZ-2 seed liquid.

[0022] 2) Take the seed liquid of FQZ-2 above, first carry out preliminary culture in 1L shake flask, then carry out step-by-step culture in 100L fermenter to obtain enough seed liquid. After that, the seed liquid is transferred to a fermenter with a capacity of 10,000L, and fed-batch fermentation is carried out under the conditions of constant temperature of 25°C and rotation speed set to 200rpm. After 5 days of fermentation, the final biomass obtained reaches 55g / L.

[0023] Figure 4 This is the growth curve of Isochrysis chrysotrichum FQZ-2 in a 10000L fermenter. Figure 4It can be seen that after Isochrysis FQZ-2 was connected to the 10000L fermenter, it could grow rapidly, quickly enter the exponential growth phase after 25 hours of cultivation, and obtain the maximum biomass of 55g / L at 96 hours, indicating that Isochrysis FQZ-2 has good potential for industrial production. Example 3

[0024] Application of Isochrysis chrysotrichum FQZ-2 in the production of algal oil by fermentation 1) The fermentation liquid of Isochrysis chrysotrichum FQZ-2 obtained in the 10000L fermenter of Example 2 was harvested by a butterfly centrifuge, and extracted by n-hexane-ethanol (1:2) method at a solid-liquid ratio of 1:20 for 5 hours at an extraction temperature of 50°C. After the extraction was completed, the n-hexane phase was collected, and after concentration and solvent recovery, the final n-hexane phase crude oil yield was 25%. Example 4

[0025] Application of Isochrysis chrysotrichum FQZ-2 in the fermentation production of DHA and EPA The crude oil in the n-hexane phase obtained in Example 3 was thoroughly mixed with ethanol at a molar ratio of 1:100, and then lipase CAL-A (added amount 2%) was added, and the mixture was reacted at 35° C. for 48 hours. After concentration and recovery of ethanol, the oil recovery rate was 45%, of which the total content of ω-3 fatty acids (DHA, EPA) reached 90%.

Claims

1. A plant of Isochrysis chrysophylla, It is characterized in that Its classification name is Isochrysis FQZ-2, and its scientific name is Isochrysis galbana FQZ-2), has been deposited in the China Type Culture Collection, Wuhan University, Wuhan, China. The deposit date is June 19, 2024, and the deposit number is: CCTCC NO: M 20241307.

2. Use of Isochrysis as claimed in claim 1 in heterotrophic fermentation.

3. Use of Isochrysis as claimed in claim 1 in fermentation production of algae oil.

4. Use of Isochrysis as claimed in claim 1 in fermentation production of DHA and EPA.

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