Culture method of seawater microalgae

Abstract

The invention provides a culture method of seawater microalgae. The culture method comprises the following steps: sequentially carrying out first-step culture, second-step culture, third-step culture and fourth-step culture on algae species of the seawater microalgae to obtain the seawater microalgae, 4, culturing in an outdoor pipeline-type photobioreactor, and carrying out shading treatment on the pipeline-type photobioreactor until the shading area is greater than or equal to 65%; wherein the light intensity of the shading part is 0-200 [mu] mol photon.m <-2 >. S <-1 >, and the light intensity of the non-shading part is 0-2000 photon.m <-2 >. S <-1 >. According to the method, the seawater microalgae can grow in a better state through a culture method of expanding a culture system step by step and finally utilizing a sealed pipeline type photobioreactor to perform fourth-step culture, and the illumination intensity at the culture stage is adjusted, so that the culture efficiency and the yield of the seawater microalgae are improved, and the yield of fucoxanthin is improved.

Description

Technical Field

[0001] The invention relates to the technical field of algae cultivation, and in particular to a method for cultivating seawater microalgae. Background Art

[0002] Microalgae is a kind of biological resource with high application value. The advantages of microalgae are its rich resources, wide variety and fast growth rate. Diatoms are a kind of marine microalgae, which are widely distributed in the ocean. They are unicellular planktonic ecological groups in marine microalgae and the main group of marine phytoplankton. The number and species can account for more than 90%. Its biomass accounts for about one-fifth of the primary productivity of the earth and two-fifths of the total primary productivity of the ocean. It is of great ecological significance. Diatoms have particularly high nutritional value and contain a variety of biologically active substances, such as enzyme inhibitors, antibiotics, and pharmaceutically active ingredients. They are especially rich in fucoxanthin and eicosapentaenoic acid (EPA) and other edible and medicinal nutrients. They also have the characteristics of fast growth rate and short cycle, and have high commercial value. Diatoms such as long-eared tooth algae have been included in the new resource food catalog in some European and American countries.

[0003] Marine diatoms that are being commercially developed include Phaeodactylum tricornutum, Thalassiosira weissflogii, Thalassiosira punctigera, Odontella aurita, Thalassiosira pseudonana, Skeletonema costatum, Pseudo-nitzschia, Chaetoceroscurvisetus and other algae species.

[0004] Fucoxanthin, also known as brown algae and fucoxanthin, is a natural pigment of the xanthophyll class in carotenoids, accounting for more than 10% of the total amount of about 700 naturally occurring carotenoids. It is orange-red to brown in color and is a pigment contained in brown algae, diatoms, golden algae and haptophytes. It is widely found in various algae, marine phytoplankton, aquatic shellfish and other animals and plants. It has beneficial pharmacological effects such as anti-tumor, anti-inflammatory, antioxidant, weight loss, and nerve cell protection. It can increase the content of ARA (arachidonic acid) and DHA (docosahexaenoic acid) in mice. It is widely used in the market as a medicine, skin care and beauty product, and health care product. Eicosapentaenoic acid, also known as EPA, belongs to the ω-3 series of polyunsaturated fatty acids and is an important nutrient that is indispensable to the human body. EPA can help reduce the content of cholesterol and triglycerides, promote the metabolism of saturated fatty acids in the body, thereby reducing blood viscosity, improving blood circulation, improving tissue oxygen supply and eliminating fatigue, preventing fat deposition in blood vessel walls, preventing the formation and development of atherosclerosis, and preventing cardiovascular diseases such as cerebral thrombosis, cerebral hemorrhage, and hypertension. Diatoms such as P. triangularis and P. longiflora can accumulate the above two beneficial functional components at the same time, which is worth large-scale cultivation and development.

[0005] Odontella aurita is a marine unicellular diatom with a cylindrical body of 10-95 μm and rich in fucoxanthin and eicosapentaenoic acid (EPA). As a highly functional carotenoid, fucoxanthin has multiple physiological functions such as antioxidant, anti-tumor, weight loss, and anti-inflammatory, and has high utilization value in food, health products and medicines. Phaeodactylum tricornutum belongs to the Phaeodactylum genus of Phaeodactylum family, Phaeodactylum order, Phaeodactylum order, Phaeodactylum family, Phaeodactylum tricornutum, which is a unicellular floating algae widely distributed in the global marine environment. The cell size is generally around 5-8 μm, and the cell wall is composed of silica. The algae is rich in protein, polysaccharides, carotenoids and polyunsaturated fatty acids (such as EPA, etc.), has high nutritional value and health functions, and can be used in food, feed, cosmetics and medicine industries. For example, the EPA extracted from it is widely believed to be beneficial to human cardiovascular health and can be made into nutritional supplements.

[0006] However, the current production and research of Long-eared Toothed Algae are mostly concentrated in the laboratory scale, and the outdoor culture reactors are mostly open runway pools. This method has low yields and is susceptible to environmental pollution, making it difficult to continuously and stably produce high-quality Long-eared Toothed Algae biomass. The overall industrial scale of the cultivation research of Triangular Brown Finger Algae is also relatively small, and it faces challenges in technical bottlenecks, cost control and market promotion.

[0007] At present, there are three large-scale cultivation methods for microalgae: photosynthetic autotrophy, heterotrophy and mixotrophy. Photosynthetic autotrophic microalgae grow by their own photosynthesis, heterotrophic microalgae need to be artificially added with organic matter to maintain their growth, and some other microalgae species can combine photosynthetic autotrophy and heterotrophic assimilation of organic matter to grow mixotrophically. The photosynthetic autotrophic method is divided into open raceway pool and closed photobioreactor culture. In large-scale microalgae production, open pool cultivation has low cost and low energy consumption, but it is particularly susceptible to contamination by other algae and protozoa. Only a few microalgae species with special adaptability can adapt to this cultivation method, such as Chlorella adapting to high-nutrient culture medium, Dunaliella adapting to high-salt environment, and Spirulina adapting to high-alkali environment. This method is not suitable for small microalgae with weak environmental adaptability, such as long-eared tooth algae and triangular brown finger algae. For example, the French Sanofi Bio-Industries company uses open pools to cultivate seawater diatoms, and the yield of fucoxanthin is only 1.86 mg / g. The main reason is that the open pond culture process is too extensive and cannot provide the growth environment required by diatom cells in stages, nor can it accurately provide the optimal growth environment required to promote unsaturated fatty acids such as fucoxanthin and EPA.

[0008] Compared with open pools, closed photobioreactors completely overcome the problem of easy pollution and can cultivate a single type of microalgae stably for a long time. They can provide the required growth environment according to the different growth stages of algae cells, including the required temperature, pH environment, light intensity, nutrients, CO 2 The current large-scale cultivation of freshwater microalgae Haematococcus pluvialis uses existing closed pipeline photobioreactors, which have significant advantages in controlling pollution and increasing production, and can provide a more stable cultivation environment and stable production. However, the cultivation of seawater microalgae in pipeline reactors is not mature and cannot meet the needs of industrialized and efficient production.

[0009] Therefore, how to provide a cultivation method that can efficiently produce seawater microalgae in a closed reactor is of great significance to the subsequent commercial development of seawater microalgae. Summary of the invention

[0010] The main purpose of the present invention is to provide a method for culturing marine microalgae, by which high value-added polyunsaturated fatty acid substances such as fucoxanthin and EPA can be stably accumulated in marine microalgae cells, so as to solve the problem that the biomass and the accumulation of fucoxanthin and EPA in the prior art of cultivating marine microalgae are both low.

[0011] In order to achieve the above-mentioned object, according to a first aspect of the present invention, a method for cultivating seawater microalgae is provided, the method comprising: using seawater microalgae species to carry out a first step of cultivation, a second step of cultivation, a third step of cultivation and a fourth step of cultivation in sequence to obtain seawater microalgae; the capacity of the cultivation system of the first step of cultivation, the second step of cultivation, the third step of cultivation and the fourth step of cultivation is increased in sequence; the fourth step of cultivation is carried out in an outdoor tubular photobioreactor, the tubular photobioreactor is subjected to a light shielding treatment, and the light shielding area is ≥65%; wherein the light intensity of the light shielding part is 0-200 μmol photon·m -2 ·s -1 The light intensity of the unshielded part is 0-2000 μmol photon·m -2 ·s -1 The marine microalgae suitable for the cultivation method include diatoms such as Long-eared Toothed Algae and Triangular Brown Finger Algae.

[0012] Further, the first step of culturing comprises: inoculating the seawater microalgae species into a culture tube, performing the first step of aeration culture, and obtaining the first step of culture solution; preferably, the inoculation density of the seawater microalgae species is 0.6-1 g / L; preferably, the mass content of fucoxanthin in the seawater microalgae species is 1.5%-2%; preferably, the air flow rate of the first step of aeration culture is 0.1-0.4 L / min; preferably, the CO 2 The flow rate is 25-40 mL / min; preferably, CO 2 The ratio to compressed air is 0.25-1%.

[0013] Furthermore, the light intensity of the first step of aeration culture is 50-100 μmol photon·m -2 ·s -1 ; Preferably, the pH of the first step of aeration culture is 8-8.5; Preferably, the temperature of the first step of aeration culture is 20-28°C; Preferably, the time of the first step of aeration culture is 8-10 days.

[0014] Furthermore, the second step of culturing includes: inoculating the first step culture solution into a 10L membrane bag reactor, and performing the second step of aeration culturing to obtain the second step culture solution; preferably, the inoculation density of the seawater microalgae in the first step culture solution is 0.5-0.8 g / L; preferably, the air flow rate of the second step of aeration culturing is 1.2-1.6 m 3 / h; preferably, the CO of the second step aeration culture 2 The flow rate is 50-100mL / min.

[0015] Furthermore, the light intensity of the second step aeration culture is 50-100 μmol photon·m -2 ·s -1; Preferably, the pH of the second step aeration culture is 8-8.5; Preferably, the temperature of the second step aeration culture is 20-28°C; Preferably, the time of the second step aeration culture is 8-10 days.

[0016] Further, the third step of culturing includes: after the second step of culturing solution is subjected to a first sedimentation, the supernatant is removed to obtain a first batch of concentrated algae species; the first batch of concentrated algae species is inoculated into a 20L membrane bag reactor or a 300L plate-type membrane bag photobioreactor, and a third step of aeration culture is performed to obtain a third step of culturing solution; preferably, the pH of the third step of aeration culture is 8-8.5; preferably, the temperature of the third step of aeration culture is 20-30°C; preferably, the time of the third step of aeration culture is 8-10 days; preferably, the light intensity of the third step of aeration culture is 50-150 μmol photon·m -2 ·s -1 Preferably, after the first batch of concentrated algae species is inoculated into the first culture medium, the inoculation density of the seawater microalgae in the culture solution to be cultured in the third step is 0.5-0.8 g / L.

[0017] Furthermore, when the third step of aeration culture is carried out in a 20 L membrane bag reactor, the air flow rate of the third step of aeration culture is 1.6-2.0 m 3 / h; preferably, when the third step of aeration culture is carried out in a 20L membrane bag reactor, the CO 2 The flow rate is 5-10 L / min; preferably, when the third step of aeration culture is carried out in a 300 L plate-type membrane bag photobioreactor, the air flow rate of the third step of aeration culture is 1.2-1.6 m 3 / h; preferably, when the third step of aeration culture is carried out in a 300L plate-type membrane bag photobioreactor, the CO 2 The flow rate is 50-100L / min.

[0018] Further, the fourth step of culturing includes: after the third step of culture solution is subjected to a second sedimentation, the supernatant is removed to obtain a second batch of concentrated algae species; the second batch of concentrated algae species are inoculated in a pipeline photobioreactor, and a fourth step of aeration culture is carried out, and the seawater microalgae in the fourth step of culture solution is harvested when the density reaches 2-2.5 g / L to obtain seawater microalgae; preferably, after the second batch of concentrated algae species are inoculated in the first culture medium, the inoculation density of the seawater microalgae in the culture solution of the fourth step of aeration culture is 0.5-0.8 g / L; preferably, the air flow rate of the fourth step of aeration culture is 1.6-2.5 m 3 / h; preferably, the CO of the fourth step aeration culture 2 The flow rate is 100-400L / h.

[0019] Furthermore, the pH of the fourth step aeration culture is 8-8.5; preferably, the temperature of the fourth step aeration culture is 20-30°C.

[0020] Further, the first culture medium is used for the first culture step, the second culture step, the third culture step and the fourth culture step; the first culture medium comprises: 0.5-1.5 g / L NaNO 3 , 0.01-0.0315g / L FeCl 3 6H 2 O, 0.5-1.781×10 -4 g / LMnCl 2 ·4H 2 O, 0.5-2.3×10 -5 g / L ZnSO 4 ·4H 2 O, 0.1-1.19×10 -5 g / L CoCl 2 6H 2 O, 0.5-2.5×10 -6 g / LCuSO 4 ·5H 2 O, 1.84×10 -6 g / L Na 3 VO 4 , 15-35g / L sea salt, 0.1-0.3g / L NaSiO 3 ·4H 2 O, 0.01-0.03 g / L NaH 2 PO 4 ·H 2 O, 0.5-2×10 -4 μg / L Vitamin B 1 , 0.1-1×10 -6 μg / L biotin and 0.1-1×10 -6 μg / L Vitamin B 12 .

[0021] By applying the technical solution of the present invention, a cultivation method is adopted in which a step-by-step cultivation system is expanded, and a closed tubular photobioreactor is used for the fourth step of cultivation, and the tubular photobioreactor is subjected to shading treatment. By adjusting the light intensity of the cultivation, the seawater microalgae can grow under a suitable state, and the cultivation efficiency and yield of the seawater microalgae can be improved, and the yield of the rich fucoxanthin and polyunsaturated fatty acids such as EPA can also be further improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0023] Figure 1 A curve diagram showing the change in dry weight of the long-eared dentata in the first step of cultivation in Example 1 as a function of cultivation time;

[0024] Figure 2 A curve chart showing the change of fucoxanthin content in the long-eared dentata cultured in the first step in Example 1 as a function of culture time;

[0025] Figure 3 A curve diagram showing the change in dry weight of the long-eared dentata cultured in the second step in Example 1 as a function of culture time;

[0026] Figure 4 A curve diagram showing the change in dry weight of the long-eared dentata cultured in the third step of Example 1 as a function of culture time;

[0027] Figure 5 A curve diagram showing the change in dry weight of the long-eared dentata cultured in the third step of Example 2 as a function of culture time;

[0028] Figure 6 A data graph showing the changes of the culture liquid temperature and the gas temperature with the culture time in the pipeline photobioreactor in the fourth step of culture in Example 1;

[0029] Figure 7 A curve diagram showing the change of the dry weight of the long-eared dentata cultured in the fourth step in Example 1 and the phycoxanthin content therein with the culture time;

[0030] Figure 8 A curve diagram showing the change in dry weight of Phaeodactylum triangularis cultured in the fourth step in Example 3-5 as a function of culture time;

[0031] Fig. 9 A curve chart showing the change of fucoxanthin content in the triangular flavonoids cultured in the fourth step in Example 3-5 as a function of culture time;

[0032] Fig.10 A curve chart showing the change in dry weight of the long-eared dentata cultured in the fourth step as a function of culture time in Example 1 and Comparative Example 1 is shown;

[0033] Fig.11 The microscopic morphology of the long-eared tooth algae cells cultured for 5 days in the fourth step in Example 1 and Comparative Example 1 is shown;

[0034] Fig.12A curve chart showing the change in dry weight of the long-eared dentata over culture time in the fourth culture using the artificial seawater culture medium and the first culture medium of the present application in Comparative Example 2. DETAILED DESCRIPTION

[0035] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0036] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0037] As mentioned in the background technology, the large-scale closed culture method for seawater microalgae is still immature. It has the problems of complex operation and poor algae growth activity as the scale of the culture water body expands. Therefore, how to provide a method that can cultivate large-scale seawater microalgae efficiently and with high quality is of great significance to the commercial development of this type of algae.

[0038] In a first typical embodiment of the present application, a method for culturing seawater microalgae is provided, the method comprising: using seawater microalgae species to perform a first step of culturing, a second step of culturing, a third step of culturing and a fourth step of culturing in sequence to obtain seawater microalgae; the capacity of the culturing system of the first step of culturing, the second step of culturing, the third step of culturing and the fourth step of culturing is increased in sequence; the fourth step of culturing is performed in an outdoor tubular photobioreactor, the tubular photobioreactor is subjected to a shading treatment, and the shading area is ≥65%; wherein the light intensity of the shading part is 0-200 μmol photon·m -2 ·s -1 The light intensity of the unshielded part is 0-2000 μmol photon·m -2 ·s -1 Marine microalgae include: Long-eared tooth algae and triangular brown finger algae.

[0039] The present application utilizes a step-by-step expansion culture system to culture seawater microalgae, and utilizes a sealed pipeline photobioreactor for the final stage of expansion culture, and adjusts the light intensity of the fourth step culture, so that the seawater microalgae can be efficiently grown and reproduced under more suitable conditions, and the closed culture environment can avoid pollution from the external environment as much as possible, including nutrients, pH value, CO 2 The culture conditions such as dosage can be precisely controlled, so that stably growing seawater microalgae products can be obtained.

[0040] In order to further obtain seawater microalgae with better growth activity, the preserved algae species in a small number are activated and cultured. The first step of culture includes: inoculating the seawater microalgae species into a culture tube, performing the first step of aeration culture, and obtaining the first step of culture solution; preferably, the inoculation density of the seawater microalgae species is 0.6-1g / L; preferably, the mass content of fucoxanthin in the seawater microalgae species is 1.5%-2%; preferably, the air flow rate of the first step of aeration culture is 0.1-0.4L / min; preferably, the CO 2 The flow rate is 25-40 mL / min; preferably, CO 2 The ratio of compressed air is 0.25-1%. It should be noted that the culture container used in the first culture of the present application can be any device that can meet the culture capacity requirements.

[0041] In order to further improve the growth state of seawater microalgae species, in a preferred embodiment, the light intensity of the first step of aeration culture is 50-100 μmol photon·m -2 ·s -1 ; Preferably, the pH of the first aeration culture is 8-8.5; Preferably, the temperature of the first aeration culture is 20-28°C; Preferably, the duration of the first aeration culture is 8-10 days. The pH value is adjusted according to the algae content in the culture solution.

[0042] In order to further obtain more seawater microalgae with better growth status, the first step culture solution in the culture column is further activated and cultured by system expansion. In a preferred embodiment, the second step culture includes: inoculating the first step culture solution into a 10L membrane bag reactor, and performing the second step aeration culture to obtain the second step culture solution; preferably, the inoculation density of the seawater microalgae in the first step culture solution is 0.5-0.8g / L; preferably, the air flow rate of the second step aeration culture is 1.2-1.6m 3 / h; preferably, the CO of the second step aeration culture 2 The flow rate is 50-100mL / min. The use of membrane bag reactors not only improves the culture efficiency but also reduces the cost, making it suitable for pilot-scale algae cultivation. 2 The flow rate is adjusted as the pH value changes. It should be noted that the culture container used in the second step of the culture of the present application can be any device that can meet the culture capacity requirements.

[0043] In order to further improve the growth rate and fucoxanthin production of seawater microalgae, in a preferred embodiment, the light intensity of the second aeration culture is 50-100 μmol photon·m -2 ·s -1Preferably, the pH of the second aeration culture is 8-8.5; Preferably, the temperature of the second aeration culture is 20-28°C; Preferably, the duration of the second aeration culture is 8-10 days. The light intensity is adjusted according to the algae content in the culture solution.

[0044] In order to cultivate seawater microalgae in a larger-scale culture system, the algae solution of the second step culture is concentrated and then further expanded. In a preferred embodiment, the third step culture includes: after the second step culture solution is subjected to a first sedimentation, the supernatant is removed to obtain a first batch of concentrated algae species; the first batch of concentrated algae species is inoculated into a 20L membrane bag reactor or a 300L plate-type membrane bag photobioreactor, and a third step of aeration culture is performed to obtain a third step culture solution; preferably, the pH of the third step aeration culture is 8-8.5; preferably, the temperature of the third step aeration culture is 20-30°C; preferably, the time of the third step aeration culture is 8-10 days; preferably, the light intensity of the third step aeration culture is 50-150 μmol photon·m -2 ·s -1 ; Preferably, after the first batch of concentrated algae species is inoculated into the first culture medium, the inoculation density of the seawater microalgae in the culture solution to be cultured in the third step is 0.5-0.8 g / L. The light intensity is controlled by a shading net, and the culture temperature is controlled by spraying water. It should be noted that the culture container used in the third step of the present application can be any device that can meet the culture capacity requirements.

[0045] In order to enable the seawater microalgae to be in a better growth state in different culture devices, in a preferred embodiment, when the third step of aeration culture is carried out in a 20L membrane bag reactor, the air flow rate of the third step of aeration culture is 1.6-2.0m 3 / h; preferably, when the third step of aeration culture is carried out in a 20L membrane bag reactor, the CO 2 The flow rate is 5-10 L / min; preferably, when the third step of aeration culture is carried out in a 300 L plate-type membrane bag photobioreactor, the air flow rate of the third step of aeration culture is 1.2-1.6 m 3 / h; preferably, when the third step of aeration culture is carried out in a 300L plate-type membrane bag photobioreactor, the CO 2 The flow rate is 50-100L / min.

[0046] In order to make seawater microalgae adapt to industrial production and to further obtain seawater microalgae with higher yield of active ingredients (such as fucoxanthin, polyunsaturated fatty acid EPA, etc.), in a preferred embodiment, the fourth step of cultivation includes: after the third step of culture solution is subjected to a second sedimentation, the supernatant is removed to obtain a second batch of concentrated algae species; the second batch of concentrated algae species are inoculated in a pipeline photobioreactor, and the fourth step of aeration culture is carried out, and the seawater microalgae in the culture solution of the fourth step are harvested when the density reaches 2-2.5g / L to obtain seawater microalgae; preferably, after the second batch of concentrated algae species are inoculated in the first culture medium, the inoculation density of the seawater microalgae in the culture solution of the fourth step of aeration culture is 0.5-0.8g / L; preferably, the air flow rate of the fourth step of aeration culture is 1.6-2.5m 3 / h; preferably, the CO of the fourth step aeration culture 2 The flow rate is 100-400 L / h. Preferably, the pH of the fourth step of aeration culture is 8-8.5; preferably, the temperature of the fourth step of aeration culture is 20-30°C.

[0047] In order to further promote the growth rate of seawater microalgae and obtain seawater microalgae and fucoxanthin with a larger yield, the culture medium components are further optimized. In a preferred embodiment, the first culture medium is used for the first step culture, the second step culture, the third step culture and the fourth step culture; the first culture medium includes: 0.5-1.5g / L NaNO 3 , 0.01-0.0315g / LFeCl 3 6H 2 O, 0.5-1.781×10 -4 g / L MnCl 2 ·4H 2 O, 0.5-2.3×10 -5 g / L ZnSO 4 ·4H 2 O, 0.1-1.19×10 -5 g / LCoCl 2 6H 2 O, 0.5-2.5×10 -6 g / L CuSO 4 ·5H 2 O, 1.84×10 -6 g / LNa 3 VO 4 , 15-35g / L sea salt, 0.1-0.3g / L NaSiO 3 ·4H 2 O, 0.01-0.03 g / L NaH 2 PO 4 ·H 2 O, 0.5-2×10-4 μg / L Vitamin B 1 , 0.1-1×10 -6 μg / L biotin and 0.1-1×10 -6 μg / L Vitamin B 12 .

[0048] In order to further obtain seawater microalgae that is easy to preserve for subsequent applications, in a preferred embodiment, after the seawater microalgae is obtained by the fourth step of culturing, the culturing method further includes: subjecting the seawater microalgae obtained by the fourth step of culturing to self-sedimentation and concentration, the sedimentation time is ≥12h, removing the supernatant, and introducing the remaining part into a plate and frame filter press through a pneumatic diaphragm pump, and maintaining the pressure of the plate and frame for 4-5h at a pressure of 15-20Mpa. After the pressure maintenance is completed, the seawater microalgae mud is cleaned from the plate and frame filter press, and finally packaged into bags and stored in a -20℃ cold storage.

[0049] Example 1

[0050] The first culture medium used in this example includes: 1.5 g / L NaNO 3 , 0.0315g / L FeCl 3 6H 2 O, 1.781×10 -4 g / LMnCl 2 ·4H 2 O, 2.3×10 -5 g / L ZnSO 4 ·4H 2 O, 1.19×10 -5 g / L CoCl 2 6H 2 O, 2.5×10 - 6 g / L CuSO 4 ·5H 2 O, 1.84×10 -6 g / L Na 3 VO 4 , 35g / L sea salt, 0.3g / L NaSiO 3 ·4H 2 O, 0.03 g / L NaH 2 PO 4 ·H 2 O, 2×10 -4 μg / L Vitamin B 1 , 1×10 -6 μg / L biotin, 1×10 -6 μg / L Vitamin B 12. And it is prepared with disinfected tap water, and the residual chlorine in the disinfected tap water is 4-6ppm. In order to meet the residual chlorine standard, the disinfected tap water can be photolyzed and 3-5ppm sodium bichlorite can be added to degrade the residual chlorine.

[0051] The pipeline photobioreactor used in this embodiment may include multiple rows of glass tubes with an inner diameter of 6 cm, driven by a circulating pump, and the total volume of the reactor may be 17.5 m 3 The 300L plate-type membrane bag photobioreactor is made of stainless steel and has a built-in PE membrane bag. The 20L membrane bag reactor is made of PE and can be used symmetrically with a single-side volume of 20L. It should be noted that the culture container used in the second step of the present application can be any device that can meet the culture capacity requirements.

[0052] Initial culture: Algae activation. Obtain the long-eared tooth algae species from the algae chamber, transfer the long-eared tooth algae species to a 500 mL culture bottle containing 250 mL of the first culture medium, and culture it statically for 5 days with an illumination of 40 μmol photon·m -2 ·s -1 , the culture temperature is 25°C (air-conditioning controlled). The density of the cultivated long-eared tooth algae is 0.8g / L, and the mass content of fucoxanthin in the long-eared tooth algae is 1.5%-2%;

[0053] First step of cultivation: 500 mL of the long-eared tooth algae obtained from the initial cultivation was inoculated into the first culture medium in a glass column tube with an inner diameter of 6 cm. The volume of the culture solution to be used for the first step of cultivation was 650 mL. The first step of aeration cultivation was performed. The air flow rate of the first step of aeration cultivation was 0.1-0.4 L / min, and the CO 2 The flow rate is 25-40 mL / min (the ratio of carbon dioxide to compressed air is 0.25-1%), and the light intensity is 50-100 μmol photon·m -2 ·s -1 , pH is 8-8.5, temperature is 25°C, the first step aeration culture time is 9 days, and the first step culture solution (containing microalgae) is obtained.

[0054] The change of dry weight of the long-eared tooth algae with the culture time in the first step of the culture process was detected. The specific method is as follows: a certain volume of algae liquid is taken, the supernatant is removed by centrifugation, and the algae mud in the centrifuge tube is dried. The dry weight of the long-eared tooth algae is deducted from the weight of the centrifuge tube (dried to constant weight in advance). The results are as follows: Figure 1 As shown in the figure, the change of fucoxanthin content in the long-eared tooth algae with the culture time was detected by ultraviolet spectrophotometry. The results are as follows Figure 2 shown.

[0055] The second step of cultivation: the first step of the culture solution was inoculated into the first culture medium of the 10L membrane bag reactor, and the volume of the culture solution to be used for the second step of cultivation was 10L, and the second step of aeration cultivation was performed. The inoculation density of the long-eared dentata in the first culture solution for inoculation was 0.8g / L, and the air flow rate of the second step of aeration cultivation was 1.2-1.6m 3 / h, CO 2 The flow rate is 50-100 mL / min and the light intensity is 50-100 μmol photon·m -2 ·s -1 , pH 8-8.5, temperature 20-28°C, and the second step of aeration culture for 10 days to obtain the second batch of culture fluid (containing microalgae).

[0056] The above-mentioned method for detecting dry weight was used to detect the change of dry weight of the long-eared dentata cultured in the second step with the culture time. The results are as follows: Figure 3 shown.

[0057] The third step of cultivation: let the second step culture solution settle for 4 hours, extract the supernatant, and obtain the first batch of concentrated algae species. Prepare the first culture solution used in the third step of cultivation in an outdoor 500L mixing tank, inoculate the first batch of concentrated algae species into the 500L mixing tank, and then divide it into a 20L membrane bag photobioreactor for the third step of aeration cultivation. The volume of the culture solution to be used for the third step of aeration cultivation is 20L. Among them, the inoculation density of the long-eared tooth algae in the solution to be used for the third step of aeration cultivation is 0.5g / L, and the air flow rate of the third step of aeration cultivation is 1.6-2.0m 3 / h, CO 2 The flow rate was 5 L / min and the light intensity was 150 μmolphoton·m -2 ·s -1 , pH is 8-8.5, temperature is 25°C, and the time of the third step aeration culture is 14 days to obtain the third step culture solution (containing microalgae).

[0058] The above-mentioned method for detecting dry weight was used to detect the change of dry weight of the long-eared dentata cultured in the third step with the culture time. The results are as follows: Figure 4 shown.

[0059] The fourth step of cultivation: The third step culture solution is stopped for aeration and sedimentation at night, and the supernatant is extracted with a pneumatic diaphragm pump the next morning to obtain the second batch of concentrated algae species. The second batch of concentrated algae species is extracted with a pneumatic diaphragm pump into a pre-disinfected and cleaned pipeline photobioreactor located outdoors, with a culture volume of 17,500 L. Then the first culture medium is added for the fourth step of aeration culture, and the volume of the culture solution to be subjected to the fourth step of aeration culture is 17,500 L. Among them, the inoculation density of the long-eared tooth algae in the solution to be subjected to the fourth step of aeration culture is 0.8 g / L, and the air flow rate of the fourth step of aeration culture is 1.6-2.5 m 3 / h, CO 2 The flow rate is 100-400 L / h, the pH is controlled at 8-8.5, and the time of the fourth step aeration culture is 10 days to obtain the fourth step culture system.

[0060] The light intensity of the pipeline photobioreactor is controlled by a shading net. The volume of the shading part is 65% of the total volume of the pipeline photobioreactor, and the light intensity of the shading part is controlled at 50-200 μmol photon·m -2 ·s -1 The light intensity of the unshielded part is the actual outdoor light intensity (varies with time between 0-2000 μmol photon·m -2 ·s -1 In addition, a 316 stainless steel overflow tank is installed above the pipeline photobioreactor, and the length of the overflow tank is not less than 30m. The spray water of the overflow tank is used to cool the pipeline photobioreactor. The temperature of the algae liquid in the fourth step of aeration culture is 20-30°C.

[0061] When the density of the long-eared dentata in the fourth step culture solution reaches 2-2.5g / L, it is harvested and the next batch of the third step culture solution is used for the next round of fourth step culture. In the fourth step of aeration culture, the online temperature probe is used to detect the changes in the culture solution temperature and gas temperature in the device over time. The results are as follows: Figure 6 As shown, the above-mentioned method for detecting dry weight was used to detect the change of dry weight of the long-eared tooth algae with the culture time in the fourth step of cultivation, and the ultraviolet spectrophotometry was used to detect the change of fucoxanthin content in the long-eared tooth algae with the culture time. The results are as follows Figure 7 shown.

[0062] Harvesting of long-eared tooth algae: transfer the algae liquid (including microalgae bodies) after the fourth step of cultivation to the harvesting bucket for self-sedimentation and concentration of the algae bodies. The sedimentation time is not less than 12 hours. The supernatant is discharged, and then the concentrated algae liquid is introduced into the plate and frame filter press through a pneumatic diaphragm pump. The plate and frame are pressurized for 4-5 hours at a pressure of 15-20Mpa. After the pressure is maintained, the long-eared tooth algae mud is cleaned from the plate and frame filter press, and finally packaged into bags and stored in a -20℃ cold storage.

[0063] Example 2

[0064] Except for the third step of the culturing phase, which is different from that in Example 1, the remaining operations in this example are the same as those in Example 1.

[0065] The specific operation of the third step of cultivation in this embodiment is as follows:

[0066] The third step of cultivation: let the second step culture solution settle for 4 hours, extract the supernatant, and obtain the first batch of concentrated algae. Prepare the first culture solution used in the third step of cultivation in an outdoor 300L plate-type membrane bag photobioreactor, inoculate the first batch of concentrated algae into the 300L plate-type membrane bag photobioreactor, and carry out the third step of aeration cultivation. The volume of the culture solution to be carried out for the third step of aeration cultivation is 300L. Among them, the inoculation density of the long-eared tooth algae in the solution to be aerated culture in the third step is 0.5g / L, and the air flow rate of the third step of aeration cultivation is 1.2-1.6m 3 / h, CO 2 The flow rate is 50-150 L / min and the light intensity is 150 μmol photon·m -2 ·s -1 , pH is 8-8.5, temperature is 25°C, the time of the third step aeration culture is 14 days, and the third step culture solution is obtained.

[0067] The above-mentioned method for detecting dry weight was used to detect the change of dry weight of the long-eared dentata cultured in the third step with the culture time. The results are as follows: Figure 5 shown.

[0068] Example 3

[0069] Except for the different species of seawater microalgae cultured in this example, the other culture conditions are the same as those in Example 1. The seawater microalgae cultured in this example is Phaeochromis triangularis. The method for detecting the dry weight and fucoxanthin content of algae in Example 1 was used to monitor the dry weight and fucoxanthin content of algae during the fourth step of culture. The results are as follows: Figure 8 and Fig. 9 The blue point values ​​and curve are shown in Figure 2.

[0070] Example 4

[0071] Except for the different species of seawater microalgae cultured in this example, the other culture conditions are the same as those in Example 2. The seawater microalgae cultured in this example is Phaeochromis triangularis. The method for detecting the dry weight and fucoxanthin content of algae in Example 1 was used to monitor the dry weight and fucoxanthin content of algae during the fourth step of culture. The results are as follows: Figure 8 and Fig. 9 The red point value and curve are shown in the figure.

[0072] Example 5

[0073] The operation of this example is the same as that of Example 3, and the experiment is repeated. The method for detecting the dry weight of algae and the content of fucoxanthin in Example 1 (UV detection method) was used to detect the dry weight of algae and the content of fucoxanthin in the fourth step of cultivation in Example 3-5. The results are as follows: Figure 8 and Fig. 9 shown.

[0074] The P. triangularis or P. longiflora harvested from each culture in Examples 3-5 were mixed and tested by high performance liquid chromatography to obtain specific data on fucoxanthin content, EPA (eicosapentaenoic acid) content and harvest dry weight (such as the detection method of algae dry weight) as shown in Table 1.

[0075] Table 1 Dry weight of Phaeodactylum triangularis, fucoxanthin and EPA content in algal cells

[0076]

[0077]

[0078] Comparative Example 1

[0079] Effects of different shading (light intensity) on the growth and fucoxanthin accumulation of Cynodon dactylon.

[0080] Except that the volume of the shading part of the tubular photobioreactor in the fourth culturing stage is different from that in Example 1, the other operations of this comparative example are the same, and the volume of the shading part of the tubular photobioreactor in this comparative example is 55% of the total volume of the tubular photobioreactor.

[0081] The long-eared tooth algae obtained by the final culture was tested by high performance liquid chromatography, and specific data on the fucoxanthin content, EPA (eicosapentaenoic acid) content and harvest rate were obtained as shown in Table 2. Among them, the detection of the fucoxanthin content by high performance liquid chromatography is as follows: the long-eared tooth algae in culture is extracted by acetone, and the absorbance value of the extract at 450nm is detected, so as to calculate the fucoxanthin content in the long-eared tooth algae.

[0082] It can be seen that the main nutrients in Comparative Example 1 have all decreased significantly, the fucoxanthin content decreased by about 31%, the EPA content decreased by about 21%, and the harvest rate decreased by about 16%. Among them, the fucoxanthin content and EPA content are the ratios of the detection value of fucoxanthin or EPA to the dry weight of Long-eared Toothed Algae, and the harvest rate is the ratio of the theoretical value of Long-eared Toothed Algae to the actual dry weight.

[0083] Table 2:

[0084] Unshaded ratio Fucoxanthin content (%DW) EPA content (%DW) Harvest rate 35% (Example 1) 1.18％ 3.88％ 89％ 45% (Comparative Example 1) 0.81％ 3.08％ 73％

[0085] During the production process, the dry weight of the long-eared dentata cultured for 0-14 days in the fourth step of the culture stage of Example 1 and Comparative Example 1 was detected using the dry weight detection method in Example 1, and the following results were obtained: Fig.10 From the data shown, it can be seen that an unshaded ratio of about 45% will have a significant impact on the biomass concentration of Long-eared Dendrobium, with the yield falling by nearly 20%.

[0086] The morphology of the long-eared dentate algae cells of Example 1 and Comparative Example 1 after the fourth step of aeration culture for 5 days was observed microscopically. Fig.11 As shown, it can be seen that the cell morphology of the long-eared tooth algae in comparative example 1 is poor, the cells are more scattered, and the cells are smaller, which makes subsequent harvesting difficult and reduces the harvest rate.

[0087] In addition, the component contents in the culture solution at each harvest of the fourth step of the continuous culture process were detected. The specific data are shown in Table 3. It can be seen that the average fucoxanthin content in the finished product of the long-eared tooth algae obtained by the culture method of the present application is about 1.1%, the average EPA content is about 3.4%, and the average biomass of the long-eared tooth algae is 2.6 g / L.

[0088] Table 3:

[0089] Harvest Date Fucoxanthin content (%DW) EPA content (%DW) Harvest dry weight (g / L) 2024 / 6 / 29 1.10％ 3.27％ 2.070 2024 / 7 / 17 1.07％ 2.81％ 2.766 2024 / 8 / 10 1.14％ 3.57％ 3.390 2024 / 9 / 1 1.31％ 3.70％ 1.870 2024 / 10 / 1 1.08％ 2.62％ 2.560 2024 / 10 / 9 1.15％ 3.86％ 2.400

[0090] Comparative Example 2

[0091] Effects of different culture medium components on the growth of Cynodon longioides and the accumulation of fucoxanthin and EPA.

[0092] Except that the composition of the culture medium used in this comparative example is different from that in Example 1, the rest of the operations are the same as those in Example 1. This comparative example uses the mL1 culture medium (artificial seawater) and natural seawater (from Sanofi Bio-Industries, France) disclosed in the patent document (application number 202111114769.2). The specific components of the mL1 culture medium are as follows: 1.5 g / L NaNO 3, 20.758g / L NaCl, 3.477g / L Na 2 SO 4 , 0.587g / L KCl, 0.17g / L NaHCO 4 , 9.395g / LMgCl 2 6H 2 O, 1.316 g / L CaCl 2 ·2H 2 O, 0.0845g / L KBr, 0.0225g / LH 3 BO 3 ,0.0027g / L NaF,0.0214g / LSrCl 2 6H 2 O, 0.00436 g / L EDTA-Na 2 , 0.0315g / L FeCl 3 6H 2 O, 1.781×10 -4 g / LMnCl 2 ·4H 2 O, 2.3×10 -5 g / L ZnSO 4 ·4H 2 O, 1.19×10 -5 g / L CoCl 2 6H 2 O, 2.5×10 -6 g / LCuSO 4 ·5H 2 O, 1.84×10 -6 g / L Na3VO4,1.94×10 -6 g / L KCrO 4 , add the above ingredients to 1L deionized water, sterilize at 121℃ for 20min, and then add 0.6g / L sterilized NaSiO 3 ·4H 2 O, 0.03 g / LNaH 2 PO 4 ·H 2 O, 2×10 -4 μg / L Vitamin B1, 1×10 -6 μg / L biotin, 1×10 -6 μg / L vitamin B12.

[0093] The fucoxanthin content and EPA content of the long-eared cysts were obtained by culturing the first culture medium of the present application, mL1 culture medium (artificial seawater) and natural seawater (from Sanofi Bio-Industries, France) as shown in Table 4 below. It can be seen that the first culture medium of the present application can be used to culture fucoxanthin and EPA with higher content than natural seawater. Fig.12 As shown, although the dry weight of the long-eared tooth algae obtained using the first culture medium of the present application and the artificial seawater culture medium is not much different, the first culture medium of the present application has simple ingredients and low cost, and is more suitable for large-scale commercial production. The cost comparison between the first culture medium and artificial seawater culture is shown in Table 5.

[0094] It can be seen that the first culture medium of the present application can simultaneously culture high content of fucoxanthin and EPA (eicosapentaenoic acid) at a lower cost, and is more suitable for commercial-scale production.

[0095] Table 4:

[0096] Culture medium Culture reactor type Fucoxanthin content (% dry weight) EPA content (% dry weight) First culture medium Pipeline Photobioreactor 1.14％±0.09％ 3.31％±0.5％ natural sea water Open track pool 0.19％ 2.06％±0.28％

[0097] Table 5:

[0098]

[0099] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: by using the cultivation method of the present application, compared with the traditional open runway pool cultivation method, by accurately controlling the conditions such as light, temperature, pH value, etc. in the cultivation environment, the long-eared tooth algae can grow in a better state at each growth stage, significantly improving the cultivation efficiency and the yield of the long-eared tooth algae, and the yield of the rich fucoxanthin can also be further improved. And the closed reactor effectively reduces the risk of environmental pollution, and can stably and continuously produce high-quality long-eared tooth algae biomass.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for cultivating seawater microalgae, characterized in that: include: Using seawater microalgae species to perform the first step of culturing, the second step of culturing, the third step of culturing and the fourth step of culturing in sequence to obtain the seawater microalgae; The capacity of the culture system of the first step culture, the second step culture, the third step culture and the fourth step culture increases in sequence; The fourth step of culturing is carried out in an outdoor tubular photobioreactor, and the tubular photobioreactor is subjected to shading treatment, and the shading area is ≥ 65%; The light intensity of the shading part is 0-200 μmol photon·m -2 ·s -1 , the light intensity of the unshielded part is 0-2000μmolphoton·m -2 ·s -1 .

2. The culture method according to claim 1, characterized in that The first step of culturing comprises: Inoculating the seawater microalgae species into a culture tube, performing a first step of aeration culture, and obtaining a first step of culture solution; Preferably, the inoculation density of the seawater microalgae species is 0.6-1 g / L; Preferably, the mass content of fucoxanthin in the seawater microalgae species is 1.5%-2%; Preferably, the air flow rate of the first step of aeration culture is 0.1-0.4 L / min; Preferably, the CO2 flow rate of the first step of aeration culture is 25-40 mL / min; Preferably, the ratio of CO2 to compressed air is 0.25-1%.

3. The culture method according to claim 2, characterized in that The light intensity of the first step of aeration culture is 50-100 μmolphoton·m -2 ·s -1 ; Preferably, the pH of the first step of aeration culture is 8-8.5; Preferably, the temperature of the first step of aeration culture is 20-28°C; Preferably, the first step of aeration culture lasts for 8-10 days.

4. The culture method according to claim 2, characterized in that The second step of cultivation includes: The first step culture solution is inoculated into a 10 L membrane bag reactor, and a second step aeration culture is performed to obtain a second step culture solution; Preferably, the inoculation density of the seawater microalgae in the culture solution in the first step is 0.5-0.8 g / L; Preferably, the air flow rate of the second step of aeration culture is 1.2-1.6 m 3 / h; Preferably, the CO2 flow rate of the second step aeration culture is 50-100 mL / min.

5. The culture method according to claim 4, characterized in that The light intensity of the second step of aeration culture is 50-100 μmolphoton·m -2 ·s -1 ; Preferably, the pH of the second step aeration culture is 8-8.5; Preferably, the temperature of the second step of aeration culture is 20-28°C; Preferably, the duration of the second step of aeration culture is 8-10 days.

6. The culture method according to claim 4, characterized in that The third step of culturing comprises: After the second step culture solution is subjected to a first sedimentation, the supernatant is removed to obtain a first batch of concentrated algae seeds; Inoculating the first batch of concentrated algae seeds into a 20L membrane bag reactor or a 300L plate-type membrane bag photobioreactor, and performing a third step of aeration culture to obtain a third step culture solution; Preferably, the pH of the aeration culture in the third step is 8-8.5; Preferably, the temperature of the third step of aeration culture is 20-30°C; Preferably, the third step of aeration culture lasts for 8-10 days; Preferably, the light intensity of the third step of aeration culture is 50-150 μmol photon·m -2 ·s -1 ; Preferably, after the first batch of concentrated algae species is inoculated into the first culture medium, the inoculation density of the seawater microalgae in the culture solution to be cultured in the third step is 0.5-0.8 g / L.

7. The culture method according to claim 6, characterized in that: When the third step of aeration culture is carried out in the 20L membrane bag reactor, the air flow rate of the third step of aeration culture is 1.6-2.0m 3 / h; Preferably, when the third step of aeration culture is carried out in the 20 L membrane bag reactor, the CO2 flow rate of the third step of aeration culture is 5-10 L / min; Preferably, when the third step of aeration culture is carried out in the 300L plate-type membrane bag photobioreactor, the air flow rate of the third step of aeration culture is 1.2-1.6m 3 / h; Preferably, when the third step of aeration culture is carried out in the 300 L plate-type membrane bag photobioreactor, the CO2 flow rate of the third step of aeration culture is 50-100 L / min.

8. The culture method according to claim 6, characterized in that: The fourth step of culturing comprises: After the culture solution in the third step is subjected to a second sedimentation, the supernatant is removed to obtain a second batch of concentrated algae seeds; inoculating the second batch of concentrated algae seeds in a pipeline photobioreactor, performing the fourth step of aeration culture, and harvesting the seawater microalgae when the density of the seawater microalgae in the culture solution in the fourth step reaches 2-2.5 g / L to obtain the seawater microalgae; Preferably, after the second batch of concentrated algae species is inoculated into the first culture medium, the inoculation density of the seawater microalgae in the culture solution to be aerated and cultured in the fourth step is 0.5-0.8 g / L; Preferably, the air flow rate of the fourth step of aeration culture is 1.6-2.5m 3 / h; Preferably, the CO2 flow rate of the fourth step aeration culture is 100-400 L / h.

9. The culture method according to claim 8, characterized in that: The pH of the aeration culture in the fourth step is 8-8.5; Preferably, the temperature of the aeration culture in the fourth step is 20-30°C.

10. The culture method according to claim 8, characterized in that: Using the first culture medium to carry out the first step culture, the second step culture, the third step culture and the fourth step culture; Preferably, the first culture medium comprises: 0.5-1.5 g / L NaNO3, 0.01-0.0315 g / L FeCl3·6H2O, 0.5-1.781×10 -4 g / L MnCl2·4H2O, 0.5-2.3×10 -5 g / L ZnSO4·4H2O, 0.1-1.19×10 -5 g / L CoCl2·6H2O, 0.5-2.5×10 -6 g / L CuSO4·5H2O,1.84×10 -6 g / L Na3VO4, 15-35g / L sea salt, 0.1-0.3g / L NaSiO3·4H2O, 0.01-0.03g / L NaH2 PO4·H2O, 0.5-2×10 -4 μg / L vitamin B1, 0.1-1×10 -6 μg / L biotin and 0.1-1×10 -6 μg / L Vitamin B 12 .

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