WO2012100583A1

WO2012100583A1 - Culturing method for microalgae with high yields

Info

Publication number: WO2012100583A1
Application number: PCT/CN2011/082336
Authority: WO
Inventors: 李元广; 韩菲菲; 黄建科; 王军; 王伟良; 章真; 李淑兰; 范建华; 陈铖; 魏鸿刚; 沈国敏
Original assignee: 华东理工大学; 上海泽元海洋生物技术有限公司
Priority date: 2011-01-27
Filing date: 2011-11-17
Publication date: 2012-08-02
Also published as: CN102154110A; CN102154110B

Abstract

Disclosed is a culturing method for microalgae which involves the step of heterotrophic culturing of microalgae cultivars and the step of light autotrophic culturing. In the step of light autotrophic culturing, it utilizes algae cells arrived from heterotrophic culturing as seeds. The method can not only provide a plenty of algae seeds, but also can accelerate light autotrophic culturing of microalgae and the forming rate of target product.

Description

Description High-yield microalgae culture method

The invention belongs to the field of bioenergy and/or microalgae biotechnology, and relates to a high yield microalgae culture party

Background technique

Microalgae cells are rich in various high-value active substances such as proteins, polysaccharides, fatty acids and carotenoids. Therefore, microalgae currently has a wide range of applications in food, feed, medicine, environmental protection and bioenergy.

The main culture methods of microalgae are photoautotrophic, mixed nutrition and heterotrophic culture. At present, microalgae (such as chlorella, spirulina, salt algae, Haematococcus pluvialis, etc.) that have been industrially applied are almost always cultivated by light self-cultivation.

The mixed nutrient culture of microalgae needs to be cultured in a sterilizable photobioreactor, and it is necessary to simultaneously ensure aseptic culture and sufficient illumination conditions, and the requirements for the culture equipment are extremely high, and the equipment is difficult to enlarge. Therefore, in the large-scale cultivation of actual microalgae, almost no mixed nutrition method is used for cultivation. Compared with photoautotrophic culture, the quality of algal cells (such as protein and pigment content) in heterotrophic culture is low and often difficult to apply. Therefore, in the above three microalgae culture modes, the algal cells cultured by photoautotrophic have high quality and have attracted much attention. Currently, researchers are keen to produce biodiesel using the oil accumulated by microalgae photoautotrophic culture (Attilio Converti, Alessandro A.Casazza, Erika Y.Ortiz, Patrizia Perego, Marco Del Borghi., Effect of temperature and nitrogen concentration on The growth and lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodesel production [J]. Chemical Engineering and Processing, 2009, 48: 1146 1151 ).

Although the photoautotrophic culture process of microalgae can accumulate a large amount of useful substances (such as oil, protein, etc.), there are still three problems: 1) low seeding density results in contamination by algae and protozoa; 2) Seed expansion is carried out by photoautotrophic culture, which has a very long cycle (generally 1 to 2 months) and is easily contaminated. Algae requires a large number of culture devices and equipment and covers an area of several months. Large; 3) Microalgae light autotrophic culture process cells grow slowly, the yield of the target product is low.

Therefore, there is still a need in the art for a high yield microalgae culture process. Summary of the invention In view of the above problems, the present invention provides an effective solution for heterotrophic cultivation of algae species in heterotrophic cultured microalgae, followed by photoautotrophic cultivation using algal cells obtained by heterotrophic culture as seeds. The method of the invention can fully exert the advantages of rapid growth of microalgae in the heterotrophic stage, and can not only provide a large number of algae species for large-scale photoautotrophic cultivation of microalgae, but also accelerate the photoautotrophic growth and the target product of the microalgae ( The formation rate of oil, protein, etc., provides an important technical means for solving the problems of long algae expansion period, slow cell growth and low yield of target products in the large-scale photoautotrophic culture of microalgae. .

Accordingly, a first aspect of the present invention provides a microalgae cultivation method comprising a heterotrophic culture step of a microalgae species and a photoautotrophic culture step of the algal cells obtained by heterotrophic culture as seeds. In a specific embodiment, the microalgae culture method further comprises the steps of harvesting algae cells, extracting active ingredients in the algal cells, and/or drying the algal cells into algal flour.

A second aspect of the present invention provides a method for producing a fat or oil, which comprises a heterotrophic culture step of a microalgae species, a photoautotrophic culture step performed as a seed by heterotrophic culture, and an algal cell harvesting step. And the steps of oil extraction. In a specific embodiment, the method further comprises: mixing all the components in the supernatant obtained by extracting the oil with the precipitate of the algae and spray-drying to obtain the algal flour.

A third aspect of the present invention provides a protein production method comprising a heterotrophic culture step of a microalgae species, a photoautotrophic culture step performed as a seed by heterotrophic culture, and an algal cell harvesting step. And the steps of protein extraction.

A fourth aspect of the present invention provides a method for producing a microalgae active ingredient, which comprises a heterotrophic culture step of a microalgae species, a photoautotrophic culture step performed as a seed by heterotrophic culture, and an algae The step of cell harvesting, and the steps of extracting active ingredients (protein, polysaccharide, chlorophyll, lutein, oil, fatty acid, chlorella growth factor, etc.).

The present invention also includes a method for producing algal flour, the method comprising the heterotrophic culture step of the microalgae species, the photoautotrophic culture step of the algae cells obtained by the heterotrophic culture as the seed, and the step of the algae cell harvesting And the step of drying the harvested algal cells into algal flour.

In a specific embodiment, the microalgae is selected from the group consisting of heterotrophic cultured microalgae.

In a specific embodiment, the microalgae is selected from the group consisting of:

Chlorella pyrmoidoscd, Chlorella vulgaris, in the genus Chlorella

(Chlorella ellipsoidea), Chlorella emersonii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella regularis, Chlorella minutissima, Chlorella protothecoides, Chlorella zofingiensis, and Brachiomonas submarina, Chlamydobonas reinhardtii, Chlamydomonas acidophila, Haematococcus pluvialis, Haematococcus lacustris, Scenedesmus obliquus, Spongiococcum exetriccium, Tetraselmis Suecica, Tetraselmis chuii, Tetraselmis tetrathele, Tetraselmis verrucosa, Micr actinium pusillum-,

Cylindrotheca fusiformis, Nitzschia laevis, Nitzschia alba, Nitzschia fonticola, Navicula incerta, Navicula pelliculosa-,

Anabaena variabilis- of Cyanophyta

Poterioochromonas malhamensis

Amphidinium carterae, Crypthecodinium cohnii;

Euglena grid lis-, and mouth

Galdieria sulphuraria of the red algae gate.

In a specific embodiment, the step of heterotrophic cultivation of the microalgae algae comprises: adding a medium having a pH of 4.0 9.0 to the bioreactor, and accessing the microalgae species according to a working volume of 0.1 to 30% for batching Culture, fed-batch culture or semi-continuous culture, culture temperature is 10~40 °C, control pH is less than 9.0, control dissolved oxygen is above 1%.

In a specific embodiment, the step of self-cultivating the microalgae comprises: inoculating the heterotrophic energy microalgae species into a photoautotrophic culture device for photoautotrophic culture at a temperature of 5 to 50 ° C, continuous illumination Or intermittent light, the light intensity is 0.1~150kk, the photoautotrophic culture period is 5 500 hours, the initial inoculation density is 0.01 10.00 g/L, and the pH is 4.0~12.0o.

In a specific embodiment, the heterotrophic medium consists of a nitrogen source, an organic carbon source, an inorganic salt, a trace element, and water; the photoautotrophic medium consists of a nitrogen source, an inorganic salt, and water.

In a specific embodiment, the heterotrophic step is carried out in a shake flask, mechanically agitated, airlifted or bubbling heterotrophic culture bioreactor, the light autotrophic culture step is shaken or selected Any open source runway pool or round pool, closed flat photobioreactor or pipeline photobioreactor or column photobioreactor, film stand pouch or hanging bag, etc. In the device, the lighting conditions are natural light or artificial light.

In a specific embodiment, when the chlorella is Chlorella vulgaris, the medium used for heterotrophy consists essentially of the following components: KN0 ₃ 5-15 g/L, glucose 10 60 g/L, KH ₂ P0 ₄ 0.3-0.9 g/L, Na ₂ HP0 ₄ 12H ₂ 0 1.0-10.0 g/l, MgS0 ₄ -7H ₂ 0 0.2-1.0 g/l, CaCl ₂ 0.05-0.3 g/l, FeS0 ₄ -7H ₂ 0 0.01~0.05 g / liter, trace element 0.5~4ml and water, wherein the composition of trace elements is H ₃ B0 ₃ 5-15 g / liter, ZnS0 ₄ '7H ₂ 0 5.0-10.0 g / liter, MnCl _r H ₂ 0 1.0-2.0 g/L, ( Η ₄ ) ₆ Μο ₇ 0 ₂₄ ·4Η ₂ 0 0.5-1.5 g/L, CuS0 ₄ '5H ₂ 0 1.0-2.0 g/L, Co(N0 ₃ ) _r 6H ₂ 0 0.1-0.9 g/l.

In a specific embodiment, when the chlorella is Chlorella chlorella, the medium used for heterotrophy consists essentially of the following components: glucose 10 60 g/L, urea 2.0 8.0 g/L, KH ₂ P0 ₄ 1.0-2.0 g/l, MgS0 ₄ .7H ₂ 0 1.0~2.0 g/l, CaCl ₂ 0.05~0.1 g/l, trisodium citrate 0.1~2.0 g/l, Fe-EDTA Solution 0.5~1 mL, A5 solution l~5mL and water; The formulation of Fe-EDTA solution is FeS (V7H ₂ 0 20-30 g/L and EDTA 20~40 g/L; A5 solution formula is H ₃ B0 ₃ 2.5- 4.0 g / liter, MnCl ₂ '4H ₂ 0 1.0-2.0 g / liter, ZnSO ₄ -7H ₂ O 0.1~0.6 g / liter, CuS0 ₄ '5H ₂ 0 0.05 0.1 g / liter, Na ₂ Mo0 ₄ 0.01~0.05 G/L.

In a specific embodiment, after the glucose in the heterotrophic culture solution is consumed, the heterotrophic culture is terminated, and the algal cells obtained by the heterotrophic culture are subjected to a photoautotrophic culture step as a seed.

In other embodiments of the present application, the above-described microalgae culture method can be carried out using the culture medium and culture conditions described in the present application.

The advantages of the present invention are described in detail by heterotrophic culture of chlorella species and algal cells obtained by heterotrophic culture as seeds for photoautotrophic culture of produced diesel fuel as an example:

(1) can greatly increase the rate of algae cultivation. The initial inoculation density of microalgae cultured in outdoor large ponds is

0.05-0. lg/l, the volume of the large pool is generally 5000L or more. To meet this requirement, 250~500g of microalgae are needed as seeds. If the traditional photoautotrophic expansion of algae species is used, it takes 1 to 2 months (the density of algae cells in the self-cultivation of algae for 10 days is generally 0.2~0.5g/l, which needs to be expanded step by step (about 10 times). The dilution rate of the left and right can only enter the large pool); while the heterotrophic culture of the 50L fermenter is used for 2 to 3 days.

(2) Compared with the traditional photoautotrophic culture of algae, it can reduce the number of devices and equipment in the cultivation process of algae, and has a small footprint and high yield per unit area (or volume). For example, the expansion of the algae species required for autotrophic culture of 5000L large pools requires 5L, 50L and 500L culture equipment for self-cultivation and algae cultivation, and 50L culture equipment for algal culture.

(3) Compared with algal species photoautotrophic culture, algal species heterotrophic culture is not affected by outdoor weather and environment. When the algae species are self-cultivated, if they are unable to continue the culture in the rainy weather, they need to move into the room and add artificial light to continue the self-cultivation of the algae species, thus increasing the cost of artificial light. In addition, the self-cultivation of algae is susceptible to contamination by predators, algae, and other predators, resulting in failure of algae cultivation and severely affecting production.

(4) When the outdoor large pool is cultivated, the larger the inoculation amount, the less likely it is to be contaminated by other algae or protozoa; the heterotrophic culture of the algae species can provide a large amount of seeds in time to meet the demand for increasing the inoculation amount during photoautotrophic cultivation. .

(5) The vigor of heterotrophic algae is better than the algae cultured by photoautotrophic culture. When the amount of the same algae cells was added for photoautotrophic culture, the algal cell density, oil yield and protein yield of the phototrophic culture process using the heterotrophic cultured cells as algal species were higher than those by photoautotrophic culture. The cells serve as corresponding values for the photoautotrophic culture process of the algae species. In addition, since the density of algae cells obtained by photoautotrophic culture with heterotrophic algae is high, the harvesting cost of the energy microalgae can be reduced.

In summary, the heterotrophic culture of the algae species of the present invention and the photoautotrophic culture mode of the algae cells obtained by heterotrophic culture as seeds can solve the problem of low efficiency and vulnerability to contamination of algae species during photoautotrophic culture. Many problems have arisen and the growth rate of microalgae in the photoautotrophic culture process is slow and the yield of the target product (such as oil, protein, etc.) is low. Therefore, the present invention provides a solution to the problem of slow cell growth and low yield of the target product during microautotrophic photoautotrophic culture. An important technical means, especially for the industrialization of the production of biodiesel using microalgae, has laid an important foundation. DRAWINGS

Figure 1 shows the growth process of Chlorella pyrenoidosa seeds in heterotrophic culture in 500 ml shake flasks and photoautotrophic culture in 2 L flasks.

Figure 2 shows the growth process of common chlorella seeds in heterotrophic culture in 500 ml shake flasks and photoautotrophic culture in 2 L flasks.

Figure 3 shows the growth process of ellipsoidal seeds in heterotrophic culture in 500 ml shake flasks and photoautotrophic culture in 2 L flasks.

Figure 4 shows the algal cell growth curve of phototrophic culture of Chlorella pyrenoidosa heterotrophic seeds and photoautotrophic seeds in an indoor 2L photobioreactor.

Figure 5 shows the oil yield of phototrophic cultures of Chlorella pyrenoidosa heterotrophic seeds and photoautotrophic seeds in an indoor 2L photobioreactor.

Figure 6 shows the algal cell growth curve of phototrophic culture of common chlorella heterotrophic seeds and photoautotrophic seeds in an indoor 2L photobioreactor.

Figure 7 shows the oil yield of phototrophic cultures of common chlorella heterotrophic seeds and photoautotrophic seeds in an indoor 2L photobioreactor.

Figure 8 shows the algal cell growth curve of photoautotrophic culture of ellipsoidella heterotrophic seeds and photoautotrophic seeds in an indoor 2L photobioreactor.

Figure 9 shows the oil yield of photoautotrophic cultures of heterotrophic chlorella heterotrophic seeds and photoautotrophic seeds in an indoor 2L photobioreactor.

Figure 10 shows the algal cell growth curve and the highest oil yield of phototrophic culture of Chlorella pyrenoidosa heterotrophic seeds in an outdoor 2L photobioreactor.

Figure 11 shows the algal cell growth curve and the highest oil yield in the photoautotrophic culture of the nucleus chlorella heterotrophic seeds in an outdoor 60L plastic pot. detailed description

Microalgae suitable for use in the present application include all microalgae which can be heterotrophically cultured, including but not limited to Chlorella pyrenoidosa in the genus Chlorella, Chlorella vulgaris, small oval Chlorella ellipsoidea, Chlorella emersonii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella regularis, Chlorella minutissima, Chlorella protothecoides, Chlorella zofingiensis, and Brachiomonas submarina, Chlamydobonas in the green algae Reinhardtii, Chlamydomonas acidophila, Haematococcus pluvialis, Haematococcus lacustris, Scenedesmus obliquus, Spongiococcum exetriccium, Tetraselmis suecica, Tetraselmis chuii, Tetraselmis tetrathele, Tetraselmis verrucosa, Micractinium pusillum-,

Cylindrotheca fusiformis, Nitzschia laevis, Nitzschia alba, Nitzschia fonticola, Navicula incerta, Navicula pelliculosa; Anabaena variabilis of Cyanophyta; Poterioochromonas malhamensis of the genus Cymbidium; Amphidinium carterae, Crypthecodinium cohnii of the genus Algae; Euglena gricilis of the genus Eucalyptus; Gate of the Galdieria sulphuraria.

In a preferred embodiment, the invention is practiced using Chlorella. In a more preferred embodiment, the invention is practiced using Chlorella pyrenoidosa, Chlorella vulgaris or Chlorella ellipsoid. In other preferred embodiments, the present invention employs Chlorella pyrenoidosa, Chlorella vulgaris or Chlorella ellipses to carry out microalgae culture with high oil yield and high protein yield.

Heterotrophic cultivation of microalgal seeds can be carried out using various media well known in the art. Generally, the heterotrophic medium contains a nitrogen source, an organic carbon source, an inorganic salt, a trace element, and water. Nitrogen sources, organic carbon sources, inorganic salts, trace elements, and the like suitable for microalgae culture are well known in the art. For example, as a nitrogen source, urea or various nitrates can be used, such as

KN0 ₃ and the like; as an organic carbon source, glucose, sucrose, glycerin or the like can be used.

Such media include HA-SK medium (Chinese patent ZL 200610024004.9), Endo medium

(Ogbonna J.C., Masui. H., Tanaka. H. Sequential heterotrophic: autotrophic cultivation an effective method of producing Chlorella biomass for health food and animal feed. J. Appl. Phycol. 1997, 9, 359-366) and the like.

The HA-SK medium used in the present invention consists essentially of KNO ₃ , glucose and inorganic salts, trace elements and water. In the above technical solution, the trace element is preferably selected from the group consisting of H ₃ B0 ₃ , ZnS0 ₄ -7H ₂ 0, MnCl ₂ H ₂ 0, ( Η ₄ ) ₆ Μο ₇ 0 ₂₄ · 4Η ₂ 0, CuS0 ₄ -5H ₂ 0, one or more or all of Co(N0 ₃ ) ₂ ·6Η ₂ 0 .

The term "consisting essentially of" means that the above medium may contain, in addition to the main component KN0 ₃ , glucose and inorganic salts, trace elements and water, some basic or novel properties to the composition (ie The microalgae can maintain a high cell density in a short culture period, and the active substance content is greatly increased compared with the conventional heterotrophic culture) there is no substantially influential component. The term "consisting of" means that the above medium consists of the specific components indicated, no other components, but may carry impurities in a usual range.

In this medium, the components of the medium can be varied within a certain range without greatly affecting the density and quality of the microalgae cells. Therefore, the amounts of these components should not be strictly limited by the examples. As is well known to those skilled in the art, inorganic salts such as magnesium sulfate, calcium chloride, ferrous sulfate, and phosphate, and trace elements such as Μη, Ζη, Β, I, M, Cu, may also be added to the culture medium. Co et al.

In the present invention, a preferred trace element component is preferably selected from the group consisting of H ₃ B0 ₃ , ZnS0 ₄ -7H ₂ 0, MnCl ₂ H ₂ 0, ( Η ₄ ) 6Μο ₇ 0 ₂ 4·4Η ₂ 0, CuS0 ₄ - One or more of 5H ₂ 0, Co(N0 ₃ ) ₂ -6H ₂ 0. Inorganic salts and trace elements The amount used can be determined based on conventional knowledge.

The HA-SK medium used in the present invention basically consists of the following components: KN0 ₃ 5~15 g/L, glucose 10~60 g/L, KH ₂ P0 ₄ 0.3-0.9 g/L, Na2HP0 ₄ ' 12H ₂ 0 1.0~10.0 g / liter, MgS0 ₄ '7H ₂ 0 0.2-1.0 g / liter, CaCl ₂ 0.05 ~ 0.3 g / liter, FeS0 ₄ .7H ₂ 0 0.01 ~ 0.05 g / liter, trace elements 0.5 ~ 4ml and water , wherein the composition of the trace elements is H ₃ B0 ₃ 5-15 g / liter, ZnS (V7H ₂ 0 5.0-10.0 g / liter, MnCl _r H ₂ 0 1.0-2.0 g / liter, ( Η ₄ ) ₆ Μο ₇ 0 ₂₄ · 4 Η ₂ 0 0.5-1.5 g / liter, CuS0 ₄ '5H ₂ 0 1.0-2.0 g / liter, Co (N0 ₃ ) 6H ₂ 0 0.1-0.9 g / liter.

In a preferred embodiment, the HA-SK medium composition of the present invention is preferably composed of the following components: KN0 ₃ 7 g/L, glucose 40 g/L, KH ₂ P0 ₄ 0.6 g/L, Na ₂ HP (12H ₂ 0 2.0 g / liter, MgS0 ₄ -7H ₂ 0 0.8 g / liter, CaCl ₂ 0.2 g / liter, FeS (V7H ₂ 0 0.03 g / liter, trace element 1.5mL and water 1000mL, the composition of trace elements H ₃ B0 ₃ 11-12 g/l, ZnS0 ₄ .7H ₂ 0 8.5-9.5 g/l, MnCl ₂ H ₂ 0 1.4-1.5 g/l, ( Η ₄ ) ₆ Μο ₇ 0 ₂₄ ·4Η ₂ 0 0.8-0.9 g/L, CuS0 ₄ '5H ₂ 0 1.5-1.6 g/L, Co(N0 ₃ ) ₂ -6H ₂ 0 0.45-0.55 g/l.

The Endo medium used in the present invention consists essentially of the following components: glucose 10 60 g / liter, urea 2 ~ 8 g / liter, KH ₂ P0 ₄ 1 ~ 2 g / liter, Na ₂ HP0 ₄ .12H ₂ 0 1.0~ 10.0 g / liter, MgS0 ₄ .7H ₂ 0 1 ~ 2 g / liter, CaCl ₂ 0.05-0.1 g / liter, trisodium citrate 0.1 ~ 2.0 g / liter, Fe-EDTA solution 0.5 1 mL, A5 solution l ~ 5mL and water; the Fe-EDTA solution is FeS (V7H ₂ 0 20-30 g / liter and EDTA 20 40 g / liter; A5 solution formula is H ₃ B0 ₃ 2.5-4.0 g / liter, MnCl _r 4H ₂ 0 1.0-2.0 g/l, ZnS0 ₄ .7H ₂ 0 0.1-0.6 g/l, CuS0 ₄ -5H ₂ 0 5-10 g/l, Na ₂ Mo0 ₄ 0.01-0.05 g/l.

In a preferred embodiment, the Endo medium consists of the following components: glucose 40 g/l, urea 6.0 g/l, KH ₂ P0 ₄ 1.5 g/l, Na2HP0 ₄ ' 12H ₂ 0 5.0 g/l , MgS0 ₄ '7H ₂ 0 1.8 g / liter, CaCl ₂ 0.05 g / liter, trisodium citrate 0.4 g / liter, Fe-EDTA solution 0.8 mL, A5 solution 2.0 mL and mouth water, wherein Fe-EDTA solution is FeS (V7H ₂ 0 25 g / liter and EDTA 33.5 g / liter, A5 solution formulation is H ₃ B0 ₃ 2.86 g / liter, MnCl _r 4H ₂ 0 1.81 g / liter, ZnS0 ₄ '7H ₂ 0 0.222 g / liter, CuS0 ₄ -5H ₂ 0 0.07 g / liter, Na ₂ MoO ₄ 0.021 g / liter.

After the medium is formulated according to the above formulation, the pH of the medium can be adjusted to 4.0-9.0 by a conventional means such as an acid or a base, and autoclaved at 115 to 120 ° C for 15 to 20 minutes. Seed heterotrophic culture can be carried out in four ways, such as batch culture, fed-batch culture, semi-continuous culture (with release) or continuous culture.

When seed heterotrophic culture is carried out, the corresponding prepared medium is added to the bioreactor, and water is added to the working volume, usually with a charging coefficient of 0.6 0.8, and then steam sterilized (121 ° C, maintained for about 20 minutes). When the temperature drops to 30~35 °C, the microalgae seeds are connected to the heterotrophic culture according to 1~15% of the working volume.

Regardless of the culture method used, during the cultivation process, the appropriate culture conditions must be controlled to make the microalgae seeds normal. Long. Usually, the control temperature is 20~35 °C, for example, 28~30 °C, the dissolved oxygen concentration is not less than 5%, and the pH is not higher than 9.0. In a preferred embodiment, the dissolved oxygen is not less than 10% of the air saturation concentration and the pH is not higher than 8.5. In other preferred embodiments, the dissolved oxygen is not less than 15% of the air saturation concentration, and the pH is not higher than 8.

During the cultivation process, the pH should not be too high or too low. Generally, as the culture progresses, the pH will rise slowly (this phenomenon is particularly obvious for ordinary chlorella). If the pH is too high, the growth of algae cells will be adversely affected. The acid (for example, 10% sulfuric acid) is adjusted so that the pH is not higher than 9.0, and the preferred pH is 6.5 to 7.5.

Heterotrophic can be carried out in a heterotrophic culture bioreactor such as shake flask, mechanical agitation, airlift, or bubbling. When the glucose in the seed heterotrophic culture solution is consumed, the seed heterotrophic culture ends. Subsequently, it was inoculated into a photoautotrophic culture apparatus for photoautotrophic culture. The initial inoculation density of photoautotrophic culture is usually 0.01 1 g / liter, temperature is 10 ~ 40 ° C, light intensity is 0.1 ~ 100kk, light and dark cycle is 24:0 6: 18, pH is 4.0 9.0, ventilation is 0.05~5vvm, the concentration of C0 ₂ is 0.03~5%.

When the growth of the algal cells is in a stable phase (i.e., when the density of the algae cells is not increased), the photoautotrophic culture is terminated, and the algal cells are harvested.

Photoautotrophic culture can be carried out using various photoautotrophic media well known in the art. Generally, the photoautotrophic medium contains a nitrogen source, a phosphorus source, an inorganic carbon source, an inorganic salt, a trace element, and water. Nitrogen sources, phosphorus sources, inorganic carbon sources, inorganic salts, trace elements, and the like suitable for microalgae culture are well known in the art. For example, as the nitrogen source, urea or various nitrates such as KN0 ₃ may be used; as the phosphorus source, for example, NaH ₂ P0 ₄ may be used _; as the inorganic carbon source, for example, CO ₂ or the like may be used.

This medium is based on F-Si medium.

The improved F-Si medium used in the present invention consists essentially of NaN0 ₃ , NaH ₂ P0 ₄ and trace elements, a small amount of vitamins and water. In the above technical solution, the trace element is preferably selected from the group consisting of FeC ₆ H ₅ O _r 5H ₂ 0, ZnS0 ₄ -7H ₂ 0 _: MnCl ₂ H ₂ 0, Na ₂ Mo0 ₄ -2H ₂ 0, CuS0 ₄ -5H One or more or all of ₂ 0, Na ₂ EDTA, CoCl ₂ .

The term "consisting essentially of "·····" means that the above medium may contain some basic ingredients for the composition in addition to the main components NaN0 ₃ , NaH ₂ P0 ₄ and trace elements, a small amount of vitamins and water. Characteristics or new characteristics (ie, the microalgae can maintain a higher cell density in a shorter culture period, and the active substance content is significantly increased compared with conventional photoautotrophic culture) Minute.

In this medium, the components of the medium can be varied within a certain range without greatly affecting the density and quality of the microalgae cells. Therefore, the amounts of these components should not be strictly limited by the examples. As is well known to those skilled in the art, inorganic salts such as magnesium sulfate, calcium chloride and ferrous sulfate, and trace elements such as Mn, Zn, B, I, M, Cu, Co and the like may be added to the medium.

In the present invention, a preferred trace element component is preferably selected from the group consisting of FeC ₆ H ₅ O _r 5H ₂ 0, ZnS0 ₄ -7H ₂ 0, MnCl ₂ H ₂ 0, Na ₂ Mo0 ₄ -2H ₂ 0, CuS0 ₄ - One or more or all of 5H ₂ 0, Na ₂ EDTA, CoCl ₂ . The amount of inorganic salts and trace elements can be determined based on conventional knowledge.

The improved F/2-Si medium used in the present invention basically consists of the following components: NaN0 ₃ 0.1 1.0 g / liter, NaH ₂ P (V2H ₂ 0 0.01 0.1 g / liter; trace elements 0.5 ~ 4 ml, of which trace The composition of the element is ZnS (V7H ₂ 0 0.02-0.2 g / liter, MnCl _r 4H ₂ 0 0.2 ~ 2.0 g / liter, CuS0 ₄ -5H ₂ 0 0.01-0.1 g / liter, FeC ₆ H ₅ O _r 5H ₂ 0 1.0-10 g / liter, Na ₂ Mo0 ₄ '2H ₂ 0 0.05-0.5 g / liter, Na ₂ EDTA 2.0-20 g / liter, CoCl ₂ 0.01-0.1 g / liter, vitamin 0.5 ~ 4ml and water, of which trace The composition of the elements is vitamin B12 0.1 1.0 mg / liter, vitamin B1 50 1000 mg / liter, vitamin H 0.1 1.0 mg / liter.

In a preferred embodiment, the improved F/2-Si medium of the present invention is preferably composed of the following components: NaN0 ₃ 0.5 g/L, NaH ₂ P (V2H ₂ 0.05 g/L; trace element 1.5 ml, The composition of the trace elements is ZnS0 ₄ -7H ₂ 0 0.1-0.15 g / liter, MnCl ₂ H ₂ O 1.0 ~ 1.5 g / liter, CuS0 ₄ -5H ₂ 0 0.03-0.07 g / liter, FeC ₆ H ₅ O _r 5H ₂ 0 3.0-4.0 g/l, Na ₂ Mo0 ₄ '2H ₂ 0 0.1-0.3 g/l, Na ₂ EDTA 10-12 g/l, CoCl ₂ 0.02-0.06 g/l, vitamin 2 ml and water 1000 mL The composition of trace elements is vitamin B12 0.03 0.05 mg / liter, vitamin B1 100 200 mg / liter, vitamin H 0.5 1.0 mg / liter.

After the medium is formulated according to the above formulation, the pH of the medium can be adjusted to 4.0 9.0 by a conventional means such as an acid or a base. Photoautotrophic culture can be carried out in four ways: batch culture, fed-batch culture, semi-continuous culture (with release) or continuous culture. Algae cell harvesting, oil extraction and comprehensive utilization of algae

After the autotrophic culture is completed, the microalgae are harvested by centrifugation to obtain a wet algae body. Methods for harvesting algae cells include, but are not limited to, high speed centrifugation, flocculation, air flotation or filtration; algae cell wall breaking methods include, but are not limited to, algae autolysis, high pressure homogenization, enzymatic hydrolysis, aqueous phase pyrolysis, etc. Broken wall method.

The method for extracting intracellular fats and oils includes, but is not limited to, organic solvent extraction, that is, drying the algae body at 80-105 ° C to a constant weight, and grinding the algal powder, and extracting the oil from the dry algae powder by using a chloroform methanol standard extraction solvent. The extraction solvent was repeatedly extracted until the color of the algal powder turned white, and the solvent was removed by rotary evaporation.

The other components in the supernatant can be gradually separated and extracted to obtain various other active ingredients of the microalgae, including but not limited to intracellular active substances such as fatty acids, proteins, polysaccharides, chlorophyll, lutein, and chlorella growth factors. Alternatively, all the components in the supernatant may be directly spray-dried with the algal precipitate to obtain chlorella powder for processing into animal feed, aquaculture bait, food, food additives, medicines and nutrients.

In the present invention, the microalgae obtained by the culture can be comprehensively utilized, and various active ingredients such as a pigment (for example, lutein), a protein, and a polysaccharide can be extracted. The order of extraction of the active ingredient is not particularly limited, but it is generally necessary to satisfy the premise that the step of first extraction cannot cause loss of the component to be extracted later. Methods for extracting proteins, polysaccharides, and the like are also well known in the art. Alternatively, the harvested algae can be used directly. For example, it can be dried (eg spray dried) and harvested. The wet algae body can be used to develop animal feed, aquaculture bait, food, food additives, medicines and nutrients.

The methods for determining the dry weight, oil content and intracellular biochemical composition of algae cells are as follows:

Determination of dry weight of algae cells: Take 50 ml of culture medium during microalgae (such as chlorella) culture, centrifuge at 8000 rpm for 10 minutes, wash the algae after centrifugation 3 times with deionized water, and transfer to a weighing bottle ( In W1 (g), it is dried in a 105 ° C oven to a constant weight W2 (g). The dry weight of algae Cx can be calculated according to the following formula: Cx (g / l) = (W2-W1) / V / lOOOo

Determination of fat: Take a certain amount of algae cells dried to constant weight in each culture stage, grind to powder form in a mortar, weigh the appropriate amount of algae powder (0.2 0.5 g), carefully transfer to a centrifuge tube, and add an appropriate amount of extraction solvent (chloroform). :Methanol = 2: 1) Ultrasonic shaking in an ultrasonic shaker for 30 min, centrifugation at 8000 rpm for 10 min, transferring the supernatant to a dry rotating evaporation vial of known weight, repeating the above steps until the supernatant is colorless. The supernatants were combined, evaporated to dryness, weighed and the oil content was calculated.

Grease content (%) Calculated as follows:

Grease (%) = (W2-W0) / W1 X 100

Where: _W1 - is the weight of algae powder, _{g; wo} - the weight of the rotary evaporation bottle for drying to constant weight, g; W2 - the weight of the evaporation bottle after evaporation of the oil extract, g.

Determination of protein content: The total protein content in cells of microalgae (such as chlorella) was determined by Kjeldahl method (Ning Zhengxiang. Handbook of Food Composition Analysis. Beijing: China Light Industry Press, 1998, 76-78). The relevant content of the present invention will be further described below by way of examples. Unless otherwise stated, the content of each component in the medium used in the present invention is expressed in grams per liter (g/L). It should be understood that "contains" and "comprises" in this application also includes the meaning of "consisting of" and "consisting of." Example 1: Study on the growth of algae cells during heterotrophic and photoautotrophic culture of Chlorella pyrenoidosa The nucleus of the nucleus of the present example was heterotrophic cultured in a 500 ml shake flask and a 2 L flask. Perform photoautotrophic culture. As the next seed of photoautotrophic culture, the growth curve of algae cells in the heterotrophic and photoautotrophic culture seeds of Chlorella pyrenoidosa was determined. The seedling density of Chlorella pyrenoidosa seeds was 0.20g/l, the temperature was 30°C, and the rotation speed was 150rmp. When cultured to 72h, the glucose in the culture solution was consumed, and the algal cell density was 6.8g/ l, the seed used for the next photoautotrophic culture; the seeding density of Chlorella pyrenoid seeds is 0.20g/l, the temperature is 25°C, the light intensity is 2000k, and the light and dark period is 24: 0, shaken 3 times a day, cultured to 120h, algae cells in the exponential growth phase, algal cell density of 0.56g / l, used for the next photoautotrophic culture of seeds (Figure 1). It can be seen that the density of seed cells in heterotrophic culture is higher than that of photoautotrophic culture (12.1 times that of photoautotrophic seeds) and the culture period is short (48 hours shorter than that of photoautotrophic seeds). Example 2: Study on the growth of algae cells during the heterotrophic and photoautotrophic culture of Chlorella vulgaris

The common chlorella in the present example was subjected to heterotrophic culture in a 500 ml shake flask and a light autotrophic culture in a 2 L flask, and as a seed for the next photoautotrophic culture, the ordinary chlorella heterotrophic was separately measured. And algae cell growth curve of seed autotrophic culture. The seedling density of the common chlorella seeds was 0.30g/l, the temperature was 30 °C, and the rotation speed was 150rmp. When cultured to 72h, the glucose in the culture solution was consumed, and the algal cell density was 8.1g/l. Seeds used for the next photoautotrophic culture; the seedling density of common Chlorella seeds is 0.30g/l, the temperature is 25 V, the light intensity is 2000k, and the light and dark cycle is 24:0. The cells were shaken 3 times, and when cultured for 120 hours, the algae cells were in the exponential growth phase, and the algal cell density was 0.65 g/l, which was used for the next photoautotrophic culture (Fig. 2). It can be seen that the heterotrophic cultured seed cells have a higher density (12.5 times that of photoautotrophic seeds) and a shorter culture period (48 hours shorter than photoautotrophic seeds) compared to seeds grown by photoautotrophic culture. Example 3: Study on the growth of algae cells in the heterotrophic and photoautotrophic culture of Chlorella ellipsoids

The chlorella ellipsoid of the present example was subjected to heterotrophic culture in a 500 ml shake flask and a light autotrophic culture in a 2 L flask. As a seed for the next photoautotrophic culture, the ellipsoidal heterotrophic growth was measured. And algae cell growth curve of seed autotrophic culture. The seeding density of chlorella chlorella seeds was 0.25g/l, the temperature was 30 °C, the rotation speed was 150rmp, and when cultured to 72h, the glucose in the culture solution was consumed, and the algal cell density was 8.8g/l. For the next step of photoautotrophic culture; the seedling density of Helicobacter ellips seeds is 0.25g/l, the temperature is 25 V, the light intensity is 2000k, and the light and dark cycle is 24:0. After shaking for 3 times, the algae cells were in the exponential growth phase at 120 h, and the algal cell density was 0.71 g/l, which was used for the next photoautotrophic culture (Fig. 3). It can be seen that the heterotrophic cultured seed cells have a higher density (12. 4 times that of photoautotrophic seeds) and a shorter culture period (48 hours shorter than photoautotrophic seeds) compared to seeds grown by photoautotrophic culture. Example 4: Phototrophic culture of Chlorella pyrenoidosa heterotrophic seeds and photoautotrophic seeds in an indoor 2L photobioreactor

In this example, the seeds of heterotrophic and photoautotrophic nucleus chlorella were cultured in an indoor 2L cylindrical airlift photobioreactor, and the heterotrophic and photoautotrophic seeds were determined in the process of photoautotrophic growth. Algae cell growth and oil yield. The inoculation density of heterotrophic and photoautotrophic seeds was 0.3g/l, the photoautotrophic culture temperature was 30 °C, and 2% of C0 _{2 was introduced} . The aeration was 0.5vvm, the light intensity was 10000k, and the light and dark. The period is 24:0. After autotrophic culture for 96 hours, the density of algal cells of heterotrophic seeds was 2.03g/l, the yield of oil was 149.55mg/l/d, and the density of algal cells of photoautotrophic seeds was only 0.93g/l. It is only 87.68 mg/l/d (Fig. 4 and Fig. 5). It can be seen that the vigor of the heterotrophic seeds is stronger than that of the photoautotrophic seeds, and the density of the algae cells in the same culture time is higher (2.2 of the photoautotrophic seeds). Double), the oil yield is higher (1.7 times that of photoautotrophic seeds). Example 5: Photoautotrophic culture of Oryza sativa seeds and photoautotrophic seeds in an indoor 2L photobioreactor

In this embodiment, the phototrophic and photoautotrophic common chlorella seeds were self-supported in an indoor 2L cylindrical airlift photobioreactor, and the heterotrophic and photoautotrophic seeds were determined in the photoautotrophic process. Algae cell growth and oil yield. The inoculation density of heterotrophic and photoautotrophic seeds was 0.3g/l, the photoautotrophic culture temperature was 30 °C, and 2% of C0 _{2 was introduced} . The aeration was 0.5vvm, the light intensity was 10000k, and the light and dark. The period is 24:0. After autotrophic culture for 96 hours, the density of algal cells of heterotrophic seeds was 2.03g/l, the yield of oil was 138.49mg/l/d, and the density of algal cells of photoautotrophic seeds was only 1.25g/l. Only 82.81 mg / l / d (Figure 6 and Figure 7). It can be seen that compared with photoautotrophic seeds, the vigor of heterotrophic seeds is stronger, the density of algae cells in the same culture time is higher (1.6 times that of photoautotrophic seeds), and the yield of oil is higher (light self-supporting) 1.7 times the seed). Example 6: Photoautotrophic culture of heterotrophic seeds and photoautotrophic seeds of Chlorella ellipticus in an indoor 2L photobioreactor

In this example, the seeds of heterotrophic and photoautotrophic chlorella were cultured in an indoor 2L cylindrical airlift photobioreactor, and the heterotrophic and photoautotrophic seeds were determined in the photoautotrophic process. Algae cell growth and oil yield. The inoculation density of heterotrophic and photoautotrophic seeds was 0.3g/l, the photoautotrophic culture temperature was 30 °C, and 2% of C0 _{2 was introduced} . The aeration was 0.5vvm, the light intensity was 10000k, and the light and dark. The period is 24:0. After autotrophic culture for 96 hours, the density of algal cells in heterotrophic seeds was 1.65 g/l, the yield of oil was 121.48 mg/l/d, and the density of algal cells in photoautotrophic seeds was only 0.91 g/l. It is only 72.29 mg/l/d (Figures 8 and 9). It can be seen that compared with photoautotrophic seeds, the vigor of the heterotrophic seeds is stronger, the density of algae cells in the same culture time is higher (1.8 times that of photoautotrophic seeds), and the oil yield is higher (light self-supporting) 1.7 times the seed). Example 7: Study on Photoautotrophic Culture of Chlorella pyrenoid Heterotrophic Seeds in Outdoor 2L Photobioreactor This example is a photoautotrophic culture of protein nucleus in an outdoor 2L cylindrical airlift photobioreactor Algae heterotrophic seeds were used to determine the algal cell growth process and the highest oil yield of heterotrophic seeds during their outdoor photoautotrophic process. The initial seeding density was 0.06 g/l, the temperature and light intensity were both outdoor actual temperature and light intensity, and the ventilation was 0.3 wm. After autotrophic culture for 83 hours, the algal cell density and oil yield reached the highest values, which were 1.44 g/l and P 123.43 mg/l/d, respectively (Fig. 10). It can be seen that the heterotrophic seeds can be rapidly autotrophically cultured and achieve high oil yield in the room, and can be rapidly autotrophically cultured outdoors and achieve high oil yield. Example 8: Study on photoautotrophic culture of heterotrophic seeds of Chlorella pyrenoidosa in outdoor 60L plastic pots. In this example, photoautotrophic culture of Chlorella vulgaris heterotrophic seeds was carried out in outdoor 60L plastic pots, and heterotrophic seeds were determined. The algae cell growth process and the highest oil yield of the seeds during their outdoor photoautotrophic process. The initial seeding density was 0.10 g/l, the temperature and light intensity were both outdoor actual temperature and light intensity, and the ventilation was 0.3 vvm. After autotrophic culture for 108 hours, algal cell density and oil yield reached the highest values of 0.75 g/l and 55.34 mg/l/d, respectively (Fig. 11). The culture volume in a 60L plastic pot is generally 50L. To achieve an initial seeding density of 0.10g/l, 5g of algae is required. For heterotrophic cultured seeds (see Figure 1), only four 500ml shake flasks are required. The liquid volume is 200ml. 5.44g of seed can be obtained after 3 days of culture, while the seed cultured by light (see Figure 1) requires 9 2L triangular flasks (with a liquid volume of 1L) for 5 days. 5.04g of seed. It can be seen that compared with the seed light autotrophic culture, the seed heterotrophic culture can not only shorten the seed culture time, but also improve the efficiency of the whole culture process, and the culture equipment required for seed culture is used less and the floor space is small. Therefore, if outdoor large-scale photoautotrophic cultivation of microalgae, only the use of heterotrophic culture seeds can meet the needs of a large number of algae in time. While the invention has been described with respect to the specific embodiments of the present invention, it will be apparent to those skilled in the art Therefore, the appended claims are intended to cover all such modifications within the scope of the invention.

Claims

A method of cultivating a microalgae, characterized in that the method comprises a heterotrophic culture step of a microalgae species and a photoautotrophic culture step of the algae cells obtained by heterotrophic culture as seeds.

2. A method for producing a fat or oil, characterized in that the method comprises a heterotrophic culture step of a microalgae species, a photoautotrophic culture step performed as a seed by heterotrophic culture, and an algal cell harvesting step. And the steps of oil extraction.

3. A method for producing a microalgae active ingredient or algal flour, characterized in that the method comprises a heterotrophic culture step of a microalgae species, and a photoautotrophic culture carried out by using the algal cells obtained by heterotrophic culture as a seed Steps, and steps of harvesting algae cells and extracting or drying the algal cells into algal flour.

The method according to any one of claims 1 to 3, wherein the heterotrophic cultivation of the microalgae species is carried out in a mode of batch culture, fed-batch culture, semi-continuous culture and continuous culture. to cultivate.

The method according to any one of claims 1 to 4, wherein the microalgae is selected from the group consisting of: Chlorella pyrenoidosa, Chlorella pyrenoidosa, Chlorella pyrenoidosa i Chlorella Vulgaris), #||l| ^J^3⁄4c3⁄4 i Chlorella ellipsoidea) , Chlorella emersonii, Chlorella sorokiniana, Chlorella saccharophila, Chlorella regularis, Chlorella minutissima, Chlorella protothecoides, Chlorella zofingiensis, and Brachiomonas submarina, Chlamydobonas reinhardtii in the green algae Chlamydomonas acidophila, Haematococcus pluvialis, Haematococcus lacustris, Scenedesmus obliquus, Spongiococcum exetriccium, Tetraselmis suecica, Tetraselmis chuii, Tetraselmis tetrathele, Tetraselmis verrucosa, Micractinium pusillum;

Cylindrotheca fusiformis, Nitzschia laevis, Nitzschia alba, Nitzschia fonticola, Navicula incerta, Navicula pelliculosa;

Anabaena variabilis

Poterioochromonas malhamemis

Amphidinium carter ae, Crypthecodinium cohnii;

Euglena grid lis;

Galdieria sulphuraria of the red algae gate.

The method according to any one of claims 1 to 5, wherein the step of heterotrophic cultivation of the microalgae algae comprises: adding a medium having a pH of 4.0 9.0 to the bioreactor, according to the work 0.1~30% of the volume is connected to the microalgae algae for batch culture, fed-batch culture, semi-continuous culture or continuous culture, and the culture temperature is 10~40 °C. The pH is controlled to be less than 9.0, and the dissolved oxygen is controlled to be above 1%.

The method according to any one of claims 1 to 6, wherein the algal cells obtained by heterotrophic culture are used as seeds for photoautotrophic culture, comprising: connecting heterotrophic cultured algae to light Self-cultivation in autotrophic culture equipment, culture temperature is 5~50 °C, continuous light or intermittent illumination, light intensity is 0.1~150kk, photoautotrophic culture period is 5-500 hours, initial inoculation density is 0.01- 10.00 g / liter, pH 4.0 ~ 12.0.

The method according to any one of claims 1 to 7, wherein the heterotrophic culture medium of the algae species is composed of a nitrogen source, an organic carbon source, an inorganic salt, a trace element and water; a photoautotrophic medium It consists of a nitrogen source, an inorganic salt and water.

The method according to any one of claims 1 to 8, wherein the heterotrophic cultivation step of the algae is carried out in a shake flask, a mechanical agitation type, an airlift type or a bubbling bioreactor, The photoautotrophic culture step is carried out in a shake flask or from an open track pool or a round pool, a closed plate photobioreactor or a pipe photobioreactor or a column photobioreactor, a film pouch or A hanging bag or the like is used in a device for self-cultivation of microalgae light, and the light condition is natural light or artificial light.

The method according to any one of claims 1 to 9, wherein when the organic carbon source in the heterotrophic culture solution is consumed, the heterotrophic culture is terminated, and the algal cells obtained by the heterotrophic culture are used as seeds. The photoautotrophic culture step is carried out.