WO2015085631A1

WO2015085631A1 - Method for culturing botryococcus spp. with high yield

Info

Publication number: WO2015085631A1
Application number: PCT/CN2013/090407
Authority: WO
Inventors: 李元广; 王伟良; 黄建科; 范建华; 王军; 梁松涛; 王俊; 潘荣华; 陈杰; 沈国敏
Original assignee: 嘉兴泽元生物制品有限责任公司
Priority date: 2013-12-13
Filing date: 2013-12-25
Publication date: 2015-06-18
Also published as: CN103642694A

Abstract

Provided are a method for culturing Botryococcus spp. with high yield and a method for producing oil and/or hydrocarbon. The method comprises heterotrophic or mixotrophic culture process of Botryococcus spp. seeds, and photoautotrophic culture process of the seeds obtained by heterotrophic or mixotrophic cultivation. The method can sufficiently exploit the advantage of the rapid growth of algal cells in the heterotrophic or mixotrophic culture phase to provide large number of algal seeds for Botryococcus spp. in the photoautotrophic culture phase, and makes Botryococcus spp. grow rapidly and accumulate oil and hydrocarbon in the photoautotrophic cultivation phase.

Description

High-yield grape algae cultivation method

The present invention is in the field of bioenergy and relates to a method for rapidly obtaining algae having high oil and/or hydrocarbon yield. Background technique

Botryococcus spp. is a group of single-celled microalgae widely distributed in freshwater areas of tropical and temperate regions, occasionally also found in brackish water or seawater (Huang et al. 1999; Volova et al. 2003). ). An important feature of grape algae is the ability to synthesize and accumulate a variety of lipids, hydrocarbons (Brown and Knights 1969; Knights et al. 1970) and ether lipids (Metzger and Casadevall 1991; Metzger 1999). The algae's hydrocarbon content can account for 15-80% of the dry weight of the cells, which is much higher than the hydrocarbon content of other microorganisms (Banerjee et al. 2002). Due to its high hydrocarbon content and its ability to multiply under certain conditions (Wake and Hillen 1980; Townsend 2001), grape algae is considered a renewable resource that can be used to produce liquid fuels (Casadevall et al. 1985).

The cells of Botryococcus bmunii have an elliptical shape, 3 to 6 micrometers in width and 6 to 12 micrometers in length. They are embedded in a layered funnel-shaped gel, often in groups of 2 or 4 cells. In the central unity, all the cells radiate more or less in a radial manner, and the cells are tightly packed close to each other, so that the entire population appears in a fairly uniform shape. The population size can reach 100 microns ~ 0.5 mm. The colonies of grape algae are clusters of cells of different sizes, resembling grapes, which are connected by transparent refractive filaments. No flagella, containing chlorophyll a, b and α, β-carotene, lutein and other pigments. According to the source, especially the structure of the hydrocarbons produced, the grape algae are divided into three strains: A, B and L. The optimum growth temperature of each strain of grape algae is different. The optimum growth temperature of strain A is 25 °C, and the growth rate is slower than 30 °C. The suitable growth temperature of strain B is relatively high, still at 35 °C. It can grow, but it produces the most hydrocarbon at 25 °C. The A strain produces C ₂₃ -C ₃₃ odd-numbered carbon unbranched linear diolefins and triolefins, and the B strain produces C _3Q -C ₃₇ terpenoids with polyunsaturated acids and branched hydrocarbons, and L strains form C ₄₍ )H ₇₈ This single tetraene hydrocarbon. At present, the research on grape algae cultivation at home and abroad mainly focuses on photoautotrophic. Although the photoautotrophic culture process of grape algae can accumulate a large amount of oils and hydrocarbons, the synthesis of high-energy oils or hydrocarbons in cells is an extremely energy-intensive process, which means that the photoautotrophic growth rate of grape algae is high. It is lower than other microalgae and has a longer doubling time than other microalgae. At present, the following problems exist in the photoautotrophic culture of grape algae: 1) The low seeding density is susceptible to the contamination of the algae and protozoa and the environmental and climatic conditions; 2) the current seed expansion is carried out by photoautotrophic Culture, the cycle is very long (if a certain amount of algae species is connected to the big pool, it usually takes at least 1~2 months), and the algae species need a large number of culture devices and equipment in the continuous expansion of several months. The area is large, and the unit culture yield is low; 3) If the algae species cultivated by outdoor light are contaminated by algae or protozoa, plankton, etc., then the algae species will be scrapped, and the algae species need to be re-applied. Expanding the cultivation work, this will lead to the next step of photoautotrophic cultivation without algae species for cultivation, which will seriously affect production.

On the other hand, heterotrophic or polyculture can obtain high cell density and high cell growth rate, so heterotrophic or polyculture can greatly increase the culture density and cell yield of the grape algae, but the obtained algae oil and hydrocarbons The content is low, so it cannot be used for large-scale cultivation of grape algae for the purpose of producing high-yield oils and hydrocarbons. In addition, in the heterotrophic or polyculture process, the medium usually selected is a common microalgae culture medium, and no growth-promoting plant growth hormone substances are added. The culture process is usually not regulated or the unoptimized feed is added by intermittent flow. Medium, this method does not consider the difference in nutrient requirements between grapevine heterotrophic and polyculture and ordinary photoautotrophic, resulting in poor culture of the culture medium and relatively slow cell growth.

Therefore, there is a need in the art for an efficient grape algae cultivation process and method. Summary of the invention

In view of the above problems, the present invention provides an effective solution by first cultivating grape algae by heterotrophic or polyculture (also known as cultivating) culture, and then using the algal cells obtained by heterotrophic or polyculture culture as seeds. Self-cultivation of light. The method of the invention can fully exert the advantages of rapid growth of algae cells in the heterotrophic or polyculture stage of the algae, and provide a large number of algae species and microalgae to rapidly grow and accumulate useful substances in the photoautotrophic stage during the photoautotrophic stage ( Oils and hydrocarbons) and high cell yield and yield of metabolites provide important technical means to solve the problem of cell growth rate and low yield of useful substances (fats and hydrocarbons) during autotrophic culture of grape algae.

Therefore, the first aspect of the present invention provides a method for cultivating a grape algae, which comprises a grapevine cell The heterotrophic or polyculture culture step and the photoautotrophic culture step performed as a seed by the algal cells obtained by heterotrophic or polyculture culture.

A second aspect of the present invention provides a method for producing a fat or oil and/or a hydrocarbon, the method comprising a heterotrophic or polyculture culture step of a grapevine cell, and a light carried out by using the algal cell obtained by heterotrophic or polyculture culture as a seed Autotrophic culture steps, as well as steps for algal cell harvesting and oil/total hydrocarbon extraction.

In a specific embodiment, the above method of the present invention comprises:

(1) Heterotrophic or polyculture culture of grape algae cells, wherein, during the culture process, the pH is kept constant by feeding and the elements such as carbon, nitrogen and/or phosphorus are stabilized within a certain concentration range, and the heterotrophic or polyculture culture is controlled at the end The concentration of nutrients such as carbon, nitrogen and/or phosphorus is low or even zero;

(2) performing photoautotrophic culture using algal cells obtained by heterotrophic or polyculture as seeds, and

(3) Harvesting algae cells, and separating and extracting oils and/or hydrocarbons.

In a specific embodiment, the species of the algae used include, but are not limited to, Botryococcus braunii, Botryococcus sudeticus, Botryococcus sp., Botryococcus spp

In a specific embodiment, the algal cells obtained by heterotrophic or polyculture can be diluted with water, and then added to the diluted algal solution by photoautotrophic culture medium for photoautotrophic culture; or directly obtained by heterotrophic or polyculture The grape algae cells are cultured in light.

In a specific embodiment, the grape algae adopts any one of three strains of Sotrjococo^ braw Y A, B and L.

In a specific embodiment, the step of heterotrophic or polyculture of the cyanobacteria comprises: adding a medium having a pH of 4.0 to 1 1.0, preferably 7.5 to 8.5, in the bioreactor, according to a working volume of 0.1 to 30 % access to algae for batch culture, fed-batch culture, repeated fed-batch culture, semi-continuous culture or continuous culture, culture temperature is 10~40 °C, control pH is less than 10.0, preferably less than 9.0, controlled dissolution Oxygen is above 1%.

In a specific embodiment, the grapevine algae species are cultured in a heterotrophic manner, and no illumination is required. When the grape algae is cultured in a polyculture, illumination is required, and the light intensity ranges from 1 to 2,500.

In a specific embodiment, the step of self-cultivating the grape algae comprises: inoculating the heterotrophic or polyculture (cultivating) grape algae species into a photoautotrophic culture device for photoautotrophy, and the culture temperature is 5~ 50 ° C, continuous light or intermittent illumination, light intensity is ¹ ~2500 molm ^-1 , photoautotrophic culture period is 1~180 days, preferably 5~40 days, initial inoculation density is 0.01~5.0 g / liter, The medium does not contain organic carbon The source has a pH of 4.0 to 9.0.

In a specific embodiment, the heterotrophic or polyculture medium consists of a nitrogen source, an organic carbon source, a plant growth hormone, an inorganic salt, a trace element, and water; a photoautotrophic medium consists of a nitrogen source, a plant growth hormone, and an inorganic salt. , trace elements and water composition.

In a specific embodiment, the heterotrophic step is carried out in a shake flask, a mechanical agitation, an airlift or a bubbling heterotrophic culture bioreactor; the algae polyculture step is in a shake flask, a steam-sterilized closed photobioreactor (transparent), and a bioreactor (including an external light source and/or an internal light source) containing a light system such as a mechanical agitation; the photoautotrophic culture step is carried out in a shake flask or Equipped from an open runway or round cell, a closed flat photobioreactor or a tubular photobioreactor or a column photobioreactor or a film stereobioreactor or an open and closed hybrid Any device that can be used for photoautotrophic cultivation of microalgae, such as a photoautotrophic culture system or an adherent culture system, is irradiated with natural light or artificial light.

In a specific embodiment, when the algae species is brown grape algae, the medium used for heterotrophic or polyculture consists essentially of the following components: KNO ₃ 0.1-15 g/l, organic carbon source 0.1 to 50 g/升, K ₂ HPO ₄ 0.1~10.0 g / liter, MgSO ₄ '7H ₂ O 0.1~10 g / liter, CaCl ₂ '2H ₂ O 0.01 ~ 5 g / liter, phytohormone 2,4-dichlorophenoxy Acetic acid 0.001-5 mg / liter, benzylaminopurine 0.001-5 mg / liter, abscisic acid 0.001-5 mg / liter, gibberellin 0.001-5 mg / liter, 3- butyl butyric acid 0.001-5 mg / liter, Naphthaleneacetic acid 0.001-5 mg / liter, brassinoin 0.001-5 mg / liter, ferric citrate 0.01 ~ 5 g / liter, citric acid 0.1 ~ 15 g / liter, trace elements 0.5 ~ 20ml and water, which trace elements The composition is H ₃ BO ₃ 0.1-10 g / liter, ZnSO ₄ -7H ₂ O 0.1-10.0 g / liter, MnCl ₂ -4H ₂ O 0.5-10.0 g / liter, (ΝΗ ₄ ) ₆ Μο ₇ Ο ₂₄ · 4Η ₂ Ο 0.01-5 g / liter, CuSO ₄ -5H ₂ O 0.01 ~ 5.0 g / liter, Co (NO ₃ ) ₂ -6H ₂ O 0.01 ~ 5 g / liter.

In a specific embodiment, the organic carbon source includes any carbon source that can be cultured as a microorganism, including but not limited to glucose, maltose, sucrose, lactose, starch, hydrolysate of cellulose hemicellulose, glycerin, sodium acetate, acetic acid. Glucose is generally preferred.

In a specific embodiment, after the organic carbon source in the heterotrophic or polyculture medium is consumed, the heterotrophic or polyculture is terminated, and the algal cells obtained by heterotrophic or polyculture are used as seeds to perform photoautotrophic culture. step.

In other specific embodiments of the present application, the culture medium and culture conditions described in the present application can be used. The above culture method of grape algae is applied.

The advantages of the present invention are described in detail by the heterotrophic or polyculture culture of the grape algae species and the photoautotrophic culture of the heterotrophic or polyculture culture as a seed for the production of oils and fats and/or hydrocarbons.

(1) can greatly increase the rate of algae cultivation. The initial inoculation density of the microalgae cultured in a large outdoor pool is 0.05 to 0.1 g/l, and the volume of the large pool is generally 5000 L. If this requirement is required, 250 to 500 g of microalgae are required. If the traditional photoautotrophic expansion of algae species is used, it takes 1 to 2 months (the algal cell density of the grape algae self-cultivation and expansion for 10 days is 0.2g/l, the culture volume is 500L); Polyculture of algae can be done in just a few days.

(2) Compared with the traditional algal species photoautotrophic culture, the number of devices and equipment in the cultivation process of algae species can be reduced, and the area is small, and the yield per unit culture area is high. The self-cultivation of algae requires 500L of culture equipment and takes a long time (1~2 months), while the heterotrophic/polyculture of algae requires only 50L of culture equipment and takes a short time (10-20 days). ).

(3) Compared with the self-cultivation of algae, the heterotrophic or polyculture of algae is not affected by outdoor weather and environment. When the algae species are self-supporting, if they are unable to continue the culture in the case of short-term rainy weather, they need to be moved into the room and artificial light is added to continue the self-cultivation of the algae species, thus increasing the need for algal cultures. Artificial lighting costs. In addition, if the algae species that are self-supporting by outdoor light are contaminated by protozoa, plankton, etc., then the algae species will be scrapped, and the expansion of algae species will be carried out again, which will lead to the next step of photoautotrophic culture without algae. The problems used for cultivation seriously affect 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. Therefore, heterotrophic or polyculture of algae species can provide a large amount of seeds required for photoautotrophic culture in time.

(5) The vigor of heterotrophic or polycultured cells as algae is better than that of photoautotrophic algae. When the amount of the same algae cells is used for photoautotrophic culture, the algal cell density of heterotrophic algae species is harvested. The oil and total hydrocarbon yields are higher than the algal cell density and oil and total hydrocarbon yield of photoautotrophic algae. Therefore, in the case of obtaining the same oil fat and total hydrocarbon yield, the use of heterotrophic or mixed algae species can make the light autotrophic equipment have a small footprint and a high area yield. In addition, since the density of algae cells obtained by photoautotrophic culture using heterotrophic or polycultured cells as algae species is large, the cost of harvesting grape algae is greatly reduced.

In summary, the photoautotrophic culture mode of the algae cells obtained by heterotrophic or polyculture culture as seeds can completely solve the problem of low efficiency and contamination of algae cultivation during photoautotrophic culture. Many problems and the slow growth rate of grape algae in the photoautotrophic stage and the low yield of oil and total hydrocarbons. Therefore, the present invention provides an important technical means for solving the problem of low yield of cells, oils and fats and total hydrocarbons during autotrophic cultivation of grape algae, and in particular, lays a solid industrial foundation for the production of liquid fuel by using grape algae. DRAWINGS

Figure 1 shows the growth process of grapevine cells in heterotrophic, polyculture and photoautotrophic culture in 500ml shake flasks.

Figure 2 shows the culture process of grapevine in a 5L fermentor. In the figure, ▲/country represents an example of controlling pH and feeding strategy, and adding plant growth hormone; Δ/port means controlling only pH, unoptimized feeding medium and feeding strategy, and no plant growth hormone added. Example; Β / Φ p _H and a control feeding strategy, auxin embodiment not added.

Figure 3 shows the algal cell growth process of photoautotrophic culture of viticulture heterotrophic seeds, polyculture seeds and photoautotrophic seeds in an indoor 2L photobioreactor.

Figure 4 shows the oil and total hydrocarbon content and yield of the autotrophic culture of the algae heterotrophic seeds, polyculture seeds and photoautotrophic seeds in an indoor 2L photobioreactor.

Figure 5 shows the algae cell growth process of photoautotrophic culture of grape seed heterotrophic seeds, polyculture seeds and photoautotrophic seeds in an outdoor 60L plastic pot.

Figure 6 shows the oil and total hydrocarbon content and yield of the autotrophic culture of the algae heterotrophic seeds, polyculture seeds and photoautotrophic seeds in an outdoor 60L plastic pot. detailed description

The term "consisting essentially of" means that the above medium may contain, in addition to the main component KNO ₃ , glucose and inorganic salts, trace elements and water, some basic or novel properties of the composition (ie The cell density was maintained at a higher level in the shorter culture period, while the hydrocarbon content was significantly increased compared to conventional heterotrophic cultures. There were no substantially influential components. 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.

The various technical solutions of the present invention can be implemented by using various grape algae known in the prior art, including but not limited to In the order of SotryococcMS braunii, Botryococcus sudeticus, Botryococcus sp., Botryococcus spp. Brown grape algae includes three strains A, B and L.

In a specific embodiment, the Botryococcus braunif A strain is used, and the optimum growth temperature of the A strain is 25 °C, and the growth rate is slower than 30 °C, and the A strain produces an odd number of c ₂₃ -c ₃₃ Carbon-branched linear diolefins and triolefins can also accumulate oil in cells. Heterotrophic or polyculture

Heterotrophic or polyculture of the grape seed can be carried out using various media well known in the art. Unlike heterotrophic no light, polyculture is illuminated. An organic carbon source is required in both heterotrophic and polyculture media. Generally, heterotrophic and polyculture media are the same, containing nitrogen sources, organic carbon sources, inorganic salts, trace elements, and water. Nitrogen sources, organic carbon sources, inorganic salts, trace elements and the like suitable for the cultivation of grape algae are well known in the art. For example, as a nitrogen source, urea or various nitrates such as KNO ₃ and the like can be used; as an organic carbon source, any carbon source which can be cultured as a microorganism can be used, including but not limited to glucose, maltose, sucrose, lactose, Glycerin, starch. However, there have been no reports of the addition of plant growth hormone to heterotrophic or polyculture media, and the inventive addition of plant growth hormone to the grapevine culture medium significantly promotes cell growth.

The plant growth hormone used in the medium of the present invention (including heterotrophic, polyculture medium and photoautotrophic medium) can significantly promote cell growth, and such hormones include, but are not limited to, 2,4-dichlorophenoxyacetic acid, benzylamino嘌呤, abscisic acid, gibberellin, 3-indolic acid, naphthaleneacetic acid and brassinolide. The medium may contain one or more plant growth hormones. The total amount of plant growth hormone in the medium may be 0.001 - 35 mg / liter of medium, usually 0.001 -20 mg / liter, more usually 0.001 - 15 mg / liter, 0.005 - 10 mg / liter, 0.01 - 10 mg / liter, ranging from 0.1 to 5 mg / liter.

In a specific embodiment, each plant growth hormone, if present, may be at a concentration of, for example, 2,4-dichlorophenoxyacetic acid 0.001 to 5 mg/liter, benzylaminopurine 0.001 to 5 mg/liter, and abscisic acid 0.001 to 5 Mg/L, gibberellin 0.001 -5 mg/L, 3-吲哚-butyric acid 0.001 -5 mg/L, naphthaleneacetic acid 0.001 -5 mg/L, brassinolide 0.001-5 mg/L. The concentration of each plant growth hormone is preferably, for example, 0.01 -4 mg / liter, 0.1 - 4 mg / liter, 0.3 - 4 mg / liter, 0.3 - 3 mg / liter, 0.5 - 2.5 mg / liter.

The plant growth hormone can be obtained from a commercially available route and then directly added to the heterotrophic/polyculture and photoautotrophic culture medium disclosed in the present invention, and examples of such a medium are as follows. The heterotrophic and polyculture medium used in the present invention consists essentially of KNO ₃ , glucose, plant growth hormone, inorganic salts, trace elements and water. In the technical solution, the trace element is preferably selected from the group consisting of H ₃ BO ₃ , ZnSO ₄ · 7H ₂ O, MnCl ₂ * H ₂ O, (NH ₄ ) ₆ Mo ₇ O ₂₄ * 4H ₂ O, CuSO ₄ · One or more or all of 5H ₂ O, Co(NO ₃ ) ₂ · 6H ₂ O.

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 Mn, Zn, B, 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 BO ₃ , ZnSO ₄ - 7H ₂ O, MnCl ₂ - H ₂ O, (NH ₄ ) ₆ Mo ₇ O ₂₄ * 4H ₂ O, CuSO ₄ * One or more of 5H ₂ O, Co(NO ₃ ) ₂ *6H ₂ O. The amount of inorganic salts and trace elements can be determined based on conventional knowledge.

The heterotrophic and polyculture medium used in the present invention consists essentially of the following components: KNO ₃ 0.1-15 g/l, glucose 0.1 to 50 g/l, K ₂ HPO ₄ 0.1 to 10.0 g/l, MgSO ₄ ' 7H ₂ O 0.1~10g / liter, CaCl ₂ '2H ₂ O 0.01~5g / liter, ferric citrate 0.01~5g / liter, citric acid 0.1~15g / liter, 2,4-dichlorophenoxy Acetic acid 0.001-5 mg / liter, benzylaminopurine 0.001-5 mg / liter, abscisic acid 0.001-5 mg / liter, gibberellin 0.001-5 mg / liter, 3-indolic acid 0.001-5 mg / liter, Naphthaleneacetic acid 0.001-5 mg / liter, brassinoin 0.001-5 mg / liter, trace element 0.5 ~ 20ml and water, wherein the trace element composition is H ₃ BO ₃ 0.1-10 g / liter, ZnSO ₄ -7H ₂ O 0.1-10.0 g/L, MnCl ₂ -4H ₂ O 0.5~10.0 g/L, (ΝΗ ₄ ) ₆ Μο ₇ Ο ₂₄ ·4Η ₂ Ο 0.01~5 g/L, CuSO ₄ '5H ₂ O 0.01~5.0 g /L, Co(NO ₃ ) ₂ -6H ₂ O 0.01 to 5 g / liter.

In one embodiment, the heterotrophic and polyculture medium employed in the present invention consists essentially of the following components: KNO ₃ 0.2 to 5 g/L, glucose 1 to 15 g/L, K ₂ HPO ₄ 0.1 to 2.0 g. /L, MgSO ₄ '7H ₂ O 0.1-2.0 g / liter, CaCl ₂ -2H ₂ O 0.05-1.0 g / liter, ferric citrate 0. 05 ~ 0 · 5 g / liter, citric acid 0.1 - 1.5 g / Liter, abscisic acid 0.001-5 mg / liter, gibberellin 0.001-5 mg / liter, 3-indolic acid 0.001-5 mg / liter, brassinoin 0.001-5 mg / liter, trace element 0.5 ~ 10ml and Water, wherein the composition of the trace elements is H ₃ BO ₃ 1-5 g / liter, ZnSO ₄ '7H ₂ O 0.1 ~ 1.5 g / liter, MnCl ₂ '4H ₂ O 0.5 ~ 5.0 g / liter, (ΝΗ ₄ ) ₆ Μο ₇ Ο ₂₄ ·4Η ₂ Ο 0·01~0·2 g/L, CuSO ₄ -5H ₂ O 0.01-0.5 g/L and Co(NO ₃ ) ₂ -6H ₂ O 0.05 to 0.5 g/L.

In a preferred embodiment, the heterotrophic and polyculture medium compositions of the present invention are preferably Composition: KNO ₃ 1.2 g / liter, glucose 5 g / liter, K ₂ HPO ₄ 0.6 g / liter, MgSO ₄ * 7H ₂ O 0.8 g / liter, CaCl ₂ 0.2 g / liter, iron citrate 0.1 g / liter , citric acid 0.6 g / liter, abscisic acid 1 mg / liter, gibberellin 0.8 mg / liter, brassinolide 2 mg / liter, trace element 6 ml and 1000 ml water, wherein the trace element composition is H ₃ BO ₃ 2~3g/L, ZnSO ₄ *7H ₂ O 0.1~0.3g/L, MnCl ₂ H ₂ O 1-2.5 g/L, (ΝΗ ₄ ) ₆ Μο ₇ Ο ₂₄ ·4Η ₂ Ο 0.01-0.04 g/升, CuSO ₄ *5H ₂ O 0.05~0.1 g / liter, Co(NO ₃ ) ₂ *6H ₂ O 0. 5~0.1 g / liter.

After the medium is prepared according to the above formulation, the pH of the medium can be adjusted to 5.0 to 11.0, preferably 7.0 to 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. The heterotrophic or polyculture culture can be carried out in a batch culture, fed-batch culture, repeated fed-batch culture, semi-continuous culture or continuous culture.

When seed heterotrophic or polyculture 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 to 0.8, and then steam sterilized (121 ° C, maintained). About 20 minutes), when the temperature drops to 20~30 °C, connect the grape algae according to the working volume of 1~15% to start heterotrophic or polyculture.

When the grapevine is cultured in heterotrophic, no light is needed. When the algae are cultured in a polyculture, light is needed, and the light can be made using natural light (sunlight) or artificial light, such as fluorescent lights, LED lights, and the like. The artificial light source can be external to the culture solution (external light source) or deep inside the culture solution (internal light source). The light intensity ranges from ^{1 to} 2500 molm-'s- ¹ , and in a preferred embodiment, the light intensity is from 1 to 900 molm" ² s ^_1 .

Regardless of any one of batch culture, fed-batch culture, repeated fed-batch culture, semi-continuous culture or continuous culture, appropriate culture conditions must be controlled during the culture to allow microalgae seeds to grow normally. . Usually, the control temperature is 20 to 35 ° C, for example, 22 to 30 ° C, the dissolved oxygen is not less than 5% of the air saturation concentration, and the pH is controlled at 6.0 to 9.0. In a preferred embodiment, the temperature is 24 to 28 ° C, the dissolved oxygen is not less than 10% of the air saturation concentration, and the pH is controlled at 7.5 to 8.0.

Heterotrophic culture can be carried out in a heterotrophic culture bioreactor such as shake flask, mechanical agitation, airlift, or bubbling. Algae polyculture can be carried out in shake flasks, steam-sterilized closed photobioreactors (transparent), and mechanically agitated bioreactors (including external sources and/or internal sources).

When the seeds are heterotrophic and the glucose in the polyculture medium is consumed, the seeds are heterotrophic or polyculture Bunch. Heterotrophic or polyculture time is usually around 3-20 days.

In the method of the present invention, during the process of heterotrophic or polyculture culture of grape algae, the pH is controlled by feeding and the elements such as carbon, nitrogen and/or phosphorus are stabilized within a certain concentration range, and at the end of heterotrophic or polyculture Control the concentration of nutrients such as carbon, nitrogen and/or phosphorus to a low or even zero concentration.

During the heterotrophic or polyculture process, the pH of the algal solution is controlled by feeding to a constant value in the range of 6.0-10.0, such as pH 8.0. The pH of the algal solution is usually controlled to a constant value in the range of 7.5 to 9.0, more preferably in the range of 7.8 to 8.5.

It should be understood that slight variations in pH are permitted. For example, a change in pH can be allowed, wherein Y 1.0, such as Y 0.2, Y^O. l o In certain embodiments, Y = 0. Thus, in one embodiment, the pH of the algal fluid is controlled to be Χ ± 通过 by feeding, wherein the 5.0 gentleman Υ 9.0. For example, in one embodiment of the present invention, the pH of the algal liquid is controlled to be within the range of 8.0 ± 0.3 by feeding.

In the process of heterotrophic or polyculture culture of microalgae, the carbon content in the algae solution can be controlled within the range of 2-20 mM by feeding, the nitrogen content is controlled within the range of 0.5-10 mM, and the phosphorus content is controlled at 0.05- Within the range of 3.5 mM.

In a specific embodiment, the carbon content in the algae liquid can be controlled in the range of 2-12 mM by feeding, the nitrogen content is controlled in the range of l-6 mM, and the phosphorus content is controlled in the range of 0.1-2.5 mM. Inside.

It is also included to control the content of magnesium in the algae liquid to a range of 0.05 to 3 mM by feeding. In a specific embodiment, the magnesium content in the algal solution is controlled to be in the range of 0.10 to 2.0 mM by feeding.

At the end of heterotrophic or polyculture, the concentration of nutrients such as carbon, nitrogen and/or phosphorus is controlled to be substantially consumed or even zero. For example, the concentration of carbon source is zero, nitrogen source and / or phosphorus source / or magnesium The concentration of the source is controlled below O.OlmM. Self-supporting

The photoautotrophic culture medium may be added to the obtained grape algae heterotrophic or polyculture medium, and diluted with water to carry out photoautotrophic culture.

Alternatively, the grapevine cells obtained by heterotrophic or polyculture can be directly subjected to photoautotrophic culture, that is, directly added photoautotrophic culture medium for photoautotrophic culture.

Subsequently, inoculation into a photoautotrophic culture device for photoautotrophic culture, that is, no organic compound is added, In the presence of essential inorganic nutrients and light energy, the CO ₂ as a carbon source is subjected to reduction assimilation to synthesize a culture mode in which all organic metabolites in the cells are cultured. The initial inoculation density of photoautotrophic culture is usually

0.01~1g/L, temperature is 5~50°C, light intensity is 1-2500 μιηοΐιη-^" ¹ , continuous illumination or intermittent illumination, ρΗ is 4.0~9.0, ventilation is 0.05~5wm, and CO _{2 is introduced.} The concentration is 0.03~10%.

When the growth of the algae 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 algae cells are harvested. The photoautotrophic time is usually 1-50 days.

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 KNO ₃ or the like can be used; as the phosphorus source, for example, K ₂ HPO ₄ can be used _{; and} as the inorganic carbon source, for example, CO ₂ or the like can be used. In order to promote rapid cell growth, the present invention creatively adds phytohormone to photoautotrophic culture media.

The photoautotrophic medium used in the present invention consists essentially of the following components: KNO ₃ 0.1-15 g / liter, K ₂ HPO ₄ 0.1~10.0 g / liter, MgSO ₄ '7H ₂ O 0.1 ~ 10 g / liter, CaCl ₂ '2H ₂ O 0.01~5g/L, ferric citrate 0.01~5g/L, citric acid 0.1~15g/L, 2,4-dichlorophenoxyacetic acid 0.001-5mg/L, benzylamino嘌呤0.001-5 mg / liter, abscisic acid 0.001-5 mg / liter, gibberellin 0.001-5 mg / liter, 3-anthic acid 0.001-5 mg / liter, naphthalene acetic acid 0.001-5 mg / liter, 芸Luciferin 0.001-5 mg / liter, trace element 0.5~20ml and water, wherein the composition of trace elements is H ₃ BO ₃ 0.1~10 g / liter, ZnSO ₄ '7H ₂ O 0.1~10.0 g / liter, MnCl ₂ ' 4H ₂ O 0.5~10.0 g / l, (ΝΗ ₄ ) ₆ Μο ₇ Ο ₂₄ ·4Η ₂ Ο 0.01-5 g / liter, CuSO ₄ -5H ₂ O 0.01~5.0 g / liter, Co(NO ₃ ) ₂ - 6H ₂ O 0.01 to 5 g / liter.

In one embodiment, the photoautotrophic medium employed in the present invention consists essentially of the following components: ΚΝΟ ₃ 0·2 〜5 g/L, Κ ₂ ΗΡΟ ₄ 0·1~2·0 g/L, MgSO ₄ -7H ₂ O 0· 1~2·0 g / liter, CaCl ₂ -2H ₂ O 0.05~1.0 g / liter, ferric citrate 0.05~0.5 g / liter, citric acid 0.1~1.5 g / liter, abscisic acid 0.001-5 mg / liter, gibberellin 0.001-5 mg / liter, 3-butyric acid 0.001-5 mg / liter, brassinoin 0.001-5 mg / liter, trace elements 0.5 ~ 10ml and water, of which trace The composition of the elements is H ₃ BO ₃ 1-5 g / liter, ZnSO ₄ -7H ₂ O 0.1~1.5 g / liter, MnCl ₂ -4H ₂ O 0.5~5.0 g / liter, (ΝΗ ₄ ) ₆ Μο ₇ Ο ₂₄ · 4 Η ₂ Ο 0.01-0.2 g / liter, CuSO ₄ '5H ₂ O 0 · 01 ~ 0 · 5 g / liter and Co (NO ₃ ) ₂ -6H ₂ O 0.05 ~ 0.5 g / liter. In a preferred embodiment, the photoautotrophic medium composition of the present invention is preferably composed of the following components: KNO ₃ 1.2 g/L, K ₂ HPO ₄ 0.6 g/L, MgSO ₄ *7H ₂ O 0.8 g/ Liter, CaCl ₂ 0.2 g / liter, ferric citrate 0.1 g / liter, citric acid 0.6 g / liter, abscisic acid 1 mg / liter, gibberellin 0.8 mg / liter, brassinomycin 2 mg / liter, trace element 6 Ml and 1000 ml water, wherein the composition of the trace elements is H ₃ BO ₃ 2-3 g / liter, ZnSO ₄ * 7H ₂ O 0.1 ~ 0.3 g / liter, MnCl ₂ * 4H ₂ O 1-2.5 g / liter, ( ΝΗ ₄ ) ₆ Μο ₇ Ο ₂₄ ·4Η ₂ Ο 0.01~0.04 g/L, CuSO ₄ *5H ₂ O 0.05~0.1 g/L, Co(NO ₃ ) ₂ * 6H ₂ O 0. 5~0.1 g/L .

After the medium is prepared according to the above formula, the pH of the medium can be adjusted to 6.0 to 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 or disinfected with sodium hypochlorite. 2 to 20 h, then neutralized with sodium thiosulfate. The photoautotrophic culture can be carried out in four ways, such as batch culture, fed-batch culture, semi-continuous culture (with release) or continuous culture. Algae cell harvesting and extraction of total hydrocarbons in cells

After the autotrophic culture is completed, the grape algae is 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, an organic solvent extraction method, that is, drying the algae body at a constant weight of 80 to 105 ° C, 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 is repeatedly extracted until the color of the algal powder turns white, and the solvent is removed by rotary evaporation.

The method for extracting total intracellular hydrocarbons includes, but is not limited to, an organic solvent extraction method, that is, drying the algae body at a constant weight of 80 to 105 ° C, and grinding the algal powder, and extracting total hydrocarbons from the dry algal powder by using hexamethylene hydride. The extraction solvent was repeatedly extracted until the color of the algal powder turned white, and the solvent was removed by blowing with nitrogen. The method of extracting total hydrocarbons also includes separating the hydrocarbons secreted from the cells directly from the culture solution.

The methods for determining the dry weight and total hydrocarbon content of algae cells are as follows:

Determination of dry weight of algae cells: Take V ml of culture medium during the culture of grape algae, centrifuge at 8000 rpm for 10 minutes, wash the algae after centrifugation 3 times with deionized water, and transfer to a weighing bottle (W1 (g)). , Dry in an oven at 80 ° C to constant weight W2 (g). The dry weight of algae Cx can be calculated according to the following formula: Cx (g / l) = (W2-W1) /V/ 1000

Determination of fat: Take a certain amount of algae cells that are dried to constant weight in each culture stage, and grind to powder in a mortar. At the end, weigh the appropriate amount of algae powder (0.2~0.5 g) carefully into the centrifuge tube, add appropriate extraction solvent (chloroform: methanol = 2: 1), ultrasonically shake for 30 min in an ultrasonic shaker, centrifuge at 8000 rpm for 10 min, and clear the supernatant. Transfer to a dry rotating evaporation vial of known weight and repeat the above steps until the supernatant is colorless. The supernatants were combined, evaporated to dryness, weighed and the fat content was calculated.

Grease content (%) Calculated as follows:

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

Where: _W1 is the weight of the algal powder, _g is the weight of the rotary evaporation bottle dried to constant weight, g; W2 is the weight of the evaporation bottle after the oil extract is evaporated to dryness, g.

Determination of total hydrocarbons: Take a certain amount of algal 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 appropriate amount of extraction solvent. (正正垸) Ultrasonic shaking in an ultrasonic oscillator for 30 min, centrifugation at 5000 rpm for 10 min, transfer the supernatant to a screw tube of known weight, and repeat the above steps until the supernatant is colorless. After combining the supernatants, the hexanes were blown dry with nitrogen at room temperature, weighed and the total hydrocarbon content was calculated.

Total hydrocarbon content (%) Calculated as follows:

Total hydrocarbon (%) = ( W2-W0 ) / W1 X 100

Where: _W1 is the weight of algae powder, _{g; wo} __ is the weight of the screw tube for drying to constant weight, _g;

W2 - the weight of the screw tube after drying the extract, g.

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 the terms "including" and "including" in this application also include the meaning of "consisting of" and "consisting of."

Example 1: Study on cell growth of algal cells during cell heterotrophic, polyculture and photoautotrophic culture of iBotryococcus braunii

The grape algae of the present example were heterotrophic, polyculture and photoautotrophic culture in 500 ml shake flasks, respectively. The inoculation density of the grape algae culture was 0.05 g/l, the temperature was 25 °C, and the rotation speed was 150 rpm. The conditions of polyculture of grape algae were consistent with that of heterotrophic culture. Except for external illumination, the light intensity was SO molm—albergia heterotrophic and polyculture cultured for 12 days. The glucose in the culture solution was consumed, and the algal cell density was 0.70 g/ l and 0.78g/L, which can be used for the next self-cultivation of seeds; the inoculation of grape algae seeds during autotrophic culture The degree is 0.05g/l, the temperature is 25°C, the light intensity is SOO molm- ¹ , continuous illumination, and the density of algae cells is only 0.20g/l when cultured for 12 days, which is used for the next seed of photoautotrophic culture. (figure 1). It can be seen that compared with photoautotrophic culture, the growth rate of algal cells in heterotrophic culture and polyculture culture is faster than that of photoautotrophic culture (3.5 and 3.9 times of growth rate of photoautotrophic culture, respectively). Example 2: Control of heterotrophic and polyculture culture processes in a 5L bioreactor under conditions of different culture conditions: Sotrj^coco^ brim Y

The heterotrophic or polyculture medium and water were added to a 5 L bioreactor to 2.5 L, followed by steam sterilization, and then when the temperature was lowered to 25 ° C, the algae were introduced to start heterotrophic or polyculture. In polyculture, the external light is OO molm - in heterotrophic or polyculture culture, the feeding is started, and the pH is kept constant at 8.0 ± 0.3 by controlling the continuous flow of the feed medium. The feed medium includes nutrient salts such as an organic carbon source (glucose), a nitrogen source (KNO ₃ ), a plant growth hormone, and an inorganic salt, and the supplemental nutrient salt is the above-mentioned corresponding medium concentrated to promote the growth of the microalgae. At the same time, timely monitor the content of carbon, nitrogen, phosphorus and magnesium in the fermentation broth, and appropriately adjust the content of the four substances in the feed medium to ensure the concentration of the four substances in the fermentation broth is stable. In the absence of hormones, other procedures and experimental conditions are the same, and only the medium does not contain plant growth hormone. In the case of unoptimized control, only the pH in the fermentation broth is monitored, the pH is kept constant at 8.0±0.3 through the feed medium, and the contents of other substances such as carbon, nitrogen, phosphorus and magnesium are not controlled, and hormonal substances are not controlled. Also not added, other experimental conditions and operations are the same.

The results are shown in Figure 2. At the end of heterotrophic culture, the pH and feeding strategy were controlled, and the heterotrophic culture of phytohormone was added, and the dry weight of the cells reached 15.6 g/1. The heterotrophic culture was only controlled by pH and feeding strategy but without adding phytohormone. The cell dry weight reached 6.5g/l; while the heterotrophic culture without optimized feeding strategy and without adding phytohormone, the dry weight of the cell was only 4.1 g/l. Therefore, the heterotrophic culture that controls the feeding strategy optimization and the addition of plant hormones has a 2.8-fold increase in cell density compared to the heterotrophic culture without pH-optimized feeding strategy and without the addition of plant growth hormone; The cell density was increased by a factor of 1.4 compared to the heterotrophic culture with pH and feed strategy but without the addition of phytohormones. At the end of polyculture, the pH and feeding strategy were controlled, and the heterotrophic culture of phytohormone was added, and the dry weight of the cells reached 16.5 g/1. The heterotrophic culture was only controlled by pH and feeding strategy but without adding phytohormone. , the cell dry weight reached 7.6g / l; and the heterotrophic culture without optimized feeding strategy and without adding phytohormone, the dry weight of the cell is only 4.8 g / lo Example 3: Study on photoautotrophic culture of vitreous heterotrophic seeds, polyculture seeds and photoautotrophic seeds in an indoor 2L photobioreactor

In this example, in the indoor 2L cylindrical airlift photobioreactor, the light-autotrophic culture of heterotrophic, polyculture and photoautotrophic grape algae seeds was determined, respectively, and heterotrophic, polytrophic and photoautotrophic seeds were determined in their light. Algal cell density, final oil and total hydrocarbon content and yield during the cultivation process. The inoculation density of heterotrophic, polyculture and photoautotrophic seeds was 0.3g/l, the photoautotrophic culture temperature was 25 °C, and 2% CO _{2 was introduced} , and the aeration was 0.25vvm. In polyculture and photoautotrophic culture, the light intensity is lll molm - ² s - continuous illumination. After autotrophic culture for 12 days, the density of algal cells of heterotrophic seeds was 1.98g/l, the total hydrocarbon yield was 50.58mg/l/d, the yield of oil was 33.61 mg/1/d; the density of algal cells of polyculture seeds At 2.1 g/L, the total hydrocarbon yield is 58.13 mg/l/d, and the oil yield is 37.35 mg/1/d; the phototrophic seed has an algal cell density of only 0.98 g/l, and the total hydrocarbon yield is only 21.08 mg / l / d, oil yield 13.62 mg / 1 / d (Figure 3 and Figure 4). It can be seen that the heterotrophic and polyculture seeds have stronger vigor than the photoautotrophic seeds, and the density of algae cells in the same culture time is higher (2.02 and 2.14 times of photoautotrophic seeds, respectively), total hydrocarbon yield. Higher (2.40 and 2.76 times for photoautotrophic seeds, respectively), higher oil yields (2.47 and 2.74 times for photoautotrophic seeds, respectively). Example 4: Study on photoautotrophic culture of grape seed heterotrophic seeds, polyculture seeds and photoautotrophic seeds in 60L (40L liquid) plastic pots

In this example, the light-autotrophic cultivation of heterotrophic, polyculture and photoautotrophic grape algae seeds in an outdoor 60L plastic pot was used to measure the algal cell growth of heterotrophic, polytrophic and photoautotrophic seeds in their photoautotrophic process. And total hydrocarbon yield. The initial inoculation density was 0.15 g/l, the temperature and light intensity were both outdoor natural temperature and light intensity, and the ventilation was 0.3 wm. Light autotrophic culture for 12 days. The density of algal cells in heterotrophic seeds was 0.92g/l, the total hydrocarbon yield was 21.85mg/l/d, the oil yield was 16.28 mg/1/d, and the algal cell density of mixed seeds was 0.95g/L. The yield was 23.32mg/l/d, the oil yield was 17.37 mg/1/d; the algal cell density of photoautotrophic seeds was only 0.45g/l, and the total hydrocarbon yield was only 9.49mg/l/d. The rate is 7.5 mg/1/d (see Figures 5 and 6). It can be seen that the heterotrophic and polyculture seeds have stronger vigor than the photoautotrophic seeds, and the density of algae cells in the same culture time is higher (1.92 and 1.98 times of photoautotrophic seeds, respectively), total hydrocarbon yield. Higher (2.30 and 2.46 times for photoautotrophic seeds, respectively), higher oil yields (2.17 and 2.31 times for photoautotrophic seeds, respectively). 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 for cultivating a grape algae, characterized in that it comprises a heterotrophic or polyculture culture step of grape algae and a photoautotrophic culture step carried out by using algal cells obtained by heterotrophic or polyculture culture as seeds.

A method for producing a fat or oil and/or a hydrocarbon, characterized in that the method comprises a heterotrophic or polyculture culture step of a grape algae species, a light carried out as a seed by algal cells obtained by heterotrophic or polyculture Autotrophic culture steps, as well as algal cell harvesting and oil and hydrocarbon extraction steps.

3. A highly efficient method for cultivating algae or a method for rapidly producing oils and/or hydrocarbons, characterized in that the method comprises:

(1) Heterotrophic or polyculture culture of grape algae, wherein, during the culture process, the pH is controlled by feeding and the elements such as carbon, nitrogen and/or phosphorus are stabilized within a certain concentration range, and carbon is controlled at the end of heterotrophic or polyculture The concentration of nutrients such as nitrogen and/or phosphorus is low or even zero;

The method according to any one of claims 1 to 3, wherein the species of the genus Chlorella includes, but is not limited to, Botryococcus bmimii, Botryococcus sudeticus, Botryococcus sp., Botryococcus spp

The method according to any one of claims 1 to 3, wherein the method is carried out in a mode of batch culture, fed-batch culture, repeated fed-batch culture, semi-continuous culture or continuous culture. Heterotrophic or polyculture.

The method according to any one of claims 1 to 3, wherein the heterotrophic or polyculture culture step comprises: adding a medium having a pH of 5.0 to 1 1.0 in the bioreactor, according to the work 0.1~30% of the volume is connected to the microalgae algae for batch culture, fed-batch culture, repeated fed-batch culture, semi-continuous culture or continuous culture. The culture temperature is 10~40 °C, and the control pH is less than 10.0. Control dissolved oxygen above 1%.

The method according to any one of claims 1 to 4, wherein the grapevine algae species are cultured in a heterotrophic manner, and no illumination is required; when the grapevine algae species are cultured in a polyculture, the illumination is required. The light intensity ranges from 1 to 2500 molm - ² s.

The method according to any one of claims 1 to 3, wherein the photoautotrophic cultivation of the algae cells obtained by heterotrophic or polyculture culture as seeds comprises: heterotrophic or polyculture culture The algae species are subjected to photoautotrophic culture in a photoautotrophic culture apparatus, and the culture temperature is 5 to 50 ° C, continuous light or intermittent illumination, and the light intensity is 1 to 2500 molm - ² s - the photoautotrophic culture period is 1~ At 180 days, the initial seeding density was 0.01 to 5.0 g/liter, and the medium contained no organic carbon source and had a pH of 4.0 to 9.0.

The method according to any one of claims 1 to 3, wherein the heterotrophic or polyculture medium of the algae species contains a nitrogen source, an organic carbon source, a plant growth hormone, an inorganic salt, a trace element, and water. Or consisting of the nitrogen source, an organic carbon source, a plant growth hormone, an inorganic salt, a trace element, and water; the photoautotrophic medium containing plant growth hormone, a nitrogen source, an inorganic salt, and water, or a plant growth hormone , nitrogen source, inorganic salt and water.

The method according to any one of claims 1 to 3, wherein the heterotrophic cultivation step is carried out in a shake flask, a mechanical agitation type, an airlift type or a bubbling bioreactor; The polyculture step is carried out in a shake flask, a steam sterilizable closed photobioreactor (transparent), and a mechanically agitated bioreactor containing a light system; the photoautotrophic culture step is in a shake flask or is selected from the group consisting of Open runway or round pool, closed flat photobioreactor or ducted photobioreactor or column photobioreactor or film pouch or open and closed hybrid photoautotrophic culture system or An adherent culture system or the like is used in any apparatus for microalgae photoautotrophic culture, and the light conditions are natural light or artificial light.