KR101477139B1 - Method for Mass Production of Hydrodictyon Algae and Preparation for Its Application - Google Patents

Abstract

The present invention relates to a process for producing high quality seeds for hydrodictyon birds which have not yet been reported on mass production techniques of freshwater algae; Manufacture / supply of a suitable medium for mass production; Promotion of production and proper management of culture conditions for high content of useful substances; Harvesting / dehydration of grown nets; Harvesting, storage, and pretreatment of the harvested biomass, including the drying / storage / pretreatment steps of the harvested biomass, and a related media composition. Since the netted bird cultivated by the present invention is an algae, which is a non-edible resource, there is no possibility of food price increase and ethical problems when the industrial demand of biomass increases, and the waste resources (wastewater and waste CO2) (Carbohydrate, protein, etc.) and easy saccharification, it can be usefully used as a main raw material for future production of biochemical products. have.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for mass production of algae,

The present invention relates to a method for the mass culture, efficient pre-treatment in the production of biomass in order to take advantage of one of the net malsok (Hydrodictyo n) of the fresh water algae of green algae to the high quality of the biomass resources.

In the face of various problems such as depletion of petroleum resources, increase of energy demand, global warming and CO ₂ emission problems, and enforcement of environmental regulations, it is essential to efficiently utilize biological resources (biomass) Technology development is needed.

The biomass should have several factors such as: 1) low value for food or feed and low concern for food price hike when used industrially, 2) high productivity or industrial value of product 3) It will not be possible to compete with cultivation factors for agricultural production by cultivating non-agricultural land or using non-agricultural water. 4) It can reduce carbon dioxide pollution or environmental pollution. And the ability to be relatively good.

Biological resources that satisfy the above factors relatively well are algae and unlike the carbohydrate / starch biomass (first generation biomass) and fibrous biomass (second generation biomass), they are classified as third generation biomass, (Chemical feedstocks). ≪ / RTI >

First, the use of land and other resources is high because birds can grow in various environmental conditions depending on the species. (Rodolfi et al., Biotechnol. Bioeng., 102: 100-112, 2009) Second, algae are photosynthetic, have a shorter growth period and faster growth rate, resulting in a higher total biomass production. The CO ₂ uptake is also high, so 1 ㎡ means that the rainforest absorbs 15 to 20 tonnes of CO ₂ per year, while microalgae of the same area absorb 30 to 40 tonnes per year. Third, one of the special advantages of algae cultivation is that biomass production and water purification effect can be obtained simultaneously by using waste carbon dioxide and using wastewater (wastewater, brackish water, gray water, etc.) as a nutrient source during cultivation. Fourth, among species of birds, there are species that can produce a relatively high amount of specific ingredients such as carbohydrates, proteins, and lipids. Therefore, if they are used well, the efficiency of recycling as an industrial raw material is very high. Fifth, algae are easier to pretreat than fibrous biomass (containing 15-30% lignin) because of the absence of lignin.

However, since not all species of birds known to exist between 30,000 and 40,000 species have the above merits, many researchers have recently discovered and improved bird species that are more valuable for use, production of algae biomass, We are putting a lot of effort into technology development. So far, the industrial application of algae has been limited to the fields of health food and functional medicines, pigment production, aquaculture feed, cosmetic materials (Karina et al., Comparative Biochemistry and Physiology, Part C 146: 60-78, 2007) However, the tendency of technology development after the rise in oil prices has led to a great worldwide boom in the study of algae for the production of bio-diesel and bio-alcohol to secure alternative energy sources (Scott, et al., Current Opinion in Biotechnology 21: 1 Renewable and Sustainable Energy Reviews 14: 217232, 2010) (Ge et al., Renewable Energy. 36: 84-89, 2011; Harun et al., Chemical Engineering Jounal 168: 1079-1084, 2011; Adams et al. , J. Appl. Phycol., 21, 569574, 2009, Brennan and Owende, Renew, Sustain Energy Rev. 14, 557577, 2010; Rojan et al., Bioresource Technology 102: 186-193, 2011). Meanwhile, various uses of algae as raw materials for producing biochemicals (bioplastics, platform chemicals for producing chemical products, etc.) in preparation for depletion of petroleum resources have been examined (Nguyen et al., Bioresource Technology 110, 552-559, 2012).

However, as population increases and demand for biomass increases, the price of agricultural products may rise. Therefore, it is necessary to utilize biomass that does not compete with agricultural products, especially for raw materials for chemical products. In order to be competitive, Development of low-energy / low-cost production methods of resource biomass is urgently required. This is because biomass feedstock costs are increasing in the production costs of each process (cultivation, harvesting / drying, pretreatment, saccharification and fermentation) from production to utilization of biomass. In other words, it means that economic production of raw materials through biomass production efficiency is very important. In addition, the important point in the production of algae biomass is that the quality of the biomass should be excellent along with the mass production of the biomass itself. In general, since algae grow quickly and have a short life cycle, it is known that the quality change range is large due to the difference of the internal physiological condition of the algae and the external cultivation environment condition. Therefore, the cultivation technique, It should be able to develop and utilize it positively.

The netted bird of the present invention is classified as Chlorophyta, Chlorophyceae, Chlorococcales, Hydrodictyaceae, Hydrodictyon.

The use of antimicrobial active material extraction (Ghazala et al., J. Biol. Biotech. 1 (3): 343-350, 2004), application as organic fertilizer or feed , J. Fish Biol. 27 (3): 327-334, 1985), methane (Legros et al., Belg. Comm. Eur. Communities (EUR 10024, Energy Biomass): 369-373, 1985; Eur. Communities (EUR 7160): 183-194, 1981), lactic acid (Nguyen et al., Bioresource Tech. 110: 552-559, 2012), fermented sugar solution (Kim et al. , 2012), and on the other hand it is reported that the dried biomass product can be used as a building material for sound insulation and insulation (US 2011/0307976 A1).

Hyrodictyon algae are easy to be harvested only after they have been cultured and can be harvested only in the dry field, so they can be harvested in the process of unicellular microalgae (chlorella, scenedesmus, etc.) The sample can be collected at a low energy / low cost. Also, the operations such as washing, dewatering, drying and pulverizing are very easy. In addition, the net barb bird does not have lignin, carbohydrate usually accounts for 30-70% by weight, and in particular, the proportion of the hexane (glucose + mannose) content most widely used for fermentation is over 90% It has been proven to be of great value as an edible biomass resource (Kim et al., Journal of Korean Society of Weed Science 32 (2) 87-96, 2012).

However, studies on physiological ecological characteristics have been carried out to some extent on the netting of birds of the net, but reports on the mass production method can produce netted birds by feeding wastewater and carbon dioxide in a slope-based culture facility (US 2011/0307976 A1), specific and actual cultivation technology reports have not yet been introduced.

The net horses are completely different from the unicellular birds such as chlorella or senedes mousse. Also, the breeding methods are different from Cladophora, Oscillatoria, etc. belonging to the same thought bird. Therefore, it is obvious that there is a limit to apply these culture methods to the net. In other words, the net has the following characteristic traits (John McReynolds, Bull Torrey Bot Club, 88 (6): 397-403, 1961).

The net horses are cylindrical single cells, usually 5-6 cells in a pentagonal or hexagonal shape, and they are connected to each other to form a net, which leads to colonial life and affluent life. Since the horses' cells do not undergo somatic cell division after mitosis (chromosomal division), they form polynuclear cells called coenocytes, and the number of nuclei continues to increase as they grow. In addition, there are many chloroplasts and pyrenoids in polynuclear cells. A well-grown multinucleate cell undergoes a series of protoplast cleavage during the night, producing an uninucleate zoospore with two monocotyledons, numbering several thousand. During the daytime, the host colonies gather in the mother cell, and after a while they spread to the cell wall and join together to form a cylindrical net. Then the cell wall of the blastocyst disappears and the young net grows bigger through the cell kidney, and the net length goes up to more than 1m. Often sometimes even sexual reproduction, oily reproduction is caused by small isoforms (isogamates) and more than a thousand cytoplasmic cytoplasm is produced. Two homozygous spouses form a zygote. The zygote becomes four motile spores by meiosis and the spore grows into a polyeder structure of each shape. The cytoplasm in the polyether is divided and zoospores are formed and these hosts collect to form a disc-shaped net.

Therefore, in order to mass-produce algae in the net, it is required that the silent breeding should be carried out at a higher speed. Various technologies such as medium composition and feeding method, growth environment control, culture tank type, culture period, . The major technologies to be secured for the mass production of netted birds and their reasons are as follows.

In the case of birds of the net, several cells gather to form a net. There are various types of net from round to cylindrical. Among them, the most productive and easy to manage is that the net shape is cylindrical and the length is longer. Therefore, it is important for mass production to have a technology capable of producing such a shape net. However, in the case of birds in the net, the seeds produced by silkworm breeding are not produced in the seeds themselves when the density of the seeds is high or the nutrient level of the medium is not appropriate. Special techniques are required for production.

2) The composition of the medium has optimum conditions according to each species of birds. Therefore, it is one of the key issues in the development of mass production technology to establish a medium composition that is best for the growth of the species and can be produced at low cost. In general, the composition of the medium is composed of a large amount of elements such as nitrogen and phosphoric acid, which are necessary for the growth but require a large amount, a trace element which is required to a small extent but absolutely necessary for growth, vitamins and other compounds which help the growth vitality. The horses have the characteristics of rapid growth and are relatively sensitive to the surrounding environment (light, temperature, pH and growth space, etc.). Therefore, in some cases, cells may suddenly disintegrate or stop growing, and vitality tends to drop easily during subculture. Therefore, establishing the culture medium, nutrient supply system, and culture management technology that comprehensively consider these are core technologies that must be essential for mass production.

3) In order to use avian biomass effectively, it is desirable to have a large amount of components accumulated in the interior as well as mass production. In order to be useful as an industrial bio-resource, it is necessary to cultivate a high-carbohydrate or protein-rich state. In order to obtain such high-quality biomass, it is essential that the nitrogen and phosphoric acid as well as various other factors be controlled in parallel.

4) On the other hand, in order to mass-produce algae biomass at low cost, it is desirable to be open to the open or open type in the open field. In this case, unwanted harmful algae may be mixed, or the occurrence of harmful organism (insect, hospital microorganism, , It is very necessary for the cultivation and management to control the plant. Especially, it is necessary to develop and apply management technology to prevent it from being easily disintegrated when large-scale proliferation of microalgae or microorganisms is observed.

5) Generally, in order to economically mass-produce birds, ultimately, the production per unit area should be increased. High-density cultivation is basically necessary for this purpose, but it is difficult to cultivate high density depending on species of algae. Even in the case of birds of the net, they belong to species that are difficult to cultivate at high density. Therefore, it is necessary to establish and operate a system to increase productivity per unit area by special culture strategy such as nutrient supply method.

6) In order to easily utilize the produced biomass as a raw material resource for production of biochemical products, various technologies related to drying, storage, and other pretreatment methods should be constructed and operated efficiently. In particular, since the dry matter of the bird is relatively light but has a large volume, it is necessary to develop a proper storage method that can be stored in a smaller space, and a pre-treatment method for preventing decay at the storage is also needed.

However, there have been no specific reports on the main technologies to be secured for the mass production and utilization of netted birds.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

SUMMARY OF THE INVENTION The present inventors have made extensive researches in order to solve the problems of the prior art described above and the technical requirements of the related art. The present invention aims at securing industrial biomass resources in preparation for depletion of petroleum resources. The present invention relates to a method for mass production of high quality seeds for nettail birds, Economical and efficient netting of barbarian birds, including the production / supply of production of appropriate media, promotion of production and proper management of culture conditions for high content of beneficial substances, harvesting / dehydrating of grown nets, drying / storage / pretreatment of harvested biomass And high quality biomass harvesting, storage and pretreatment, and a related medium composition. As a result, the present invention has been completed by developing harvesting, storing and pretreatment methods for efficiently and economically mass-producing algae that have not yet been reported on mass production technology and for efficient utilization of biomass .

Accordingly, it is an object of this invention to provide a mass production method of the net malsok (Hydrodictyon) algae (algae).

Another object of the present invention to provide a method for producing superior seed (seed) of the net malsok (Hydrodictyon) algae (algae).

A further object of the present invention to provide a culture method of the net malsok (Hydrodictyon) algae (algae).

Carbohydrates of the present invention - and to provide a method for containing the culture nets malsok (Hydrodictyon) algae (algae).

It is another object of the present invention the protein-to provide a culture process containing a high net malsok (Hydrodictyon) algae (algae).

Another object of the present invention is to provide a composition for culturing Hydrodictyaceae algae.

Another object of the present invention is to provide a harvesting and storing method for efficient utilization of biomass.

It is still another object of the present invention to provide a method for producing a dry-edged net-like bird with a discolored whitened flame and a dried body manufactured thereby.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for mass production of a Hydrodictyon algae comprising the steps of:

(a) preparing a good seed of a Hydrodictyon algae; Wherein the superior seed is a net-like bird cell population with a net-like, green-colored, long-cylindrical cytomorphic character without net disintegration; And

(b) Inoculating and culturing the medium with a high-quality seed of the above-mentioned nettle bird.

SUMMARY OF THE INVENTION The present inventors have made extensive researches in order to solve the problems of the prior art described above and the technical requirements of the related art. The present invention aims at securing industrial biomass resources in preparation for depletion of petroleum resources. The present invention relates to a method for mass production of high quality seeds for nettail birds, Economical and efficient netting of barbarian birds, including the production / supply of production of appropriate media, promotion of production and proper management of culture conditions for high content of beneficial substances, harvesting / dehydrating of grown nets, drying / storage / pretreatment of harvested biomass And high quality biomass harvesting, storage and pretreatment, and a related medium composition. As a result, the present invention has been completed by developing harvesting, storing and pretreatment methods for efficiently and economically mass-producing algae that have not yet been reported on mass production technology and for efficient utilization of biomass .

The method for mass production of the bird of the present invention will be described step by step as follows:

Step (a): Step of producing a high-quality seed

The first step of the present invention is a method for producing a high quality seed of a nettle bird, wherein the high quality seed has a green (preferably bright green) color without net disintegration and a long cylindrical morphology It is a group of bird cells in the net.

The production process of the high-quality seeds will be described in detail as follows:

First, the birds in the net are cultured in a first incubation tank containing the medium to prepare a first bird net in the net.

The culture in the first culture tank is a culture for selecting a morphologically superior individual while achieving high density growth.

According to a preferred embodiment of the present invention, a suitable number (e.g., 10-200, 30-150, and 50-100, etc.) of said nettles (e.g., small seeds of nettles) (For example, a mDM medium) (having a volume and a planar area smaller than that of the second and third culturing tanks below, for example, a 0.1-square-meter area of 10 L volume) -10 days (preferably 1-8 days, more preferably 2-6 days, even more preferably 2-5 days, even more preferably 2-4 days).

The initial inoculated individuals used in the culture in the first culture tank are preferably 0.5 to 3.0 cm in size, more preferably 0.5 to 2.0 cm in size, and even more preferably 0.5 to 1.0 cm in size.

In this first culture tank, a primary algal net is obtained by culturing. In this case, when the primary algal net is inoculated with a very small net horse of 0.5-1.0 cm and raised three days, it is usually about 3- 4 cm in size.

Next, a second algae net is manufactured by growing the algae net at a low density by hitting the first algae net in a second culture tank containing the medium.

The culture in the second culture tank is a culture for rapidly growing algae in a netting medium at a low density in an appropriate medium to produce a good individual.

The appropriate medium used in the culture in the second culture tank is preferably mDB supplemented with SBC (sodium bicarbonate), and the amount of supplemented SBC is preferably 10-200 ppm, more preferably 30-100 ppm , And even more preferably 40 to 60 ppm.

According to a preferred embodiment of the present invention, the appropriate number (e. G., 10-100, 20-80 and 30-70, etc.) of the primary algae nets is maintained in a medium (e.g., containing 50 ppm sodium bicarbonate (preferably 1 to 8 days, more preferably 2 to 6 days, more preferably 2 to 6 days, and most preferably 2 to 6 days) in a second incubation tank (for example, More preferably 2-5 days, even more preferably 2-4 days).

The culture in the second incubation tank is carried out by growing the algal net at a low density. The term " low density " used in reference to the secondary culture refers to a culture medium containing 10-100 nets, preferably 20-80, more preferably 30-70, most preferably Means culturing 40-50 nets (the number of birds at the time of culture can be increased or decreased as the net size varies).

On the other hand, the first culture is performed by growing a small seed at a higher density than the second culture. The term " high density " used in reference to the primary culture refers to a culture volume of 50-200 nets, preferably 60-150 nets, more preferably 70-130 nets, Most preferably 90-110 nets.

In this secondary culture tank, a secondary algal net is obtained by incubation, in which case the secondary algal net typically has a size of about 10-20 cm.

Then, in the third culture tank containing the medium, the first bird net or the second bird net (preferably, the second bird net) is dug up to grow the bird net at a low density, A seed is obtained by induction.

The culture of the third culture tank is a culture for further culturing in a proper medium (for example, mDM) to induce a normal seed.

According to a preferred embodiment of the present invention, a suitable number (e.g., 5-50, 10-50, and 10-30, etc.) of said second algal nets is treated with a third And then cultured for 3-5 days in a tea incubator (for example, a 1.5-square-meter-wide area of 100 L volume) to obtain a good seed.

Cultivation in the third culture tank is carried out by growing a bird net at a low density. The term " low density " used in reference to the tertiary culture means that when cultivating an algae of about 10-20 cm in size based on a culture vessel 1.5 m 2 wide, 5-60 nets, preferably 5-50 More preferably 20 to 40 nets (the number of birds at the time of cultivation may be more or less than the number given above as the net size varies).

Through the above three steps, we obtain a good seed of the algae. Good seeds have a length of 0.5-2 cm in individual (net) units.

Thus, the term " good seed " used in reference to nettles refers to a net having a green (preferably vivid green) color without disintegration and having a long cylindrical cytomorphological character, (Or cell group) of netting having a length of 2 cm.

The collected high-quality seeds are used for mass culture while being stored at a low temperature (preferably 4-15 ° C) below 15 ° C.

On the other hand, the medium used for producing the high-quality seeds can be described with reference to the medium used in the following step (b).

Step (b): Inoculation and culture step

Next, a high-quality seed of the above-mentioned nettle bird is inoculated and cultured in a medium.

The cultivation of the algae according to the present invention can be carried out without special solution stirring apparatus while feeding the culture medium of step (a) at least once to various water tanks, but it is preferable to use a flat type culture tank And it is more preferable to add a device for raising the water temperature at a low temperature by spreading a heat ray on the water).

According to a preferred embodiment of the present invention, when the above-mentioned net-like algae are cultured in a culture vessel provided with a stirring device, the dehydrated fresh weight is increased by about 35%. This is because the efficiency of nutrient utilization is higher due to the agitation treatment and the underwater environmental condition is improved. (Reference: Example 14)

As used herein, the term " dehydrated fresh weight " refers to the weight of a net-like algae that has been dehydrated by obtaining a net-like algae cultured according to the method of the present invention.

Preferably, the optimal seeding density of the high-quality seed for mass production of the nettle bird of the present invention is in the range of 0.01-50 g / 0.1 m 2 dehydrated, more preferably 0.02-10 g / 0.1 m 2 dehydrated, Most preferably, 0.01-5 g / 0.1 m < 2 > is dehydrated in vivo (see Example 12).

The cultivation of the high quality seeds of the present invention is carried out by adjusting the medium temperature, pH, light period, nitrogen source content, carbon source content and phosphoric acid content.

Preferably, when seeding and culturing the high-quality seed of the present invention, the medium is carried out at a temperature range of 10-40 캜. Preferably, the cultivation is carried out at a temperature range of 15-38 占 폚, and more preferably at a temperature range of 20-35 占 폚.

According to a preferred embodiment of the present invention, the present inventors cultured the strains of the present invention and found that the dehydrated organisms were found to be higher in the order of 25 ° C> 20 ° C> 30 ° C> 25 ° C> 15 ° C, The cultivation temperature was found to be 35 ° C> 30 ° C> 25 ° C> 20 ° C> 15 ° C in the order of 35 ° C> 30 ° C> 25 ° C> 20 ° C> (Note: Example 2-1).

Preferably, when seeding and cultivating the high-quality seed of the present invention of the present invention into the medium, the daytime or night culture temperature is preferably 20 ° C or higher. More preferably, the daytime incubation temperature is in the range of 25-40 占 폚, and the night incubation temperature is in the range of 20-35 占 폚. Most preferably, the daytime incubation temperature is in the range of 30-35 ° C, and the overnight incubation temperature is 20-30 ° C.

According to a preferred embodiment of the present invention, when the high-quality seed of the present invention is cultured under the conditions of the weekly incubation temperature of 30-35 ° C and the night culture temperature of 20-30 ° C, the daytime incubation temperature is 25 ° C and night incubation temperature The degree of glucose production in the nettle algae is higher than in the case of culturing at 15 ° C (Reference: Example 2-2).

Preferably, the pH of the medium is suitably from pH 6.5 to pH 11.9 when the medium is seeded and cultured in a medium with a high-quality seed of the present invention. More preferably, the pH of the medium is between pH 7.5 and 11.7, most preferably between pH 8.5 and pH 11.5.

According to a preferred embodiment of the present invention, when the high-quality seed of the present invention is cultured for 10 days by replacing the medium on days 3, 5 and 8 so that the pH is maintained, the pH of the medium is adjusted to pH 10 (N / m) of 19.58 mg / net and a net length of 5.78 +/- 1.31 (cm / net) when the medium was maintained at a pH of 11 and a dehydrated fresh weight of 19.84 mg / net and a net length of 5.72 +/- 0.83 / Net) (Reference: Example 3).

Preferably, when the seeds of the present invention are inoculated and cultured in a medium containing a high-quality seed of a bird of the present invention, the amount of glucose produced increases with an increase in photoperiod of the photoperiod of the culture, except for the continuous shoot condition. More preferably, the incubation is a photoperiod condition of 6-20 hours, and most preferably a photoperiod condition of 10-20 hours.

According to a preferred embodiment of the present invention, when the high-quality seed of the present invention is cultured under the light condition of 6 hours to 24 hours, the glucose accumulation amount in the order of 18 hours> 12 hours> 24 hours> (Note: Example 4).

Preferably, the carbon source is additionally added under the above-described photoperiod condition when the medium is seeded and cultured in the medium with the high-quality seed of the present invention.

According to a preferred embodiment of the present invention, when 200 ppm of sodium bicarbonate is additionally added to the medium under the 14-hour photoperiod condition, dehydrated fresh weight and glucose production amount are lower than those in the case of no addition of sodium bicarbonate under the same conditions 20% or more (Reference: Example 4).

Preferably, when seeding and culturing the medium with a high quality seed of the present invention, the culture is carried out under the condition of 10-1500 μmolm ^-2 s ^-1 luminous intensity. More preferably 50-1000 μmolm ^-2 s ^-1 light, and most preferably 80-500 μmol m ^-2 s ^-1 light intensity.

The nematodes of the present invention are typically cultured in a medium containing a carbon source and a nitrogen source. The culture medium was Allen's medium, BG11, Waris-H, BBM (Bold's Basal Medium), CM (Modified Closterium Medium, Watanabe et al., 2000, NIES Collection List of Strains. HR-v1 or AW-v1 may be used, preferably DMDM, HR-v1 or AW-v1. The compositions of the Allen medium, BG11, Waris-H, BBM, CM, DM, modified DM (mDDM), HR-v1 and AW-v1 are shown in Table 26.

The medium may be prepared by using at least one kind of water selected from distilled water, deaerated tap water, distilled water, ground water, rainwater, discharged water, river water and the like.

As the nitrogen source in the medium used in the present invention, at least one nitrogen source selected from the group consisting of ammonium, nitrate, urea and ammonium nitrate can be used in the concentration range of 1-50 ppm. The concentration is preferably 5-45 ppm, more preferably 10-30 ppm, and most preferably 15-25 ppm. Further, the carbon source may be used in a concentration range of 1-500 ppm of one or more carbon sources selected from the group consisting of bicarbonate, carbonate and carbon dioxide. Preferably, the carbon source is at a concentration of 50-450 ppm, more preferably at a concentration of 100-400 ppm, and most preferably at a concentration of 150-350 ppm. Phosphorus monovalent phosphate or divalent phosphate can be used in the concentration range of 1-30 ppm. Preferably, the phosphate is used at a concentration of 2-28 ppm, more preferably at a concentration of 4-26 ppm, and most preferably at a concentration of 5-25 ppm.

In particular, since the bird has a property of growing better in the alkaline solution, it is preferable to add an appropriate amount of KOH or sodium metasilicate in addition to NaOH in order to control the alkalinity of the medium. Preferably, when the alkaline solution is NaOH, it is used in a concentration range of 1-5 mg / l, more preferably in a range of 1-4 mg / l, most preferably in a range of 1-2 mg / l Range. Preferably, when the alkaline solution is sodium metasilicate, it is used in a concentration range of 40-80 mg / l, more preferably in a concentration range of 50-70 mg / l, and most preferably 55-75 mg / l concentration range.

In the present invention, in case of seeding and culturing a high-quality seed of a wild-type bird in a culture medium, a carbon source, a nitrogen source and a phosphorus (P) source may additionally be added to the culture.

The carbon source, the nitrogen source, and the source may be the carbon source, nitrogen source, and phosphorus described in the step (a).

Preferably, when seeding and culturing the medium with a high quality seed of the present invention, the culture maintains a carbon source content of 0-400 ppm. More preferably a carbon source content of 50-350 ppm, and most preferably a carbon source content of 100-300 ppm.

According to a preferred embodiment of the present invention, when the sodium bicarbonate as the carbon source has a carbon source content of 300 ppm, the growth increases by 30% or more as compared with the untreated group. (Reference Example 10)

Preferably, when seeding and culturing the medium with a high quality seed of the present invention, the medium maintains the concentration of the nitrogen source and phosphorus (P) source.

According to a preferred embodiment of the present invention, when 50-300 ppm of potassium nitrate and 200 ppm of sodium bicarbonate are simultaneously treated with the nitrogen source, potassium nitrate is compared with only 50-300 ppm alone To about 20% (Example: 5).

The growth of nettles of the present invention is more dependent on the nitrogen content than the phosphate content in the medium. This means that the accumulation of carbides in the medium of the nettles algae is more important than the appropriate ratio of nitrogen and phosphorus content.

According to a preferred embodiment of the present invention, when the high-quality seed of the present invention is inoculated and cultured on the medium, the content of phosphorus (potassium sulphate, PP) in the 1 x mDM medium is 6.2-24.8 ppm, nitrogen Calcium nitrate, CN) content of 5-20 ppm is suitable (see Example 6).

The medium of the present invention of the present invention does not necessarily contain silicon and vitamin.

According to a preferred embodiment of the present invention, in order to examine the necessity of silicon known as a trace element required for promotion of growth and normal maintenance of cell morphology during algal culture, when sodium methasilicate is treated at various concentrations of 0-64 ppm , There was no difference in glucose production between sodium methasilicate treatment concentrations. In addition, in order to investigate the need for vitamin, 0.04 ppm of cyanocobalamin, 0.04 ppm of thiamine-HCl, and 0.04 ppm of biotin were treated, and no significant increase in the dehydrated fresh weight and net length was observed compared with the untreated group. These results indicate that large-scale production of nettle algae can be carried out by using an economical medium containing no silicon or vitamin, which is an expensive medium composition (see Examples 7 and 8).

According to another aspect of the invention, the present invention provides a method for producing superior seed (seed) of the net malsok (Hydrodictyon) algae (algae) comprising the following steps:

(a) culturing the algae in a first culture tank containing a medium to produce a first alga net in the net;

(b) growing the algae net at a low density in a second incubation tank containing the medium to produce the second alga net; And

(c) inducing a first seedling net or a second seedling net at a low density and growing a birdnet in a third culture tank containing the medium to induce a good seed of the endemic bird; The superior seeds of the above-mentioned net-like birds are net-like bird cells with long-cylindrical cytomorphic characteristics with a green color without disassembling the net.

The method for producing a high quality seed of a bird of the present invention is described in detail in a step (a) of " a method for mass production of a net-like bird. &Quot; The description common to both is omitted in order to avoid the excessive complexity of the present specification.

According to a further aspect of the present invention, there is provided a method of preparing a photoperiod having a water temperature of 15-35 占 폚, a luminous intensity of 10-1500 占 퐉 ^-2 s ^-1 , a pH of 7.0-11.9, a 4-18 hour photoperiod, It provides a culture method of the net malsok (Hydrodictyon) algae (algae), comprising the step of culturing algae in terms of fresh weight seeds /0.1 ㎡ of dehydration.

The method of cultivating a net-like bird of the present invention is described in detail in steps (a) and (b) of "Method for mass production of a net-like bird". The description common to both is omitted in order to avoid the excessive complexity of the present specification.

Preferably, the water depth is 1-20 cm, more preferably 3-17 cm, and most preferably 5-15 cm.

According to another aspect of the present invention, the present invention provides a process for the preparation of a compound of formula I in the presence of a carbon source, 5-60 ppm nitrogen (based on CN, calcium nitrate) and 6.2-74.4 ppm phosphorus (based on PP, dipotassium phosphate) , light intensity 80-500 μmolm ^-2 s ^-1 and a carbohydrate comprising the steps of culturing at 25-35 ℃ conditions and provides a culture method of containing net malsok (Hydrodictyon) algae (algae).

The method for culturing a carbohydrate-rich, high-molecular-weight bird of the present invention can be carried out by the above-described method of mass production of a bird of the present invention. Accordingly, the description common to both is omitted in order to avoid the excessive complexity of the present specification.

In the method of culturing a carbohydrate-rich, high-molecular-weight algae of the present invention, the nitrogen and phosphorus contents used can also be expressed as 0.5 to 7.0 ppm nitrogen (based on N molecules) and 1.0-13.2 ppm phosphorus have.

Preferably, the amount of nitrogen (based on CN, calcium nitrate) used in the culture is 10-40 ppm, more preferably 15-30 ppm.

Preferably, the amount of phosphorus (based on PP, based on dipotassium phosphate) used in the culture is 6.2-50.0 ppm, more preferably 6.2-40.0 ppm, even more preferably 6.2-30.0 ppm.

The carbon source content is preferably 2.4-50 ppm carbon source (based on C molecules).

The term " carbohydrate-rich content " used in reference to the method of cultivating a nethorse algae of the present invention means that the content of glucose, based on 100 g of the dry weight (DW) of the nethorse algae obtained by the culture method of the present invention, Means 20 g or more (e.g., 20-60 g), preferably 30-60 g, more preferably 35-53 g, and the content of reducing sugar is 30-65 g, preferably 40-65 g .

In accordance with another aspect of the present invention, the present invention provides a process for the preparation of a pharmaceutical composition comprising a pH of 7.0-11.9 and 25-35 in the presence of a carbon source, 10-300 ppm nitrogen (based on calcium nitrate) and 2.0-24.8 ppm phosphorus (based on dipotassium phosphate) ℃ protein comprising the step of culturing in conditions and provides a culture method of containing net malsok (Hydrodictyon) algae (algae).

The method for culturing the protein-rich, high-molecular-weight algae of the present invention can be carried out by the above-described method of mass production of algae in the net. Accordingly, the description common to both is omitted in order to avoid the excessive complexity of the present specification.

In the method of culturing a protein-rich netting bird of the present invention, the content of nitrogen and phosphorus used can also be expressed as 2.0 to 40 ppm of nitrogen (based on N molecules) and 1.0 to 4.5 ppm of phosphorus (based on P molecules) have.

Preferably, the amount of nitrogen (based on CN, calcium nitrate) used in the culture is 10-100 ppm, more preferably 10-50 ppm.

Preferably, the amount of phosphorus (based on PP, based on dipotassium phosphate) used in the culture is 2.0-20.0 ppm, more preferably 5.0-15.0 ppm.

The carbon source content is preferably 15-150 ppm carbon source (2.4-24 ppm based on C molecules).

The term " protein-containing content " used in reference to the method of cultivating a net-like algae of the present invention means that the protein content is 20-40 wt%, preferably 20-40 wt%, in dry weight of the net- Means 24-30% by weight.

Preferably, the net-like bird is a hydrodiction reticulatum reticulatum ), Hydrodictyon africanum ) and hydrodictyon patenaeforme . The term " hydrocortisone & quot ;

In accordance with another aspect of the invention, the invention is calcium nitrate, potassium phosphate, magnesium sulfate, sodium bicarbonate, EDTA Na _2, FeNa · EDTA, NaOH, H ₃ BO _3, Mn ^{2 +} ions and (NH _{4) 6} including Mo ₇ O _24, and provides a pH of 9.2-9.6 net malsok (Hydrodictyon) algae (algae) composition for culture.

Preferably, the composition for the culture of endosperm algae comprises 10-30 mg / l calcium nitrate, 5-20 mg / l potassium phosphate, 15-35 mg / l magnesium sulfate, 10-400 mg / l sodium bicarbonate _{, 1-5 ㎎ / l EDTA Na 2} , 1.5 ㎎ / l FeNa · EDTA, 1-3 ㎎ / l NaOH, 1-5 ㎎ / l H 3 BO 3, Mn 2 + ions and 0.5-1.5 ㎎ / l ( NH ₄ ) ₆ Mo ₇ O ₂₄ , pH 9.2-9.6, more preferably 15-25 mg / l calcium nitrate, 10-15 mg / l potassium phosphate, 20-30 mg / l magnesium sulfate , 50-300 mg / l sodium bicarbonate, 2-3 mg / l EDTA Na ₂ , 2-3 mg / l FeNa.EDTA, 1-2 mg / l NaOH, 2-3 mg / l H ₃ BO ₃ , Mn ^{2 +} ion and 0.3-1.3 mg / l (NH ₄ ) ₆ Mo ₇ O ₂₄ , and the pH is 9.2-9.6.

According to a preferred embodiment of the present invention, increased dehydrated fresh weight is shown compared to Allen's medium and BG11, the existing algal medium. On the other hand, Allen's medium and BG11 showed a tendency of occurrence of undesired microalgae, which is not suitable as a medium for nettles algae.

In addition, as compared with the Waris-H medium, the mDM medium shows dehydrated fresh weight and dry weight similar to Waris-H even in simple ingredient composition not containing vitamins and silicon, It is a medium suitable for mass production.

In accordance with another aspect of the invention, calcium nitrate, potassium phosphate, ammonium phosphate, magnesium sulfate, sodium chloride, ferric citrate, sodium bicarbonate, EDTA Na _2, FeNa · EDTA, sodium silicate, H ₃ BO _3, Co ^The present invention provides a composition for culturing a Hydrodictyaceae algae containing ^{2 +} , Cu ^{2 +} , Mn ^{2 +} , Zn ^{2 +} ions and (NH ₄ ) ₆ Mo ₇ O ₂₄ and having a pH of 9.2-9.6 .

Preferably, an aqueous solution containing 30-50 mg / l calcium nitrate, 5-20 mg / l potassium phosphate, 5-15 mg / l ammonium phosphate, 40-60 mg / l magnesium sulfate, 40-60 mg / l sodium chloride, 10 to 20 mg / l peric citrate, 50 to 350 mg / l sodium bicarbonate, 1-5 mg / l EDTA Na ₂ , 1-5 mg / l FeNa. EDTA, 40-80 mg / l sodium silicate, _{-5 ㎎ / l H 3 BO 3} , Co 2 +, Cu 2 +, Mn 2 +, Zn 2 + ions and _{0.5-1.5 ㎎ / l (NH 4)} 6 comprises a Mo ₇ O _24, pH 9.2-9.6 L calcium phosphate, 45-55 mg / l magnesium sulfate, 45-55 mg / l potassium phosphate, 8-12 mg / l ammonium phosphate, and more preferably 45-55 mg / L sodium bicarbonate, 2-3 mg / l EDTA Na ₂ , 2-3 mg / l FeNa.EDTA, 50-70 mg / l sodium chloride, 13-17 mg / l ferric citrate, 100-300 mg / l sodium bicarbonate, comprises a _{silicate, 2-3 ㎎ / l H 3 BO} 3, Co 2 +, Cu 2 +, Mn 2 +, Zn 2 + ions and _{0.3-1.3 ㎎ / l (NH 4)} 6 Mo 7 O 24, pH is pH 9.2-9.6.

Preferably, the mDM medium is suitable for short-term (3 ± 1 day) culture, and HR-v1 medium is suitable for long-term (6 ± 1 day) culture.

According to a preferred embodiment of the present invention, it is suitable to cultivate mDM medium every 3 +/- 1 days and HR-v1 medium every 6 +/- 1 days for 7-15 days.

According to a preferred embodiment of the present invention, the medium of the present invention comprises cyanocobalamin (Vit B12), thiamin (Vit B1) and biotin (Vit H) contained in conventional media used for culturing algae I never do that.

According to another aspect of the invention, potassium nitrate, potassium phosphate, ammonium chloride, calcium chloride, sodium bicarbonate, NaOH, Cu ^{2 +,} Fe ^{3 +,} Mg ^{+ 2} and Zn ^{2 +} ions, and including, pH 9.2- 9.6 < / RTI > of Hydrodictyaceae algae.

Preferably, an aqueous solution containing 35-55 mg / l potassium nitrate, 2-6 mg / l potassium phosphate, 15-30 mg / l ammonium chloride, 0.01-0.3 mg / l calcium chloride, 50-150 mg / l sodium bicarbonate , 0.5-2.0 ㎎ / l NaOH, Cu ^{2 +,} Fe ^{3 +,} Mg ^{+ 2} and Zn ^{+ 2} includes the ion, and a pH 9.2-9.6, more preferably, 40-50 ㎎ / l potassium nitrate, 3-5 ㎎ / l potassium phosphate, 20-25 ㎎ / l ammonium chloride, 0.05-0.25 ㎎ / l calcium chloride, 70-120 ㎎ / l sodium bicarbonate, 1.0-1.8 ㎎ / l NaOH, Cu 2 +, Fe ^{3 +} , Mg ^{2 +} and Zn ²⁺ ions, and pH 9.2-9.6.

According to a preferred embodiment of the present invention, the medium of the present invention comprises cyanocobalamin (Vit B12), thiamin (Vit B1) and biotin (Vit H) contained in conventional media used for culturing algae I never do that.

Preferably, the culture composition of the present invention can be used by doubling to 2 x or 3 x to increase the accumulation of a useful substance (e.g., carbohydrate or protein) (see Example 11-4).

In accordance with another aspect of the invention, the present invention provides a net malsok (Hydrodictyon) storage of birds comprising the steps of:

(a) dewatering the algae in the net;

(b) spraying an acid solution, an alkali solution or a starch solution onto the result of step (a); And

(c) leaving the result of step (b) as it is or pressing it and drying it at a high temperature.

Preferably, the step (a) further comprises the step of obtaining (for example, by culturing) the net-like algae before the step (a). The harvesting is carried out by using a sieve having a pore size of 30 탆 or more, preferably by using a key having a pore size of 100 탆 or more, by filtration, and dewatering the obtained nettle bird .

The starch used in the present invention is preferably 0.1 to 20% by weight of the starch.

Preferably, the starch solution of step (b) is prepared by heating the starch solution, diluting the starch solution with distilled water or alcohol, treating the starch solution at 80 ° C for 10-30 minutes or in a microwave oven for 1-3 minutes, Solution.

The acid solution used in the present invention is preferably a weak acid solution, more preferably a weak acid solution in a pH range of 1 to 4.0.

The alkali solution used in the present invention is preferably a weak alkali solution, more preferably a weak alkali solution in a pH range of 10.0 to 12.0.

Preferably, the high temperature of step (c) is 40-90 占 폚, more preferably 43-48 占 폚.

According to a preferred embodiment of the present invention, step (c) of the present invention is hot air dried at 45 DEG C for 2 days.

The method for storing algae of the present invention is a method of storing algae in the net, which may be corrupted due to moisture absorption during storage at room temperature. To prevent this, a pretreatment process such as saccharification or dipping in a buffer solution, weak acid or weak alkali solution, (B) or step (b).

According to another aspect of the invention, the present invention provides a whitening (discoloration) net malsok (Hydrodictyon) algae dried body in the manufacturing method comprising the steps of:

(a) dewatering the algae in the net; And

(b) illuminating the result of step (a) with natural light or ultraviolet light (UV) to break down chlorophyll, bleach and dry.

Preferably, the algae used in the present invention are harvested by the above-described culturing method of the present invention.

Preferably, the step (b) is a step of exposing the substrate to a natural or ultraviolet light in a solution of a compound generating active oxygen at an appropriate concentration (for example, a solution of hydrogen peroxide, peracetic acid, sodium hypochlorite, hypochlorous acid, ozone, titanium dioxide, And then bleached and dried.

According to another aspect of the invention, the present invention provides a bleaching net malsok (Hydrodictyon) algae dried body produced by the method described above.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to a method for producing high quality seeds for hydrodictyon algae which have not yet been reported on mass production techniques of freshwater algae; Manufacture / supply of a suitable medium for mass production; Promotion of production and proper management of culture conditions for high content of useful substances; Harvesting / dehydration of grown nets; Harvesting, storage and pretreatment of the harvested biomass, including the drying / storage / pretreatment steps of the harvested biomass, as well as related media compositions for large-scale cultivation and high-quality biomass, harvesting, storage and pretreatment.

(b) The netted bird cultured according to the present invention is an algae, which is a non-edible resource. Therefore, when the industrial demand of biomass is increased, there is no possibility of raising food prices and ethical problems, CO ₂ ) can be produced at a low cost, and it is possible to supply resources stably. Since the content of useful components (carbohydrate, protein, etc.) is high and it is easy to be saccharified, it is useful as a main raw material for future production of biochemical products It is worth exploiting.

FIG. 1 is a diagrammatic representation of a process from seeding for mass production of algal biomass to high quality biomass mass production, harvesting, dehydrating, drying and storing according to the present invention.
FIG. 2 is a graph showing the effect of various types of medium for microalgae growth on the growth rate of HR indoor.
FIG. 3 is a graph showing the change over time of carbohydrate accumulation during the incubation period when HR was cultured in 1 × mDM medium and HR-v1 medium.
FIG. 4 is a graph showing the growth rate of HR in a room according to a feeding method (timing and amount) of 1x mDM medium. M1, M2, M3 and M4 mean that the basic medium was replaced every 1, 2, 3 and 4 days after the initial medium was supplied, and M5 was cultured for 9 days without any replacement.
5 is a graph showing the degree of carbohydrate accumulation of HR grown according to the feeding method (timing and amount) of 1 x mDM medium. The description of M1 to M5 is shown in Fig.
FIG. 6 is a graph showing the change over time of the degree of accumulation of carbohydrates according to the difference in throughput of sodium bicarbonate during HR culture in a large greenhouse culture tank.
FIG. 7 is a graph showing a change over time in the degree of accumulation of carbohydrates according to the pH adjustment of the culture medium during HR culture in a large-scale greenhouse culture tank.
Fig. 8 shows photographs of the harvested HR biomass after crush drying (left) and after general drying (right).
FIG. 9 is a photograph showing a partially decolorized (left) and a completely decolored and whitened (right) image when the harvested HR biomass is placed in a natural light condition.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Production of superior seeds of Hydrodictyon algae

In the case of a horse horse bird (HR), several cells gather to form a net, and there exist various shapes of the net ranging from a circular shape to a cylindrical shape. Among them, the most productive and easy to manage is that the net has a cylindrical shape and the length is longer. Therefore, it is important for mass production to have a technology capable of producing such a shape net. However, in the case of seeds produced by silkworm breeding, when the seed density is high or the nutrient level of the medium is not proper, the seeds themselves are not produced, or even if they are made, most of them are defective. Special techniques are required for production. In the preliminary experiment, it was confirmed that the seed density was high or the growth environment was poor during the culture, and the seeding rate was high even when seeding did not occur or occurred at the high concentration nutrient condition at the seed induction stage. In addition, since it is not an aseptic condition, it is very advantageous to produce a high-quality seed in a state where the propagation of other microalgae mixed is minimized. Therefore, this study was carried out to establish a proper method to produce high quality seeds of nettles.

1-1 Derivation of HR seed according to nutrient concentration and composition

The size to investigate whether the HR induced growth and excellent seed of the nutrient concentration in the medium of 4-8 cm HR two kinds of culture medium (adjusted to the deaerated water mDM 1x and 2x mDM) is filled tray (1.6m ² / 200L) . Day for 14 hours, 30 ° C / night for 10 hours, and 25 hours. As a result, growth was good in both media, but seed induction occurred only in 1x mDM (Table 1). In another experiment, the addition of SBC 100 (100 ppm bicarbonate) promoted HR growth but had a negative effect on seed induction and showed good results with SBC 50 (50 ppm bicarbonate) treatment. Addition of N / P or 0.1% sucrose to 1x mDM also had a negative effect on HR seed (results not shown). These results suggest that after the HR growth, the nutritional excess in the induction of the seeds has a rather negative effect, and that an appropriate balance of nutrients is required in particular.

Days after inoculation (DAI) Growth and seed induction rate of HR according to nutrient concentration 1x mDM 2x mDM 3 - Length of object 15-20cm,
- 30% seed induction - object length 12-15cm,
- 1-2% seed induction 5 - Growth suspension,
- 100% seed induction - object length 15-25cm,
- 10% seed induction 7 - 0.5-1.0 cm long seed collection - Growth suspension, seed induction suspension

1-2. Induced HR Seed by Culture Temperature

HR was adjusted to growth and precipitation seed derived whether to investigate the size of 2-3 cm in HR degassed water according to culture temperature difference 1x mDM + SBC50 is filled tray (1.5m ² / 100L) was inoculated to 500 4 at (Primary culture). Thereafter, a 9-10 cm-long HR specimen grown on the small tray was picked and transferred to a 1.6 m ² tray containing 200 L of 1x mDM + SBC50 (heat wire was installed on the bottom) in a greenhouse, (Secondary culture). As a result, it was confirmed that when the incubation temperature is high, the seed induction period is accelerated and the seed induction ratio is also increased (Table 2). These results reaffirmed that the effect is more pronounced when the incubation temperature is increased to 30-35 ° C.

Days after inoculation (DAI) Growth and seed induction rate of HR according to incubation temperature Light 26-27 ℃ / Dark 22-23 Light 28-29 / Dark 24-25 4 - The object length is 9-10cm, - Length of object 7-15cm, 6 - object length 15-22cm
- Normal form - object length 15-22cm
- CEL (cell elasticity loosening) symptoms 8 10% seed induction 90% seed induction 10 90% seed induction, collection 100% seed induction, collection

1-3. HR seed induction in HR-v1 medium

It was experimented to see if the initial nutrient supply was sufficient and then the cultivation in the abundant nutrient supply condition would be better for seed induction. In other words, about 100 HR of 2-3 cm in size was injected into 100 L of HR-v1 medium, and after 4 days of inoculation, the seeds were transferred to different media (1 × mDM or Tap water) . As a result, when HR-v1 medium was continuously cultivated, the HR seed induction proceeded well but the degree of unevenness was high. In particular, it was difficult to obtain a clean seed because of the occurrence of other green algae in the late culture period. When seeding was induced by growing HR-v1 medium for 4 days and then transferring it to tap water, seed induction proceeded well and uniformity was excellent. However, after seed induction, the growth was slowed, the degree of greenness was reduced, and isolation from the mother was not easy. When seeding was induced by HR-v1 medium for 4 days and transferred to 1x mDM, seed induction proceeded well, uniformity was good, and HR initial growth and greenness after seed induction were also good (Table 3). As a result, it was considered that it would be most preferable to induce seeds by growing HR-v1 for 4-5 days and transferring it to 1x mDM or 1 / 2x mDM. However, since HR-v1 is more expensive than 1x mDM, comprehensive economic feasibility should be considered.

After incubation (DAI) Growth and seed induction rate of HR according to media HR-v1 HR-v1 -> 1x mDM ^{1 )} HR-v1 -> Tap water ^{1 )} 0 Object length 2-3cm, 4 Object length 8-10cm Object length 8-10cm Object length 8-10cm 5 30-40% seed induction 70% seed induction 70% seed induction 6 80% seed induction 95% seed induction 95% seed induction 7 95% seed induction 100% seed induction 100% seed induction ^{2 )} 8 0.5-1.8cm of new seed 1.5 ± 0.3cm of new seed 0.7 ± 0.2cm of new seed

 1) Transfer the growth from HR-v1 for four days to fresh medium.

2) The color of the new seed is relatively light green, and isolation from the mother is difficult.

1-4. large tray In two step  Depending on culture HR Seed  Judo

Two step cultivation was performed to produce HR seeds on a larger scale. First, contains a 1X + mDM SBC50 medium made of deaerated water in a small tray (1.5m ² / 100L) a large amount of HR of 2-3 cm in size were grown inoculation 3 days (primary culture). HR (5-7cm in length), a small tray to 3 days growth was transferred to 100 each cultured on a large tray (6.48m ² / 500L) is mDM 1X + SBC50 medium containing one made of deaerated water. As a result, seed induction was 30% at the 7th day and 90-95% at the 9th day after the initial inoculation, and grown to a size (0.5-1.5cm) suitable for collection on the 11th day. From one large tray, about 600 g of HR seed was obtained on dehydration basis.

1-5. Two step  Depending on the culture density in culture HR Seed  Judo

To investigate the difference in seed induction according to the culture density in a medium having a relatively low nutrient concentration. That is, about 100 of 3-4 cm HRs were inoculated into 100 L of 1x mDMV medium, and the seeds of 13-18 cm in length were grown for 4 days in three different culture densities in the freshly prepared medium. The mDMV medium is an mDM medium supplemented with vitamins.

As shown in Table 4, when the HR net was sufficiently grown, seed induction occurred at the same time even when the density of net entities after 2 times (40/100 L) was high, and the induced new HR seed remained excellent form (Table 4). The lower the density, the faster the growth after seed induction. This may be because the remaining nutrients are relatively abundant. From these results, three-stage cultivation seemed to be desirable for inducing good seeds. In the first stage, high-density growth is performed, and morphologically superior individuals are selected. In the second stage, HR is rapidly grown in a proper medium to make a good individual. In the third culture, the density is higher than that in the second culture, I think it would be a good idea.

Days after treatment (DAT) ^{1 )} HR seed induction rate according to culture density 10 HR / 100L 20 HR / 100L 40 HR / 100L 0 Object length 13-18cm Object length 13-18cm Object length 13-18cm 2 Object length 25-30cm
10% seed induction Average length 20cm
15% seed induction Average length of object 15cm
20% seed induction 3 100% seed induction 100% seed induction 100% seed induction 5 Very good seed collection Good seed collection Good seed collection

1) Transfer HR for 4 days in 1x mDMV (13-18 cm length) to fresh medium (1x mDM).

1-6. Three step  In culture HR Seed  Judo

In order to induce high-quality seeds more efficiently, three-step cultivation was considered to be preferable. To prove this, three-step cultivation was used to induce HR seeding. The HR used in the experiment was 0.5-1.0 cm in size and very young. The type of medium used for each step and the incubation period are shown in Table 5 below. As a result, as expected, HR seed could be produced more efficiently in 10 days after the initial inoculation (Table 5). In the first step, the HR seeds are grown at high density for 2-3 days (HR seed 2-4 cm), and the morphologically superior individuals are selected. In the second step, the low density culture is carried out in the appropriate medium for 3-4 days, And 3 days after seeding. In the case of the third culture, a strategy of inducing normal seeds while culturing them within 3 days was desirable.

Incubation step Date of vaccination
(DAI) HR growth and seed induction rate ^{1 )} 1 ^st step,
100 ea / 10L 0 0.5-1.0 cm 3 3.0-4.0 cm 2 ^nd step,
40 ea / 100 L 0 3.0-4.0 cm 3 15 ± 2 cm, 0% seed induction 3 ^rd step,
40 ea / 100 L 0 15 ± 2 cm, 0% seed induction 2 85% seed induction, Normal type 3 100% seed induction, Normal type 4 Dehydrated seeds of about 400g in good fresh weight (seed length 0.5-1.5cm)

1) The medium used was 1x mDMV, 1x mDM + SBC 50, and 1x mDM when cultured at 1, 2, and 3, respectively.

1-7. Collected directly outdoors HR Seed  Judo

Those grown outdoors have a much larger HR net size than those that have been cultivated in the greenhouse. In fact, they experimented to see what difference they had in growing greenhouses and seed induction. The young HR (about 1-2 cm in size) collected outdoors was selected and cultured in 1 × mDMV medium. After that, step cultivation was performed in the same manner as the previous experiment. As a result, the growth tended to be very rapid, and thus the size of one individual was also about five times larger. However, there was no significant difference in seed induction period or induction rate, and the shape and size of the final HR seeds were similar to those grown in greenhouse conditions (Table 6).

Incubation step Post-vaccination date (DAI) HR growth and seed induction rate ^{1 )} 1 ^st step,
100 ea / 10L 0 1.0-2.0 cm 3 7.0-10.0 cm 2 ^nd step,
25 ea / 100L 0 7.0-10.0 cm 3 A tray was 35.0 ± 6.6cm, B tray was 42.3 ± 8.4cm,
0% seed induction 3 ^rd step,
15 ea / 100 L 0 A tray was 35.0 ± 6.6cm, B tray was 42.3 ± 8.4cm,
0% seed induction 2.0 A tray was 64.8.0 ± 15.9cm, B tray was 73.6 ± 14.2cm,
CEL (cell elasticity loosening) symptoms observed 3.0 A tray was 70.30 ± 16.9 cm, B tray was 73.6 ± 14.2 cm,
HR seed induction rate: A tray 70%, B tray 95% 4.0 HR seed induction rate: A tray 95%, B tray 99% 5.0 Seed collection (length 0.5-2.0cm)

1) The medium used was 1x mDMV, 1x mDM + SBC 50, and 1x mDM when cultured at 1, 2, and 3, respectively.

Example  2: Temperature Net horse ( HR ) Effect on growth

2-1. Effect of thermostat treatment

To investigate the effect of temperature on HR growth, To a 5 L vinyl plastic bottle was added 1 L of SBC100 (sodium bicarbonate 100 ppm) culture medium containing 1 x mDM (1-fold modified Diatom Medium) (prepared with tap water) and stored at 15 ° C The HR seeds (individual length 1-2 cm) were taken out, and 0.2 g of the dehydrated fresh weight in which water was removed with a tissue was taken and administered to the culture. The cells were incubated for 5 days in a multi-chamber (15, 20, 25, 30, 35 ℃ constant temperature, 14 hour photoperiod, fluorescent light 50-70 μmol m ^-2 s ^-1 ) . After 5 days of incubation, the dehydration was investigated in fresh weight and in dry weight. The dry matter was dried in a 45 ℃ hot air drier for 3 days. As a result, the fresh weight was higher in the order of 25 ° C> 20 ° C> 30 ° C> 35 ° C> 15 ° C. In the case of buildings, however, the ratio of building to building was higher in the order of 35 ° C> 30 ° C> 25 ° C> 20 ° C> 15 ° C. These results indicate that the growth of HR can be performed satisfactorily in the temperature range of 20-35 ℃. We also compared the amount of glucose production (useful for storing carbohydrates in biomass) that is useful for fermentation from these biomass. Glucose production from HR biomass was performed in the same manner as in the previously reported method (Kim et al., Korean Society of Weed Science 32 (2): 85-97, 2012). As a result, the amount of glucose production was higher in the order of 35 ° C> 30 ° C = 25 ° C> 20 ° C> 15 ° C, and increased with increasing temperature (Table 7).

Temperature HR growth after 5 days of culture Glucose
(g / 100 g DM) Dehydrated fresh weight
(FW, g / L) In building
(DW, mg / L) DW / FW (%) 15 ℃ 0.807 + - 0.10 63.5 ± 8.3 7.87 25.61 + 0.04 20 ℃ 1.430 0.04 161.8 ± 3.3 11.31 38.45 + 0.36 25 ℃ 1.537 + 0.06 223.1 ± 3.5 14.52 45.43 + - 0.21 30 ℃ 1.350 + 0.03 229.2 ± 8.2 16.98 45.13 + - 0.28 35 ℃ 1.280 + 0.08 257.4 ± 15.8 20.11 47.69 + 0.07

2-2. Heat treatment effect

HR growth and glucose production were investigated at the temperature of day and night. The experimental method was the same as the previous experiment and only the temperature condition was different. As a result, growth temperature and growth of glucose were higher than that of daytime 25 ° C / night 15 ° C at 30-35 ° C / night and 20-30 ° C at night when temperature difference was fixed at 10 ° C (Table 8). On the other hand, when the daytime temperature was fixed at 30 ° C and the nighttime temperature was changed in the 15-30 ° C range, the growth and glucose production tended to increase as the nighttime temperature increased.

Thermoconditioning conditions HR growth after 5 days of culture Glucose
(g / 100 g DM) Light condition
(14 hr) Cancer Conditions
(10 hr) Dehydrated fresh weight
(FW, g / L) In building
(DW, mg / L) DW / FW
(%) 25 ℃ 15 ℃ 1.615 + 0.06 231.4 + - 4.4 14.33 41.61 ± 0.32 30 ℃ 20 ℃ 1.698 + 0.08 269.2 ± 9.3 15.85 45.40 ± 0.32 35 ℃ 25 ℃ 1.523 + 0.127 262.0 + - 8.3 17.20 46.33 ± 0.26 30 ℃ 30 ℃ 1.693 + - 0.155 290.6 ± 17.5 17.16 46.16 + - 0.67

Taking all the above results into consideration, in order to mass-produce high-quality HR biomass, it was considered to be preferable to adjust the temperature of daytime or nighttime to be at least 20 ° C or more, preferably 25 ° C or more.

Example  3: culture solution pH end Net horse ( HR ) Effect on growth

To investigate the pH range on HR growth, DM medium (see Table 26) was used as a basic medium for culturing. The pH of the medium was adjusted to pH 4, pH 5, pH 6, pH 7, pH 8, pH 9, pH 10 and pH 1 with 1 N HCl or 1 N NaOH 11, and then 70 ml was added to a 125 ml Erlenmeyer flask. After that, 5 HR seeds of a certain size were added, and then the length and weight (in dehydrated fresh weight) of the experimental group were measured after culturing for 7 days in a 25 ° C. incubator for 14 hours. As a result, as shown in Table 9, decolorization was not achieved at pH 4 and pH 5, and good growth was observed at pH 7 to pH 10 at higher pH. Similar results were obtained at pH 10 and pH 11, indicating that the HR samples of this study were alkaliphyllic algae.

The pH of the DM medium Dehydrated fresh weight (㎎ / net) Net length (cm / net) 4 0.14 0.56 + 0.11 5 0.20 0.94 + 0.21 6 2.68 1.22 0.31 7 10.89 3.36 ± 1.64 8 15.84 4.58 ± 0.85 9 18.38 4.78 ± 0.50 10 19.58 5.78 ± 1.31 11 19.84 5.72 ± 0.83

On the other hand, the effect of medium pH on HR growth was investigated under larger greenhouse experimental conditions. Into a rectangular box (41x25.5 cm, 0.1 m ² ), 1 L DM medium prepared with tap water was injected 5 L per box. A previously prepared HR seed (net length 1.5-2.5 cm) was added 0.8 g per vessel in dehydrated and cultured for 10 days under greenhouse conditions. The pH in the medium was adjusted to pH 5.5, pH 7.5, and pH 9.5 with 10 N HCl and maintained for 10 days at 3, 5, and 8 days with fresh media of the same composition Is known to generally increase the pH of the medium). After culturing for 10 days, HR samples were harvested and dehydrated fresh weight and dry weight were investigated. The glucose content accumulated in the HR sample was then investigated as in Example 2.

As a result, on the 2nd day of cultivation, the greenness of the HR individuals was weak and the growth was poor at pH 5.5 treatment. In the case of growth after 10 days of culture, there was a great difference between the treatments, and the growth rate was higher in the case of higher pH and in the case of higher glucose, . Therefore, in order to mass-produce high-quality HR biomass, it was considered preferable to start the culture by adjusting the medium pH to 9.0 to pH 9.5 or more.

The pH of the medium HR growth after 10 days of culture Glucose
(g / 100 g DM) Dehydrated fresh weight
(FW, g / 0.1 m ² ) In building
(DW, g / 0.1 m ² ) DW / FW
(%) 5.5 11.69 ± 1.35 0.65 ± 0.05 5.6 14.34 ± 0.29 7.5 21.26 ± 1.00 1.67 + 0.04 7.9 21.85 ± 0.46 9.5 30.71 + - 0.88 2.21 ± 0.06 7.2 27.18 + - 0.63

Example  4: Guangzhou Giga HR  Effects on Growth and Carbohydrate Accumulation

HR growth and carbohydrate accumulation. Add 1 L of SBC100 culture medium (prepared with tap water) containing 1 × mDM to a 5 L clear vinyl plastic bottle, remove the HR seed (1-2 cm) stored at 15 ° C, and remove dehydrated tissue from the tissue. And administered to the culture solution. The cells were incubated for 5 days in a 30 ° C constant temperature multi-chamber (6, 12, 18, 24 hour photoperiod, fluorescent light 50-70 μmol m ^-2 s ^-1 ) I shook it by -2 times. After 5 days of culture, glucose production from dehydrated fresh, dry and HR biomass was investigated as in Example 2. As shown in Table 11, HR growth was higher with longer photoperiod when the continuous condition was excluded. However, the live weight and dry weight were lower than that of the 18 - hour light period and higher than that of the 6 - hour light period. On the other hand, the rate of building increased with the long period of Gwangju period. Glucose production from HR biomass was higher in the order of 18>12>24> 6 hours, and carbohydrate accumulation tended to be higher when the photoperiod time was longer (Table 11).

Guangzhou
(Weekly, hr) HR growth after 5 days of culture Glucose
(g / 100 g DM) Dehydrated fresh weight
(FW, g / L) In building
(DW, mg / L) DW / FW (%) 6 0.55 + 0.02 478.7 ± 0.0 14.3 22.6 + 0.04 12 1.15 ± 0.11 9195.7 ± 0.5 17.0 36.7 ± 1.39 18 1.43 + 0.03 281.6 ± 7.1 19.7 44.0 ± 0.69 24 1.11 + 0.06 251.2 ± 17.7 22.6 38.4 ± 0.21

On the other hand, we investigated the effect of Gwangju on HR growth and carbohydrate accumulation under SBC (Sodium Bicarbonate) throughput. 5 L each of SBC 200 containing 1 x mDM medium and 1 x mDM prepared with tap water was injected into a rectangular shaped living box (41 x 25.5 cm, 0.1 m ² ) container. The previously prepared HR seed (net length 2.0-3.0 cm) was put into dehydrated fresh weight 0.8 g per box and cultured in an indoor growth chamber for 7 days. The environmental conditions of the growth chamber were 25 ℃ constant temperature, 24hr / night 0hr or 14hr / night 10hr photoperiod and 90-120μmolm ^-2 s ^-1 light intensity. After incubation for 7 days in the growth chamber under the above conditions, HR samples were harvested and the glucose content produced in dehydrated fresh, dry and HR samples was examined as in Example 2. As a result, the results were slightly different according to the composition of the medium. In the case of 1 × mDM medium, there was no significant difference in the HR growth between the photoperiod and the carbohydrate accumulation was slightly higher in the photoperiod of 14 hours than 24 hours of continuous illumination Respectively. On the other hand, when 200 ppm of SBC was added to 1 × mDM medium, HR growth was significantly increased compared with no addition, but there was no significant difference between the photoperiod treatments and the 14 hours photoperiod. However, in the case of carbohydrate accumulation, the results were significantly higher in the 14-hour photoperiod than in the 24-hour continuous illumination (Table 12). In other words, the photoperiod effect was remarkable in the carbohydrate accumulation than in the growth, and the degree of carbohydrate accumulation was significantly different according to the SBC throughput. Ultimately, in order to mass produce high quality HR biomass, It is necessary to cultivate with SBC addition.

Guangzhou badge HR growth after 7 days of culture Glucose
(g / 100 g DM) Dehydrated fresh weight
(FW, g / 0.1 m ² ) In building
(DW, g / 0.1 m ² ) DW / FW
(%) Daytime 14h / Nighttime 10h 1 x DM 8.85 ± 0.35 1.15 ± 0.03 13.0 40.21 + - 1.75 1 x DM + S200 10.84 0.40 1.69 ± 0.05 15.6 50.40 ± 2.16 Daytime 24h / Nighttime 0h 1 x DM 7.83 + - 0.55 1.24 ± 0.11 14.8 36.03 + - 1.12 1 x DM + S200 9.04 + - 0.38 1.53 + - 0.12 16.9 37.88 + - 0.66

Example  5: Adding nitrogen source to the greenhouse HR  Growth and Sugar content  Effect

5-1. Ammonium nitrate ( Ammonium nitrate ) In this greenhouse culture HR  Effect on growth

The effect of ammonium nitrate addition on the HR growth and carbohydrate accumulation as a nitrogen source for algal culture was investigated. SBC200 medium containing 1 x mDM, prepared with tap water, was injected into rectangular rectangular living box (41 x 25.5 cm, 0.1 m ² ) containers, each 5 L per box. Ammonium nitrate was then added to several concentrations. A previously prepared HR seed (net length 3.0-4.0 cm) was added 0.8 g per vessel in dehydrated fresh weight and cultured in the greenhouse for 7 days. The environmental conditions of the greenhouse were 25-30 ° C / night at 20-25 ° C, 14 hours light period, 90-500μmolm ^-2 s ^-1 brightness. After 7 days of incubation in the greenhouse under the above conditions, the HR samples were harvested and the glucose content produced from dehydrated fresh, dry and HR samples was examined in the same manner as in Example 2. As a result, in the case of addition of ammonium nitrate, the HR growth tended to increase slightly until 30 ppm, then gradually decrease thereafter. However, in the case of carbohydrate accumulation, the higher the treatment concentration of ammonium nitrate was, the lower the result was. Overall, the decrease in glucose production due to increased nitrogen content was greater than the increase in biomass due to the addition of ammonium nitrate (Table 13).

Ammonium nitrate (mg / L) After 7 days of incubation, HR growth (g / 0.1 m ² ) Amount of glucose production
(g / 100 g DM) Dehydrated fresh weight (FW) Building (DW) DW / FW (%) 0 12.68 + - 0.11 1.644 ± 0.053 13.0 42.27 + 1.04 (100.0%) 15.6 13.81 + 0.07 1.668 ± 0.061 12.1 33.11 + - 0.95 (78.3%) 31.2 13.96 + - 0.10 1.675 ± 0.000 12.0 27.56 + - 0.47 (65.2%) 62.5 12.34 + - 1.12 1.585 + 0.094 12.8 27.08 + - 0.54 (64.1%) 125 9.72 + - 0.72 1.312 + 0.012 13.5 25.52 + - 0.43 (60.4%) 250 3.78 + - 0.74 0.517 + 0.132 13.7 22.95 + 1.71 (54.3%) 500 0.71 + 0.02 0.099 ± 0.002 13.9 nd

5-2. Urea  In greenhouse cultivation HR  Effect on growth

The effect of urea addition on HR growth and carbohydrate accumulation as a nitrogen source in algal culture was investigated. 5 L each of SBC150 medium containing 1 x mDM or 1 x mDM prepared with tap water was injected into a rectangular living box (41 x 25.5 cm, 0.1 m ² ). Urea was then added to give various concentrations. The previously prepared HR seed (net length 3.0-4.0 cm) was put into dehydrated fresh weight 0.82 g per vessel and cultured in the greenhouse for 7 days. The environmental conditions of the greenhouse were 25-30 ° C / night at 20-25 ° C, 14 hour light period, 90-350 μmolm ^-2 s ^-1 light intensity. After 5 days of incubation in the greenhouse under the above conditions, the HR samples were harvested and the glucose content produced in the building and HR samples was investigated as in the above-mentioned method. As a result, as shown in Table 14, SBC150 treatment induced an increase of 28.4% in dryness compared to SBC-treated at 1 × mDM. The increase of the urea treatment in the building was 14-18% higher than the untreated condition in the condition of SBC treatment but only 6-12% higher than the untreated condition in the condition without SBC. Overall, the increase during drying due to urea treatment was relatively weak and the effect was not increased above 50 ppm. On the other hand, SBC150 treatment induced a 17.4% glucose increase in 1 × mDM compared with SBC untreated control in the study of the effect of urea on carbohydrate accumulation. Glucose production by urea treatment was 80-87% of the untreated condition under the condition of SBC, but it was decreased by 76-83% compared to the untreated condition (Table 14). Glucose decline with increasing treatment concentration was not observed above urea 50 ppm. Therefore, it is considered that a concentration of 50 ppm or less is preferable when an additional supply of urea is desired.

Urea
(mg / L) In the building (g / 0.1 ㎡) Glucose (g / 100 g DM) No treatment of SBC150 SBC150 processing No treatment of SBC150 SBC150 processing 0.0 0.95 + - 0.03 (100.0) 1.22 + - 0.01 (100.0) 36.19 + - 1.57 (100.0) 42.47 + - 0.64 (100.0) 50 1.01 0.00 (106.3) 1.44 + - 0.01 (118.0) 28.70 + -0.53 (79.3) 35.90 + - 1.29 (84.5) 100 1.07 ± 0.00 (112.6) 1.42 + - 0.03 (116.4) 29.95 + - 0.54 (82.8) 33.85 + - 0.90 (79.7) 200 0.97 + - 0.10 (102.1) 1.44 + - 0.04 (118.0) 27.32 + - 0.92 (75.5) 34.15 + -0.87 (80.4) 300 0.96 + 0.11 (101.1) 1.40 ± 0.00 (114.8) 28.65 + - 2.59 (79.2) 37.05 + - 0.19 (87.2)

5-3. KNO ₃ In the greenhouse culture HR  Effect on growth

The effect of KNO ₃ addition on the HR growth and carbohydrate accumulation as a nitrogen source in algal culture was investigated. 5 L each of SBC200 medium containing 1 x mDM or 1 x mDM prepared with tap water was injected into a rectangular shaped living box (41 x 25.5 cm, 0.1 m 2) container. KNO _{3 was} then added to achieve various concentrations. The previously prepared HR seed (net length 2.0-3.0 cm) was added to dehydrated fresh weight 0.80 g per vessel and incubated in the greenhouse for 5 days. The environmental conditions of the greenhouse were 25-30 ° C / night at 20-25 ° C, 14 hour light period, 90-350 μmolm ^-2 s ^-1 light intensity. After 5 days of incubation in the greenhouse under the above conditions, the HR samples were harvested and the glucose content produced from fresh, dry and HR samples was investigated as described above. First, observation of the live weight change with various concentrations of KNO _3, the fresh weight was increased effects of the KNO ₃ treatment higher tendency when SBC200 the addition than without this SBS200. On the other hand, the effect of KNO ₃ treatment seems to be different depending on the composition ratio in the culture medium. That is, in the absence of SBC200, growth of about 10% was observed at 50-200 ppm, but at 300 ppm, growth was not improved as in the control. On the other hand, in the SBC200 treatment, about 20% of the fresh weight gain tendency was observed without any difference in concentration up to 50-300 ppm (Table 15).

KNO ₃
(Mg / L) HR dehydration in 5 days culture Fresh weight (g / 0.1 m 2) No processing of SBC200 SBC200 processing 0.0 13.0 + - 0.14 (100.0) 9.53 + - 0.23 (100.0) 50 14.5 ± 1.47 (111.3) 11.47 + - 0.43 (120.4) 100 14.3 ± 0.72 (110.2) 11.24 + -0.94 (117.9) 200 14.2 ± 0.50 (109.1) 11.38 + - 0.82 (119.4) 300 13.4 ± 0.61 (103.4) 11.57 + - 1.02 (121.4)

These results suggest that treatment of KNO _{3 at} 50 ppm or more did not have a significant effect on growth. Therefore, the effects of 50 ppm of KNO ₃ on carbohydrate accumulation were investigated. Except that the size of the HR seed used in the experiment was 1.5-2.5 cm. As a result, SBC200 treatment induced an increase in 22.2% glucose production in 1 x mDM compared to SBC200 untreated. The amount of glucose produced by the addition of 50 ppm of KNO ₃ was about 80% of the untreated state under both conditions, which was about 20% reduced (Table 16).

KNO ₃
(Mg / L) Glucose production (g / 100 g DM) No processing of SBC200 SBC200 processing 0.0
50 34.75 + - 0.60 (100.0)
28.32 + - 0.76 (81.5) 42.47 ± 1.09 (100.0)
33.84 + - 1.35 (79.7)

Example  6: Nitrogen and phosphorus content HR  Effects on growth and carbohydrate accumulation

HR algae culture, and the effects of Ca (NO ₃ ) ₂ 4H ₂ O (CN) and K ₂ HPO ₄ (PP) as phosphate sources on HR growth and carbohydrate accumulation. SBC200 medium containing 1 x mDM prepared with tap water in a rectangular living box (41 x 25.5 cm, 0.1 m 2) container was injected with 5 L each per box. The concentrations of nitrogen and phosphorus were then adjusted so as to be combinations of various concentrations. The previously prepared HR seed (net length 2.0-3.0 cm) was put into dehydrated fresh weight 0.80 g per vessel and cultured in a greenhouse for 5 days. The environmental conditions of the greenhouse were 25-30 ° C / night at 20-25 ° C, 14 hour light period, 90-350 μmolm ^-2 s ^-1 light intensity. After 4 days of incubation in the greenhouse under the above conditions, the HR samples were harvested and the glucose content produced in the building and the HR samples was investigated as described above. As a result of observing changes in the structure according to the concentration of nitrogen (CN) treatment, the concentration of nitrogen (CN) contained in the basic 1 × mDM medium was 20 ppm, (CN) concentrations were reduced to 5 ppm and 10 ppm, respectively, to 75.1% and 87.8% of the 20 ppm treatments, respectively. The concentration of phosphorus (PP) contained in the basic 1 × mDM medium was 12.4 ppm, which was increased to 24.8 ppm or decreased to 3.1 ppm and 6.2 ppm Even though the building did not show a big change. Thus, HR growth was found to be more dependent on nitrogen content than the phosphorous content in the culture (Table 17).

When the concentration of nitrogen (CN) contained in the basic 1 × mDM medium was 20 ppm and increased to 40 ppm, the glucose production was increased to 83.2% . When the concentration of nitrogen (CN) was decreased to 5 ppm and 10 ppm, 92.4% and 99.1% of 20 ppm treatment, respectively, showed a very low decrease in glucose content. The concentration of phosphorus (PP) contained in the basic 1 × mDM medium was 12.4 ppm, which was increased to 24.8 ppm or 3.1 ppm and 6.2 ppm Glucose content was 93.8%, 79.6% and 94.1%, respectively, when they were decreased, respectively (Table 17). Therefore, the proper concentration of carbohydrate accumulation in HR is more important than the appropriate ratio of nitrogen (CN) and phosphorus (P), and the content of phosphorus (PP) is 6.2 ppm to 24.8 ppm and the concentration of nitrogen (CN) is 5 ppm to 20 ppm level was found to be a favorable condition for carbohydrate accumulation.

Nutrient (mg / L) In the building (g / 0.1 ㎡) Glucose (g / 100 g DM) Ca (NO ₃ ) ₂ .4H ₂ O (CN) K ₂ HPO ₄
(PP) CN / PP 5
10
20
40 12.4
12.4
12.4
12.4 0.4032
0.8065
1.6129
3.2258 0.89 0.028 (75.1)
1.04 0.042 (87.8)
1.19 ± 0.007 (100.0)
1.20 0.028 (101.3) 33.28 占 1.17 (92.4)
35.70 + - 0.32 (99.1)
36.03 + - 1.65 (100.0)
29.96 + -0.44 (83.2) 20
20
20
20 3.1
6.2
12.4
24.8 6.4516
3.2258
1.6129
0.8065 1.22 ± 0.042 (102.1)
1.17 + - 0.021 (97.5)
1.20 ± 0.007 (100.0)
1.21 + - 0.014 (101.3) 30.59 + - 0.49 (79.6)
36.17 + - 0.33 (94.1)
38.43 + - 0.85 (100.0)
36.03 + - 1.58 (93.8)

Example  7: Silicate ( Silicate ) Depending on the amount added HR  Growth and Sugar content  Effect

Silicon is known to be a trace element in algae cultivation for promoting growth and maintaining normal cell morphology. It is an indispensable element in diatom culture. The effect of addition of sodium metasilicate (SMS), which is frequently used as a silicon source, on HR growth and carbohydrate accumulation was investigated. SBC200 medium containing 1 x mDM, in which the SMS was removed by tap water, was injected into each rectangular box (41 × 25.5 cm, 0.1 ㎡). Then, the pH of each treatment solution was adjusted to be constant from pH 9.5 to pH 9.8 with 1 N NaOH while SMS was added so that the final concentration was 0 ppm to 64 ppm. The previously prepared HR seed (net length 2.0-3.0 cm) was added to dehydrated fresh weight 0.81 g per vessel and cultured in a greenhouse for 6 days. The environmental conditions of the greenhouse were 25-30 ° C / night at 20-25 ° C, 14 hour light period, 90-350 μmolm ^-2 s ^-1 light intensity. After culturing in the greenhouse for 6 days, the HR samples were harvested and the glucose content produced in the building and HR samples was investigated as described above. As a result, there was no difference in the SMS treatment concentration in fresh weight. In the case of buildings, there was a tendency to slightly increase in treatments above 32 ppm, and in the ratio of live weight to building weight, the building ratio tended to increase from 16 ppm or more (Table 18). This is thought to be due to the introduction of silicon into cells. On the other hand, there was no difference in glucose accumulation between SMS treatment concentrations. From these results, it is considered that the supply of silicon is not essential for the culture of HR (SMS is a high-priced ingredient in the medium composition), and thus it is considered that HR can be mass-produced at a lower cost of the medium.

NaSiO ₃ .9H ₂ O
(ppm) HR growth after 6 days of culture
(g / 0.1 ㎡ L) Amount of glucose production
(g / 100 g DM) Dehydrated fresh weight (FW) Building (DW) DW / FW (%) 0 11.20 ± 0.42 1.230 + 0.06 10.98 35.05 ± 0.52 One 10.82 + - 0.45 1.175 + 0.03 10.86 35.37 ± 0.29 2 10.58 ± 0.39 1.194 + 0.07 11.29 37.50 ± 2.08 4 11.15 ± 0.42 1.276 + 0.06 11.44 32.67 ± 1.36 8 11.28 0.95 1.253 + 0.08 11.11 34.70 + - 0.66 16 10.26 ± 0.35 1.191 + 0.02 11.61 32.30 ± 0.71 32 10.47 + - 0.47 1.241 + 0.02 11.85 34.09 ± 1.30 64 10.82 0.20 1.295 + - 0.01 11.97 35.05 ± 0.86

Example  8: Within the culture medium  Presence or absence of vitamins Net horse ( HR ) Effect on growth

The effect of vitamin on HR growth was examined. As a basic medium for culturing, 1 × DM medium was used, and a medium supplemented with or without vitamin was prepared, and then 70 ml was added to a 125 ml Erlenmeyer flask. The final concentration of added vitamins was 0.04 ppm of cyanocobalamin, 0.04 ppm of thiamine-HCl, and 0.04 ppm of biotin (vitamin B complex). After that, 5 pieces of HR seeds of a certain size were added, and then the length and weight (dehydrated fresh weight) were measured after culturing for 7 days with a photoperiod for 14 hours in a constant temperature room at 25 ° C. As a result, as shown in Table 19, HR growth did not occur without vitamin addition. This means that there is no need to add expensive vitamins to the culture, which indicates that more economical production is possible.

Basic badge Vitamins ^{1 )} Dehydrated fresh weight (㎎ / net) HR net length (cm / net) 1 x mDM No treatment 4.62 3.25 0.30 1 x DM process 4.40 3.50 + - 0.41

1) 0.04 ppm of vitamin B12 + 0.04 ppm of vitamin B1 + 0.04 ppm of biotin

Example  9: NaCl  When the treatment concentration is HR  Effects on Growth and Carbohydrate Accumulation

The effects of NaCl on HR growth and carbohydrate accumulation were investigated. SBC100 medium containing 1 x mDM (prepared by adding 1.5 times the basic medium concentration in the case of nitrogen and phosphoric acid) was prepared in a rectangular living box (41 × 25.5 cm, 0.1 ㎡). NaCl was then added to a final concentration ranging from 0 ppm to 5000 ppm. Prepared HR seeds (net length 2.0-3.0 cm) were put into fresh dehydrated 1.0 g per vessel, covered with 3 openings for ventilation, cultured for 6 days in a growth chamber, and shaken 1-2 times a day gave. The environmental conditions of the growth chamber were 25 ℃ constant temperature, 14 hours light period, 60-120 μmolm ^-2 s ^-1 light intensity. After incubation for 6 days in the growth chamber under the above conditions, the HR samples were harvested and the glucose content produced in the dehydrated fresh, dry and HR samples was investigated as described above. As a result, growth inhibition was not observed up to 1000 ppm of NaCl and growth was inhibited from the above concentration. At 300 ppm NaCl, fresh weight and dry weight increased by 25.4% and 7.5%, respectively (Table 20). On the other hand, there was no significant change in the amount of glucose produced up to 1000 ppm of NaCl, but at higher concentrations, the amount of glucose production decreased with increasing concentration (Table 20), which was related to the fact that there was little increase in pH in the culture medium during culture Thought. In conclusion, NaCl was slightly increased in fresh weight and dry weight compared to the control at the appropriate concentration (around 300ppm), but was not considered to have a direct effect on carbohydrate accumulation.

NaCl
(ppm) HR growth after 6 days of culture (g / 0.1 ㎡) Glucose
(g / 100 g DM) After cultivation, the pH of the medium Dehydrated fresh weight
(FW) In building
(DW) DW / FW (%) 0 11.0 + - 0.76 1.60 + 0.03 14.55 37.69 + - 2.56 11.32 ± 0.01 100 12.6 ± 0.27 1.70 + 0.08 13.49 37.91 + 1.67 11.31 + 0.02 300 13.8 ± 0.35 1.72 + - 0.01 12.46 37.66 ± 1.05 11.18 + 0.04 1,000 13.2 ± 0.93 1.60 0.11 12.12 38.71 + 1.34 11.01 + - 0.01 3,000 6.0 ± 0.93 0.57 ± 0.12 9.5 24.36 + 2.02 9.43 ± 0.08 5,000 1.2 ± 0.52 0.15 + 0.06 12.5 15.96 + - 0.38 9.64 + - 0.10

Example  10: Sodium bicarbonate ( Sodium bicarbonate ; SBC , NaHCO ₃ ) Treatment in greenhouse culture HR  Effect on Growth Enhancement and Carbohydrate Accumulation

The main inorganic carbon source for algal culture is CO ₂ . This means that CO _{2 ,} HCO ₃ and CO ₃ And it affects the growth of birds. In general, birds fed the CO _2, the gas phase during culture in this case has been reported that the use efficiency is not sufficiently soluble in water up to within 25%. Therefore, there is a need for a technique for converting and supplying water in a form of high water solubility (for example, NaHCO ₃ ). In this study, sodium bicarbonate (NaHCO ₃ ; SBC) were used to determine if growth and carbohydrate accumulation were promoted.

Into a rectangular living box (41 x 25.5 cm, 0.1 m 2) container, 1 L mDM medium prepared with tap water was injected into each 5 L of each box. SBC was then added to give a final concentration of 0 ppm to 300 ppm. The HR seeds (1.0-2.0 ㎝, 3.0-4.0 ㎝ and 5.0-10.0 ㎝, based on net length) prepared beforehand were added in dehydrated fresh weight to 0.8 g per vessel and cultured in a greenhouse for 5 days. I shook it by -2 times. The environmental conditions of the greenhouse were 25 ℃ constant temperature, 14 hour photoperiod and 90-350 μmolm ^-2 s ^-1 luminous intensity. After 5 days of incubation in the greenhouse under the above conditions, the HR samples were harvested and the glucose content produced in dehydrated fresh, dry and HR samples was examined as in Example 1. [

When SBC was injected into young kidneys with HR net length of 1-2 ㎝, HR growth was accelerated up to the final concentration of 200 ppm and increased by 47.7% at 200 ppm. However, at 300 ppm, it increased by 33.7% compared to the untreated and tended to be lower than that of 200 ppm. When SBC was administered at a growth period of 3-4 ㎝ in HR net length, HR growth was accelerated in proportion to the treatment concentration up to 300 ppm and increased by 40.2% at 300 ppm. When SBC was administered at a growth period of 5-10 ㎝ in HR net length, HR growth was accelerated up to 300 ppm and increased by 36.1% at 300 ppm. Growth enhancement effect was relatively low compared to the treatment when HR net length was 3-4 ㎝ (Table 21). However, in the case of high cloudiness, SBC compound treatment effect was low and growth inhibition could occur at high concentration. These results indicate that SBC is more effective in the growth stage and in the condition of weathering and photosynthesis. In this experimental condition, at least 40% of growth promotion effect could be obtained by adding SBC.

NaHCO ₃
(mg / L) After 5 days of culture, HR growth (g / 0.1 m ^{2 in} dehydrated fresh weight) Growth phase I (1-2 cm) Growth phase II (3-4 cm) Growth Stage III (5-10 cm) 0.0
50
100
200
300 8.6 ± 0.57 g (100.0%)
9.9 ± 0.88 g (115.1%)
11.2 ± 0.70 g (130.2%)
12.7 ± 0.99 g (147.7%)
11.5 ± 0.51 g (133.7%) 9.8 ± 0.72 g (100.0%)
10.4 ± 0.72 g (105.8%)
11.5 ± 0.17 g (117.8%)
12.8 ± 0.80 g (130.3%)
13.7 ± 0.65 g (140.2%) 8.0 ± 0.56 g (100.0%)
9.0 ± 0.11 g (112.3%)
9.2 ± 0.08 g (115.3%)
10.2 ± 0.39 g (128.5%)
10.9 ± 0.50 g (136.1%)

Example  11: HR  For mass production Appropriate medium  Produce

In the case of HR, the media used for physiological ecological studies and species maintenance were Waris-H, McFadden & Melkonian, Phycologia 25: 551-557, 1986 and Hill's medium, Pickett-Heaps, Protoplasma 70 : 325-347, 1970). However, there has been no report on selection or production of a suitable medium for mass production. Therefore, this study was conducted to produce a new medium for the production of low cost / high quality HR biomass.

11-1. In greenhouse cultivation HR  On growth Culture medium  Effects by type (1)

This study was carried out to investigate the difference in the growth rate of HR in the indoor cultivation of microalgae. Five liters of SBC200 medium containing Allen's medium (1 ×), BG11 (1 ×), and 1 × mDM prepared in distilled water was injected into each rectangular box (41 × 25.5 cm, 0.1 ㎡) in a rectangular living box. The previously prepared HR seeds (2.0-3.0 cm by net length) were put into fresh dehydrated 0.2 g per vessel, incubated in the greenhouse for 5 days, and shaken 1-2 times a day. The environmental conditions of the greenhouse were 20 - 30 ° C / night at 20-25 ° C, 14 - hour light period, 90-350 μmolm ^-2 s ^-1 brightness. After 5 days of incubation in the greenhouse under the above conditions, HR samples were harvested and examined for dehydrated fresh weight as in Example 2. As a result, as shown in FIG. 2, the best growth was observed in the mDM medium, followed by Allen medium and BG11. On the other hand, in Allen culture medium and BG11, undesired microalgae were more likely to occur than HR, and this was thought to be caused by nutrient overload.

11-2. In greenhouse cultivation HR  On growth Culture medium  Effects by type (2)

This study was conducted to compare the effect of 1 × mDM produced by the present research team on the HR growth between the existing HR-growth medium for HR growth. 5 liters of each experimental medium containing distilled water was injected into a rectangular living box (41 × 25.5 cm, 0.1 ㎡). The previously prepared HR seeds (3.0-4.0 cm in net length) were added in dehydrated fresh weight to 0.8 g per vessel, incubated in the greenhouse for 5 days, and shaken 1-2 times a day. The environmental conditions of the greenhouse were 20 - 30 ° C / night at 20-25 ° C, 14 - hour light period, 90-350 μmolm ^-2 s ^-1 brightness. After 5 days of incubation in the greenhouse under the above conditions, the HR samples were harvested and dehydrated fresh weight and dry weight were investigated as in Example 2. H + Si + SBC200 = Waris-H + Si + SBC200 and mRNA> Waris-H + Si, mDM + SBC200 = Waris-H + Si + SBC200 in the case of growth on the third day after the incubation. mDM + SBC200>mDM> Waris-H + Si (Table 22). Larissa-H medium contains more kinds of components and vitamins than mDM medium, which is expensive to manufacture. However, the mDM medium had a simple composition and did not contain vitamins, and there was no significant difference in HR growth. Therefore, it was considered that mDM would be preferable for mass production of HR.

badge SBC
(μg / mL) Dehydrated fresh weight (g / 0.1 ㎡) In the building (g / 0.1 ㎡) Mean ± SD Relative value% Mean ± SD opponent % Modified DM (mDM) 0 9.98 ± 0.26 100.0 ± 2.6 1.12 ± 0.01 100.0 ± 1.0 200 12.09 + - 1.04 121.1 + - 10.4 1.51 + 0.06 135.1 + - 10.4 Waris-H + Si 0 13.40 ± 0.73 100.0 ± 5.5 1.03 0.02 100.0 ± 2.0 200 16.47 + - 0.66 122.9 ± 4.9 1.60 + 0.03 155.0 ± 2.4

11-3. In greenhouse cultivation HR  On growth By badge type  Effects (3)

This study was carried out to compare the effect of HR-v1 medium on the HR growth of the 1 × mDM medium and the HR-v1 medium. Table 26 shows the composition of the medium. Each of the above two media prepared with distilled water was injected into a rectangular living box (41 × 25.5 cm, 0.1 ㎡) container in an amount of 5 L per box. The previously prepared HR seeds (3.0-4.0 cm in net length) were added in dehydrated fresh weight to 0.8 g per vessel, incubated in the greenhouse for 5 days, and shaken 1-2 times a day. The environmental conditions of the greenhouse were 20 - 30 ° C / night at 20-25 ° C, 14 - hour light period, 90-350 μmolm ^-2 s ^-1 brightness. After 5 days of incubation in the greenhouse under the above conditions, the HR samples were harvested and dehydrated fresh weight and dry weight were investigated as in Example 2. As a result, the cultures using HR-v1 medium were higher than those of 1 × mDM + SBC200 in both live weight and building weight (Table 23).

badge SBC
(μg / ml) Dehydrated fresh weight (g / 0.1 ㎡) In the building (g / 0.1 ㎡) Mean ± SD opponent % Mean ± SD opponent % mDM 200 10.57 ± 1.03 100.0 9.7 1.31 + 0.08 100.0 0.7 HR-v1 250 15.52 ± 0.27 146.8 ± 2.6 1.67 ± 0.05 127.2 ± 0.4

On the other hand, the carbohydrate accumulation in HR biomass was investigated over time while cultured for 9 days under the above conditions. The glucose content produced from the HR samples harvested during the culture was examined as in Example 2 to assess the degree of carbohydrate accumulation. As a result, the HR of the HR cultured in the mDM + SBC200 medium showed almost no change in glucose production from about 5 days to 9 days (about 44 g / 100 g DM) (HR was increased in the mDM medium without SBC200 supplementation The yield was 37.6 ± 2.6 g / 100 g DM. However, the HR cultured in the HR-v1 (containing SBC250) medium tended to increase gradually as 27.4, 34.9 and 47.7 g / 100 g DM produced glucose from the samples on the 5th, 7th and 9th days of culture 3). At the end of incubation, the amount of glucose produced was slightly higher in HR-v1 than in mDM + SBC200. Therefore, in order to produce high-quality HR biomass under short incubation period, the mDM medium is good and the HR-v1 medium is good for producing high-quality HR biomass under a longer incubation period.

11-4. mDM  When the concentration of the medium is HR  Effects on Growth and Carbohydrate Accumulation

The effects of mDM medium concentration and SBC treatment concentration on HR growth and carbohydrate accumulation were investigated. (1 x mDM), 2-fold concentration mDM (2 x mDM) and 3-fold concentration mDM (3 x mDM) medium were prepared in a rectangular living box (41 x 25.5 cm, 0.1 m 2) , SBC concentrations of 0, 50, 100, and 200 ppm, respectively, and then 5 L of each was injected per living box container. A previously prepared HR seed (net length 2.0-3.0 cm) was put into dehydrated fresh weight 2.0 g per vessel and incubated in the greenhouse for 5-7 days. Greenhouse environmental conditions of clear weather were 25-30 ℃ / night at 20-25 ℃, 14 hours light period, 90-350 μmolm ^-2 s ^-1 light intensity, but cloudy weather during the experiment. The HR samples were harvested after 5 days for 1 × mDM treatment and 7 days for 2 × mDM and 3 × mDM treatment in the greenhouse under the above conditions. The resulting glucose content was investigated.

As a result, when the degree of growth was compared, it was found that the higher the mDM concentration was, the higher the dry weight was. When SBC was added to each mDM culture medium, the concentration of SBC treatment increased with increasing SBC concentration. Therefore, the highest growth rate of HR growth was 3 × mDM + SBC200, which was 2.27 g / 0.1 ㎡ in 7 days culture (Table 24).

SBC
(Mg / L) mDM concentration, in the building (g / 0.1m ² ) 1 x mDM, 5 days culture 2 x mDM, 7 days culture 3 x mDM, 7 days culture 0.0
50.0
100.0
200.0 1.28 + 0.04
1.49 + 0.03
1.53 ± 0.06
1.57 ± 0.09 1.74 ± 0.01
1.92 + 0.08
1.98 ± 0.06
2.15 + 0.02 1.87 + 0.07
2.17 ± 0.06
2.26 + 0.04
2.27 ± 0.12

On the other hand, according to the results of glucose production from HR cultured samples, glucose production was decreased with increasing mDM concentration in the case of SBC treatment. It is estimated that this is related to the increase of nitrogen content. SBC treatment showed slightly different responses between mDM concentrations. That is, when SBC was added to 2 × mDM or 3 × mDM medium, the amount of glucose production tended to increase in proportion to the SBC treatment concentration. However, in 1 × mDM, glucose production was increased by SBC treatment, but there was no significant difference between SBC 50-200 ppm treatments (Table 25). These results show that in the case of carbohydrate accumulation a proper balance between HR algae physiology, culture environment, nutrient content (especially N / P content) and SBC content in the medium is very important.

SBC
(Mg / L) mDM concentration, glucose production amount (g / 100 g DM) 1 x mDM, 5 days culture 2 x mDM, 7 days culture 3 x mDM, 7 days culture 0.0
50.0
100.0
200.0 34.03 + - 0.86
39.35 + 0.13
39.25 + - 0.10
38.54 + - 0.54 25.97 + - 0.82
33.02 + - 2.65
33.91. + -. 3.47
34.29 + - 0.95 28.13 + - 1.42
29.68 + - 0.11
34.69 + - 0.67
35.36 ± 0.21

As a result of the above-mentioned results, in the present invention, three media compositions were prepared for the mass culture of HR, and the specific ingredients and contents thereof are shown in Table 26. HR growth was poor in BBM and CM medium, and HR growth was not good and carbohydrate accumulation was low in allen and BG11 due to high concentration of nutrients and pH incompatibility. Since DM (pH 6.9) was developed as a medium suitable for diatom culture, it was limited to green algae growth and diatom was highly polluted during outdoor culture. Wariss-H is good for HR growth but is not suitable for production of biomass with high carbohydrate content. In the present invention, a medium suitable for mass culture of HR was developed considering growth and accumulation of useful components (carbohydrate, protein). For the characteristics of the three media prepared, mDM was cultured for 4-7 days for a short period of time, Is suitable for securing a large quantity of high-quality HR biomass that can produce glucose of at least 40 g / 100 g DM in accordance with the above-mentioned method (when culturing under the same conditions using Allen's medium, BG11, Only about 25 g / 100 g of DM tend to be produced). On the other hand, HR-v1 is a medium suitable for producing high quality HR biomass that can produce glucose of at least 40 g / 100 g DM according to this experimental method while culturing for 9 days or more for a relatively long period of time. AW-1, on the other hand, is a useful medium for producing large quantities of biomass in a very simple composition, regardless of carbohydrate-rich biomass production.

In addition to the degassed tap water, the culture medium may be prepared using at least one kind of water selected from distilled water, ground water, rainwater, discharged water, river water and the like. The components suggested as nitrogen sources may be replaced by other components and may be supplied in concentrations ranging from 1 to 50 ppm in the form of ammonium, nitrate, urea and ammonium nitrate. In addition to bicarbonate as a carbon source, it can be supplied in the range of 1-300 ppm in the form of carbonate or carbon dioxide. In particular, since the net-like algae grow better in the alkaline solution, it is possible to control the alkalinity of the medium by adding an appropriate amount of KOH or sodium metasilicate in addition to NaOH.

Example  12: Growth titration  density

To investigate the effect of initial population (individual density) on final biomass production during HR culture. A 1 × mDM + SBC200 medium was prepared from tap water into a rectangular living box (41 × 25.5 cm, 0.1 ㎡), and 5 L of each was injected per living box container. The previously prepared HR seeds (net length 1.5-2.5 cm) were added 0.4-2.4 g per vessel in dehydrated fresh weight, and cultured in the greenhouse for 9 days, and the medium of the same medium composition was newly changed on the 5th day of culture. The greenhouse environmental conditions of clear weather were 25-30 ° C / night at 20-25 ° C, 14 hours light period, 90-350 μmolm ^-2 s ^-1 brightness. After completion of the culture, dehydrated fresh weight and dry weight were investigated.

As a result, the amount of final biomass produced increased with increasing initial density (Table 27). In the linear regression equation, y = 6.8521x + 13.512 (R² = 0.978), where y is the final dehydrated fresh weight, and x is the initial introduced dehydrated fresh weight. That is, when 0.8 g of HR seed is added under the experimental conditions, about 19 g of biomass can be obtained in the dehydrated living body. On the other hand, when linear regression equations were obtained from purely increased buildings, y = 0.6929x + 1.5567 (R² = 0.95), where y is the final building and x is the initial seeded dehydration fresh weight. Therefore, considering that the initial population density is too high, the production amount will be decreased. If the cultivation period is about 10 days, less than 3 g / 0.1 m 2 of dehydrated live weight is appropriate.

The initial HR density (dehydrated fresh weight g / 0.1 m2) HR growth after 9 days of culture (g / 0.1 ㎡) ¹⁾ Dehydrated fresh weight In building 0.4 16.18 ± 1.85 1.75 + 0.16 0.8 19.61 ± 2.12 2.08 ± 0.16 1.2 23.02 ± 0.24 2.43 + 0.07 1.6 27.41 ± 0.05 2.86 ± 0.07 2.0 29.39 + - 0.87 2.97 ± 0.08 2.4 31.42 ± 0.85 3.07 ± 0.09

1) After 5 days of culture, the medium was replaced with new medium

Example  13: According to the method of feeding the medium HR  Productivity Difference

HR growth and carbohydrate accumulation will be influenced not only by the culture environment and medium composition, but also by how the medium is supplied. Therefore, in case of mass production of HR, it is very important to supply medium (timing and quantity) to obtain high-quality biomass while increasing production per unit area. The purpose of this study was to investigate how the growth and quality of HR varied with the number of media supplied during the culture period.

A 1 × mDM + SBC200 medium was prepared from tap water into a rectangular living box (41 × 25.5 cm, 0.1 ㎡), and 5 L of each was injected per living box container. The previously prepared HR seeds (net length 1.5-2.5 cm) were put into the dehydrated fresh weight 0.8 g per vessel and cultured in the greenhouse for 9 days. The medium of the same composition was replaced at regular intervals as shown in Table 21. The greenhouse environmental conditions of clear weather were 25-30 ° C / night at 20-25 ° C, 14 hours light period, 90-350 μmolm ^-2 s ^-1 brightness. After completion of the culture, the HR samples were harvested and examined for the glucose content produced in dehydrated fresh, dry and HR samples as in Example 2.

Way When the medium is replaced (date after initiation of culture) One 2 3 4 5 6 7 8 9 M1 o o o o o o o o Research M2 o o o o Research M3 o o Research M4 o o Research M5 Research

As a result, in the case of dehydrated fresh weight examined on the 6th day, the method (M1) was changed from day to day and the next was changed every 2 days (M2) and 3 days (M3) The lowest. On the other hand, when cultured for 9 days, M1, M2 and M3 were higher in the order of M4> M5 (FIG. 4). The similarities of M1, M2 and M3 are assumed to be due to the saturation of nutrients relative to individual densities for M1 and M2, and other microalgae were more prevalent in M1. M3 was the highest (32.6% increase compared to M5), followed by M2>M1>M4> M5 when the samples were incubated for 9 days. As a result of examining the amount of glucose produced in these samples, M5> M3 and M4>M2> M1 were shown. In general, the shorter the medium replacement period, the lower the glucose production (carbohydrate accumulation) (FIG. Therefore, when mass production of high quality HR using 1 × mDM medium is desired, it is most preferable to use the M3 method in which the medium is replaced or supplemented every 3 days.

Example  14: HR  Badge for growth Stirring treatment  effect

Whether or not the culture liquid is stirred And the effect on the degree of HR growth. 90 g of AW-1 medium (14.5 ㎝ in height) was supplied to a raceway-shaped algae culture tank and 36 g / 90 L / 0.6 ㎡ of HR seed (2-4 ㎝) , In a greenhouse condition (night 20-25 / day 25-30, 14 hours photoperiod, natural light 90-300 μmolm ^-2 s ^-1 ). One of the cultures was artificially stirred once a day, instead of allowing the medium to flow by rotating the stirrer at a rate of 7.5 rpm per minute to allow the medium to flow and the agitator was not used on the other side. After a total of 7 days of culture, the HR grown with one layer of miracloth was collected and dehydrated fresh weight and dry weight were investigated as in Example 2. As a result, there was almost no difference in dehydrated organisms in the presence or absence of stirring, but in the case of the building, the stirring tendency was increased by about 35%. This is due to the higher efficiency of nutrient utilization and the better underwater environmental conditions due to the agitation process. However, individuals did not die but continued to grow even in the absence of antioxidants.

Agitation of media HR growth after 7 days of culture (g / 0.6 ㎡) ¹⁾ Dehydrated fresh weight (A) In building (B) B / A (%) 0 rpm / min 129.1 13.8 10.72 7.5 rpm / min 123.0 18.7 15.18

1) Cultured for 7 days under conditions of 13 hours photoperiod (natural light 90-300 μmolm ^-2 s ^-1 ).

Example  15: In the indoor culture room HR  massive production

Based on the results of the above experiment, mass production of HR was tried in an indoor culture room. A 1 × mDM culture medium was prepared from tap water in a rectangular plastic container (41 × 25.5 cm, 0.1 ㎡) made of transparent plastic, and 6 L of each was injected per living box container. 3.0 g of each of the prepared HR seeds (net length 2.0-3.0 cm) was put into the dehydrated whole body, and 3.0 g of the medium was put into the vessel. The medium was covered with a thin transparent film to prevent the medium from being excessively evaporated. After 3 days, 5 ml of 1 × mDM + SBC100 storage medium (1000 times) per container was added. The medium was shaken once or twice a day during the culture. The environmental conditions of the growing room were 25 ℃ constant temperature, 14 hours photoperiod (fluorescent lamp) and 50-100 μmolm ^-2 s ^-1 luminous intensity. After completion of the culture, the HR samples were harvested and examined for the glucose content produced in dehydrated fresh, dry and HR samples as in Example 2. On the other hand, the reducing sugar content of the enzyme hydrolysates of the HR samples was further investigated by the reported method (Nguyen et al., Bioresource Tech. 110: 552-559, 2012).

As a result, the productivity of HR biomass was 19.8 g, 19.1 g and 21.3 g in Experiment A, B and C, respectively, based on the weight of dry matter per 1.0 ㎡ area, and the average 20 g / It varied slightly from sample to sample, but was in the 10.7% to 14.4% range. Productivity was less in the lower greenhouse conditions incubation, which was considered to be a factor of greater intensity. On the other hand, the amount of glucose produced was 47.9 g, 44.4 g, and 42.1 g, respectively, in average of 44.8 g / 100 g DM per 100 g dried matter (DM) in Experiments A, B and C (Table 30). On the other hand, the content of reducing sugar was 50.5 ± 0.52 by mixing the biomass of Experiment B and Experiment C. This means that about 50% of the dry matter weight is biomass containing fermented sugars, so the HR biomass produced by this method can be evaluated as a very good quality biomass. As a result, the indoor incubation method can be used as a suitable method to obtain high quality HR biomass at low cost (easy management) for a short period of time.

Experiment number After 7 days of culture, HR growth (g / 1.0 ㎡) carbohydrate
(g / 100 g DM) Dehydrated fresh weight (FW) In building DW / FW (%) Glucose Reducing sugar One 137.3 19.83 14.44 47.88 + 5.01 - 2 131.4 19.06 14.50 44.36 ± 0.29 50.5 ± 0.52 3 199.5 21.26 10.66 42.12 ± 1.97

Example  16: Greenhouse Large In culture  High quality (carbohydrate High content ) HR  massive production

16-1. SBC  Throughput Variance ( HR - d37 )

To investigate the difference in HR productivity and carbohydrate accumulation according to SBC throughput difference when cultivating HR in greenhouse condition near to natural state. Cultivation was carried out by a two-step culture method. That is, in the case of the first stage culture, 100 L of 1 × mDM + SBC50 medium was supplied to a rectangular small tray (0.75 × 1.99 m = 1.5 ㎡) made of wood, and the prepared HR seed was inoculated And cultured for 3 days. After that, 500 L of medium was supplied to a large rectangular tray (3.6 m × 1.8 m = 6.48 ㎡) made of vinyl, and HR was transferred (about 180 g in dehydrated fresh weight) And cultured. The water level was about 7.5 ㎝ were cultured in low average temperature 28 ℃ / night Average temperature 22 ℃, 13 sigan greenhouse conditions of photoperiod and light intensity 150-500 μmol m ² s ^-1 for 7 days. I shot the shade when the light was on. During the two-step culture, the nutrients were supplied three times, that is, at the start of the culture, the second day after the start of the culture, and the fourth day after the start of the culture. Each of the medium compositions was 2 × mDM + SBC50 , SBC50 , 2 × mDM NaSiO ₃₎ + it was SBC100 and SBC200. During the incubation period, the broth was maintained at a level of stirring once a day to circulate the culture. After the cultivation, HR after growth was collected in a paddy field, washed once with tap water, dehydrated, and dehydrated fresh weight was measured and dried in a hot air drier at 45 ° C for 2 days. On the other hand, the HR samples during the culturing were taken in the same manner as above, and the amount of glucose produced was examined in each period. In the case of glucose content in HR biomass, enzymatic hydrolysis was first performed using four enzyme complexes, and the resulting glucose content was analyzed by a biochemical analyzer (YSI 2700 SELECT ^™ , YSI Life Sciences, USA) See Example 2).

As a result, the biomass productivity when SBC100 or SBC200 was treated with the third nutrient in the second stage culture was 12% higher than that with SBC100 treatment in dehydrated fresh weight, and 3.4% (Table 31).

Culture number When supplying tertiary nutrients
SBC concentration HR growth after completion of culture (g / 1.0 m2) Dehydrated fresh weight (FW) Building (DW) DW / FW (%) HR-d37A 100ppm 253.5 30.00 11.84 HR-d37B 200ppm 283.8 31.02 10.93

On the other hand, the degree of carbohydrate accumulation between two SBC treatments was compared. As a result, glucose production was higher in the case of high SBC throughput, and sugar content tended to be higher in later harvest (FIG. 6). This means that the SBC contributes to the transition to the party. Overall, SBC treatment positively contributes to HR biomass growth and carbohydrate accumulation. From these results, it was assumed that the lower sugar content of HR-d33 was related to the higher nitrogen / phosphorus concentration due to the 2-fold mDM supply during nutrient supply during culture.

16-2. pH  Adjustment difference ( HR - d38 )

To investigate the difference of HR productivity and carbohydrate accumulation according to the presence or absence of pH adjustment in the culture medium when cultivating HR in a greenhouse condition close to the natural state. Cultivation was carried out by a two-step culture method. That is, in the case of the first stage culture, 100 L of MDM + SBC50 medium was supplied to a small rectangular tray (0.75 × 1.99 m = 1.5 ㎡) made of wood and the prepared HR seed was inoculated for 3 days Lt; / RTI > After that, 500 L of medium was supplied to a large rectangular tray (3.6 m × 1.8 m = 6.48 ㎡) made of vinyl, and HR was transferred (about 200 g in dehydrated fresh weight) And cultured. The water level was about 7.5 ㎝, and it was cultured for 10 days at a day average temperature of 28 ° C / night at 22 ° C, 13 hours of light period and 150 - 500 μmol m ² s ^-1 of light intensity, It was cloudy weather. I shaded the shade at the time of the strong light. Nutrients during stage 2 cultures five times i.e., culture started after 0, 3, 3.67, 5.69, bar hayeotneun supplied to the 8.67 days, respectively, of the medium composition is 2 × mDM + SBC100, SBC100, 2 × mDM (only K ₂ HPO ₄ is 1 x, NaSiO ₃ No addition) + SBC100, SBC200 and SBC50. During the incubation period, the broth was maintained at a level of several times a day for circulation of the culture. During the incubation, the pH of the culture medium was adjusted to 10 N HCl solution so as to be between pH 10.0 and pH 10.3. After the cultivation, HR after growth was collected in a paddy field, washed once with tap water, dehydrated, and dehydrated fresh weight was measured and dried in a 45 ° C hot air drier for 2 days. On the other hand, the HR samples during the culturing were taken in the same manner as above, and the amount of glucose produced was examined in each period.

As a result, no significant difference was found between the two treatments when comparing the biomass productivity between the pH of the culture medium and the control (Table 32).

Culture number PH adjustment during incubation HR growth after completion of culture (g / 1.0 m2) Dehydrated fresh weight (FW) Building (DW) DW / FW (%) HR-d38A Adjusted 342.7 37.2 10.90 HR-d38B No adjustment 372.5 38.1 10.20

On the other hand, the degree of carbohydrate accumulation between the two treatments was compared. As a result, the amount of glucose produced in the pH regulator was about 15% higher, and its content was excellent as 45-48 g / 100 g of glucose DM. The amount of glucose produced from the 8th day after the second stage of culture to the end of the culture was almost the same, indicating that the carbohydrate accumulation had already reached saturation (FIG. 7). Therefore, it was considered that the pH control in the culture medium was important when mass production of high quality HR was performed. It is considered that the harvesting time of this experiment condition could be advanced within 8 days after the start of the second stage cultivation.

16-3. Medium composition  Change and timing of supply HR Of greenhouse cultivation HR - d33A )

When HR was cultivated in a greenhouse condition closer to the natural state, the productivity and carbohydrate accumulation were examined by controlling the composition of the medium and the timing of feeding. Cultivation was carried out by a two-step culture method. That is, in the case of the first stage culture, 100 L of 1 × mDM + SBC50 medium was supplied to a rectangular small tray (0.75 × 1.99 m = 1.5 ㎡) made of wood, and the prepared HR seed was inoculated And cultured for 3 days. After that, 500 L of medium was supplied to a large rectangular tray (3.6 m × 1.8 m = 6.48 ㎡) made of vinyl, and HR was transferred (about 200 g in dehydrated fresh weight) And cultured. The water level was about 7.5 ㎝ were cultured for 10 days in a low average temperature 26 ℃ / night Average temperature 22 ℃, 13 sigan greenhouse conditions of photoperiod and light intensity 80-500 μmol m ² s ^-1. During the two-step culture, the nutrients were supplied three times, that is, three days after the start of the culture, three days after the start of the culture, and six days after the start of the culture. The composition of each medium was 1 x mDM + SBC50 + KNO ₃ 20 ppm + KH ₂ PO ₄ 4 ppm, 1 × mDM (NaSiO 3 _{Additive-free) + SBC100 + KNO 3 20 ppm} + KH 2 PO 4 4 ppm and 1 × mDM (NaSiO ₃ No addition) + SBC110. During the incubation period, the broth was maintained at a level enough to stir the culture several times a day to circulate the culture. After the cultivation, HR after growth was collected in a paddy field, washed once with tap water, dehydrated, and dehydrated fresh weight was measured and dried in a 45 ° C hot air drier for 2 days. Analysis of the composition of these samples was commissioned by Korea Food Research Institute. The components were analyzed by Korea Food Research Institute (KFDA). The moisture content was determined by the method of the atmospheric pressure heating method (2009), the crude lipid was determined by the food cycle (2009), the ether extraction method, the protein by Kjeldahl method, Respectively. As a result, the composition of the sample (HR-d33) was 69.9% of carbohydrate, 12.7% of protein, 2.5% of total lipid, 3.3% of moisture and 11.6% of ash.

On the other hand, the amount of glucose and the amount of reducing sugar in the same sample were examined as in the previous experiment. As a result, the glucose and reducing sugar contents from HR biomass were very high, 52.0 ± 0.29 and 59.7 ± 2.38 g, respectively, per 100 g of dry matter (Table 32). The productivity of biomass was 35.3 g per 1 m2 of dry matter (Table 33). This was twice as much as that in the indoor condition and the carbohydrate accumulation was also increased. Therefore, it was reaffirmed that it is possible to produce higher quality HR with less energy input and management cost than the facility cost and operation management cost (especially energy and effort required for microalgae harvesting) required for single cell microalga cultivation. Management technology was expected to enable the production of a greater quantity of high quality HR.

Culture number HR growth after completion of culture (g / 1.0 m2) carbohydrate
(g / 100 g DM) Dehydrated fresh weight (FW) Building (DW) DW / FW (%) Glucose Reducing sugar HR-d33A 290.3 35.3 12.17 52.0 ± 0.29 59.7 ± 2.38

Example  17: High protein content HR  production( HR - PT1 )

Higher protein content among algae biomass components increases the value of utility as feed, fertilizer or food. This study was conducted to develop the culture technology required to produce HR biomass with relatively high protein content.

200 L of 1 × mDM, SBC50, 20 ppm of KNO ₃ and 4 ppm of KH ₂ PO _{4 was} supplied to a rectangular tray (2.0 m × 0.8 m = 1.6 ㎡) made of wood and made of vinyl resin HR seeds with a net length of 2-3 cm were deemed to be 3-4 g / L based on dehydrated fresh weight. Then, the cells were cultured for 7 days at a day-average temperature of 25-30 ° C / night at an average temperature of 20-25 ° C, a 14-hour photoperiod and a light intensity of 80-300 μmol m ² s ^-1 . On the 4th day after the initiation of the culture, nutrients were additionally supplied, the amount of the supplemented medium was 1/1000 of the remaining culture medium, and the final concentrations of the components of the supplied medium were 1 × mDM, SBC50, KNO ₃ 20 ppm and KH ₂ PO ₄ 4 ppm. The final pH of the culture was then adjusted to pH 8.5 by the addition of 1 N HCl and the culture was maintained at the level of several times a day for circulation throughout the culture. After the cultivation, HR after growth was collected in a paddy field, washed once with tap water, dehydrated, and dehydrated fresh weight was measured and dried in a 45 ° C hot air drier for 2 days. Analysis of the composition of these samples was commissioned by Korea Food Research Institute. Moisture was determined according to the food circulation (2009), atmospheric pressure heat drying method, crude lipid was determined according to the food circulation (2009) ether extraction method, protein was measured using Kjeldahl method and ash was determined according to Food Release (2009) ash test method. The composition of HR - PT1 was 56.1% of carbohydrate, 24.4% of total protein, 1.5% of total lipid, 3.7% of moisture, 14.3% of ash, , The protein content was about twice as high as the other samples (see Example 16), while the carbohydrate content was able to produce lower HR biomass.

Example  18: Dry storage methods to save drying space

Since HR is a net-shaped entity, it exists in swollen state after dehydration. Therefore, it requires considerable space to dry it, and it may take a lot of dry energy. Fortunately, HR seemed to be somewhat sticky on the surface of the cell, so it would seem that the volume would be significantly reduced if it was put into the natural state or sprayed with a small amount of natural adhesive and dried. Therefore, we tried to test the possibility.

The dehydrated HR was put in a basket without any treatment, and the basket was obtained by pressing and fixing by using a rubber band. Meanwhile, the 10% starch solution was dissolved by heating, and a small amount of the cooled solution was sprayed with an atomizer. Then, it was hot-air dried at 45 ° C for 2 days. The crush-dried HR mat (matt) was cut to a certain size to make a block, and the size and weight thereof were measured. Then, water was added to make 100% moisture, and then the block size and weight were measured .

As a result, it was possible to drastically reduce the drying volume even by crushing and drying the dehydrated HR itself without starch solution treatment (FIG. 8), and it was possible to save at least 5 times the drying space. The volume recovery and moisture retention of the crushed and dried HR were increased 4.73 times by volume and 18.07 times by weight, respectively. That is, it was confirmed that the pressed HR had the ability to absorb and retain water about 18 times its own weight (Table 34). The above results are also shown in the case of starch solution treatment.

Compressed HR sample Water supply After watering Volume (cm < 3 & Weight (g) Volume (cm < 3 & Weight (g) One 3.2 x 4.0 x 0.3 = 3.84 0.93 3.3 x 4.1 x 1.3 = 17.59 16.61 2 3.0 x 4.0 x 0.3 = 3.60 0.96 3.0 x 4.0 x 1.5 = 18.00 17.01 3 3.2 x 3.8 x 0.3 = 3.65 0.97 3.3 x 3.8 x 1.7 = 16.83 18.06

In addition to the method of reducing the storage volume through squeeze drying, the possibility of the vacuum packaging method was examined. When using a conventional vacuum packing machine, the storage volume could be reduced by about 20 times as compared with the case without vacuum packaging. Therefore, a vacuum storage method is considered to be very useful for efficiently storing a large amount of high-quality biomass for a long period of time.

On the other hand, in the case of storage at room temperature, there is a fear of being corrupted during storage due to moisture absorption. To prevent this, dehydrated HR biomass was immersed in a weak acid or weak alkali solution, then dehydrated again, dried, and then compressed and dried or vacuum-preserved as described above, whereby high quality biomass could be stored for a long period without corruption.

Example  19: White Pig HR  Acquired dry matter

The HR biomass produced is green when dried by itself. However, bleaching it to obtain a whitening HR dried body may facilitate glycosylation, or the whitened HR dried body itself may be used for other purposes. Therefore, in order to obtain HR biomass decomposed by chlorophyll, it is dehydrated and then dried in sunlight or UV for 10 days or more (FIG. 9), and a compound generating active oxygen (hydrogen peroxide, peracetic acid, sodium hypochlorite, hypochlorous acid, Ozone, titanium dioxide, etc.) 0.1% to 10% concentration, HR biomass was immersed and exposed to natural light or UV for 0.1-10 days to obtain completely bleached biomass.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (18)

A method for mass production of a Hydrodictyon algae comprising the steps of:
(a) culturing (i) said net-like algae in a first culture tank containing a medium to produce a first alga net in the net;
(Ii) A second algae net containing the medium was grown in the second algae net to grow the algae net at a low density (10-100 ng of 1.5-m 2 flat area of the culture tank of 100 L volume) Fabricating a net; And
(Iii) inducing a first seedling net or a second seedling net at a low density and growing a birdnet in a third culture tank containing the medium to induce a good seed of the endemic bird Preparing a good seed of a Hydrodictyon algae; Wherein the superior seed is a net-like bird cell population with a net-like, green-colored, long-cylindrical cytomorphic character without net disintegration; And
(b) Inoculating and culturing the medium with a high-quality seed of the above-mentioned nettle bird.
delete delete delete delete The method according to claim 1, wherein said nettles are selected from the group consisting of Hydrodictyon reticulatum , Hydrodictyon africanum and Hydrodictyon patenaeforme Or a mixed bird of at least two species.
delete delete delete delete delete delete The method of claim 1, wherein the steps (a), (b) or (a) and (b) are performed at a temperature of 15-35 ° C, a luminous intensity of 10-1500 μmol ^-2 s ^-1 , a pH 7.0-11.9, Wherein the culturing is carried out under the conditions of an hour light photoperiod, a water depth of 0.5 cm or more, and 0.01 to 50.00 g of dehydrated algae seed / 0.1 m2.
The method of claim 1, wherein said step (a), (b) or (a) and (b) comprises a carbon source, 5-60 ppm nitrogen (based on calcium nitrate) and 6.2-74.4 ppm phosphorus Wherein the culturing is carried out at a pH of 9.0-11.9, a light intensity of 80-500 μmolm ^-2 s ^-1 and a temperature of 25-35 ° C. in the presence of the enzyme.
The method of claim 1, wherein the steps (a), (b) or (a) and (b) comprise a carbon source, 10-300 ppm nitrogen (based on calcium nitrate) and 2.0-24.8 ppm phosphorus Wherein the step of culturing is carried out under the conditions of pH 7.0-11.9 and 25-35 ° C in the presence of a culture medium.
The method of claim 1, wherein the step (a), (b) or (a) and (b) comprises 10-30 mg / L calcium nitrate, 5-20 mg / L potassium phosphate, 15-35 mg / magnesium sulfate, 10-400 ㎎ / L sodium _{bicarbonate, 1-5 ㎎ / L EDTA Na 2} , 1-5 ㎎ / L FeNa · EDTA, 1-3 ㎎ / L NaOH, 1-5 ㎎ / LH 3 BO 3 , Mn ²⁺ ions, and 0.5-1.5 mg / L (NH ₄ ) ₆ Mo ₇ O ₂₄ .
The method of claim 1, wherein the step (a), (b), or (a) and (b) comprises 30-50 mg / L calcium nitrate, 5-20 mg / L potassium phosphate, 5-15 mg / L ammonium sulfate, 40-60 mg / L magnesium sulfate, 40-60 mg / L sodium chloride, 10-20 mg / L ferric citrate, 50-350 mg / L sodium bicarbonate, 1-5 mg / L EDTA Na ₂ , 1-5 ㎎ / L FeNa · EDTA , 40-80 ㎎ / L sodium _{silicate, 1-5 ㎎ / LH 3 BO 3} , Co 2+, Cu 2+, Mn 2+, Zn 2+ ions and 0.5-1.5 Wherein the composition is cultured in a composition for culturing a net horseshoe algae containing ₁₀ mg / L (NH ₄ ) ₆ Mo ₇ O ₂₄ .
The method of claim 1, wherein the step (a), (b) or (a) and (b) comprises 35-55 mg / L potassium nitrate, 2-6 mg / L potassium phosphate, Sodium chloride, 50-150 mg / L sodium bicarbonate, 0.5-2.0 mg / L NaOH, Cu ²⁺ , Fe ³⁺ , Mg ²⁺ and Zn ²⁺ ions Wherein the composition is cultured in a composition for culturing a barnyard algae.

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