KR20150081038A - Method for Conversing Low-Carbohydrate to High-Carbohydrate Hydrodictyon Algae - Google Patents

Abstract

The present invention relates to a method for converting low carbohydrate hydrodictyon algae to high carbohydrate hydrodictyon algae, the invention comprising the steps of: (a) inoculating low-carbohydrate hydrodictyon algae to a culture medium of low-carbohydrate hydrodictyon aglae comprising a carbon source, a nitrogen source and a phosphorus source; and (b) culturing the end product in the step (a) and converting to high-carbohydrate hydrodictyon algae. The high-carbohydrate hydrodictyon algae cultured by the present invention is non-edible algae and thus, there is no ground to cause a food price soaring and ethical problems if an industrial demand for biomass rises. In addition, the high-carbohydrate hydrodictyon algae can be produced at a low cost by using waste resources (wastewater, waste CO_2) and thus, a stable supply of resource is possible. Further, the high-carbohydrate hydrodictyon algae has characteristics of a high content of useful components (protein, carbohydrate) and an easy saccharification conversion and therefore, can be valuable and usefully used as a main material for biochemical products in the future.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for converting a hydrodictyon bird into a carbohydrate-

The present invention relates to a method for converting a carbohydrate-low containing Hydrodictyon algae into a carbohydrate-rich high netting algae.

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.

In recent years, many researchers have been putting a great deal of effort into finding and improving algae species that are more valuable for use, producing algae biomass, and developing technology for exploiting them. 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. Rojan et al., Bioresource Technology 102: 186-193, 2011), which is incorporated herein by reference in its entirety. 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 low-cost 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.

Hyrodictyon algae, which are the object of the present invention, can be used only in the dry and dry conditions when collected after cultivation, and thus can be used as a single-celled microalgae [chlorella, scenedesmus ) And the like], the sample can be collected at 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).

In the present invention, studies on the physiological ecological characteristics have been carried out to some extent in the present invention. However, it has been reported that the technology related to the mass production method is capable of producing net-like birds by supplying wastewater and carbon dioxide to the culture facility using the slope US 2011/0307976 A1). However, in general, algae require a large amount of water when culturing, and the cost of nutrients supplied to the algae is high. Therefore, in order to reduce these costs, wastewater having a high nitrogen / phosphorus content is used. The carbohydrate content is 20-30% or less as compared with the dry weight, and thus low-quality biomass is produced. There is no report yet on how to convert these low quality nettles into very high quality nettles with high carbohydrate content.

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.

The present inventors have made extensive efforts to achieve the conventional problems and the technical demands of the related art. The present invention relates to a method for developing a high-quality mass production and high-quality mass production of a Hydrodictyon algae having a large variation in quality according to an external cultivation environment, and is capable of rapidly producing a carbohydrate- The present inventors have completed the present invention by developing a method for converting the biomass into a high-yielding biomass.

Accordingly, it is an object of the present invention to provide a method for converting carbohydrate-low netting barley birds into carbohydrate-rich high netting barley birds.

It is another object of the present invention to provide a carbohydrate-rich, net-like algae.

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 of converting a carbohydrate-low containing Hydrodictyon algae into a carbohydrate-rich high netting algae comprising the steps of:

(a) inoculating a carbohydrate-low netting bait bird into a culture medium of a carbohydrate-low netting bait bird containing carbon source, nitrogen source and phosphorus; And

(b) culturing the resulting product of step (a) and converting it into a carbohydrate-rich, net barley bird.

The present inventors have made extensive efforts to achieve the conventional problems and the technical demands of the related art. The present invention aims to develop a method for mass production and quality improvement of a net barb bird having a large variation in quality depending on an external cultivation environment and cultivating a net barb bird with a carbohydrate- To develop a method for

The method of converting the carbohydrate-low netting bird of the present invention into the carbohydrate-rich net netting bird is described in detail in each step.

Step (a): Carbohydrate- Low content Netting  Inoculation of birds

First, the present invention is to inoculate a carbohydrate-low netting bait bird into a culture medium of a carbohydrate-low netting net equilibrium containing carbon source, nitrogen source and phosphorus.

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. Microalgae and Protozoa), DM (Diatom Medium), modified DM (mDM), HR-v1 or AW-v1.

The culture medium of the present invention is a culture medium containing a carbon source, a nitrogen source and a phosphorus source.

The carbon source may be any carbon source conventionally used in the art. For example, the carbon source may be selected from bicarbonate, carbonate or carbon dioxide. According to an embodiment of the present invention, the carbon source is bicarbonate.

The culture medium has a concentration of 10 to 30 mg / L of the carbon source based on the bicarbonate, and can be additionally supplemented with the carbon source to convert it into a carbohydrate-rich high netting bird. According to one embodiment of the present invention, the final concentration is 100-400 mg / L, based on sodium bicarbonate, and in other embodiments, 180-350 mg / L.

The nitrogen source may be any nitrogen source conventionally used in the art. For example, the nitrogen source may utilize at least one nitrogen source selected from the group consisting of nitrate, ammonium, urea and ammonium nitrate, , Said source of nitrogen is potassium nitrate.

Wherein the culture medium has a concentration of 10-30, 13-27, or 15-25 mg / L of the nitrogen source based on calcium nitrate, and contains the nitrogen source at a half concentration to convert it into a carbohydrate- can do. According to one embodiment of the invention, it has a concentration of 5-15, 7-12 or 9-11 mg / L, based on calcium nitrate.

The personnel may be any personnel conventionally used in the art, for example, the personnel may be selected from potassium primary phosphate or secondary potassium phosphate, according to one embodiment of the present invention, 2 potassium.

The culture medium has a concentration of 5-20, 7-17, or 10-14 mg / L based on potassium phosphate, and converts the population into a carbohydrate- Concentration. According to one embodiment of the invention, it has a concentration of 3-10, 4-8 or 5-7 mg / L based on potassium secondary phosphate.

The culture medium additionally contains at least one ingredient selected from the group consisting of magnesium sulfate, EDTA Na ₂ , EDTA sodium iron, sodium hydroxide, boron, manganese, zinc, molybdenum and combinations thereof in addition to the carbon source, do.

The culture medium of the present invention can be prepared using distilled water, distilled water other than degassed tap water, ground water, rainwater, river water or a mixture thereof.

In addition, since the method of converting the carbohydrate-rich high-net-content bird of the present invention into an alginic acid medium, it is preferable to add sodium hydroxide, potassium hydroxide, sodium carbonate or a mixture thereof to the medium in order to impart the alkalinity of the medium, .

In order to convert the carbohydrate-rich, high-molecular-weight algae of the present invention, the above culture medium may be prepared in the range of x 1 to x 2. According to one embodiment of the present invention, the culture medium is x 1 to x 1.5.

A carbohydrate-low containing net barbarian algae is inoculated into the culture medium.

As used herein, the term 'carbohydrate-low netting barley bird' means that the glucose content is less than 30 g based on 100 g dry weight of net barren bird, and the 'carbohydrate-rich net barley bird' Means that the glucose content is 30-60 g, 35-55 g or 35-50 g and the content of reducing sugar is 40-65 g, 45-65 g or 50-65 g based on 100 g of the dry weight of the composition.

According to one embodiment of the present invention, the 'carbohydrate - low content net barbarian' is a net barb bird used for sewage treatment.

The net barn bird which can be applied to the method of converting the carbohydrate-low content net barn birds of the present invention into carbohydrate-rich high net barn birds is the hydrodiction species.

The hydrodiction species may be selected from the group consisting of Hydrodictyon reticulatum , Hydrodictyon africanum, africanum ) and hydrodictyon patenaeforme . The term " hydrocortisone "

According to one embodiment of the present invention, the net bargain is a hydrodiction reticulatum.

The method of converting a carbohydrate-low netting bird of the present invention into a carbohydrate-high netting bird is converted into a carbohydrate-rich netting bird with photosynthesis while the carbohydrate- It is preferable to supply a culture medium of 2-40 L, 2-30 L or 2-20 L per unit area (m 2).

According to one embodiment of the present invention, the water depth is 0.2-10 cm, 0.4-10 cm, or 0.6-10 cm.

When the density of the carbohydrate-low-content netting bird inoculated on the culture medium is high, the efficiency of photosynthesis of the netting bird is lowered, so the inoculum should be inoculated at an appropriate density.

According to one embodiment of the present invention, the inoculum of step (b) is inoculated with a net barren bird of 1-35 g / 0.1 m 2, 1-30 g / 0.1 m 2 or 5-25 g / 0.1 m 2 on dehydrated fresh weight basis .

In order to increase the photosynthetic efficiency of the carbohydrate-low containing net barb bird inoculated on the culture medium, cultivation is carried out using a culture tank provided with a reflecting film on the bottom surface.

Wherein the reflective film is made of a metal selected from the group consisting of aluminum, silver, gold, copper, stainless steel, titanium nitride and nickel. According to one embodiment of the present invention, the reflective film is a reflective film composed of aluminum.

In order to further increase the production amount per unit area of the bird-bearing algae of the present invention, the culture system of the culture tank may be appropriately equipped and cultured.

Step (b): Carbohydrate- High content Netting  Conversion to birds

The resulting product of step (a) is then cultured and converted to carbohydrate-rich, net barley algae.

The culture is carried out at a suitable pH. According to one embodiment of the present invention, the optimum pH is 9.8-11.9, 9.8-11.0, or 10.0-10.5.

The culture is carried out at an appropriate temperature. According to one embodiment of the present invention, the suitable temperature is 20-40 캜, 22-38 캜 or 25-36 캜. According to another embodiment of the present invention, the daytime and nighttime temperatures can be set differently.

The cultivation is carried out under an appropriate light. According to one embodiment, the proper light intensity is ^{50-500 μmol m -2 s -1, 50-350} μmol m -2 s -1 or 60-300μmol m ^-2 s ^-1.

The incubation is carried out under appropriate light period. According to one embodiment of the invention, the photoperiod is 4-18 hours, 8-16 hours or 12-15 hours.

The culture is carried out under a suitable culture period. According to one embodiment of the present invention, the incubation period is 2-5 days, 3-4 days, or 3 days depending on the culture temperature and the light intensity.

According to another aspect of the present invention, the present invention provides a carbohydrate-rich highland barley bird produced by the method.

The carbohydrate-rich high molecular weight algae of the present invention can be prepared by the above-mentioned " method of converting the " carbohydrate-low content Hydrodictyon algae into carbohydrate-rich high netting algae. &Quot; Accordingly, the description common to both is omitted in order to avoid the excessive complexity of the present specification.

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

(a) Nematode algae is a marine algae that has high growth potential with high growth and easy carbohydrate accumulation, and has excellent advantages as an industrial biomass because it has very easy glycosylation properties.

(b) Carbohydrate-rich, high-netting bird cultured by the present invention is an algae, which is a non-edible resource. Therefore, when the industrial demand for biomass is increased, there is no possibility of food price increase and ethical problems, (Carbohydrate, protein, etc.) and easy glycosylation because it can be produced at low cost by using the wastewater and waste CO ₂ . It is worthy of being usefully utilized as a raw material.

Figure 1 schematically illustrates a method for converting carbohydrate-low netting birds grown in an outdoor or water quality purification process according to the present invention to HR biomass of high quality (high carbohydrate content) at a lower cost and at a lower cost.
FIG. 2 is a graph showing the growth and carbohydrate accumulation rate of algae in the net when the algae were cultured at various pHs.

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: Effect of temperature on growth of horses (HR) and carbohydrate accumulation

1-1. Effect of thermostat treatment

HR growth and carbohydrate accumulation. To a 5 L vinyl plastic bottle was added 1 x mDM [1-fold modified Diatom Medium, 20 mg / L Ca (NO ₃ ) ₂ 4H ₂ O, 12.4 mg / L K ₂ HPO ₄ , MgSO ₄ * 7H _{2 O 25 ㎎ / L, NaHCO} 3 16 ㎎ / L, EDTA Na 2 2.25 ㎎ / L, FeNa * EDTA * 2H 2 O 2.25 ㎎ / L, NaOH 1.41 ㎎ / L, H 3 BO 3 2.5 ㎎ / L, MnCl ₁ L of SBC100 (sodium bicarbonate 100 ppm) culture solution containing ₂ * 6H ₂ O 1.4 mg / L and (NH ₄ ) 6 Mo ₇ O ₂₄ * 4H ₂ O 1.0 mg / L) The HR seeds (individual length 1-2 cm) stored at -20 ° C were taken out and 0.2 g of the dehydrated fresh weight obtained by removing the water with a tissue was taken and administered to the culture solution. 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. However, in the case of buildings, the ratio of building to building was higher in the order of 35 ° C> 30 ° C> 25 ° C> 20 ° C> 15 ° C, and the proportion of the building increased with temperature and reached about 20% at 35 ° C (Table 1). 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 1). In Table 1, 'DM' stands for Dried Matter and means dry matter or building with moisture content around 5%.

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

1-2. 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, when the day and night temperature difference was fixed at 10 ℃, the growth temperature and growth of glucose were higher than those of 25 ℃ / night at 15 ℃ and 30 ~ 35 ℃ / night at 20-30 ℃. 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, it is considered to be preferable to adjust the daytime or nighttime temperature to be at least 20 ° C or more, preferably 25 ° C or more in order to increase carbohydrate accumulation in HR biomass production.

Example  2: culture solution pH end Net horse ( HR ) Effect on growth and carbohydrate accumulation

The pH of the culture medium in the algal culture has a significant effect on the growth and metabolic processes, and the degree is known to vary from species to species. This experiment was conducted to investigate the effect of culture pH on biomass productivity and carbohydrate accumulation in cultured horses (HR).

The HR material used for the experiment was HR seeds grown in 1 × mDM + SBC50 medium for 3 days to collect 2-4 ㎝ of net length and stored at 15 ℃. The circulation culture tank (0.643 m 2) was filled with tap water and deaerated for 2 days or more, and then a culture medium was prepared so as to have a 1.5 × mDM + SBC200 medium composition. The pH was then adjusted by running an automatic pH controller containing 2 N hydrochloric acid and 2 N KOH solution and kept constant throughout the incubation period. The prepared HR plant material was inoculated with 45 g in a 100 L culture medium based on dehydrated fresh weight. Culturing conditions were incubated for 7 days under greenhouse conditions with constant stirring (7.5 rpm / min) at 26-27 ° C, 14 h light intensity, and 60-400 μmol ^-2 s ^-1 light intensity using a stirring device. About 5-6 L of tap water was added daily to the culture tank to supplement the evaporated water content. After one week incubation, the HR was collected in a guar gang, dehydrated by a dehydrator, and dehydrated fresh weight (FW) was measured and dried in a 45 ° C hot air drier for 2 days. In order to investigate the degree of glucose production from HR biomass, HR was hydrolyzed by enzymatic hydrolysis in a previously reported method (Kim et al., Applied Biosystems, 32: 85-97, 2012), and the glucose content in the hydrolyzate was measured by a biochemical analyzer And analyzed.

 As a result, HR biomass productivity was the lowest at pH 7.0 and the highest at pH 10.5. That is, as the pH was increased up to pH 7- 10.5, the biomass productivity increased proportionally. But decreased at pH 11. The ratio of fresh weight to fresh weight was proportional to the increase of pH in the range of pH 7 - 11.0. On the other hand, if the carbohydrate productivity is expected to be obtained through the glucose content in the hydrolyzate, the glucose production is increased proportionally as the pH is increased in the range of pH 7.0-11.0 of the present experimental conditions.

Therefore, in order to increase the carbohydrate accumulation while continuing the growth, it is preferable to cultivate the culture while adjusting the pH of the culture liquid to pH 10.0-10.5, and if it is desired to enhance the carbohydrate accumulation regardless of the growth, . However, at pH 11.5-11.8 and above, caution was needed because the activity of HR cells could be rapidly decreased due to high alkali.

Example  3: Nitrogen and phosphorus content HR  Effects on growth and carbohydrate accumulation

The effect of HR ratio on the HR growth and carbohydrate accumulation was investigated by adding the main nitrogen source of HR algae culture and Ca (NO ₃ ) ₂ 4H ₂ O (CN) and K ₂ HPO ₄ (PP) 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 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 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. Therefore, HR growth was more dependent on nitrogen content than phosphorus content in culture (Table 3).

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 decreased to 83.2 %. When the concentration of nitrogen (CN) was decreased to 5 ppm and 10 ppm, the decrease of glucose content was very low as 92.4% and 99.1% of 20 ppm treatment, 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 3.1 ppm and 6.2 ppm Glucose content was 93.8%, 79.6% and 94.1%, respectively, when they were decreased, respectively (Table 3). Therefore, the proper concentration of carbohydrate accumulation in HR is more important than the appropriate ratio of nitrogen (CN) and phosphorus (PP), and the content of phosphorus (PP) is 6.2 ppm to 24.8 ppm and the concentration of nitrogen (CN) It was found that preparation of 5 ppm to 20 ppm level was favorable 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 12.4 0.4032 0.89 0.028 (75.1) 33.28 占 1.17 (92.4) 10 12.4 0.8065 1.04 0.042 (87.8) 35.70 + - 0.32 (99.1) 20 12.4 1.6129 1.19 ± 0.007 (100.0) 36.03 + - 1.65 (100.0) 40 12.4 3.2258 1.20 0.028 (101.3) 29.96 + -0.44 (83.2) 20 3.1 6.4516 1.22 ± 0.042 (102.1) 30.59 + - 0.49 (79.6) 20 6.2 3.2258 1.17 + - 0.021 (97.5) 36.17 + - 0.33 (94.1) 20 12.4 1.6129 1.20 ± 0.007 (100.0) 38.43 + - 0.85 (100.0) 20 24.8 0.8065 1.21 + - 0.014 (101.3) 36.03 + - 1.58 (93.8)

Example  4: Sodium bicarbonate ( Sodium bicarbonate ; SBC , NaHCO ₃ ) Treatment on HR growth and carbohydrate accumulation in greenhouse culture

The main inorganic carbon source for algal culture is CO ₂ . This means that as the pH of the solution increases in the solution _, 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) to investigate the concentration range of HR growth and carbohydrate accumulation.

The HR material used for the experiment was HR seeds grown in 1 × mDM + SBC50 medium for 3 days to collect 2-4 ㎝ of net length and stored at 15 ℃. The autoclave was filled with degassed tap water and incubated in a circulating culture tank (0.643 ㎡) to prepare a culture medium having a composition of 1.5 × mDM. Then, SBC was added to 100-400 ppm and then an automatic pH control device containing 2 N hydrochloric acid and 2 N KOH solution To adjust the pH to 10.5 and to remain constant throughout the incubation period. The prepared HR plant material was inoculated with 45 g in a 100 L culture medium based on dehydrated fresh weight. Culturing conditions were incubated for 6 days under a greenhouse condition with stirring at a constant speed (7.5 rpm / min) using an agitator at a constant temperature of 26-27 ° C, a 14-hour photoperiod and a light intensity of 60-500 μmolm ^-2 s ^-1 . About 5-6 L of tap water was added daily to the culture tank to supplement the evaporated water content. After one week incubation, the HR was collected in a guar gang, dehydrated by a dehydrator, and dehydrated fresh weight (FW) was measured and dried in a 45 ° C hot air drier for 2 days. In order to investigate the degree of glucose production from HR biomass, HR was hydrolyzed by enzymatic hydrolysis in a previously reported method (Kim et al., Applied Biosystems, 32: 85-97, 2012), and the glucose content in the hydrolyzate was measured by a biochemical analyzer And analyzed.

As a result, in the case of HR growth, the higher the concentration of SBC treatment, the higher the growth rate. In the case of carbohydrate accumulation, glucose production was increased with SBC treatment concentration up to 300 ppm SBC treatment, but slightly lower than SBC 400 ppm treatment with 300 ppm treatment (Table 4 and Table 5). Therefore, considering the growth and carbohydrate accumulation, when HR is cultured at pH 10.5, SBC treatment concentration should be cultured within 200-300 ppm.

SBC (ppm) Initial dose
(FW g / 0.643 m ² ) HR growth during 3 days culture
(g / 0.643 m ² ) Glucose
(g / 100 g DM) Dehydrated fresh weight (A) In building (B) B / A (%) 100 45 g 196.3 19.59 9.98 23.64 ± 0.23 200 45 g 264.0 25.63 9.71 27.19 + - 0.12 300 45 g 303.1 29.55 9.75 37.03 + - 0.14 400 45 g 314.7 32.79 10.42 36.44 + 0.21

SBC (ppm) Initial dose
(FW g / 0.643 m ² ) HR growth during 6 days culture
(g / 0.643 m ² ) Glucose
(g / 100 g DM) Dehydrated fresh weight (A) In building (B) B / A (%) 100 45 g 255.4 25.41 9.95 32.05 + - 0.35 200 45 g 286.6 28.53 9.95 32.56 ± 0.60 300 45 g 294.3 32.28 10.97 40.97 + - 0.32 400 45 g 309.6 36.13 11.67 38.86 + - 0.43

Example  5: Depth of culture medium ( water depth )this HR  Effects on Growth and Carbohydrate Accumulation

To investigate the difference of HR growth and carbohydrate accumulation according to the depth of culture medium during high - quality induction culture. (0.1 ㎡) supplied with 5 L or 10 L of culture medium of 1.0 × mDM + SBC200 (sodium bicarbonate 200 ppm) in various concentrations of HR (2-3 cm net length, building ratio 8.5% ) Were added and cultured for 5 days in an indoor growth chamber. The culture conditions were as follows: Fluorescent light intensity of 50-70 μmolm ^-2 S ^-1 , 25 ° C constant temperature, 14 hours light period. As a result, it was higher in water depth of 10 ㎝ than in 5 ㎝ depth when expressed as area unit (g / 0.1 ㎡) in growth increment, but higher in water depth of 5 ㎝ than 10 ㎝ depth in terms of volume unit (g / L) . In the 5 ㎝ depth cultivation, the growth rate was higher in the order of 5 g> 3 g> 1 g than in the initial inoculation amount. In the case of 10 ㎝ culture, the growth rate was increased in the order of 6 g> 2 g> 10 g Table 6). On the other hand, in the case of carbohydrate accumulation, the accumulation of sugar was decreased when the initial inoculation amount was the same, and the accumulation of sugar was decreased when the depth of water was same even in the same inoculation amount. Therefore, in order to obtain a high amount of HR biomass of carbohydrate, it is desirable to cultivate by adjusting the initial inoculation amount and adjusting the depth of culture medium as low as possible.

depth of water
(Medium volume) Initial dose HR growth after 5 days of culture (g in the building) Glucose productivity (g / 100 g DM) g / 0.1m ² g / 5L g / 0.1m ² g / 5L 5 cm
(5L) One One 1.51 + - 0.01 (1.425) ¹⁾ 1.51 + - 0.01 (1.425) ¹⁾ 46.75 ± 2.00 3 3 2.07 ± 0.09 (1.815) 2.07 ± 0.09 (1.815) 40.73 + 2.01 5 5 2.32 + - 0.07 (1.895) 2.32 + - 0.07 (1.895) 39.68 + - 0.66 10 cm
(10L) 2 One 2.19 0.08 (2.020) 1.10 + - 0.04 (1.010) 35.70 ± 1.13 6 3 2.86 + - 0.13 (2.350) 1.43 + - 0.07 (1.175) 34.44 ± 2.67 10 5 3.08 0.03 (2.230) 1.54 + - 0.02 (1.115) 33.72 + 0.64

1) The parentheses represent the actual increased growth during cultivation (day 5 - initial dose).

Example  6: At normal water depth HR  Carbohydrate accumulation by initial dose (5 cm  Water depth, 5L / 0.1 ㎡ `)

HR biomass with a glucose production of 27.0 g / 100 g DM or more was added to a living box (0.1 m 2) supplied with 5 L culture medium, and 10, 20, 30, 40 and 50 g based on fresh weight were added thereto and cultured under greenhouse conditions. At this time, the depth of 5 ㎝ culture was about 5 ㎝. The culture media used were 1 × mDM + SBC200 (in tap water) and cultured in the greenhouse for 5 days. HR biomass was collected and the growth and carbohydrate accumulation were measured by conventional methods. Environmental conditions for greenhouse cultivation are 90-350 μmolm ^-2 s ^-1 , day 25-30 ° C / night 20-25 ° C, and 14 hours light period.

As a result, there was no difference in the initial inoculation amount of HR in the case of the growth, and 2.1-2.9 g in the purely increased amount of culture during the incubation period of 5 L. However, in the case of glucose and reducing sugar production, the lower the initial inoculation amount, the higher the tendency. This is because the higher the initial inoculation density of HR, the higher the light transmittance and the more active the carbohydrate accumulation of carbon source.

Initial inoculation dose (FW g / 0.1m ² ) ¹⁾ HR growth (g / 0.643m ² ) ²⁾ Glucose
(g / 100 g DM) Reducing sugar
(g / 100 g DM) Fresh weight (A) In building (B) B / A (%) 10 16.78 ± 0.12
(6.78) * 2.91 ± 0.08
(2.142) * 17.34 46.82 ± 0.31 60.60 + - 4.72 20 25.89 + - 0.46
(5.89) * 4.19 ± 0.09
(2.654) * 16.18 46.57 + - 0.40 60.49 + - 0.93 30 34.28 + - 0.50
(4.28) * 5.23 ± 0.02
(2.926) * 15.26 40.31 + - 0.75 54.11 + - 0.55 40 42.28 + - 0.38
(2.28) * 6.01 ± 0.06
(2.938) * 14.21 39.86 ± 1.07 51.80 ± 1.76 50 51.05 + 0.78
(1.05) * 6.78 + 0.13
(2.230) * 13.28 38.85 + 1.54 49.61 + - 4.52

¹⁾ Medium Composition: 1 x mDM + SBC200 prepared in tap water

²⁾ Greenhouse conditions (14 hours photoperiod, natural light, 70-350 μmolm ^-2 s ^-1 )

* Figures in parentheses indicate freshly grown live / dry during the incubation period after the initial inoculation.

Example  7: Difference in culture composition at normal water depth HR  Effect of growth and carbohydrate accumulation (5 ㎝ depth, 5 L / 0.1 ㎡)

HR biomass with a glucose production of 25.5 g / 100 g DM or more was added to a living box (0.1 m 2) supplied with 5 L culture medium. At this time, the depth of 5 ㎝ culture was about 5 ㎝. In the case of nutrient supply, three conditions were set, T1 was Ca (NO ₃ ) ₂ 4H ₂ O and K ₂ HPO ₄ in 1 × mDM + SBC200 medium (10 mg / L and 6.2 mg / L, respectively, corresponding to 1/2 of the concentration), T2 is the medium excluding Ca (NO ₃ ) ₂ 4H ₂ O and K ₂ HPO ₄ in the 1 × mDM + SBC200 medium And T3 is a medium containing only SBC 200 in the deaeration water. On the 3rd and 5th day of cultivation, HR biomass was collected and the degree of growth and carbohydrate accumulation was examined by a conventional method. The environmental conditions for greenhouse cultivation are 90-350 μmolm ^-2 s ^-1 , day 25-30 ° C / night 20-25 ° C, and 14 hours (70-350 ^-2 m ^-2 s ^-1) photoperiod.

In the case of glucose production, the amount of glucose production was 34.3 g / 100 g DM and 33.88 g T3 in 3 days and T1 in 5 days, respectively. / 100 g DM. However, the difference in glucose production between the media was not significant. In conclusion, when the HR growth and the carbohydrate accumulation are to be further enhanced, the nitrogen source and the phosphorus content are increased by a factor of 1/2 (1 × mDM) compared to the normal (1 × mDM) based on Ca (NO ₃ ) ₂ 4H ₂ O and K ₂ HPO ₄ (Table 8). ≪ tb >< TABLE > ND means 'Not Determined'.

Cultivation period (days) process HR growth (g / 5L / 0.1m ² ) Glucose
(g / 100 g DM) Reducing sugar
(g / 100 g DM) Fresh weight (A) In building (B) B / A
(%) 0 T1 10.00 ± 0.00 1.26 ± 0.00 12.60 25.56 ± 0.27 31.37 ± 0.03 T2 10.00 ± 0.00 1.25 ± 0.00 12.50 26.38 ± 1.99 32.41 + 0.02 T3 10.00 ± 0.00 1.28 ± 0.06 12.80 24.79 + 0.17 32.26 + - 0.33 3 T1 17.15 + 0.36 2.32 ± 0.06 13.53 33.06 + - 0.53 38.10 ± 2.34 T2 15.48 ± 0.56 2.20 ± 0.07 14.21 32.07 ± 0.87 38.10 + - 0.48 T3 14.80 ± 0.59 2.11 + - 0.11 14.26 34.99 ± 1.36 39.24 + - 3.38 5 T1 18.80 + - 0.82 2.76 ± 0.09 14.68 33.88 ± 1.34 ND T2 16.59 ± 0.20 2.59 ± 0.05 15.61 31.89 ± 1.22 ND T3 16.12 ± 0.16 2.50 ± 0.06 15.51 30.28 + - 0.46 ND

Example  8: Normal depth of water in the greenhouse in a small tray HR  Carbohydrate accumulation induction (depth 5 cm, culture liquid 100 L / 1.46 ㎡)

HR produces low quality HR biomass with relatively low carbohydrate content at low pH and temperature, high nutrient conditions (especially nitrogen), low light intensity and high density culture, which is a problem in application to industrial biomass. Therefore, in order to overcome these problems, it is necessary to develop a technology to convert low-quality HR biomass into high-quality HR biomass with high carbohydrate content. In this study, various experiments were conducted on small trays in a greenhouse condition in order to establish a technique to increase carbohydrate accumulation in the body of low - quality HR biomass of various origins.

HR  Effect of Initial Inoculation and Culture Volume

HR biomass with a glucose production of 24.83 g / 100 g DM was added to a small tray (1.46 ㎡, 1.6 ㎡) supplied with 100 L or 200 L culture medium, and 300 g or 600 g of fresh weight was added thereto. At the time of 100 L and 200 L culture, the water depths were 5 ㎝ and 10 ㎝, respectively. In the case of nutrient supply, the culture medium of 1 × mDM + SBC200 composition was used first, and 1 × mDM (Ca (NO ₃ ) ₂ 4H ₂ O 10 mg / L; 1 x mDM) + SBC200 were added. On the 6th day of cultivation, HR biomass was collected and the degree of growth and carbohydrate accumulation was examined by a conventional method. Environmental conditions for greenhouse cultivation are 90-350 μmolm ^-2 s ^-1 , day 25-30 ° C / night 20-25 ° C, and 14 hours light period.

As a result, as shown in Table 9, the growth rate of pure water was 600 g / 200 L> 300 g / 100 L> 600 g / 100 L in that order. However, in the case of sugar accumulation, it was higher in the order of 300 g / 100 L> 600 g / 100 L> 600 g / 200 L, and 19.46 g / 100 g DM, 13.49 g / 100 g DM and 7.12 g / 100 g DM were additionally accumulated . The reason why the sugar accumulation was 300 g / 100L> 600 g / 100L was due to the low density and 600 g / 100L> 600 g / 200L was attributed to the fact that the nutrients were doubled and the total nitrogen amount was larger than the density . Considering the total amount of water and nutrients used, the initial dose of HR and culture medium is preferably 300 g / 100 L (FW 200 g / 70 L / 1.0 ㎡, about 5 cm in depth) on a 1.46 ㎡ tray to induce carbohydrate accumulation (Table 9).

Culture conditions
(Initial inoculation volume / medium volume / culture area) HR growth after 6 days of culture
(g / 1.46m2) Glucose
(g / 100 g DM) Fresh weight (A) In building (B) B / A (%) 0 Day 6 Day 300g / 100L / 1.46m ²
(205.5 g / 68.5 L / 1.0 m ² ) 695.3
(395.3) ^a) 72.75 10.46 24.83 + - 0.95 44.29 + - 0.64 600g / 100L / 1.46m ²
(411.0 g / 68.5 L / 1.0 m ² ) 931.6
(331.6) ^a) 96.44 10.35 24.83 + - 0.95 38.32 + 0.83 600g / 200L / 1.60m ²
(375.0 g / 125 L / 1.0 m ² ) 1145.3
(545.3) ^a) 104.61 9.13 24.83 + - 0.95 31.95 +/- 1.12

a) Pure dehydrated fresh weight in each tray during 6-day incubation, except for the initial dose.

HR  Influence of initial dosing and culture method

HR biomass with a glucose production of 29.61 g / 100 g DM was added to a small tray (1.46 m 2) supplied with 100 L culture medium, and 300 g or 450 g of fresh weight was added thereto and cultured under greenhouse conditions. At this time, the depth was about 5 cm. In the case of nutrient supply, two conditions were set. Condition 1 was cultured for 6 days without addition of nutrients after using 1 × mDM + SBC200 culture medium. In case of condition II, 1 × mDM + SBC200 composition , The nutrients of 1 x mDM (Ca (NO ₃ ) ₂ 4 H ₂ O 10 mg / L) + SBC 200 were added on the third day after the start of the culture. On the 6th day of cultivation, HR biomass was collected and the degree of growth and carbohydrate accumulation was examined by a conventional method. Environmental conditions for greenhouse cultivation are 90-350 μmolm ^-2 s ^-1 , day 25-30 ° C / night 20-25 ° C, and 14 hours light period.

As a result, as shown in Table 10, there was no significant difference between the initial doses of HR in the case of the increase in the pure growth rate, but slight differences were observed between nutrient supply conditions. That is, there was an increase tendency of 20-30 g / tray on dehydrated fresh weight basis under Condition II in which nutrient supplementation was made. In the case of glucose production, there was no difference between the initial doses of HR, but there was a slight difference between nutrient feeding conditions, and the glucose production was slightly decreased in condition II in which nutrient supplementation was performed (Table 10). Therefore, the addition of nutrients on the third day after initiation of culture did not contribute significantly to carbohydrate accumulation, and initial HR doses could be increased from 300 g / 100L to 450 g / 100L.

Culture conditions
(Initial inoculation volume / medium volume / culture area) How to feed the medium HR growth after 6 days of culture (g / 1.46 ㎡) Glucose (g / 100 g DM) Fresh weight (A) In building (B) B / A (%) 0 days 6 days 300g / 100L / 1.46m ²
(205.5 g / 68.5 L / 1.0 m ² ) Condition I 521.2
(221.2) ^a) 60.56 11.62 29.61 + - 0.16 39.39 ± 0.09 Condition II 542.3
(242.3) ^a) 51.77 11.39 29.61 + - 0.16 37.36 ± 0.33 450g / 100L / 1.46m ²
(308.2 g / 68.5 L / 1.0 m ² ) Condition I 661.7
(211.7) ^a) 75.82 11.46 29.61 + - 0.16 39.74 ± 0.26 Condition II 690.6
(240.6) ^a) 76.87 11.13 29.61 + - 0.16 36.62 ± 0.90

a) Pure dehydrated fresh weight in each tray during 6-day incubation, except for the initial dose.

Culture tank  Light on the floor Reflection film (Aluminum foil) installation influence

HR biomass with a glucose production of 39.4 g / 100 g DM was added to a small tray (1.46 m 2) supplied with 100 L culture medium, and 450 g of dehydrated standard was added thereto and cultured under greenhouse conditions. At this time, an aluminum foil was laid on the bottom of the incubation chamber, and the effect of the light reflection membrane was examined. In the case of nutrient supply, SBC200 was added on the third day after initiation of culture using 1 × mDM + SBC200 culture medium. On the 6th day of cultivation, HR biomass was collected and the amount of growth and carbohydrate accumulation were examined by a conventional method. Environmental conditions for greenhouse cultivation are 90-350 μmolm ^-2 s ^-1 , day 25-30 ° C / night 20-25 ° C, and 14 hours light period.

As a result, when the light reflecting film using aluminum foil was installed, the net growth rate (in the building) was increased and the glucose production was also increased (Table 11). This is thought to be due to the increase in the amount of light that HR can utilize due to the reflection of incident light.

Presence of aluminum foil After 6 days of incubation, HR growth (g / 1.46m2) Glucose (g / 100 g DM) Fresh weight (A) In building (B) B / A (%) 0 days 6 days No 743.3
(293.3) ^a) 75.36 10.14 39.41 + - 0.23 44.00 ± 1.15 Yes 733.0
(283.0) ^a) 77.13 10.52 39.41 + - 0.23 49.31 + - 0.62

a) Pure dehydrated fresh weight in each tray during 6-day incubation, except for the initial dose.

Used in sewage treatment HR  Experiment of high quality of sample

HR biomass used in actual sewage treatment was collected and high quality experiment was conducted. HR biomass with a glucose production of 18.7 g / 100 g DM was added to a small tray (1.46 m 2) supplied with 100 L culture medium and 300 g of dehydrated standard was added thereto and cultured under greenhouse conditions. In the case of nutrient supply, 1 × mDM (Ca (NO ₃ ) ₂ 4H ₂ O and K ₂ HPO ₄ (concentration of 1 × mDM + SBC200) ) Plus SBC200 was added. On the 6th day of cultivation, HR biomass was collected and the amount of growth and carbohydrate accumulation were examined by a conventional method. Environmental conditions for greenhouse cultivation are 90-350 μmolm ^-2 s ^-1 , day 25-30 ° C / night 20-25 ° C, and 14 hours light period.

As a result, a significant increase in net growth (in the building) and glucose production was recognized. That is, when the HR sample with 8.2% of the building ratio was inoculated with 300 g (24.6 g in the building), 82-92 g was produced during the drying and 3.3-3.7 times was increased. The reason for the increase in the growth rate compared to other experiments is probably related to the relatively high nitrogen source in the biomass. After the high-quality culture, the sample with 18.7 g glucose production increased to 46.7-47.0 g, and HR biomass with about 150% improvement in carbohydrate accumulation was obtained (Table 12).

HR sample HR growth (g / 1.46 m ² ) Glucose (g / 100 g DW) Fresh weight (A) In building (B) B / A (%) Just before cultivation begins After 6 days of culture EPS (2) TC1 682.5 g 92.25 13.52 18.68 + - 0.34 46.66 + - 1.23 EPS (2) TC2 664.0 g 82.21 12.38 18.68 + - 0.34 47.02 + - 0.25

Example  9: High quality at very low water depths HR Biomass  Culture method for production (Thin layer culture , TLC  Method) development

Experiments performed in Examples 6 to 8 are the results of inducing HR quality enhancement when cultured at a depth of 5 cm for 6-7 days. Practically lowering the depth of water and shortening the incubation period will produce high quality HR biomass at a lower cost / higher efficiency (hereinafter referred to as TLC). To confirm the possibility and to establish the optimum condition of the TLC method,

TLC in HR  Depending on the vaccination density HR  Growth and carbohydrate accumulation differences

HR biomass with a glucose production of 23.6 g / 100 g DM was added to a square plastic box (0.1 ㎡) supplied with 2 L culture medium, dehydrated 5, 10, 12.5, 15 and 20 g based on fresh weight, ) Or in a growth room (GR). The medium was supplied with 1 x mDM + SBC200 adjusted to pH 10.1 and the depth was about 0.8 ㎝. In the greenhouse culture, the environmental conditions are 90-250 μmolm ^-2 s ^-1 , day 25-35 ° C / night 20-25 ° C, 14 hour light period and environmental conditions 80-120 μmolm ^-2 s ^-1 , A constant temperature of 25-30 ° C, and a 14-hour photoperiod. During the incubation, the top of the box was covered with a transparent film to prevent evaporation. After culturing for 3 days under the above conditions, growth and carbohydrate accumulation were examined as in Example 8.

As a result, the growth of GR was better than that of GH, because the temperature of GR was high at night but that of GH was low. As a result of comparing the degree of growth with the inoculation density, the highest growth rate was observed during 10 g inoculation. After 3 days of incubation, the carbohydrate accumulation was significantly increased and the level was higher in GH than in GR (Table 13). This seems to be related to the relatively high light intensity in greenhouse conditions. Glucose production by inoculation density was higher in 10 g inoculation than in 5 g in both GH and GR cultures, but the inoculation density tended to decrease with increasing density. The low glucose production in the initial 5 g inoculation seems to be due to the relatively low carbohydrate accumulation due to the high nutrients (TN, TP) compared to the inoculum, and the inoculum is higher than 15 g in that the low glucose production is due to high density, In the culture medium. These results show that optimization of nutrients and light intensity is very important for the production of high quality HR biomass. When the 1 × mDM + SBC200 medium is divided into 2 L at 0.1 ㎡, The 10 g inoculation showed the best results.

Growing place Initial dose
(FW g / 2L / 0.1 m 2) After 3 days of culture, HR growth (g / 0.1m ² ) Glucose (g / 100 g DM) Fresh weight (A) In building (B) IDW (g) for 3d ¹⁾ 0 days 3 days greenhouse
(GH) 5.0 12.33 + - 0.80 1.29 + 0.03 0.851 23.60 ± 0.31 45.78 ± 0.95 10.0 14.98 + 0.49 1.74 ± 0.03 0.862 47.83 + - 0.62 12.5 16.41. + -. 1.41 1.93 ± 0.05 0.833 44.99 ± 1.18 15.0 16.93 + - 1.45 2.15 + 0.04 0.833 42.51 + - 0.94 20.0 20.59 ± 0.53 2.55 + 0.04 0.794 39.12 + - 0.88 Indoor Growth Room
(GR) 5.0 14.25 + 0.13 1.49 + 0.04 1.051 23.60 ± 0.31 34.07 ± 1.24 10.0 16.86 ± 0.63 1.96 + 0.02 1.082 41.10 ± 0.54 12.5 19.76 ± 1.25 2.15 + 0.08 1.053 37.95 +/- 0.79 15.0 22.22 + 1.81 2.24 ± 0.03 0.923 37.60 ± 1.28 20.0 24.22 + 1.87 2.62 ± 0.06 0.864 36.23 + - 0.72

1) Increased dry weight (IDW) during 3 days of culture.

To further confirm this, we applied it to a greenhouse 100 L tray (1.46 ㎡). HR biomass with a glucose production of 23.6 g / 100 g DM was added to a rectangular tray (1.46 m 2) supplied with 30 L culture medium, and 150 g of dehydrated glucose was added for 3 days. The medium was supplied with 1 x mDM + SBC200 adjusted to pH 10.1 with a depth of approximately 0.8 cm. Environmental conditions for greenhouse cultivation are 90-250 μmolm ^-2 s ^-1 , day 25-35 ° C / night 20-25 ° C, and 14 hours light period. As a result, it was confirmed that the TLC method was useful in inducing the HR quality improvement, as the glucose productivity was increased to 43.51 g / 100 g DM in addition to the increase in the pure building of 11.6 g / ㎡.

TLC in In the medium composition  Following HR  Growth and carbohydrate accumulation differences

The basic medium for TLC is 1 x mDM + S200. The possibility of reducing the nitrogen source and the personnel supply in the medium by half, if the same result would be obtained, would further reduce the nutrient supply cost, so we examined the possibility. HR biomass with a glucose production of 28.0 g / 100 g DM was put into a rectangular plastic box (0.1 m 2) supplied with 2 L culture medium, and 10 g of fresh weight was added to the greenhouse (GH) GR). The medium contained 1 x mDM (Ca (NO ₃ ) ₂ 4 H ₂ O and K ₂ HPO ₄ at 20 mg / L and 12.4 mg / L respectively) + SBC200 (1 × AB) or 1 × mDM (NO ₃ ) ₂ 4H ₂ O and K ₂ HPO ₄ were supplied at 120 mg / L and 6.2 mg / L respectively) + SBC200 (0.5 × AB), respectively. In the greenhouse culture, the environmental conditions are 90-250 μmolm ^-2 s ^-1 , day 25-35 ° C / night 20-25 ° C, 14 hours light period, environmental conditions 80-120 μmol, 25-30 Lt; 0 > C, and 14 hours photoperiod. During the incubation, the top of the box was covered with a transparent film to prevent evaporation. After culturing for 3 days under the above conditions, the amount of growth and carbohydrate accumulation were examined as in Example 8.

As a result, the growth of GR was better than that of GH, because the temperature of GR was high at night but that of GH was low. A slight increase in growth was observed at 0.5 × AB compared to 1 × AB (Table 14). In addition, the amount of glucose production was also increased at 0.5 × AB than at 1 × AB (Table 15). Therefore, the TLC method can be used as a method of producing HR biomass of high quality at a low cost / high efficiency since the water depth can be further reduced to reduce water consumption and the cultivation period can be shortened by 3 days.

Growing place Initial dose
(FW g / 2L / 0.1 m 2) 1 x AB 0.5 × AB Fresh weight
(g / 0.1 m 2) In building
(g / 0.1 m 2) IDW (g) for 3d ¹⁾ Fresh weight
(g / 0.1 m 2) In building
(g / 0.1 m 2) IDW (g) for 3d ¹⁾ Greenhouse (GH) 10 17.89 ± 0.75 2.30 ± 0.05 1.16 18.45 ± 1.99 2.32 ± 0.06 1.18 Indoor Growth Room
(GR) 10 19.49 + 1.33 2.38 ± 0.05 1.24 20.70 ± 0.73 2.51 + 0.08 1.37

1) Increased dry weight (IDW) during 3 days of culture.

Growing place Initial dose
(FW g / 2L / 0.1 m 2) 1 x AB 0.5 × AB In building
(g / 0.1 m 2) Glucose
(g / 100 g DM) In building
(g / 0.1 m 2) Glucose
(g / 100 g DM) Greenhouse (GH) 10 2.30
± 0.05 43.05
± 1.05 2.32
± 0.06 47.32
± 1.72 Indoor Growth Room (GR) 10 2.38
± 0.05 39.14
± 1.17 2.51
± 0.08 41.75
± 0.72

To further confirm this, greenhouse cultivation was performed on a larger tray (1.46 ㎡). The medium was supplied with 1 x mDM + SBC250 adjusted to pH 10.1 and the depth was about 0.8 ㎝. The environmental conditions for greenhouse cultivation are 90-500 μmolm ^-2 s ^-1 , day 25-35 ° C / night 20-25 ° C, and 14 hours light period. When cultured for 3 days under the above conditions, it was confirmed that very high quality carbohydrate-rich HR having a glucose production amount of 52-53 g / 100 g DM was produced.

TLC  In culture pH  As a coordinating substance Na ₂ CO ₃ Availability as

It is necessary that the pH of the culture prepared in the TLC culture is adjusted to pH 10.0-10.5 and that the SBC concentration is maintained at 200-300 ppm. In the meantime, NaOH or KOH was used for pH adjustment, but Na ₂ CO ₃ was used for cost reduction and additional carbon source supply. This is because Na ₂ CO ₃ is alkaline and can be present as CO ₂ , HCO ₃ ^-1 , and CO ₃ ^-2 depending on the pH in the solution, and thus it is considered that it can sufficiently serve as a carbon source in HR culture .

1 x mDM + SBC100 was used as the primary medium and the pH adjustment was adjusted to pH 10.1 with NaOH or Na ₂ CO ₃ . HR biomass with a glucose production of 31.8 g / 100 g DM was added to 2 g of the prepared culture liquid (0.1 m 2) in a rectangular plastic box (about 0.8 ㎝ in depth) and 10 g of dehydrated fresh weight was added thereto. Lt; / RTI > In the greenhouse culture, the environmental conditions were 25-35 ° C / night at 15-20 ° C for 14 hours, 80-500 μmol ^-2 s ^-1 for 14 hours, and after culturing for 3 days under the above conditions, growth and carbohydrate accumulation Respectively.

As a result, the optimum conditions for HR growth and glucose production were Na ₂ CO ₃ , pH 10.1, NaOH, and pH 10.1, respectively. .

PH of medium
(pH adjusting reagent) After 3 days of culture, HR growth (g / 0.1m ² ) Glucose productivity
(g / 100 g DM) FW DW 9.5 (control) 13.24 1.91 40.75 ± 0.65 10.1 (NaOH) 14.09 2.00 42.43 + - 0.34 10.1 (Na ₂ CO ₃₎ 15.03 2.08 46.31 ± 0.21

Therefore, it is confirmed that TLC culture method is suitable for producing high quality HR biomass than existing culture method, and it is a resource saving type culture method, which can shorten culture time by about 2 times, and water and nutrient final use amount by 2.5 times. The TLC culture method is considered to be more suitable for improving the quality of primary production HR (for example, water purification) by intensive methods because the initial inoculation amount of HR should be increased about 3 times as compared with that of normal culture. In particular, since the concentration of nitrogen in the water is high in the purification of water, the HR produced here has a low carbohydrate content. Therefore, when the TLC culture method is applied, the carbohydrate accumulation can be improved in a short time by using the minimum amount of water. It will be a very effective method for

Comprehensively, when the initial HR inoculation is lowered (1-3 g / 0.1 m 2) and cultivation is continued for a longer time to obtain high-quality biomass, the same method as in Example 8 (5 cm or more in depth and 6 (More than 7 days). However, when it is desired to obtain high quality biomass at a higher rate than the initial HR inoculation amount (10 g / 0.1 m 2), the TLC method as in Example 9 (0.8 cm in depth, 3 days) was preferably used. In particular, since the TLC method has a small amount of water, it is possible to intensively cultivate indoors using a multistage lathe (Fig. 3), and also to provide a multi-stage rotary culture apparatus It is possible to remarkably increase the production per unit area.

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 (8)

WHAT IS CLAIMED IS: 1. A method for converting a carbohydrate-low-containing Hydrodictyon bird to a carbohydrate-rich nethorse bird, comprising the steps of:
(a) inoculating a carbohydrate-low netting barley algae into a culture medium of a carbohydrate-low netting barley algae containing carbon source, nitrogen source and phosphorus; And
(b) culturing the resulting product of step (a) and converting it into a carbohydrate-rich, net barley bird.
The method according to claim 1, wherein the netted bird is a hydrodictyon reticulatum reticulatum ), Hydrodictyon wherein the mixture is one or more mixed birds selected from the group consisting of africanum and hydrodictyon patenaeforme .
The method of claim 1, wherein the carbohydrate-low netting nutrient culture medium is selected from the group consisting of magnesium sulfate, EDTA Na ₂ , EDTA sodium iron, sodium hydroxide, boron, manganese, zinc, molybdenum and combinations thereof RTI ID = 0.0 > 1, < / RTI > further comprising at least one component.

2. The method of claim 1, wherein the carbon source of the carbohydrate-low containing net barley culture medium has a concentration of 100-400 ppm based on sodium bicarbonate.
The method of claim 1, wherein the nitrogen source has a concentration of 5-15 mg / L based on calcium nitrate and the population has a concentration of 4-8 mg / L based on potassium phosphate.
The method of claim 1, wherein the medium has a depth of 0.2-10 cm.
2. The method according to claim 1, wherein the inoculation of step (a) is carried out by inoculation with a Hydrodictyon bird of 1-35 g / 0.1 m 2 based on dehydrated fresh weight.
The method according to claim 1, wherein the culturing is performed in a culture tank provided with a reflecting film.

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