KR101124466B1 - Method for manufacturing culture medium dunaliella using natural seawater and pretreated seawater - Google Patents

Abstract

PURPOSE: A method for preparing culture liquid for Dunaliella sp. using natural seawater and pretreated seawater is provided to obtain the culture liquid with rich chlorophyll a and carotinoid. CONSTITUTION: A method for preparing culture liquid for Dunaliella sp. comprises: a step of adding charcoal and NaOH to natural seawater to remove a material which generates milky turbidity; a step of oxidizing natural seawater; and a step of mixing the pretreated seawater and oxidized natural seawater to adjusting pH concentration and salinity by 7.4-7.6 and 55-65psu, respectively.

Description

Method of manufacturing Dunaliella culture using natural seawater and pretreated seawater {METHOD FOR MANUFACTURING CULTURE MEDIUM DUNALIELLA USING NATURAL SEAWATER AND PRETREATED SEAWATER}

The present invention relates to a method for producing dunaliella sp., And more particularly, to effectively produce dunaliella using natural seawater and pre-treated seawater, including growth enhancement, total carokinoid material production, and fat production. It relates to a Dunaliella culture medium production method that can be.

Microalgae research is becoming increasingly popular for human food, biofuel, pharmaceutical and cosmetic applications.

Among the microalgae, Dunaliella sp. Is known as marine microalgae that can be used for various purposes such as health food market use and pharmaceuticals.

Currently, Dunaliella is manufactured in a modified Johnson culture (1968, F / 2 Guilard, Hejazi et al. 2003), and natural seawater is not well used as a culture for Dunaliella culture. When natural seawater is used as the Dunalella culture, there are various problems such as milky turbidity generation, detection of nutrients, clumping of microalgae, HPO ₄ ² -decrease in solubility, formation of white precipitate in the culture, and the like.

Among them, milky turbidity is mainly produced by magnesium ions (Mg ²⁺ ) or calcium ions (Ca ²⁺ ). When phosphate and carbonate are added to natural seawater for the cultivation of microalgae, the culture becomes turbid. Occurs. This milky turbidity is the cause of photosynthesis, growth, cleavage and fat content in Dunaliella culture.

In addition, the production of white precipitate in the culture medium also causes a decrease in photosynthesis, growth, cleavage and fat content.

On the other hand, the availability of carbon along with other chemicals is the most important factor in Dunaliella growth. In general, carbon is supplied as carbonates (NaHCO ₃ and Na ₂ CO ₃ ) in all reported Dunaliella culture media, but the above-mentioned problems arise after the addition of carbonate to natural seawater.

Therefore, in Dunaliella culture, there is a need for a method that can utilize natural seawater.

An object of the present invention can reduce the manufacturing cost of the Dunaliella culture using pre-treated seawater and natural seawater, and the production method of Dunaliella culture that can increase the photosynthesis, growth, cleavage and fat content in the culture of Dunaliella To provide.

Dunaliella sp. Culture method using the pre-treatment seawater according to an embodiment of the present invention for achieving the above object is the pre-treatment by adding coal and NaOH to the natural seawater, milky turbidity generating material is removed Preparing sea water; Oxidizing natural sea water; And mixing the oxidized natural seawater with the pretreated seawater to adjust pH 7.4 to 7.6 and a salt concentration of 55 to 65 psu.

At this time, NaCl may be added to adjust the salt concentration of the mixed solution of the natural seawater and pretreated seawater.

In addition, KNO ₃ 1.2 ± 0.1 g / L, MgCl ₂ .6H ₂ O 1.0 ± 0.1 g / L, MgSO ₄ −7H ₂ O 0.35 ± 0.05 g / LK ₂ SO ₄ 0.25 ± 0.05 g / L, KH ₂ PO ₄ 0.15 ± 0.05 g / L, ZnSO ₄ ? 7H ₂ O 0.55 ± 0.1 g / L, MnCl ₂ ? 4H ₂ O 0.55 ± 0.1 g / L, H ₃ BO ₃ 6.5 ± _{0.5 g / L, FeCl 3?} 6H 2 O 7.5 ± 0.5 g / L, Co (NO 3) 2? 6H 2 O 0.5 ± 0.1 g / L, CuSO 4? 5H 2 O 0.01 ± 0.1 g / L, Na 2 MoO ₇ -2H ₂ O 0.03 ± 0.005 g / L, Cyanocobalamin (Vitamin B ₁₂ ) 0.1 ± 0.05 g / L, D-Biotin (Vitamin H) 7.5 ± 0.5 g / L, and Thiamine-HCl (Vitamin B ₁ ) 10.0 More ± 1.0 g / L may be added.

In addition, hydrochloric acid may be used for the oxidation treatment of the natural seawater.

In addition, the coal is characterized in that the bituminous coal.

In addition, the coal may be added at a concentration determined by Equation 1 according to the salt concentration (psu) of the natural seawater.

[Equation 1]

RA _C (g / L) = natural sea salt concentration X μ _c

(Equation 1, RA _C is the concentration of coal required, μ _c is the concentration of coal that is transparent when coal is added to 1 L of natural seawater having a specific salinity concentration, and that no white precipitate is present when the salt concentration is 60.0 psu. Divided by the specific salinity)

In addition, the NaOH may be added at a concentration determined by the following formula 2 according to the salt concentration (psu) of the natural sea water.

[Equation 2]

RA _NaOH (g / L) = natural sea salt concentration X μ _NaOH

(In Formula 2, RA _NaOH is the required concentration of _NaOH , μ _NaOH is clear when NaOH is added to 1 L of natural seawater having a specific salinity concentration, and the concentration of NaOH where no white precipitate is present at a salt concentration of 60.00 psu) Divided by the specific salinity)

In the method of manufacturing a Dunaliella culture according to the present invention, although the manufacturing cost is more than twice as low as that of a conventional J / M culture, a Dunaliella culture having a higher chlorophyll-a and carotenoid content in the culture of Dunaliella is produced. It can work.

1 shows the concentration of Dunaliella according to the culture period when Dunaliella were cultured for 16 days using the culture medium according to the Examples and Comparative Examples.
Figure 2 shows the chlorophyll a concentration of Dunaliella according to the culture period when Dunaliella was cultured for 16 days using the culture medium according to the Examples and Comparative Examples.
Figure 3 shows the total carotenoid concentration according to the culture period when Dunaliella was incubated for 16 days using the culture medium according to the Examples and Comparative Examples.

BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention, and how to accomplish them, will become apparent with reference to the embodiments and drawings described in detail below.

However, it is to be understood that the present invention is not limited to the disclosed embodiments, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. It is intended that the disclosure of the present invention be limited only by the terms of the appended claims.

Hereinafter, a method for producing Dunaliella culture medium using pre-treated seawater according to the present invention will be described in detail.

Dunaliella sp. Culture method according to the present invention includes a natural seawater pretreatment step, natural seawater oxidation step and mixing step.

Natural Seawater Pretreatment

In the natural seawater pretreatment step, pretreatment of the seawater is performed to prepare pretreated seawater from which milky turbidity generating material is removed. For pretreatment of natural seawater, coal and NaOH are added to the natural seawater in the present invention. As a result, milky turbidity generating substances such as magnesium ions and calcium ions precipitated out.

Coal may be charcoal or bituminous coal, but in the case of charcoal, it may be a factor of depleting forest resources, so it is more preferable to use bituminous coal.

For component evaluation of pretreated seawater, 27.60 g / L bituminous coal and 7.75 g / L NaOH were added to natural seawater having a pH of 8.20 and a salt concentration of 31.00 psu, and then maintained at a temperature of 29 ° C. for one week.

For pretreated seawater, approximately 92% was a supernatant and the rest was sediment. In addition, from natural seawater to pretreated seawater, the pH increased from 8.20 to 13.14, and the salt concentration increased from 31.00 psu to 45.00 psu.

Table 1 shows the contents of organic carbon and various components contained in the supernatant of natural seawater and pretreated seawater.

In Table 1, total organic carbon (OC) in natural seawater and pretreated seawater was evaluated using an organic carbon analyzer (TOC-5000A, manufactured by Shimadzu). In addition, the concentrations of Ca, Mg, Na, As, Cd, Cr, Pb, Mg, K, Sr and Hg in natural seawater and pretreated seawater, and also the concentrations of Ca and Mg in the sediment were measured at the Korea Testing Laboratory.

[Table 1]

Referring to Table 1, organic carbon was seven times higher in pretreated seawater than organic carbon in conventional natural seawater. Na, K and Sr were augmented with natural seawater pretreated. On the other hand, heavy metals such as Hg, As, Cd, Cr and Pb were not detected in the pretreated seawater.

In particular, Mg and Ca in natural seawater were reduced to 0.80 and 337.90 mg / L in pretreated seawater, whereas 874.40 and 500.70 mg / L. Mg and Ca in natural seawater were detected 99.45% and 32.52% in the sediments, respectively (see Table 1). The Mg content in the detection was 22.28 g / L.

CO ₂ or CO contained in coal may react with NaOH to form Na ₂ CO ₃ or NaHCO ₃ , and Na ₂ CO ₃ detects Ca and Mg from seawater at high pH. High pH is also characterized by the ability to kill organisms present in the pretreated seawater, thus preventing contamination by other species.

On the other hand, there is a need to optimize the amount of coal and NaOH added according to the salt concentration (psu) of natural seawater.

First, it is preferable that coal is added at the density | concentration determined by following formula (1).

[Equation 1]

RA _C (g / L) = natural sea salt concentration X μ _c

(Equation 1, RA _C is the concentration of coal required, μ _c is the concentration of coal that is transparent when coal is added to 1 L of natural sea water having a specific salinity concentration, and that the white precipitate does not exist when the salt concentration is 60.00 psu. Divided by the specific salinity)

For example, if it is clear that 27.60 g / L of coal is added at a salt concentration of 31 psu and it is known that no white precipitate is present at 60 psu, μ _c is 27.6 / 31, which is approximately 0.89. Thus, on the basis of this, approximately 17.8 g / L of coal is required for pretreatment in natural seawater with a salt concentration of 20 psu.

Next, NaOH is preferably added at a concentration determined by the following formula (2).

[Equation 2]

RA _NaOH (g / L) = natural sea salt concentration X μ _NaOH

(In Formula 2, RA _NaOH is the required concentration of _NaOH , μ _NaOH is transparent when NaOH is added to 1 L of natural seawater having a specific salinity concentration, and the concentration of NaOH where no white precipitate is present at a salt concentration of 60.00 psu) Divided by the specific salinity)

For example, if you know that you need 7.75 g / L of NaOH at a salt concentration of 31 psu, μ _NaOH is 7.75 / 31, which is approximately 0.25. Thus, based on this, approximately 5 g / L of coal is required for pretreatment in natural seawater with a salt concentration of 20 psu.

In fact, when the bituminous coal and NaOH concentrations according to Equations 1 and 2 were applied to natural seawater having 10.00 to 50.00 psu, transparency of pretreated seawater, transparency of Dunaliella culture and white precipitate formation did not occur in all cases. .

Oxidation and Mixing of Natural Seawater

In the oxidation treatment step of natural seawater, the natural seawater is oxidized in order to maintain a pH of 7.4 to 7.6, which is well-cultured by Dunaliella after mixing with the pretreated seawater.

Oxidation can utilize most acidic chemicals, typically nitric acid or hydrochloric acid.

Then, after mixing the pre-treated seawater and oxidized natural seawater, it is maintained for about a day for a certain time.

On the other hand, the salinity is well known 55-65psu in Dunaliella culture. To this end, NaCl may be added to adjust the salt concentration.

Dunaliella culture contains KNO ₃ 1.2 ± 0.1 g / L, MgCl ₂ .6H ₂ O 1.0 ± 0.1 g / L, MgSO ₄ –7H ₂ O 0.35 ± 0.05 g / LK to increase organic carbon content and provide nutrients. ₂ SO ₄ 0.25 ± 0.05 g / L, KH ₂ PO ₄ 0.15 ± 0.05 g / L, ZnSO ₄ ? 7H ₂ O 0.55 ± 0.1 g / L, MnCl ₂ ? 4H ₂ O 0.55 ± 0.1 g / L, H ₃ BO _{3 6.5 ± 0.5 g / L,} FeCl 3? 6H 2 O 7.5 ± 0.5 g / L, Co (NO 3) 2? 6H 2 O 0.5 ± 0.1 g / L, CuSO 4? 5H 2 O 0.01 ± 0.1 g / L , Na ₂ MoO ₇ -2H ₂ O 0.03 ± 0.005 g / L, Cyanocobalamin (Vitamin B ₁₂ ) 0.1 ± 0.05 g / L, D-Biotin (Vitamin H) 7.5 ± 0.5 g / L, and Thiamine-HCl (Vitamin B ₁ ) 10.0 ± 1.0 g / L may be added further.

As a result of the addition of the components of the above concentration, it was possible to incubate Dunalella with higher chlorophyll-a and carotenoid content than the existing J / M culture.

J / M culture solution (comparative example) having a composition shown in Table 2 and having a salt concentration of 60.00 psu, and a composition shown in Table 3, according to the present invention, natural seawater and pretreated seawater were mixed at a volume ratio of 4: 1, Dunaliella was incubated in a culture medium (Example) having a pH of 7.5 and 25 g / L of sodium added to a salt concentration of 60.00. The culture temperature was 30.00 ℃, the light intensity of the light was 7000 lx fluorescence, the light / dark cycle (light / dark cycle) was a time (h) ratio of 12:12.

Incubation was carried out for 16 days in a 5 L flask containing 4.5 L of the culture medium, and three cultures were performed for each condition. 1.02 g / L Dunaliella seeds were inoculated and stirred with an air injection device. Samples were analyzed every 4 days to assess growth and pigment content, and samples were analyzed on the last 16 days for biochemical analysis. Pigment content was analyzed by a spectrophotometer (PerkinElmer, Lamda 35 uv / vis spectrometer, USA).

For the measurement of Dunaliella's growth, the samples were filtered through pre-weighed filter paper. The filter paper was soaked in distilled water and then dried at the same time for use as a blank. The filter paper was kept at 55 ° C. in an oven and dried, after which the weight was measured and the dry weight was g / L It is calculated as and represented by the growth curve. The specific growth rate (μ) of Dunaliella was defined as the increase in concentration of Dunaliella (X0 → X1) per unit time (t0 → t1), and was calculated using Equation 3 below.

[Equation 3]

[Table 2] (Unit: g / L)

[Table 3] (Unit: g / L)

1 shows the concentration of Dunaliella according to the culture period when Dunaliella were cultured for 16 days using the culture medium according to the Examples and Comparative Examples.

Μ _max according to the comparative example and the example _? d ^-1 and Dunalella concentrations were 0.039 and 0.051 d ^-1 , 1.89 and 1.95 g / L. Although μ _{max ?} Although d ⁻¹ was significantly higher in the examples than the comparative medium, biomass production did not differ significantly.

Figure 2 shows the chlorophyll a concentration of Dunaliella according to the culture period when Dunaliella incubated for 16 days using the culture medium according to the Examples and Comparative Examples, Figure 3 is according to the Examples and Comparative Examples When Dunaliella was incubated for 16 days using the culture medium, total carotenoid concentrations were shown according to the culture period.

Referring to FIG. 2, the chlorophyll a content was 1.40 and 2.73 mg / L, respectively, when the culture solution according to the comparative example and the example was used, which means that the chlorophyll a content was higher in the example.

3, the total carotenoid content was 2.93 and 1.31 mg / L in Examples and Comparative Examples, respectively. The carotenoid content of Dunaliella grown in the Examples was significantly higher than that of Dunaliella grown in the Comparative Examples.

Table 4 shows the biochemical composition of Dunaliellae prepared in the culture medium according to the Examples and Comparative Examples.

[Table 4]

Referring to Table 4, Dunaliella photosynthesis, growth, cleavage, and fat content were higher in the culture medium according to the comparative example than in the culture medium according to the Example, while the lipid content was 2.5 in the case of the Example It was twice as high. There was no significant difference in CHO content in biomass produced in both medium.

Although the present invention has been described with reference to the embodiments shown in the drawings, this is merely exemplary, and those skilled in the art to which the art belongs can make various modifications and other equivalent embodiments therefrom. I will understand.

Therefore, the true technical protection scope of the present invention will be defined by the claims below.

Claims (7)

Adding coal and NaOH to natural seawater to prepare pretreated seawater from which milky turbidity generating material has been removed;
Oxidizing natural sea water; And
Dunaliella (Dunaliella sp.) Culture method comprising the step of mixing the oxidized natural seawater to the pre-treated seawater to adjust the pH 7.4 ~ 7.6, salt concentration 55 ~ 65psu.
The method of claim 1,
Dunaliella culture method characterized in that the NaCl is added to control the salt concentration of the mixed solution of the natural seawater and pre-treated seawater.
The method of claim 1,
In the mixed solution of the natural sea water and pre-treated sea water
KNO ₃ 1.2 ± 0.1 g / L, MgCl ₂ .6H ₂ O 1.0 ± 0.1 g / L, MgSO ₄ ? 7H ₂ O 0.35 ± 0.05 g / LK ₂ SO ₄ 0.25 ± 0.05 g / L, KH ₂ PO ₄ 0.15 ± 0.05 g / L, ZnSO ₄ ? 7H ₂ O 0.55 ± 0.1 g / L, MnCl ₂ ? 4H ₂ O 0.55 ± 0.1 g / L, H ₃ BO ₃ 6.5 ± 0.5 g / L, FeCl ₃ ? 6H ₂ O 7.5 ± 0.5 g / L, Co (NO 3) 2? 6H 2 O 0.5 ± 0.1 g / L, CuSO 4? 5H 2 O 0.01 ± 0.1 g / L, Na 2 MoO 7? 2H 2 O 0.03 ± 0.005 g / L, Cyanocobalamin (Vitamin B ₁₂ ) 0.1 ± 0.05 g / L, D-Biotin (Vitamin H) 7.5 ± 0.5 g / L, and Thiamine-HCl (Vitamin B ₁ ) 10.0 ± 1.0 g / L Dunaliella culture preparation method.
The method of claim 1,
Oxidation of the natural sea water
Dunaliella culture method of producing an acidic compound.
The method of claim 1,
The coal is
A method for producing a Dunaliella culture, characterized in that it is selected from bituminous coal, lignite, anthracite, powdered coal and charcoal.
The method of claim 1,
The coal is
Dunaliella culture solution production method characterized in that it is added at a concentration determined by the following formula 1 according to the salt concentration (psu) of the natural sea water.
[Formula 1]
RA _C (g / L) = natural sea salt concentration X μ _c
(Equation 1, RA _C is the concentration of coal required, μ _c is the concentration of coal that is transparent when coal is added to 1 L of natural seawater having a specific salinity concentration, and that no white precipitate is present when the salt concentration is 60.0 psu. Divided by the specific salinity)
The method of claim 1,
NaOH is
Dunaliella culture method according to the salinity (psu) of the natural sea water is added at a concentration determined by the following formula 2.
[Formula 2]
RA _NaOH (g / L) = natural sea salt concentration X μ _NaOH
(In Formula 2, RA _NaOH is the required concentration of _NaOH , μ _NaOH is clear when NaOH is added to 1 L of natural seawater having a specific salinity concentration, and the concentration of NaOH where no white precipitate is present at a salt concentration of 60.00 psu) Divided by the specific salinity)

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