WO2013058431A1

WO2013058431A1 - Method for manufacturing a culture medium of dunaliella using natural seawater and pretreated seawater

Info

Publication number: WO2013058431A1
Application number: PCT/KR2011/009366
Authority: WO
Inventors: 강도형; 아부아판; 허수진; 오철홍; 전선미; 김태호; 예보람
Original assignee: 한국해양연구원
Priority date: 2011-10-18
Filing date: 2011-12-06
Publication date: 2013-04-25
Also published as: KR101124466B1

Abstract

Provided is a method for manufacturing a culture medium of Dunaliella using natural seawater and pretreated seawater. The method for manufacturing a culture medium of Dunaliella includes: a step of adding coal and NaOH into natural seawater to manufacture pretreated seawater from which the materials for the generation of white turbidity are removed; and a step of mixing oxidized natural seawater with the pretreated seawater to adjust the mixture to have a pH of 7.4 to 7.6 and a salt concentration of 55 psu to 65 psu.

Description

Method for preparing Dunaliella culture 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 a marine microalgae that can be used for various purposes such as health food market and medicine.

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.

Meanwhile, 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 Dunalella 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 production cost of Dunaliella culture by using pre-treated seawater and natural seawater, and the production method of Dunaliella culture that can increase 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 in the mixed solution of natural seawater and pretreated seawater. 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 ThiamineHCl (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]

(Equation 1, μ _c is transparent when coal is added to 1 L of natural sea water having a specific salinity concentration, and when the salt concentration is 60.0 psu, the concentration of coal without white precipitate is divided by the corresponding salinity concentration)

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]

(In formula 2, μ _NaOH is transparent when NaOH is added to 1 L of natural seawater having a specific salt concentration, and when the salt concentration is 60.00 psu, the concentration of NaOH where no white precipitate is present is divided by the corresponding salt concentration)

Although the method for producing a Dunaliella culture according to the present invention requires more than twice as low cost as the 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.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments and drawings described below in detail.

However, the present invention is not limited to the embodiments disclosed below, but can be implemented in various different forms, only the embodiments are to make the disclosure of the present invention complete, it is common in the art It is provided to fully inform those skilled in the art of the scope of the invention, which is to be defined only by the scope of the 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 the usage-amount of coal is added in the density | concentration determined by following formula (1).

[Equation 1]

(Equation 1, μ _c is transparent when coal is added to 1 L of natural sea water having a specific salinity concentration, and when the salt concentration is 60.00 psu, the concentration of coal without white precipitate is divided by the salinity concentration)

For example, if it is known 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]

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 concentration is well known in Dunaliella culture is known to 55 ~ 65psu. 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 further added.

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 / LDunaliella 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 wetted 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 / LIt 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)

The μ _{max ·} d ⁻¹ and Dunaliella concentrations according to Comparative Examples and Examples were 0.039 and 0.051 d ⁻¹ , 1.89 and 1.95 g / L. Although μ _{max ·} d ⁻¹ was significantly higher in the examples than the comparative media, 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. Will understand.

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

Claims

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.

[Equation 1]

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

(Equation 1, μ c is transparent when coal is added to 1 L of natural sea water having a specific salinity concentration, and when the salt concentration is 60.0 psu, the concentration of coal without white precipitate is divided by the corresponding salinity concentration)
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.

[Equation 2]

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

(In formula 2, μ NaOH is transparent when NaOH is added to 1 L of natural seawater having a specific salt concentration, and when the salt concentration is 60.00 psu, the concentration of NaOH where no white precipitate is present is divided by the corresponding salt concentration)