KR101520864B1 - Culture media composition of microalgae by using salt underground water or bedrock underground water including calcium ion - Google Patents

Abstract

The present invention relates to a medium composition for culturing microalgae (for example, Dunaliella salina), characterized in that distilled water (or ground water) is contained in salt ground water or ground water.
Accordingly, the culture composition of the present invention can very effectively cultivate microalgae including Dunaliella salina by mixing distilled water into salt ground water containing a large amount of calcium ions.

Description

Technical Field [0001] The present invention relates to a microalgae culture medium using calcium ion-containing salt groundwater or rock groundwater,

The present invention relates to a medium composition for culturing microalgae (for example, Dunaliella salina), and more particularly, to a composition for promoting the growth of microalgae including Dunaliella salina to increase the culture efficiency.

The microalgae, Dunaliella salina, is a salt-loving, single-celled microorganism that is known as a marine microalgae that can be used for various applications such as health food market applications, medicines, and cosmetics.

However, most Dunaliella salinas have high costs and high energy consumption required to cultivate microalgae as cultured in modified Johnson medium (1968, F / 2 Guilard, Hejazi et al. 2003) Because of the unproductive factors, the cost and energy consumption of most cultivation methods exceeds the revenues and energies derived from microalgae culture processes. In addition, microalgae culture processes are inefficient to cultivate microalgae with efficiency in a relatively short period of time.

The conventional method of culturing Dunaliella salina is disclosed in Patent Registration No. 10-1124466, in which coal and sodium hydroxide (NaOH) are added to seawater to prepare pretreated seawater from which milk turbidity generating material is removed, And adjusting the pH of the pretreated seawater to a pH of 7.4 to 7.6 and a salt concentration of 55 to 65 psu by mixing the oxidized natural sea water with the pre-treated seawater. Despite the cost of production, techniques have been pre-registered to allow the production of chlorophyll-a and a carnitine-rich culture of Dernalella during cultivation of Dnalella.

However, natural sea water has a low concentration of calcium ion (Ca 2+), which is a metabolic stimulant necessary for promoting the growth of Dunaliella salina, and thus it is ineffective for mass production of highly efficient microalgae in a short period of time and is very unavailable industrially.

As described above, a method for mass-producing microalgae useful in Nannochloropsis including Dunaliella salina does not exist at present. In particular, microalgae of Nannochloropsis are used for industrial cultivation due to decrease in yield, contamination of medium and instability of biomass during mass culture There is a problem that it is very unlikely to be used. In addition, if microalgae having a very high utilization value as biomaterial among the microalgae of the Nannochloropsis are developed, they can be effectively used industrially.

Accordingly, it is an object of the present invention to provide a Dunaliella salina microalgae culture composition for mass production of highly efficient microalgae in an effective manner with low production cost and low energy demand, There is a purpose.

In order to achieve this object, a feature of the present invention is characterized in that distilled water or ground water is contained in salt ground water or ground water, and the salt ground water or rock water in the rock water has a calcium ion concentration of 3000 to 8000 mg / L .

At this time, the distilled water or the ground water is contained in 30 to 50% based on the volume of salt groundwater or rock groundwater.

In addition, BM (bioactive water) is contained in a volume ratio of 3 to 7% in the salt ground water or underground water culture medium diluted by the distilled water or the ground water.

In the above, salt groundwater or rock groundwater is rich in minerals because seawater and fresh water penetrate into the ground through the rock as water mixed with underground water. Because of the water mixed with seawater, it is called rock groundwater because it is obtained from salt groundwater or rock.

In addition, Bacteria Mineral Water (reference: Hiroshi N. 1998. Bacterium finds the earth - Challenge of BMW technology, published in book Blue Peace, 216pp.) Is an active water recycling livestock wastewater such as pig urine Which is the final treated water which is fermented by aerating rocks and corrosion pellets into a container into which livestock wastewater has been introduced.

According to the above-described composition and action, the culture medium composition of the present invention can be prepared by mixing distilled water or ground water with salt water or ground water containing a large amount of calcium ions, calcium ions (calcium ions) necessary for accelerating the growth of microalgae including Dunaliella salina Ca2 +) concentration is high, microalgae can be cultured very effectively in a short period of time.

In addition, since such microorganisms can be mass-cultured in such a short period of time, there is an effect that these microorganisms can be used for commercial purposes such as cosmetics.

1 is a graph showing the results of a color change experiment of D. salina according to dilution concentration of groundwater in the South Sea according to an embodiment of the present invention;
FIG. 2 is a diagram showing the results of a first experiment to change the cell absorbance of D. salina according to dilution concentration of salt groundwater according to an embodiment of the present invention. FIG.
FIG. 3 is a diagram showing the results of a second experiment of changing the cell absorbance of D. salina according to dilution concentration of salt groundwater according to an embodiment of the present invention. FIG.
FIG. 4 is a diagram showing the results of a third experiment of varying cell density according to dilution concentration of the Southern Sea salt groundwater of D. salina cultured in each 0.5-ton water tank according to an embodiment of the present invention. FIG.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

The water quality analysis of the Namhae groundwater was conducted at the Gyeongbuk Marine Biotechnology Research Institute, which is a water quality analysis institute, and the salinity, pH, etc., of the groundwater or rock groundwater N, N, Na, Mg, Ca, K, Fe, Pb, Mn, Hg, Zn, As, Ag, B, Bi, Cr, Al, F, Cl-, Br, and SO42- were measured with an ICP analyzer and the results are shown in Table 1.

Item Analysis Remarks Default item salt 175% YSI 63 Hydrogen ion concentration 7.82 Nutrients (mg / L) Nitrate nitrogen 2.05 Nitrite automatic analyzer Nitrite nitrogen ND Phosphate 0.042 Total nitrogen (T-N) 2.44 Total (T-P) 0.15 Major elements (mg / L) Sodium (Na) 1396.5 ICP analyzer Magnesium (Mg) 52.1 Calcium (Ca) 5238.5 Hardness: 13,305 Potassium (K) 6.7 Hazardous and Ionic Substances (mg / L) Iron (Fe) 0.054 ICP analyzer Lead (Pb) 0.006 " Manganese (Mn) 0.02 " Mercury (Hg) ND " Zinc (Zn) 0.048 " Arsenic (As) ND " Silver (Ag) 0.007 " Boron (B) 0.415 " Bi (Bi) 0.006 " Chromium (Cr) 0.0004 " Aluminum (Al) ND " Fluorine (F) 0.086 IC analysis Chlorine ion (Cl-) 122.2 " Bromine ion (Br) 0.745 " Sulfate ion (SO4) 6.421 " Hardness 11.3 Photometer

Table 2 shows the results of saline groundwater analysis in other regions.

Item Analysis Remarks Surface water
(Ulleungdo) Deep sea water Ulleungdo 1,500m Gangwon Goseong 300m Gangwon Goseong 500m basic
Item Salt 33.8 32.2 32.1 31.8 YSI 63 Hydrogen ion concentration 7.90 7.68 7.61 7.64 Coliform group - - - - Deep sea water
Water quality process
Experimental Method nutrition
Salts
(mg / L) Nitrate nitrogen 1.35 0.473 0.243 0.321 Bran + Luebbe
AACS V
(Nutrient analyzer) Phosphate 0.124 0.017 0.026 0.048 Silicon silicate - 0.184 0.324 0.124 Nitrogen percentage
(N / P) 10.89 27.8 9.31 6.69 main
element
(mg / L) Sodium (Na) 4,866 10,357 10,264 10,367 ICP analysis Magnesium (Mg) 1,314 1,271 1,272 1,258 " Calcium (Ca) 401 374 367 389 " Potassium (K) 230 276 243 282 " Constant composition ratio 21.1: 5.7: 1.7: 1.0 37.5: 4.6: 1.3: 1.0 42.2: 5.2: 1.5: 1.0 36.7: 4.4: 1.3: 1.0 Na: Mg: Ca: K harmfulness
effect
matter
(mg / L) Cadmium (Cd) - - - - ICP analysis Lead (Pb) - - - - " Copper (Cu) - - - - " Mercury (Hg) - - - " Hardness
(Hardness) 6258 6019 6005 6004 Using the conversion formula
(2.5 x Ca + 4 x Mg)

As shown in Table 1 and Table 2, the concentration of Ca ion (5238.5 mg / L) in the groundwater of the Namhae basin was higher than that of surface water (401 mg / L), deep ocean water (374 mg / 389 mg / L).

The hardness of the Namhae groundwater was 13,305, which was twice as high as that of general seawater and deep seawater (6004 ~ 6258), and was measured to be 1,6 times higher than that of Ariimum (8,117).

It was also higher than nitrate nitrogen and total Indian sea water, which are food and nutrient sources of microalgae.

Therefore, it can be concluded that Namhae groundwater is a competitive natural mineral resource even if it is analyzed only by physicochemical data.

Since the calcium ion contained in the salt groundwater serves as a metabolic stimulation factor necessary for the growth of microalgae, the concentration of calcium ion contained in the salt groundwater is preferably 3000 to 8000 mg / L.

Table 3 below shows the results of the analysis of the radionuclides of the South Sea groundwater, and the radiation was below the reference value or not measured. Therefore, it is possible to use it as potable water because it is not in conflict with the salt ground water standard law. (Radiation analysis of Cs-137, I-131, K-40, etc. was conducted by the Korea Radiation Technology Research Institute.)

Analyte nuclide Sample type Radioactivity concentration (mBq / L) MDA (mBq / L) Gamma nuclide (Cs-137) South Sea saltwater N.D. - Gamma nuclide (I-131) South Sea saltwater N.D. - Gamma nuclide (K-40) (natural radioactivity) South Sea saltwater 138.44 17.19 - Sr-90 South Sea saltwater Less than MDA 0.56 Tritium South Sea saltwater Less than MDA 1.444 Analysis Institution: Korea Radiation Technology Institute N.D: Not Detected MDA: Minimum Detectable Activity

Experiments to cultivate Dunaliella salina marine algae using the salt ground water are as follows.

1. Measuring Growth Rate

The growth rate of microalgae was determined by measuring a single cell of a microalgae using a hematocytometer at a constant interval from the day when the microalgae were inoculated, and calculating the cell division ratio ( K ) by the following equation (1) (Guillard 1975 ).

[Equation 1]

Where N _o is the initial population, N ₁ is the final population, t ₀ is the initial day of culture, and t ₁ is the final day of culture.

In this experiment, the concentration of chlorophyll a was measured by using a spectrophotometer to calculate the concentration of chlorophyll a. The amount of chlorophyll a was measured by filtration of 3 mL of the sample with glass fiber filter paper (GF / C, 45 ㎜) 1, and 10 mL was added. The crushed sample was put in a centrifuge tube, sealed, and centrifuged (4000 rpm, 20 min) in a dark place at 4 ° C.

After centrifugation, a part of the supernatant was transferred to an absorbing cell, and the solution was taken with a spectrophotometer (Cary 50 Conc, Varian, USA). As a control solution, acetone: water (9: Measure the absorbance of the test solution at 630 nm and 750 nm and calculate the concentration of chlorophyll a according to Equation 2 based on Standards Methods (APPA 1995).

&Quot; (2) "

Y = amount of chlorophyll a (L L ^-1 ) = 11.64 X ₁ - 2.16 X ₂ + 0.10 X ₃

X ₁ = OD ₆₆₃ - OD ₇₅₀

X ₂ = OD ₆₄₅ - OD ₇₅₀

X ₃ = OD ₆₃₀ - OD ₇₅₀

The photosynthetic efficiency of chlorophyll a is measured by fluorescence spectrometer (Phyto-PAM, Walz, Germany) at 470 ㎚, 535 ㎚ and 620 ㎚ at the maximum quantum in PSII.

&Quot; (3) "

Fv / Fm = (Fm'-Ft) / Fm '= dF / (Ft + dF)

In Equation (3), Fv / Fm is a threshold value of the fluorescence value emitted from PSII, which means the photosynthetic efficiency of the photosystem, dF is the increase in fluorescence efficiency, Ft is the instantaneous fluorescence and Fm 'is the maximum fluorescence.

The control medium is D medium, the test medium is different from the nitrogen content in the medium, and the salt medium ground medium of the present invention is utilized.

The inoculated microalgae were cultured at a constant light intensity (14 h / L, 10 h / D) and at 24 ° C and 130 μmol m ^-2 s ^-1 .

In addition, 0.5 ton of the culture tank was used to culture at a light amount of 330 μmol m ^-2 s ^-1 .

<Control medium D medium composition used in this experiment>

2. Culture test of D. salina using saline groundwater

1) Pigment changes of D. salina by dilution concentration of saltwater

As shown in the first experiment in which D. salina was inoculated at different concentrations as shown in FIG. 1, the cell density was most dramatically increased in a culture medium diluted with 40% salt water (FIG. 2).

At this time, the salt ground water is diluted using distilled water, and the dilution ratio is 30 to 50% based on the volume of salt ground water, but it is preferable that the diluted water is diluted so that the salt groundwater is diluted 40%.

As a result of the second experiment, the final density was increased in the medium supplemented with 3 to 7% (preferably 5%) of BM (bioactive water) in a culture medium diluted with 40% salt water (FIG. 3)

3, SW represents salt groundwater and W represents distilled water.

In addition, in the third experiment, the cell activity of D. salina was highest in the medium diluted with 33% salt water, the cell size was relatively large, and the cell density and pigment were high. (Figure 4)

As such, the salt concentration of the salt water (for example, the Namhae groundwater) is 14 times higher than that of the general seawater and the deep sea water. Therefore, the actual biomass is the D and 270% more than salina.

Accordingly, the saltwater ground medium of the present invention for reducing the mass production cost of D. salina can be expanded to the field demonstration culture scale, or it can be used as a medium for mass culture or mass culture demonstration culture field in connection with genetically modified species have.

Claims (3)

The concentration of calcium ion is 3000 ~ 8000mg / L salt groundwater or rock groundwater,
Wherein the distilled water is added to the salt ground water or the rock groundwater. The method according to claim 1,
Wherein the distilled water contains 30 to 50% by volume of salt groundwater or rock groundwater volume. 3. The method of claim 2,
Characterized in that it contains 3 to 7% of bioactive water (BM) in salt-ground water or rocky groundwater medium conditions diluted by the distilled water.

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