KR100799065B1 - Medium composition obtained from fermented animal wastewater including a natural substitute chelator - Google Patents

Abstract

A medium composition comprising a natural substitute chelator is provided to reduce preparation costs, maximize production of microalgae such as green algae, and minimize or inhibit toxicity by removing use of synthetic chelating agents such as EDTA(ethylene diamine tetraacetic acid). A medium composition for production of microalgae comprises 20-60% of bacteria mineral water as a natural substitute chelator which is prepared by adding rocks and humus soil pallet into pig urine in a fermentation vessel, and fermenting the mixture with aeration, wherein the microalgae are Chlorophyceae, especially Chlorella ovalis(KPCCI P-M-2) and Dunaliella parva(KPCCI P-M-6).

Description

Medium composition obtained from Fermented Animal Wastewater Including a Natural Substitute Chelator}

1 is a graph showing the change in the content of chlorophyll a of microalgae

2A to 2D are graphs showing photosynthetic efficiency

The present invention relates to a medium composition using bioactive water (Bacteria Mineral Water, BM), and more specifically, to a known medium, bioactive water is added to the known medium instead of the chemical chelating agent (EDTA), thereby making the bioactive water natural. The present invention relates to a medium composition containing bioactive water as a chelating agent, which is used as a chelating agent so that the growth of microalgae is excellent and the side effects due to toxicity are not generated.

Microalgae are studied as indicators for water quality determination and are a source of high value-added materials such as vitamins, beta-carotene, polyunsaturated fatty acids, proteins, oils, and methane gas. It is being spotlighted as a biotechnological research and development material such as food, living organism, fertilizer and fuel.

Recently, microalgae have been applied for bioremediation and used in the environmental industry to remove environmentally friendly water pollutants and remove CO ₂ from the atmosphere.

In order to industrialize these microalgae, basic research to increase cell density per unit area is essential, and the growth of microalgae according to various culture conditions (medium type, light quantity, mineral quality, temperature, presence of specific inorganic elements, etc.) A number of basic studies were conducted (Kim and Giraud 1989; Kim and Dubacq 1997; Kim and Smith 2001).

Especially with wastewater, Chlorella , Scenedesmus and Spirulina Large scale culture studies of platensis have been applied for the purpose of water purification or as a high protein feed source.

As a technique for mass-producing microalgae and purifying water quality, Korean Patent Registration No. 500333 discloses a medium composition in which 3% bioactive water is added to a commercial medium, and a method of manufacturing the same.

In the above, Bacteria Mineral Water (Reference: Hiroshi N. 1998. Bacteria Saves the Earth-Challenges of BMW Technology. Blue Peace, 216 pp.) Is an active water recycled from livestock wastewater such as pig urine. As a final treatment, the livestock wastewater is introduced into a vessel into which the rock and humus pellets are aerated and aerated for fermentation.

However, the conventional microalgae culture medium (f / 2, ASW, etc.) including a medium composition using the conventional biologically active water includes a chemical synthetic chelating agent, which is necessarily added to the medium composition. Traces of heavy metals (Zn, Cu, Co, etc.) do not dissolve with other components and function to prevent the toxic expression of these ions.

As the chelating agent, EDTA (etylenediaminetetraacetic acid) is usually used, and this EDTA is a hardly decomposable synthetic amino acid used as a heavy metal antidote and preservative. Has been reported to promote the growth of microalgae (Sung et al. 1998).

However, EDTA is a chemical synthetic material that is produced and sold at a high price. Therefore, when EDTA is used for mass production of microalgae, the production cost is increased, and the product's market competitiveness becomes inferior. There are many disadvantages to the side effects.

For example, the chelating agent is used for the treatment of chronic fatigue syndrome, islet muscle pain, lupus, cancer, arthritis, high blood pressure, etc. in alternative medicine of chelation theraphy, but low blood calcium level, allergy, hypoglycemia, etc. The same side effects are emerging, and these chelating agents not only act selectively on harmful metals, but also bind to trace metal elements, enzymes, etc. necessary for human metabolism and inhibit their actions, resulting in severe side effects on the living body.

Therefore, the development of eco-friendly natural chelating agents to reduce the production cost and increase the mass production efficiency for the industrialization of microalgae is required, as well as the development of chelating agents harmless to the human body.

In view of the above demands, an object of the present invention is to add bioactive water to a commercial medium instead of a chemical synthetic chelating agent so that it can be used as a natural chelating agent.

Another object of the present invention is to provide a medium composition rate that can maximize the productivity of green algae by using a natural chelating agent that is not toxic, to enable mass production of green algae.

A feature of the present invention for achieving the above object is that the bioactive water is added as a chelating agent added 3-50% to the weight of the total medium in the known commercial medium containing no chemical synthetic chelating agent In the medium composition.

An embodiment of the present invention having the features as described above will be described below.

First, look at in detail with respect to the materials and culture methods used in the present invention.

Culture condition

The marine microalgae used in the embodiment of the present invention is Chlorella obalis (Chlorrophyceae). ovalis ) (KPCCI PM-2) and Dunaliella parva (KPCCI PM-6) were distributed by the Korea Industrial Plankton Material Bank (KPCCI) at the Youngnam University Marine Science Research Center.

The control medium was f / 2 medium composed of the composition of Table 1 (Guillard 1975), and the experimental medium was 6% of B + medium (E + 3) and BM added with 3% of bioactive water (BM) to f / 2 medium. BM 3% added to the medium (-E + 3) in which the chelator Na ₂ EDTA was removed from the added medium (E + 6) and f / 2 medium constituents (f / 2-E) And 6% medium (-E + 6) and 10%, 20%, 30%, 40% and 50% BM medium (-E + 10, -E + 20, -E + 30) , -E + 40, -E + 50) were inoculated with the algae described above.

Table 1 shows the composition of f / 2 medium containing EDTA as a chemical chelator.

The inoculated microalgae were incubated for 14 days at a period of high cell division in a shaking incubator at a constant contrast cycle (14h / L, 10h / D) and light at 24 ° C and 130μmol · m ⁻² · s ⁻¹ . In order to measure the photosynthetic efficiency with chlorophyll a, the cells were completely incubated in the medium, and cultured for 27 days after entering the cell division plateau.

How to measure

Growth rate measurement of microalgae

Every two days from the day when two green algae were inoculated into the medium, a single cell of microalgae was measured by using a hematocytometer, and the cell division rate (K) was calculated according to Equation 1.

N _o is the initial population, N ₁ is the final population, t ₀ is the culture day when the initial population, and t ₁ means the culture day when the final population.

Quantitative and Qualitative Analysis of Chlorophyll a

The content of chlorophyll a was measured by filtering 3 mL of cultured green algae with glass fiber filter paper (GF / C, 45 mm), and then adding 10 mL of a mixture of acetone and water in a ratio of 9: 1 to the filter paper, and then grinding the sample. Was placed in a centrifuge tube, sealed, and centrifuged (4000 rpm, 20 min) in a dark place at 4 ° C.

A portion of the supernatant was transferred to an absorbing cell and tested using an absorbance photometer (Cary 50 Conc, Varian, USA).

Take a mixed solution of acetone: water mixed with 9: 1 as a test solution, and use the control solution as the reference solution. Measure the absorbance of the sample solution at 663 nm, 645 nm, 630 nm and 750 nm, and the concentration of chlorophyll a is determined by the Standards Methods (APPA) 1995).

The photosynthetic efficiency of chlorophyll a was measured by a fluorescence analyzer (Phyto-PAM, Walz, Germany) by measuring the maximum quantum at PSII at wavelengths of 470 nm, 535 nm and 620 nm by the method of Equation 3.

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

Fv / Fm is a threshold value of the fluorescence emitted from PSII, which means the photosynthetic efficiency of the light system, dF is the increase in fluorescence efficiency, Ft is the instantaneous fluorescence rate, and Fm 'is the maximum fluorescence rate.

Porcine urine treated water ( Biologically active water , Water quality analysis of BM)

The biologically active water used in the present invention is the final purified water (BM) in the swine urine purification plant facility in Chuncheon Soyanggang bioactive water (BM) farm. Water quality was analyzed.

Turbidity was measured with a digital turbidity meter (2100N, HACH, Korea), hydrogen ion concentration, DO and water temperature were pH / DO meter (D-55, Horiba, Japan), and electrical conductivity was measured with 712 electrical conductivity meter (Metrohm, Switzerland). It measured using. COD, SS, T-N and T-P were analyzed according to the water pollution process test method.

Quantitative and qualitative analysis to measure changes in heavy metals (Fe, Mg, Cu, Ti, Mn) of raw water and treated water of BM is performed by taking an appropriate amount of samples and adding 4 mL of H ₂ O ₂ and 3 mL of HNO ₃ . After digestion using (microwave), it was diluted with 2-3% HNO ₃ and analyzed by Inductively Coupled Plasma Mass Spectrometer (ICP-MS) (PQ3, VG, UK).

Look at the results according to the embodiment of the present invention as described above.

BM Water Quality Changes

Comparative analysis of water quality by collecting appropriate amounts of wastewater, fermentation for 10days, and fermentation for 40days before fermentation by soil microorganisms (such as actinomycetes and wild yeast) The results are shown in Table 2 and Table 3.

* Bactareia mineral water originated from the BM Farm of Chunchon,

Table 2 shows the physico-chemical characteristics of BM, and it can be seen that the total nitrogen (T-P) and total phosphorus (T-P) levels decrease as fermentation proceeds than in raw water.

This indicates that in the process of raw water fermentation by soil microorganisms, microorganisms decompose organic substances composed of nitrogen or phosphorus contained in pig urine into another organic acid including natural chelating agent.

Table 3 shows the variation of heavy metal concentrations in original swine urine and treated BM.The results of quantitative and qualitative analysis of heavy metals in raw water and 40 days fermented water (BM) Looking at, the copper (Cu) and iron (Fe) content was reduced as the fermentation process in raw water, magnesium (Mg) and manganese (Mn) was increased. In particular, magnesium content increased 15 and 12 times in fermented water than in raw water at 10 and 40 days of fermentation, respectively.

Magnesium ions, which make up the chlorophyll of microalgae, are central metal ions chelated with nitrogen and complex ions in one of the four pyrroles, and the redox potential is controlled in the photochemical reaction of photosynthesis. It plays an important role (Kim and Thomas 1991; et al. 2004).

Therefore, it is confirmed that BM contains a large amount of complex ions capable of inducing chlorophyll synthesis of microalgae, and it can be seen that the increase of magnesium ions in BM acts as a facilitating factor for chlorophyll synthesis.

Iron (Fe) ions decreased 41% in pig urine livestock wastewater after 10 days of fermentation, and 78% of BM fermented in 40 days. It can be seen that iron ions are used in the synthesis of natural chelating agents to replace the function of EDTA by binding to secondary metabolites produced by microorganisms.

Changes in Cell Growth Rate According to BM Concentration

Marine algae Chlorella The cell density of ovalis was increased with reference to Table 4, and cell growth rate and cell density increased in the medium added with BM 3% at f / 2 (E + 3), compared to the control medium added with EDTA. (1.1-fold), while cells grown in medium (E + 6) added BM 6% at f / 2 were reduced (0.6-fold).

On the other hand, the cell density of 14 days cultured in -E + 3 and -E + 6 cultures from which EDTA was removed from f / 2 medium was higher than that of f / 2 medium (2.5 and 1.8 times, respectively).

Cells of C. ovalis had much higher growth rates (-E + 30: 8-fold; -E + 40: 9-fold; -E + 50: 19-fold) in medium with high BM concentration (30-50%).

Dunaliella on the other hand Parva showed no significant change in cell density in the range of 3-5% of BM concentration in Table 4, but showed high cell density in -E + 3 medium without EDTA.

In the -E + 10 medium, the two microalgae showed a similar increase rate of 1.21 and 1.24 times, respectively, compared to the f / 2 control medium, but in -E + 20 medium, compared to C. ovalis (1.3 times) grown in the f / 2 control medium. The cell growth rate of D. parva (3 times) was much higher.

D. parva showed lower cell division rate with increasing BM concentration than C. ovalis . However, as EDTA was removed and the concentration of BM increased, the cell density increased with C. ovalis cells.

These results indicate that -E + 50 medium with EDTA removed and 50% BM added to f / 2 medium was the optimal medium for C. ovalis and D. parva cultures, and BM could replace the role of chemical synthetic EDTA. In addition to increasing the cell division rate of up to 19-fold ( C. ovalis ) and 7-fold ( D. parva ) in both green algae cultured in f / 2 medium, microalgae industrialization, water purification and atmospheric It can be applied to the practical use of CO ₂ control technology and mass production for the industrialization of food organisms.

Table 4 shows the variation of cellular division rates and cell densities of Chlorella ovalis and Dunaliella of two green algae cultured in various media for 14 days. parva cultured for 14 days in each medium).

Changes in Chlorophyll a Content

Chlorophyll a is a medium through which microalgae converts light energy into bioenergy through photosynthesis and is a primary producer of animal plankton and fish, which is an aquatic consumer, and an indicator that can grasp the flow and aquatic environment of aquatic ecosystems. do.

In order to analyze the primary productivity of the microalgae, the change in the content of chlorophyll a shown in FIG. 1 is compared with the change in the cell density of the microalgae.

Chlorophyll a was analyzed by comparing the pigment content on day 27 of the culture, and the content of chlorophyll a was different depending on the presence of EDTA in the medium.

Chlorophyll a concentration in C. ovalis decreased in the order of -E + 3-> E + 6-> f / 2-> E + 3-> -E + 6 medium, and D. parva chlorophyll a. The a content was decreased in the order of -E + 6-> -E + 3-> E + 6-> E + 3-> f / 2 medium.

Thus, both green algae showed the increased rate of chlorophyll a in the medium from which EDTA was removed from f / 2 medium as in the change of cell density.

Chlorophyll a content of C. ovalis with increased BM concentration was the highest at -E + 50 (1,527 mg L ^-1 ) and D. parva was highest at -E + 50 (15,004 mg L ^-1 ). .

Therefore, the optimal synthesis medium of chlorophyll a of two green algae was -E + 50 medium which removed EDTA and added 50% of BM.

Maximum photosynthetic efficiency comparison

Phyto-PAM (phytoplankton fluorescence induction measuring instrument), Phytoplankton Analyzer, was used to measure the primary productivity. 470 nm (when photoreceptor pigment through blue filter is chlorophyll b), 535 nm (photoreceptor pigment through green filter) In the case of carotenoids), the threshold of fluorescence was measured at 620 nm wavelength (in case of photoreceptive dye through orange filter) and the highest photosynthetic efficiency was measured in photosystem II of C. ovalis and D. parva .

C. ovalis showed the same type at each wavelength of cells cultured in f / 2, E + 3, E + 6, -E + 3, -E + 6 medium after 27 days of culture. As shown in FIG. 2B, the maximum photon counts of 535 nm and 620 nm increased with D. parva , while 470 nm decreased.

This indicates that the photosynthetic efficiency of carotenoids increased due to the increase in the number of D. parva cells, depending on the composition of the medium, compared to the light harvesting pigment antennas by chlorophyll b. Judging.

C. ovalis in the medium group (f / 2-E, -E + 30, -E + 40, -E + 50) to which more BM was added in FIGS. 2C to 2D was obtained at 470 nm as shown in FIG. 2C. Cells grown in E + 30 medium had the highest photon number (photosynthetic efficiency), and 535nm and 620nm showed higher photoene counts in -E + 40 medium. As the concentration of BM added to the medium increased, the carotenoid photoreceptor pigment increased. The phenomenon showed the same result as D. parva shown in FIG. 2D.

This suggests that as the concentration of BM is increased in the medium without EDTA in the culture of two green algae, the photosynthetic efficiency is increased by increasing the chlorophyll a content and the photoreceptor pigment photoinduced by the carotenoid than chlorophyll b in the auxiliary pigment. .

Therefore, BM was added more than f / 2 control medium by combining the results of cell density of chlorophyll a and photon number of PSII by wavelength (470nm, 535nm, 620nm) according to the presence of EDTA and BM medium composition. The growth rate and photosynthesis rate increased according to the composition ratio, and the medium with the addition of 50% BM was removed as the most efficient medium by removing the chemical chelating agent Na ₂ EDTA added to the f / 2 medium composition.

According to the present invention configured as described above, it can be used as a culture medium of microalgae without using a chemical synthetic chelating agent, so that side effects due to the toxicity of the chemical synthetic chelating agent do not occur, There is a significant savings effect.

In addition, since livestock wastewater is used for microalgae cultivation, there is an effect of resource recycling with the effect of environmental purification.

Furthermore, when 50% of the biologically active water is added to the medium as described above, the growth rate of the green alga is very high, thereby greatly improving the productivity.

Claims (2)

In biologically active water fermented by adding and aeration of rocks and humus pellets into a vessel into which swine urine is introduced, the biologically active water is used as a chemical synthesis killing agent to be used as an antiseptic and heavy metal detoxifying agent to replace the function of chemical synthesis chelating agent. A medium composition containing bioactive water as a chelating agent, characterized in that it is added to a known commercial medium that is not added a rating agent. [Claim 2] The medium composition of claim 1, wherein the bioactive water is added to the total weight of the medium by 20 to 60%.

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