KR20100094965A - Development of low-cost media for mass culture of spirulina - Google Patents

Abstract

The present invention relates to the establishment of an economical medium composition and optimal culture conditions capable of increasing the biomass of microalgae Spirulina capable of producing bioenergy. More specifically, the present invention aims to increase the content of biomass and constituents of spirulina by establishing optimal culture conditions by utilizing human urine as a nutrient medium. Through this, cultured spirulina can be harvested and crushed to produce carbohydrates, which are the raw materials of biodiesel and lipids. Therefore, human urine can be utilized as an economical medium for bioenergy production by culturing microalgae.

Description

Development of economic medium for mass production of bioenergy resource spirulina {Development of low-cost media for mass culture of Spirulina}

The present invention relates to the establishment of an economical medium composition and optimal culture conditions capable of increasing the biomass of microalgae Spirulina capable of producing bioenergy.

(1) bioenergy

Biomass generated by consuming organic matter and photosynthesis using sunlight can be utilized as an energy source. Biomass is a renewable and environmentally friendly alternative energy that has no problem of exhaustion. If bioenergy is produced using plants (especially grains) as raw materials, there may be a situation where there may be competition with food and feed problems, but there is no problem when using algae or green algae. Cyanobacteria and green algae are cheaper than plants and are a natural resource for bioethanol, biodiesel and biohydrogen.

There is a growing interest in the development of resources and technologies related to bioenergy production around the world. Global bioethanol production has been growing at an average annual rate of 20% since 2000, and Brazil and the United States account for most of the world production. In the US, we expect gasoline consumption to decrease by 20% by 20% and bioethanol use gradually by 2017, with an average growth rate of 5% by 2030 (Energy Information Administration, 2007). have. Since the Kyoto Protocol, the share of bioenergy among major advanced countries' alternative energy consumption has increased to 30-50%, and the EU market has shown an annual increase of 50%. In 2005, bioenergy produced in the EU was 391.4 million tons, an increase of 65.8% from 2004, and by 2020, the company plans to increase its share of the transportation fuel market to 10%. In Japan, the production of bioethanol was 30 kW / year at the end of 2005 and 400-5000 kW / year of biodiesel, but by 2030, 80% of the oil dependence on the transport sector is set.

In Korea, the market accounted for only 3.7% of domestic alternative energy use (as of 2005). Currently, biodiesel is attracting the most attention among domestic biofuels. Currently, 16 companies are registered as producers, and the annual output is only 400,000 tons. Most of the existing biodiesel producers were small and medium-sized enterprises, but in 2007, large companies such as SK Chemicals and Aekyung Petrochemical are obtaining and participating in the producers. The current outlook for bioethanol is opaque, and there is no record of it being used for fuel due to storage and distribution infrastructure construction costs and raw material import issues. Therefore, it is absolutely necessary to secure and disseminate bioethanol production technology in the future. The biggest problem of the domestic industry is the high overseas dependence of bioenergy raw materials. At present, there is almost no domestic raw material supply, which is defenseless against sudden changes in international raw material prices. Recently, vegetable oil prices are soaring due to increasing interest in bioenergy around the world. Even if some raw materials such as rapeseed, soybean and corn are grown, there is no economic feasibility due to lack of land, wages and labor. Therefore, the development of bioenergy alternative raw materials is very necessary.

(2) price

Currently, the wholesale price of aquaculture microalga Nannochloropsis is $ 18,000 (US) per tonne and the retail price of aquaculture Chlorella Vulgaris is $ 36,000 (US) per tonne. The selling price is set at a high price because it can be used as a source of food, feed, and bio-energy of southern algae and green algae. As a result, the competition for the establishment of economic mass system for producing algae and green algae is intensifying.

(3) spirulina

Spirulina, suggested by NASA as an ideal food for astronauts, contains proteins, vitamins (AE), polyunsaturated fatty acids, polysaccharides, and essential minerals (Calcium, Iron, Sodium). Magnesium, Zinc, etc.) are abundant. Spirulina is a living, single-celled organism that converts light energy into bioenergy and is distributed in a variety of environments, absorbing carbon and releasing more oxygen than higher plants. It also contains natural pigment carotenoids that promote redness of livestock meat and yellowing of poultry egg yolk when used as animal feed, and also contains alternative natural ingredients that can prevent misuse of antibiotics, growth promoters and chemicals added to existing animal feed. Doing. Therefore, when used as an animal feed is known as live bacteria that can enhance growth promotion, sexual maturity and fertility. Spirulina can be cultivated in solid and liquid media, but in Korea, it is currently imported from New Zealand, the US, China and Taiwan due to lack of mass culture facilities and economic problems such as production cost.

Accordingly, the present inventors have completed the present invention by finding out that human waste is valuable as an economical mass production medium while developing a spirulina medium composition capable of effectively producing bioenergy.

In order to achieve the above object, the present invention is the growth of microalgae and containing proteins, lipids, carbohydrates using urine (human urine), which is a physiological waste of humans rich in minerals N, P, K We want to measure the change in. Through this, we will explore the possibility of using as an economical medium for microalgae cultivation and bioenergy production for the purpose of nutrition recycling. Hereinafter, the solution of the present invention will be described in more detail.

Therefore, the present invention was used to cultivate spirulina using human urine as a nutrient medium, harvested and crushed cells to measure the change in lipid content as a raw material of biodiesel and carbohydrate content as a raw material of bioethanol.

In this project, spirulina was cultivated using 1 / 200-fold diluted urine as a medium, and total lipid content and total carbohydrate content, which are raw materials of bioenergy, were produced. Thus, urine, a waste product of humans, can be usefully used as an economic nutrition medium for mass production of microalgae. Hereinafter, the specific method of the present invention will be described in detail with reference to Examples. However, the scope of the present invention is not limited to the following examples.

Urine Medium Concentration, Culture Temperature and Additive Growth Curves

{Example}

1. Inoculation and culture of synthetic medium and spirulina

The composition of the control (SOT) medium (aka Zarrouk's Medium) of the experiment is as follows.

NaHCO ₃ 18.0g, NaNO ₃ 2.5g, K ₂ HPO ₄ 0.5g, K ₂ SO ₄ 1.0g, NaCl 1.0g, CaCl ₂ 0.04g, Na ₂ EDTA 0.08g, MgSO ₄ 7H ₂ O 0.2 g, FeSO ₄ 7H 0.01 g ₂ O, 1.0 mL of trace elements (TE) (pH 8.2, titrated with 1 mM NaOH)

The composition of TE (g / L) contained above is as follows.

H ₃ BO ₃ 2.86, (NH ₄ ) ₆ Mo ₇ O ₂₄ 0.02, MnCl ₂ 4H ₂ O 1.8, Cu ₂ SO ₄ 0.08, ZnSO ₄ 7H ₂ O 0.22

The standard medium was inoculated with spirulina at 0.05 g / L-0.01 g / 200 ml and shaken at 18 rpm.

In the standard medium, the culture temperature directly affects the spirulina growth rate. In the standard medium, the growth starts at 20 ° C., and at 37 ° C. or higher, the cells die. In this study, cultures were conducted at 30 ° C and 35 ° C to determine the temperature at which biomass, protein, lipid and carbohydrate concentrations were affected.

2. Biomass Measurement

Dry weight was used by Vonshak et al. (1982). OD 670 nm of dry weight (g / L) was measured to make a standard curve, and then measured and compared the OD value of each treated sample and the growth curve was determined.

Experimental results of measuring the growth of spirulina and the change of biomass according to the pH change of standard medium (8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0) are as follows. Incubation temperature of 30 ° C showed the maximum growth rate at pH 8.5 (635.3 mg in the building), the next highest value of 590.2 mg in dry matter at pH 8.0 (Fig. 1 and Table 3). In general, the stronger the pH, the lower the growth rate. At pH 11, the cells died.

In the case of the culture temperature of 35 ° C. showed a maximum growth rate at pH 8.0 (445.3 mg in the building), followed by 9.0 and 9.5 (Fig. 2 and Table 4). The average growth rate decreased due to the stress higher than the incubation temperature of 30 ° C, and the cells were killed at pH above 10.0.

In order to determine the appropriate light intensity required for the growth of spirulina in the standard medium, a fluorescence 40W was tested in a 12-hour / 12-hour-cycle cycle in the 1200-3000 lux range and the maximum growth of spirulina was achieved at 3000 lux. Seemed. Too low light showed photo-limitation and too high photo-inhibition.

3. Dilution Ratio and Growth of Waste Recycling Medium

Urine was collected for 24 hours in adults to dilute 1/100, 1/150, 1/200 was used as a medium experiment. As a result, spirulina grown in medium diluted 1/200 of the urine stock at 30 ° C. showed the highest growth rate of 465.3 mg in dry matter (FIG. 3 and Table 5). However, in the medium diluted with 1 / 100-1 / 50 of the urine stock solution at the culture temperature of 30 ° C, cells did not grow and died due to toxicity. Growth rate was inhibited by adding VitB12 to urine at 30 ° C incubation temperature. The dilution 1/200 and the dilution 1/200 added with VitB12 were slightly grown at the culture temperature of 35 ° C., but the cells showed signs of death 9 days after the culture, and thus could not be used in the experiment (FIG. 4 and Table 5).

Therefore, diluting the urine stock solution to 1/200 proved to be the most effective dilution rate for spirulina growth. Comparing the above results, it was reduced to 73.2% of spirulina cultivated at 30 ° C and standard medium (pH 8.5), but 4.5% higher than that of 35 ° C and standard medium (pH 8.0). Increased. The growth rate was slightly lower than that of spirulina grown at the standard medium (pH 8.5) and the culture temperature of 30 ° C.

4. Determination of Biomass Components

① Total protein content was measured by Lowry method. Crushed harvested spirulina sample (10 mg / ml) was reacted with 250 μl [Spirulina 5 μl (50 μg) + 145 μl distilled water] and 800 μl Reagent (A + B + C) for 20 minutes at room temperature. It was. 100 μl Folin-Ciocalteau`s Phenol reagent was added to the reaction solution, and the resultant was treated at room temperature for 1 hour, and the OD value was measured at a light absorption value of 560 nm by using a spectrophotometer.

Reagent for determination of total protein content Reagent A: 2% Na ₂ CO ₃ /0.1N NaOH
B: 1g CuSO ₄ 5H ₂ O / 100ml Distilled Water
C: 2g C ₄ H ₄ KNaO ₆ 4H ₂ 0 / 100ml distilled water A (12.25 ml) +
B (125 μl) +
C (125 μl)
Folin-Ciocalteau`s Phenol reagent
Dilution with distilled water (1: 1)
Standard (0.1% BSA)

Experimental results of measuring the change of total protein of spirulina according to the pH change of the standard medium (8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0) are as follows. At cultivation temperature 30 ℃ spirulina grown in a suitable standard medium at pH 8.0 showed a maximum protein content of 73.91% (Fig. 5 and Table 3). Similar to growth rate, the higher the pH, the lower the protein content. The maximum protein content was 78.11% in the standard medium titrated at pH 8.0 at 35 ° C (4.2% increase over 30 ° C) and 14.19% increase at 30 ° C even at pH 9.5 (67.68%) (Figure 5 and table). 4).

Experimental results of the waste recycling medium presented above showed a maximum protein content of 61.91% (118.9 mg in the building) of spirulina grown at a culture temperature of 30 ° C. and a urine dilution 1/150. On the other hand, 52.66%, 1/200 diluent + vitB12 was 52.62% in 1/200 dilution (Fig. 6 and Table 5).

② Total lipid content was measured by sulfo-phospho-vanillin method. A brief description of the method follows. Pulverized harvested spirulina (10 mg / ml) was mixed with 200 μl [100 μl of spirulina (1 mg) + 100 ml distilled water] and 750 μl organic solvent (1: 2, CHCl ₃ : MeOH) and then vortexed. It was. 250 μl of CHCl ₃ was added, vortexed, centrifuged at 1,000 rpm for 5 minutes, the supernatant was transferred to a new tube, dried, and 500 μl of sulfuric acid (concentrated) was added and vortexed. The tube heated at 100 ° C. for 10 minutes was cooled on ice and then treated at room temperature. 100 μl of the reaction solution was transferred to a new tube, 900 μl of phospho-vanillin was added, and the resultant was stirred at room temperature for 30 minutes to form a color. The absorbance at 530 nm was measured.

The result of measuring the total lipid content by the above method is as follows.

The total lipid content of spirulina grown at 30 ° C. and standard medium (pH 8.0) was measured as the maximum lipid content (Fig. 7 and Table 3). The highest lipid content was 7.71% at 35 ° C. and pH 9.5. (Figure 7 and Table 4). Therefore, the culture temperature of 30 ℃ and standard medium (pH 8.0) was found to be the most efficient medium to increase the lipid content.

Experimental results of the waste recycling medium presented above showed the highest total lipid content, 8.9% of spirulina grown in dilution 1/200 at the incubation temperature of 30 ° C (Fig. 8 and Table 5). This measurement was 0.61% lower than the total lipid content of spirulina grown at 30 ° C. and standard medium (pH 8.0), but 1.19% higher than 35 ° C. and standard medium (pH 9.5).

③ Total carbohydrate content was measured by Anthrone method (Osborne and Voogt, 1978). A brief description of the method follows. The harvested spirulina (10 mg / ml) was ground and mixed with 500 μl of extract (spirulina 5 mg) and 200 μl of 2% sodium sulfate, followed by centrifugation at 13,00 rpm for 5 minutes. 100 μl of the supernatant was transferred to a 15 ml tube, then 2 ml of Anthrone solution was added and heated at 100 ° C. for 12 minutes. After treatment with ice, the absorbance was measured at 625 nm.

Reagents for Determination of Total Carbohydrate Content % sodium sulfate
0.2% Anthrone solution
12 mg Anthrone + 5 ml sulfuric acid + 1 ml distilled water
Standard (0.1% glucose)

The result of measuring the total carbohydrate content by the above method is as follows.

Spirulina grown at 30 ° C incubation temperature and standard medium (pH 10.0) contained the highest carbohydrate content (3.09%) (Figure 9 and Table 3), but higher carbohydrate content at culture temperature 35 ° C and standard medium (pH 8.5) (4.62%) was determined to contain (Figure 9 and Table 4). Therefore, in the case of standard medium, the culture temperature of 35 ℃ was observed as the optimum temperature.

Experimental results of the waste recycling medium presented above showed the highest total carbohydrate content of 7.88% in spirulina grown in diluent 1/200 at 30 ° C. (FIG. 10 and Table 5). This measurement is 1.7 times larger than the maximum content in the standard medium. Therefore, the optimal medium for increasing the total carbohydrate content of spirulina is effective in diluting 1/200 human urine.

None

Claims (2)

Technology to cultivate microalgae using human urine as nutrient medium
According to claim 1, Dilution ratio and optimal culture temperature of urine capable of culturing spirulina

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