CN102453685B - A method of utilizing carbon dioxide to cultivate marine green algae to accumulate starch - Google Patents

Abstract

A method of using _CO2 to cultivate seawater green algae to accumulate starch involves marine green algae to cultivate and accumulate starch, specifically a cultivation method that utilizes carbon dioxide in the gas and adds nutrient salts to cultivate seawater green algae in a light bioreactor and accumulate starch. Using the marine green alga Tetraspermia subcardiac as the experimental material, in an air-lift flat-plate photobioreactor, CO ₂ in the gas is used to add nutrient salts to seawater, and the concentration of KNO ₃ in the medium is controlled at 0.4-0.6 g/L and continuous light, after 4 to 6 days of culture, the algal cells in the stable phase are harvested, the starch content reaches 45% to 55% of the dry weight of the algae, and the starch yield reaches 0.25 to 0.31g·L ^-1 ·d ^-1 . The method for cultivating marine green algae in the invention has the advantages of low cost, quick accumulation of starch, and ability to reduce CO ₂ emissions.

Description

A method of utilizing carbon dioxide to cultivate marine green algae to accumulate starch

technical field

The present invention relates to the cultivation and accumulation of starch of marine green algae, specifically a cultivation method utilizing carbon dioxide (CO ₂ ) in the gas (including CO ₂ in the tail gas discharged from the factory) and adding nutrient salts. In the light bioreactor, Adopt suitable light, temperature, pH and aeration conditions to cultivate seawater green algae and accumulate starch. It can be used in CO2 _emission reduction and recycling, algae cultivation, starch production and energy, food, medicine and other related fields using starch as raw material.

Background technique

Human beings are currently facing increasingly serious environmental problems. Large-scale industrial development has brought about excessive emissions of carbon dioxide. The greenhouse effect and global climate change caused by CO ₂ are gradually threatening the survival and development of human beings. The reduction of CO ₂ emission has become the focus of attention all over the world.

Marine green algae (Marine Green Algae) are low-level green plants that grow in the ocean. They can convert CO ₂ and sunlight into biomass and release oxygen through photosynthesis. Marine green algae are considered to be one of the very feasible methods for biologically fixing _CO2 because of their fast growth rate, high _CO2 fixation efficiency, strong environmental adaptability, no occupation of cultivated land, easy manual control, and continuous production. The high added-value substances it produces make it economically viable.

Starch is the accumulated product of photosynthesis in green algae, which is stored in the cells in the form of starch granules. Many green algae that can accumulate starch in large quantities are concentrated in freshwater algae, such as Chlorella (Chlorellavulgaris) can accumulate starch accounting for more than 50% of dry weight when protein synthesis is hindered; When mixed culture, it can accumulate more than 40% to 60% of dry weight starch; Chlamydomonas reinhardtii can also achieve more than 50% starch content. However, there are few reports that marine green algae can accumulate starch in large quantities. The use of seawater, which accounts for 97% of the earth's water volume, to produce seaweed starch can greatly reduce the demand for fresh water resources. In today's tight fresh water resources, its environmental protection significance is particularly prominent. In addition, most of the current microalgae cultivation methods that can accumulate starch are heterotrophic or mixed cultures with the addition of organic matter, which makes the production cost of starch higher, and the use of CO ₂ , especially the use of ethanol fermentation tail gas or flue gas and other industrial waste If CO ₂ is the only carbon source for microalgal starch accumulation, the production cost will be significantly reduced.

Contents of the invention

The purpose of the present invention is to provide a method for cultivating seawater green algae and accumulating a large amount of green algae starch using _CO2 . In order to achieve the above object, the technical scheme adopted in the present invention is:

Mix CO ₂ with air and pass it into the light reactor for algae cultivation. By controlling the cultivation conditions such as nutrients, light, temperature and pH, the marine green algae can accumulate a large amount of starch in a photoautotrophic way and harvest the algae at the right time. cell.

Using the marine green algae Tetraselmis subcordiformis as the experimental material, in a photobioreactor, using CO ₂ in the gas and adding nutrient salts, continuous light is used to make the algal cells grow and accumulate starch.

You can follow the steps below to specifically operate:

1) Obtaining algae cell seed solution

Preparation of algae culture medium: add nutrient salts to the seawater filtered by a microfiltration membrane with a pore size of 0.3 μm and its final concentration is: NaNO ₃ 100 mg/L, NaH ₂ PO ₄ 2H ₂ O 20.0 mg/L, EDTA-Na ₂ 45.0mg/L, H ₃ BO ₃ 33.6mg/L, MnCl ₂ 4H ₂ O 0.36mg/L, FeCl ₃ 6H ₂ O 1.3mg/L, ZnCl ₂ 0.21mg/L, CoCl ₂ 6H ₂ O 0.20mg/L, (NH ₄ ) ₄ Mo ₇ O ₂₄ 4H ₂ O 0.09mg/L, CuSO ₄ 5H ₂ O 0.20mg/L, KNO ₃ 1g/L, KH ₂ PO ₄ 0.05g/L, Tris 0.81g/L, _{CH3COOH0.346g} /L. Sterilize by high pressure steam at 110°C for 15 minutes, and cool down for later use.

Cultivation of algae cell seed solution: Pick a single algae colony of Tetraselmiss subcordiformis isolated and purified from a flat plate, and culture it step by step in a shaker flask. The culture conditions are: temperature 25-30°C, light intensity 30-70 μmol E·m ^-2 ·s ^-1 , white fluorescent lamp, light-to-dark ratio 14-24h: 10-0h, static cultivation, manual shaking more than 3 times a day.

2) Photobioreactor cultivation

Preparation of medium: add nutrient salts to the seawater filtered by microfiltration membrane with pore size of 0.3 μm and its final concentration is: NaH ₂ PO ₄ 2H ₂ O 20.0 mg/L, EDTA-Na ₂ 45.0 mg/L, H ₃ BO ₃ 33.6mg/L, MnCl ₂ 4H ₂ O 0.36mg/L, FeCl ₃ 6H ₂ O 1.3mg/L, ZnCl ₂ 0.21mg/L, CoCl ₂ 6H ₂ O 0.20mg/L, (NH ₄ ) ₄ Mo ₇ O ₂₄ ·4H ₂ O 0.09mg/L, CuSO ₄ ·5H ₂ O 0.20mg/L; and KNO ₃ was added at a final concentration of 0.4-0.6g/L. Sterilize by high pressure steam at 110°C for 15 minutes, and cool down for later use.

Cultivation of algae cells: The sterilized reactor is soaked in NaClO, cleaned with sterile water, and inserted into the culture medium and algae cell seed solution. The culture conditions are: the initial dry weight of cells inoculated is 0.4-0.5g/L, the temperature is 25-30°C, the pH of seawater is 6-8, the light intensity is 100-250μmol E·m ^-2 ·s ^-1 , continuous light, and air The flow rate is 0.125-0.375VVM, the _CO2 content is 1%-3%, and the algal cells are harvested when the cells enter the stationary phase after 4-6 days of culture. The starch content of the cells reaches 45%-55% of the dry weight of the algae. It can be performed intermittently or semi-continuously. nourish. The starch yield in the whole process can reach 0.25～0.31g·L ^-1 ·d ^-1 .

The seawater is natural seawater or artificial seawater; the formula of artificial seawater is: NaCl 27g/L, MgSO ₄ 7H ₂ O 6.6g/L, MgCl ₂ 6H ₂ O 5.6g/L, CaCl ₂ 2H ₂ O 1.5g /L, NaHCO ₃ 0.04g/L.

Compared with the prior art, the present invention has the following advantages:

1. Environmental friendliness: The cultivation process of marine green algae is pollution-free, and CO ₂ can be used as the only carbon source for photosynthetic autotrophy, and the utilization efficiency of CO ₂ is high, so as to realize CO ₂ emission reduction and alleviate the greenhouse effect.

2. The sustainability of starch production is good: using abundant seawater to produce starch through green algae, reducing the consumption of fresh water resources; at the same time, green algae starch production can be carried out in areas that are not suitable for food planting and human habitation, solving the problem of starch production Competing with others for food and land.

3. Low cultivation cost and rapid accumulation of starch: the formula of marine green algae culture medium is simple, the source of required nutrient elements is wide, and the cost is low; starch accumulates rapidly in algae cells, and the production cycle is short.

In a word, the present invention utilizes carbon dioxide in the gas (including CO ₂ in factory exhaust tail gas) to cultivate marine green algae to accumulate starch, realize CO ₂ emission reduction and recycling, and can be widely used in starch production and energy and food using starch as raw material , medicine and other related fields.

Description of drawings

Fig. 1 is the schematic diagram of green algae cultivation device of the present invention; Note: 1. Photobioreactor main body; 2. Light source; 3. Baffle plate; 4. Aeration pipe; 5. Gas filter; 6. Gas flowmeter; 7. Air compressor; 8. Gas pressure reducing valve; 9. _CO2 gas cylinder; arrows represent the direction of air flow.

Fig. 2 is the growth kinetics curve of green algae cell under different light-dark time ratios of the present invention;

Fig. 3 is the green algae starch accumulation kinetics curve under different light-dark time ratios of the present invention;

Fig. 4 is the kinetic curve of the ratio of green algae starch per unit cell dry weight under different light-dark time ratios of the present invention;

Fig. 5 is the growth kinetics curve of green algae cell under different _KNO3 concentration of the present invention;

Fig. 6 is the kinetic curve of green algal starch accumulation under different _KNO concentrations of the present invention;

Fig. 7 is a kinetic curve of the proportion of green algae starch per unit dry weight of cells under different KNO ₃ concentrations in the present invention.

Detailed ways

The method and results of the present invention will be described below through specific examples. The present invention uses marine green algae Tetraselmis subcordiformis as the experimental material, and uses _CO2 in the gas and a culture method of adding nutrient salts in a light bioreactor to continuously illuminate the green algae to accumulate starch.

The photobioreactor is an air-lift flat-plate photobioreactor. An aeration tube is inserted in the middle of the bottom of the reactor to disperse the introduced gas into small bubbles to improve the gas-liquid mass transfer efficiency. The source is connected; there are two parallel baffles suspended in the reactor, and there is a gap between the baffle and the bottom of the reactor. The algae liquid in the reactor does not pass through the baffle. The middle of the baffle rises, driving the algae liquid in the reactor to rise in the middle of the baffle and descend on both sides of the baffle to form a cycle and avoid dead ends; the material used in the reactor is transparent organic glass bonded by methylene chloride; the two sides of the reactor Both are provided with white fluorescent lamps; the gas source is an air compressor and _a CO gas cylinder, the flow of which is controlled by a gas pressure reducing valve and a gas flow meter arranged on the pipeline; the air produced by the air compressor and the CO gas from the gas cylinder ₂ After the flow is adjusted to reach the required _CO2 concentration, it is introduced into the reactor.

Example 1

The effect of light-dark cycle on algal cell growth and starch accumulation was investigated. Tetraselmis subcordiformis was provided by Dalian Fisheries Research Institute of Liaoning Province, and was purified by plate in our laboratory.

1) Obtaining algae cell seed solution

Preparation of nutrient salt mother liquor: prepare 1000 times nutrient salt mother liquor with deionized water, the formula is: NaNO ₃ 100g/L, NaH ₂ PO ₄ 2H ₂ O 20.0g/L, EDTA-Na ₂ 45.0g/L, H ₃ BO ₃ 33.6g/L, MnCl ₂ 4H ₂ O 0.36g/L, FeCl ₃ 6H ₂ O 1.3g/L, ZnCl ₂ 0.21g/L, CoCl ₂ 6H ₂ O 0.20g/L, (NH ₄ ) _{4Mo 7} O ₂₄ ·4H ₂ O 0.09g/L, CuSO ₄ ·5H ₂ O 0.20g/L. Sterilize by high pressure steam at 110°C for 15 minutes, and cool down for later use.

Preparation of algae culture medium: natural seawater was filtered through a microfiltration membrane with a pore size of 0.3 μm. Add 1mL of the above nutrient salt mother solution, KNO ₃ 1g, KH ₂ PO ₄ 0.05g, Tris 0.81g, CH ₃ COOH 0.346g to every liter of filtered seawater. Sterilize by high pressure steam at 110°C for 15 minutes, and cool down for later use.

The cultivation of algae cell seed liquid: Pick the single algae colony of Tetraselmiss subcordiformis isolated and purified from the plate, and culture them step by step in test tubes, 100mL shake flasks, 1000mL shake flasks and 3000mL shake flasks. The culture conditions are as follows: : temperature 25°C, light intensity 50 μmol E·m ^-2 ·s ^-1 , white fluorescent lamp, light-dark ratio 14h:10h, static culture, manual shaking more than 3 times a day.

2) Photobioreactor cultivation

Preparation of nitrogen-free nutrient salt mother solution: prepare 1000 times nitrogen-free nutrient salt mother solution with deionized water, the formula is: NaH ₂ PO ₄ 2H ₂ O 20.0g/L, EDTA-Na ₂ 45.0g/L, H ₃ BO ₃ 33.6g/L, MnCl ₂ 4H ₂ O 0.36g/L, FeCl ₃ 6H ₂ O 1.3g/L, ZnCl ₂ 0.21g/L, CoCl ₂ 6H ₂ O 0.20g/L, NH ₄ ) ₄ Mo ₇ O ₂₄ ·4H ₂ O 0.09g/L, CuSO ₄ ·5H ₂ O 0.20g/L. Sterilize by high pressure steam at 110°C for 15 minutes, and cool down for later use.

Preparation of culture medium: natural seawater was filtered through a microfiltration membrane with a pore size of 0.3 μm. 1 mL of the above nitrogen-free nutrient salt mother solution and 0.4 g of KNO ₃ were added to each liter of filtered seawater. Sterilize by high pressure steam at 110°C for 15 minutes, and cool down for later use.

Cultivation of algae cells: The sterilized reactor is soaked in NaClO, cleaned with sterile water, and inserted into the culture medium and algae cell seed solution. The culture conditions are as follows: the initial dry weight of cells inoculated is 0.5g/L, the temperature is 25°C, the pH of seawater is 7, the light intensity is 140μmol E·m ^-2 ·s ^-1 , the air flow rate is 0.25VVM, the CO ₂ content is 3%, light The dark time (h) ratios were set at 24:0, 14:10 and 12:12, respectively.

As shown in Figure 1, the photobioreactor is an air-lift flat-plate photobioreactor. The size of its main body 1 is that the length of the illuminated surface is 80mm, the height is 750mm, the light path is 40mm, and the liquid volume is 1.6L. The bottom of the reactor is inserted with an aeration tube 4, which is 25 mm above the bottom of the reactor, and can disperse the introduced gas into small bubbles, thereby improving the efficiency of gas-liquid mass transfer. The reactor has two built-in baffles 3 with a height of 510mm. The lower edge of the baffles is 26mm higher than the bottom of the reactor, which can make the gas rise in the middle, drive the algae liquid in the reactor to rise in the middle, and descend on both sides, forming a cycle and avoiding dead ends. The material used in the reactor is transparent plexiglass bonded by dichloromethane. White fluorescent lamps 2 are arranged on both sides of the reactor. The gas source is an air compressor 7 and a _CO gas cylinder 9, the flow of which is controlled by a gas pressure reducing valve 8 and a gas flow meter 6. The air produced by the air compressor 7 and the _CO2 from the gas cylinder 9 are regulated by the flow rate of the gas flow meter 6 to reach the required _CO2 concentration, and then introduced into the reactor after being sterilized by the air filter 5.

It can be seen from Figure 2 that the above-mentioned photobioreactor is used to cultivate marine green algae. Under the condition that the initial inoculum size, temperature, light intensity, ventilation rate, _CO2 concentration and nutrient supply are all the same, continuous light is used. (Light-to-dark ratio 24:0) The dry weight of algae in the reactor increased from the initial 0.5g/L to 2.1g/L within 4 days, while the dry weight of algae in the reactor was 14:10 and 12:12. The heavy ascent speed is significantly lower than that of continuous light. Using continuous light can make algae cells fix more _CO2 and grow rapidly in a short period of time.

As can be seen from Figure 3 and Figure 4, using the above-mentioned photobioreactor to cultivate marine green algae, under the same culture conditions such as initial inoculum size, temperature, light intensity, ventilation rate, _CO concentration and nutrient supply, The starch concentration in the reactor using continuous light (light-to-dark ratio 24:0) rose from the initial 0.05g/L to 1.18g/L within 4 days, and the starch content of the algae cells rose from 10% at the time of inoculation to 55%. When the light-dark ratio was 14:10 and 12:12, the increase rate of starch concentration in the reactor was significantly lower than that of continuous light. After 4 days of continuous light culture, it enters into a stable period, and the algae cells are harvested. The starch content of the cells reaches 55% of the dry weight of the algae, and the starch yield can reach 0.28g·L ^-1 ·d ^-1 .

Example 2

The effect of initial KNO ₃ concentration on algal cell growth and starch accumulation was investigated. Tetraselmis subcordiformis was provided by Dalian Fisheries Research Institute of Liaoning Province, and was purified by plate in our laboratory.

The preparation of the algae cell seed liquid and the preparation of the nitrogen-free nutrient salt mother liquid are the same as in "Example 1".

Preparation of culture medium: Artificial seawater was filtered through a microfiltration membrane with a pore size of 0.3 μm. The artificial seawater formula is: NaCl 27g/L, MgSO ₄ ·7H ₂ O 6.6g/L, MgCl ₂ ·6H ₂ O 5.6g/L, CaCl ₂ ·2H ₂ O 1.5g/L, NaHCO ₃ 0.04g/L. Add 1mL of nitrogen-free nutrient salt mother solution to every liter of filtered seawater. Sterilize by high pressure steam at 110°C for 15 minutes, and cool down for later use.

Cultivation of algae cells: The sterilized reactor is soaked in NaClO, cleaned with sterile water, and inserted into the culture medium and algae cell seed solution. The culture conditions are as follows: initial dry weight of cells inoculated at 0.4g/L, temperature at 25°C, pH 7, light intensity of 140μmol E·m ^-2 ·s ^-1 , continuous light, air flow rate of 0.25VVM, CO ₂ content of 3% , adding KNO ₃ to make its concentration 0, 0.12, 0.6 and 1.2g/L respectively.

As can be seen from Figure 5, under the same culture conditions such as initial inoculum size, temperature, light intensity, light cycle, aeration rate and _CO2 concentration, the reactions with _KNO3 concentration of 0.6g/L and 1.2g/L The dry weight of algae in the container increased from the initial 0.4g/L to 3.4g/L and 3.8g/L within 6 days, which was significantly higher than the experimental group with a lower KNO ₃ concentration, indicating that the higher KNO ₃ concentration had It is beneficial for algae cells to fix _CO2 and accumulate biomass. However, as can be seen from Figure 6 and Figure 7, the starch content can only reach 35% when the _KNO3 concentration is 1.2g/L; although the starch content of algae cells rises rapidly and can reach 48% when the _KNO3 concentration is low, However, the starch content decreased rapidly after two days of cultivation, and due to the limitation of biomass, the starch concentration in the reactor was far lower than that of the experimental group whose KNO ₃ concentration was 0.6g/L. When the concentration of KNO ₃ was 0.6g/L, the algal cells entered the stationary phase after 6 days of culture, the starch content increased from the initial 6% to 45%, and the starch yield reached 0.25g·L ^-1 ·d ^-1 .

Claims (3)

1. one kind is utilized CO ₂the method of cultivating marine green microalgae accumulation starch, is characterized in that:

In illumination bio-reactor, carry out marine green algae cultivation, with KNO ₃for nitrogenous source, utilize airborne CO ₂for sole carbon source, in adding the seawater of nutritive salt, continuous illumination is cultivated, and frustule is grown and accumulate starch, cultivates after 4～6 days and when cell enters stationary phase, gathers in the crops frustule; Pass into air gas flow 0.125～0.375VVM, CO in air ₂volume content 1%～3%, KNO in seawater ₃concentration 0.4～0.6g/L;

Marine green microalgae used is sub-heart type four slit bamboo or chopped wood algaes (Tetraselmis subcordiformis);

The ultimate density of adding nutritive salt in described seawater is: NaH ₂pO ₄2H ₂o20.0mg/L, EDTA-Na ₂45.0mg/L, H ₃bO ₃33.6mg/L, MnCl ₂4H ₂o0.36mg/L, FeCl ₃6H ₂o1.3mg/L, ZnCl ₂0.21mg/L, CoCl ₂6H ₂o0.20mg/L, (NH ₄) ₄mo ₇o ₂₄4H ₂o0.09mg/L, CuSO ₄5H ₂o0.20mg/L.

2. it is characterized in that in accordance with the method for claim 1: described seawater is natural sea-water or artificial seawater.

3. it is characterized in that in accordance with the method for claim 1:

The culture condition of described illumination bioreactor culture marine green algae is: dry weight 0.4～0.5g/L during cell initial inoculation, 20～30 ℃ of temperature, seawater pH=6～8, intensity of illumination 100～250 μ mol Em ^-2s ^-1, continuous illumination, passes into air flow quantity 0.125～0.375VVM, CO in air ₂content 1%～3%, KNO ₃concentration 0.4～0.6g/L, cultivates after 4～6 days and when cell enters stationary phase, gathers in the crops frustule, and cell starch content reaches 45%～55% of algae dry weight, carries out intermittence or semicontinuous cultivation; Whole process starch productive rate can reach 0.25～0.31gL ^-1d ^-1.

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