CN104046566B

CN104046566B - Method for rapidly preparing high-density and high-purity algae

Info

Publication number: CN104046566B
Application number: CN201310082447.3A
Authority: CN
Inventors: 李运广; 罗玮; 汪靓; 伍阳; 朱艳红
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-03-15
Filing date: 2013-03-15
Publication date: 2020-01-14
Anticipated expiration: 2033-03-15
Also published as: CN104046566A

Abstract

The invention discloses a method for quickly preparing high-density and high-purity algae seeds, belonging to the technical field of algae application. It comprises a high-efficiency culture medium and a preparation method thereof. By using the high-efficiency culture medium, the algae are cultured by shaking in a triangular flask and introducing air into a cylindrical glass tube and a flat photobioreactor in sequence. According to the density change of the algae in the culture process, the light intensity (200 plus 10000 lux) is gradually increased; CO regulation of the introduced CO₂The amount and the pH value of the culture solution are kept between 6.5 and 8.5, so that the requirements of illumination and carbon sources of the algae in different growth periods are better met, the harm of high-intensity light to the growth of the algae is avoided, and the optimal growth state is kept, thereby improving the photosynthesis efficiency. 1.2g and 0.65g of dry algae can be produced per liter of culture medium per day in the cylindrical glass tube and the flat plate bioreactor, and the final dry algae weight per liter of culture medium can respectively reach more than 12g and 8 g. The invention can greatly shorten the large-scale culture period of the algae, reduce the probability of algae pollution in the culture process and is an effective scheme for obtaining high-purity algae products.

Description

Method for rapidly preparing high-density and high-purity algae

Technical Field

The invention belongs to the technical field of algae application, and particularly relates to a culture method for quickly obtaining high-density and high-purity algae. In particular to a method for preparing high-density and high-purity algae seeds by regulating and controlling light intensity and ventilation and utilizing different culture devices in a short time, which are used as inocula for large-scale culture and are used for producing initial feed, functional food, secondary metabolites with high added values, biodiesel and the like for fishes, shrimps and shellfish.

Background

Algae is a kind of photoautotroph with simple structure, and can efficiently utilize light and CO in the presence of water and some inorganic nutritive salts₂Synthesizing organic matters. Algae has high economic value, such as chlorella is a high-quality plant protein source, haematococcus pluvialis can obtain a large amount of astaxanthin after being induced, spirulina is a good functional food, dunaliella can be used for producing carotenoids in a large amount, some marine microalgae are main plant sources of EPA and DHA, and a plurality of micro-algae are usedThe algae can be directly used as initial feed for young aquatic economic animals such as fish, shrimp, shellfish, crab, etc. Microalgae can also rapidly remove organic and inorganic nutrients in sewage, and the production of biodiesel by using microalgae is a hotspot in the current biological energy research.

Compared with higher plants, the algae has the characteristics of small size, high photosynthetic efficiency, rapid cell division, wide environment adaptation range, high yield per unit area and the like. At present, the growing shortage of grain, energy and land resources, the algae scale culture and comprehensive development and utilization have good application prospect in solving a plurality of problems faced by human beings. In algal scale culture, the number of "seeds" limits the scale of production, while its quality and purity determine the yield and quality of the harvest. Therefore, how to rapidly obtain a large amount of high-purity algae with good activity in a short time is a key technology in the large-scale cultivation and application of algae.

Two factors that are critical to algal growth are light and carbon source (CO)₂). Insufficient light energy supply can reduce the growth rate of algae, while excessive light energy supply can produce photoinhibition, damage to algae cells and death of algae cells. In the process of algae culture, the density of algae cells is constantly changed, the light demand is increased, and how to adjust the light intensity according to the density change of the algae cells makes the light demand of the algae not only be met, but also no harm is generated, and the maximum algae biomass can be obtained. In algae cultivation, CO₂Not only is an important substrate for photosynthesis, but also is an important pH regulator. When the cell density of algae is higher, if no additional CO is added₂The pH will rise above 11, and the optimal pH for most algae growth is 7-8, which severely inhibits algae growth. Thus, CO₂The function as a pH regulator is particularly important when algal cells are cultured at high density. When algae cultivation is carried out according to the conventional method, a single container, a single illumination intensity and a single CO are often adopted₂The concentration method not only causes energy and nutrition waste, but also causes the yield of algae to be low, and the yield of the final harvest is more than 2g (dw) L^-1The following. It is to this point that the present invention is directedAnd the novel production flow is adopted, the yield of algae in unit time and unit volume is improved, and technical support is provided for development and utilization of related algae.

Disclosure of Invention

The invention provides a method for quickly obtaining high-density and high-purity algae. Inoculating algae cultured on solid culture medium into a high-efficiency culture medium, sequentially performing shake culture in a triangular flask, introducing and adding CO into a cylindrical glass tube and a flat plate photobioreactor₂Air culture of (2). In the process, the light intensity is gradually increased according to the density change of the algae; simultaneously, the introduced CO is adjusted through the feedback of pH₂The amount of the algae is enough to meet the requirements of illumination and carbon sources of the algae in different growth periods, the harm of high-intensity light to the growth of the algae is avoided, and the algae is kept in the optimal growth state, so that the photosynthesis efficiency of the algae is improved, and the rapid proliferation of algae cells is realized. The technology provided by the invention can obtain a large amount of high-purity algae seeds in a short time, and is widely applied to the algae seed preparation link of algae-related industries.

The invention firstly provides a high-efficiency liquid culture medium which comprises the following components: KNO₃0.125-1.0 gL^-1，NaNO₃0.125-1.0 gL^-1，KH₂PO₄0.04-0.5 gL^-1，Na₂HPO₄0.05-0.5 gL^-1，MgSO₄7H₂O 0.06-0.3gL^-1，CaCl₂0.02-0.1 gL^-1，NaHCO₃0.02-3 gL^-1, FeCl₂3-20 mgL^-1，Na₂EDTA 1-10 mgL^-1,H₃BO₃1-10 mgL^-1，MnCl₂4H₂O 0.5-5 mgL^-1,ZnSO₄7H₂O 0.1-0.6 mgL^-1, CuSO₄5H₂O 0.02-0.2 mgL^-1, Na₂MoO₄2H₂O 0.01-0.8 mgL^-1，CoCl₂6H₂O 0.02-4 mgL^-1. When preparing the culture medium, distilled water or tap water left for 12 hours may be used.

The preferred amounts of the nutrient salts of the liquid medium are as follows: KNO₃0.44 gL^-1，NaNO₃0.60 gL^-1，KH₂PO₄0.136 gL^-1，Na₂HPO₄0.142 gL^-1，MgSO₄7H₂O 0.06-0.3 gL^-1，CaCl₂0.03gL^-1，NaHCO₃0.2 gL^-1, FeCl₂7.542 mgL^-1，Na₂EDTA 5 mgL^-1,H₃BO₃3.15 mgL^-1，MnCl₂H₂O 2 mgL^-1,ZnSO₄7H₂O 0.222 mgL^-1, CuSO₄5H₂O 0.08 mgL^-1, Na₂MoO₄2H₂O 0.041 mgL^-1，CoCl₂6H₂O0.04 mgL^-1。

For convenient taking, the nutrient salt is divided into four groups to be prepared into mother liquor with different concentrations, and each mother liquor is as follows: KNO₃、NaNO₃、KH₂PO₄And Na₂HPO₄Sequentially dissolving in distilled water to prepare 100

Mother liquor 1, CaCl₂And FeCl₂Sequentially dissolving in distilled water to obtain 1000

Mother liquor 2, MgSO₄·7H₂O and NaHCO₃Sequentially dissolved in distilled water to prepare 1000Mother liquor 3, Na₂EDTA,H₃BO₃、MnCl₂·4H₂O、ZnSO₄·7H₂O、CuSO₄·5H₂O、Na₂MoO₄·2H₂O and CoCl₂·6H₂Dissolving O in distilled water in turn to prepare 1000

And 4, mother liquor. Sterilizing the mother solutions respectively during preparation of culture medium, cooling, and collecting 10ml mother solution 1, 1ml mother solution 2, 1ml mother solutionAdding solution 3 and 1ml solution 4 into 700ml water, stirring thoroughly, adding the other one to prevent precipitation, and adding water to 1L.

The culture process of the algae provided by the invention is as follows.

1) Sterilization of laboratory instruments

Glass instruments such as triangular flasks, glass tubes and the like used in the experiment are sterilized by a high-temperature high-pressure method, the photobioreactor is cleaned and dried in the air, then is moved into a culture chamber sterilized by ultraviolet rays, 75% alcohol is sprayed on the inner wall of the culture chamber for sterilization, and after the culture chamber is dried in the air, the culture chamber is sealed for later use. The culture medium used for seed preservation, activation and culture in the cylindrical glass tube is sterilized by high temperature and high pressure. The culture medium is filtered and sterilized under negative pressure in a flat plate type reactor, and the aperture of a filter membrane is 0.45 mu m.

2) Preparing a solid culture medium containing 1.5% agar powder by using the optimized culture medium for algae seed preservation, streaking or plating the algae, and culturing at 20-30 ℃ under 500lux illumination.

3) Algal seed activation algal seeds seeded on solid medium were transferred to sterilized 50-500ml Erlenmeyer flasks. The volume of culture broth in the flask did not exceed 2/3 of its capacity. The sterilization condition is 103.5 kPa,121 ℃, 20 min. The unicellular green algae or blue algae can be directly inoculated, and the algae forming a larger population is lightly ground by a sterilized homogenizer or mortar under the condition of not adding auxiliary substances such as quartz sand and the like, and then inoculated into a unicellular or smaller population. After inoculation, the mixture is placed on a shaking bed, the culture condition is 20-30 ℃, light is supplied for 24h, the light intensity is 200-^-1Shaking and controlling the rotation speed to ensure that the bottle does not have obvious cell precipitation. Just when the culture medium is transferred into a triangular flask from the solid culture medium, the culture light intensity is 200lux, after 2-3 days of culture, the culture solution is obviously green, the light intensity is increased to 600lux, the light intensity is increased to 1200lux after two days of culture, and the culture solution can be inoculated into the cylindrical glass tube after two days of culture. The light intensity is realized by adjusting the power of the light source (such as the number of fluorescent tubes) or adding a filter screen for shading.

4) Fast culture of algae seed, inoculating the activated algae seed in the cylindrical transparent glass culture tube in the volume ratio of 1 to 5, with the inoculation amount not exceeding that of the cylindrical transparent glass culture tubeThe glass tube is preferably 75% of the total volume to avoid the algae being flushed out by gas during the cultivation process. The cylindrical glass culture tube has a diameter of 2-5cm, preferably 3.5cm, a length of 30-80cm, and a U-shaped or pointed triangular cone bottom. The culture conditions were: the temperature is controlled at 20-30 ℃, light is supplied for 24h, the light intensity is 1000-. Introducing gas filtered by filter membrane with pore diameter of 0.45 μm, and flow rate of 0.04-2L/L culture medium for min^-1To minimize aeration to prevent cell sedimentation. The introduced gas is added with 0.2-2% of CO₂Of air, CO₂Can be from high pressure steel cylinder (99%) or other high concentration CO₂Such as treated flue gas from a thermal power plant. Introduction of CO₂The volume ratio was adjusted by a flow meter, and the flow rate was increased when the pH of the culture solution was higher than pH8.5 and decreased when the pH was lower than pH 6.5.

Initial light intensity is 1000lux, increased to 3000lux after 2 days, increased to 6000lux after 2 days of culture, and cultured for 2 days, wherein the algae biomass can reach 4-12g (dw) L^-1And then the culture medium can be transferred into a flat plate photoreactor for mass culture.

5) Mass culture of algae species the algae cultured in the cylindrical glass tube were transferred to a flat photobioreactor at a volume ratio of 1:10, with an initial inoculum size of 0.4-1 g (dw) L^-1Preferably, the total volume of the inoculum is not more than 80% of the total volume. The light path of the flat-plate photobioreactor is 2-6cm, preferably 3.5cm and the depth is 50-150 cm. Culturing at 20-30 deg.C for 24 hr under 2000-10000lux light intensity and pH 7-12, and culturing under aeration. The introduced gas is added with 0.2-2% of CO₂Of the air of (2). CO 2₂Can be from high pressure steel cylinder (99%) or other high concentration CO₂Such as treated flue gas from a thermal power plant. Introduction of CO₂The volume ratio was adjusted by a flow meter, and the flow rate was increased when the pH of the culture solution was higher than pH8.5 and decreased when the pH was lower than pH 6.5. The initial light intensity in the flat plate photobioreactor is set to 2000lux, the light intensity is increased to 4000lux after 2 days of culture, the light intensity is increased to 8000lux after 2 days of culture, the algal biomass can reach 4-8g (dw) L after two days of culture^-1At this time, can be harvestedFor analysis or seeding into a larger volume bioreactor for production.

Compared with the prior art, the method for quickly obtaining the high-density and high-purity algae seeds has the following characteristics:

1 the obtained algae has high density, and after 6 days of photoautotrophic growth, the biomass of the algae can reach 12g (dw) L^-1。

2 dynamically regulating and controlling illumination and CO according to the change of the cell density and pH value of algae in the culture process₂So that they can meet the requirement of algae growth but do not cause harm, and simultaneously save energy and resources.

3 according to the characteristics of different growth stages of algae, different reactors are adopted for stage-type culture, so that the algae cells can grow rapidly.

4, enough high-purity algae seeds can be obtained in a short time and used for high-density inoculation of outdoor semi-open or open scale culture, the culture period is shortened, the growth of hybrid algae and other organisms is effectively inhibited, and a high-purity culture is obtained.

Drawings

FIG. 1 shows Chlorella vulgarisChlorella vugaris) Growth and pH change in cylindrical glass tubes (25 ℃, 1.5% CO)₂）。

FIG. 2 shows Chlorella vulgarisChlorella vugaris) Growth and pH Change in a Flat photobioreactor (25 ℃, 1.5% CO)₂）。

Detailed Description

The invention provides a method for quickly preparing high-concentration and high-purity algae seeds. Specifically, by using the high-efficiency liquid culture medium provided by the invention, algae are cultured in a triangular flask in a shaking way and are subjected to aeration culture in a cylindrical glass culture tube and a flat plate type photobioreactor in sequence. Different illumination intensities and different CO are provided in different culture stages₂Concentration, meeting the requirements of the algae growth process on illumination and carbon source, avoiding light inhibition and carbon limitation, and keeping the algae in an optimal growth state so as to obtain the maximum biomass yield. The invention is further illustrated by the following specific examples.

Examples

Chlorella (Chlorella vulgaris)Chlorella vugaris) Purchased from the fresh water algae species collection center of the institute of aquatic organisms, academy of sciences, china.

The culture medium is preferably KNO (KNO)₃0.44 gL^-1，NaNO₃0.60 gL^-1，KH₂PO₄0.136 gL^-1，Na₂HPO₄0.142 gL^-1，MgSO₄7H₂O 0.06-0.3 gL^-1，CaCl₂0.03gL^-1，NaHCO₃0.2 gL^-1,FeCl₂7.542 mgL^-1，Na₂EDTA 5 mgL^-1,H₃BO₃3.15 mgL^-1，MnCl₂H₂O 2 mgL^-1,ZnSO₄7H₂O0.222 mgL^-1, CuSO₄5H₂O 0.08 mgL^-1, Na₂MoO₄2H₂O 0.041 mgL^-1，CoCl₂6H₂O 0.04 mgL^-1). The culture medium is prepared by using distilled water, sterilizing at 121 deg.C under 103.5 kPa for 20min, and cooling.

Selecting healthy algae from the plate, inoculating into a 50ml triangular flask, and placing at 100 rmin^-1The primary activation is carried out on a shaking table at 25 ℃ under 24h illumination. The initial culture light intensity is 200lux, after 2-3 days of culture, the culture solution is obviously green, the light intensity is increased to 600lux, the light intensity is increased to 1200lux after two days of culture, and the ratio of 1: 3 ratio of the seed into a glass tube with a diameter of 3cm and a length of 60 cm.

During the culture in the glass tube, the temperature was also controlled at 25 ℃. The initial light intensity is 1000lux, the light intensity is increased to 3000lux on the third day, the light intensity is increased to 6000lux on the fifth day, and the chlorella vulgaris biomass can reach 7.32g (dw) L after 2 days of culture^-1(FIG. 1A); if the cultivation is continued, the dry weight of the biomass per liter of culture broth on the eighth and tenth days may reach 9.86g and 10.7g, respectively.

For example, on the sixth day of culture, the cells were transferred at a ratio of 1:10 into a flat photobioreactor having an optical path of 3.5cm and a depth of 80cm, and aerated culture was carried out at 25 ℃. Initial light intensity was set at 2000luxThe light intensity is increased to 4000lux on the third day, the light intensity is increased to 8000lux on the fifth day, and the algal biomass reaches 4.96 g (dw) L on the sixth day^-1(FIG. 2A), up to 6.04g (dw) L on day eight^-1。

The glass tube and the plate photoreactor were charged with 1.5% CO₂The pH of the culture solution can be maintained between 6.5 and 8.5 by the air (FIG. 1B, FIG. 2B).

Claims

1. A method for rapidly preparing high-density and high-purity algae seeds is characterized by comprising the following steps: transferring the algae seeds on the solid culture medium to a sterilized triangular flask, a cylindrical transparent glass culture tube and a flat plate photobioreactor in sequence, shaking or ventilating for culture, and introducing CO₂And air, wherein CO₂The volume ratio of the carbon dioxide is 0.2-2%, and the volume ratio is adjusted by adjusting air and CO₂Flow rate controlled by a flow meter, CO₂The gas comes from high-pressure steel cylinder or other high-concentration CO₂Of gas of (CO)₂The ratio of (A) to (B) is preferably controlled so that the pH of the solution is in the range of 6.5 to 8.5, below which the concentration is reduced and above which the concentration is increased; the temperature is controlled to be 20-30 ℃, the light is supplied for 24 hours, and the light intensity is 5-100 mu mol photons m^-2s^-1pH 7-12; the high-efficiency liquid culture medium is prepared by adding the following nutrient salts into distilled water or tap water which is placed for 12 hours: KNO₃0.125-1.0gL^-1，NaNO₃0.125-1.0gL^-1，KH₂PO₄0.04-0.5gL^-1，Na₂HPO₄0.05-0.5gL^-1，MgSO₄·7H₂O 0.06-0.3gL^-1，CaCl₂0.02-0.1gL^-1，NaHCO₃0.02-3gL^-1,FeCl₂3-20mgL^-1，Na₂EDTA 1-10mgL^-1,H₃BO₃1-10mgL^-1，MnCl₂·4H₂O 0.5-5mgL^-1,ZnSO₄·7H₂O 0.1-0.6mgL^-1,CuSO₄·5H₂O 0.02-0.2mgL^-1,Na₂MoO₄·2H₂O 0.01-0.8mgL^-1，CoCl₂·6H₂O 0.02-4mgL^-1(ii) a KNO at the time of preparation₃、NaNO₃、KH₂PO₄And Na₂HPO₄Dissolving in distilled water in turn to prepare 100X mother liquor 1, MgSO₄·7H₂O、CaCl₂And FeCl₂Dissolving in distilled water in turn to prepare 1000X mother liquor 2, NaHCO₃Preparing 1000X mother liquor 3, Na₂EDTA,H₃BO₃、MnCl₂·4H₂O、ZnSO₄·7H₂O、CuSO₄·5H₂O、Na₂MoO₄·2H₂O and CoCl₂·6H₂Dissolving O in distilled water in sequence to prepare 1000X mother liquor 4; when preparing a culture medium, sequentially adding 10ml of mother liquor 1, 1ml of mother liquor 2, 1ml of mother liquor 3 and 1ml of solution 4 into 600ml of water, adding the other one after fully stirring to prevent the generation of precipitates, and finally supplementing water to 1L;

the algae is unicellular green algae or blue algae, or green algae or blue algae forming a colony; for algae forming larger colonies, gently ground with a sterilized homogenizer or mortar without the addition of aids such as quartz sand to obtain an inoculum of single cells or smaller colonies.

2. The method for rapidly preparing high-density and high-purity algae seeds according to claim 1, wherein the method comprises the following steps: the triangular flask and the glass tube are sterilized by adopting a high-temperature high-pressure method, the photobioreactor is moved into a culture chamber subjected to ultraviolet sterilization after being cleaned and dried, 75% of alcohol is sprayed on the inner wall of the photobioreactor for sterilization, and after the photobioreactor is dried, the culture chamber is sealed for later use.

3. The method for rapidly preparing high-density and high-purity algae seeds according to claim 1, wherein the method comprises the following steps: the size of the triangular flask is 50-500 ml; the diameter of the cylindrical glass culture tube is 2-5cm, the length is 30-80cm, and the bottom is U-shaped or sharp triangular cone-shaped; the light path of the flat-plate photobioreactor is 2-6cm, and the depth is 50-100 cm.

4. The method for rapidly preparing high-density and high-purity algae seeds according to claim 1, wherein the method comprises the following steps:the liquid culture medium is prepared by adding the following nutrient salts into distilled water or tap water which is placed for 12 hours, wherein the dosage of each nutrient salt is as follows: KNO₃0.44gL^-1，NaNO₃0.60gL^-1，KH₂PO₄0.136gL^-1，Na₂HPO₄0.142gL^-1，MgSO₄·7H₂O0.06-0.3gL^-1，CaCl₂0.03gL^-1，NaHCO₃0.2gL^-1,FeCl₂7.542mgL^-1，Na₂EDTA 5mgL^-1,H₃BO₃3.15mgL^-1，MnCl₂·H₂O 2mgL^-1,ZnSO₄·7H₂O 0.222mgL^-1,CuSO₄·5H₂O 0.08mgL^-1,Na₂MoO₄·2H₂O 0.041mgL^-1，CoCl₂·6H₂O 0.04mgL^-1。

5. The method for rapidly preparing high-density and high-purity algae seeds according to claim 1, wherein the method comprises the following steps: the algae is cultured in a triangular flask by shaking at 20-120rmin^-1Introducing gas filtered by a filter membrane with the pore diameter of 0.45 mu m into a glass tube or a photobioreactor, wherein the flow rate is 0.04-2Lmin per liter of culture medium^-1。

6. The method for rapidly preparing high-density and high-purity algae seeds according to claim 1, wherein the method comprises the following steps: the CO from the steel cylinder₂The purity of the gas is more than or equal to 99 percent.

7. The method of claim 1, wherein the method comprises the steps of: the other component contains CO at a high concentration₂The gas is treated flue gas of a thermal power plant.

8. The method for rapidly preparing high-density and high-purity algae seeds according to claim 1, wherein the method comprises the following steps: the light intensity is 200 plus 1200lux for the triangular bottle, 1000 plus 6000lux for the transparent glass tube and 2000 plus 10000lux for the flat-plate photobioreactor; the intensity is realized by adjusting the power of the light source or adding a filter screen for shading.

9. The method for rapidly preparing high-density and high-purity algal species according to claim 8, wherein the method comprises the following steps: the power of the light source is adjusted by the number of the fluorescent tubes.

10. The method for rapidly preparing high-density and high-purity algae seeds according to claim 1, wherein the method comprises the following steps: the optical stress is mainly based on the cell density of algae, when the algae is transferred from a solid culture medium into a 100ml triangular flask, the culture light intensity is 200lux, after 2-3 days of culture, the culture solution is obviously green, the light intensity is increased to 600lux, the light intensity is increased to 1200lux after two days of culture, and the culture is carried out for two days according to the following formula 1: inoculating 5 proportion into glass tube with diameter of 3cm and length of 60cm, initial light intensity is 1000lux, increasing to 3000lux after 2 days, increasing to 6000lux after 2 days of culture, and culturing for 2 days, wherein the algae biomass can reach 4-12gL^-1(ii) a Transferring into a flat photobioreactor with optical path of 3.5cm at a ratio of 1:10, setting initial light intensity to 2000lux, increasing to 4000lux after 2 days of culture, increasing to 8000lux after 2 days of culture, and culturing for two days until algae biomass can reach 4-8gL^-1。