Screening method of floating microalgae suitable for floating culture and floating culture method of microalgae

Abstract

The invention relates to the field of microalgae, and discloses a floating microalgae screening method suitable for floating culture and a microalgae floating culture method.The screening method comprises the steps that microalgae is inoculated and then cultured for 24 h or above, then standing is conducted for 6-12 h, the proportion of the dry weight of algae cells floating within 1 cm of the liquid level to the total dry weight of algae is measured, and the total dry weight of the algae is calculated according to the ratio of the dry weight of the algae cells floating within 1 cm of the liquid level to the total dry weight of the algae; screening microalgae with the proportion of more than 25wt% to obtain floating microalgae; wherein the culture time enables the OD (optical density) of the algae in the algae liquid to reach more than 0.2; wherein the standing condition is as follows: the illumination intensity is 20000 to 40000 lux. The screened spirulina can be used for floating culture, so that the biomass equivalent to that under a stirring culture condition can be obtained while the energy consumption caused by driving the spirulina liquid is greatly reduced.

Description

Technical Field

[0001] The invention relates to the field of microalgae, and in particular to a method for screening floating microalgae suitable for floating culture and a method for floating culture of microalgae. Background Art

[0002] Microalgae is a kind of low-level plant that grows in water and is extremely widely distributed. It is a cell factory driven by sunlight. Through the efficient photosynthesis of microalgae cells, it absorbs CO2, converts light energy into chemical energy of carbohydrates such as fat or starch, and releases O2. The use of microalgae to produce bioenergy and chemicals can simultaneously achieve the three goals of "replacing fossil energy, reducing CO2 emissions, and purifying waste gas and sewage." The advantages of "microalgae biotechnology" are as follows: ① Microalgae is the most photosynthetic primitive plant, and its yield per unit area is dozens of times higher than that of crops. ② Microalgae can grow in water bodies with high salt and high alkaline environments, and can make full use of tidal flats, saline-alkali land, and deserts for large-scale cultivation. It can also be cultivated using non-agricultural water such as seawater, saline-alkali water, and industrial wastewater. Therefore, microalgae can compete with crops for land and water. ③ High oil production rate. The oil content of microalgae stem cells can be as high as 70%. Microalgae do not have the cell differentiation of roots, stems, leaves, etc. of higher plants. Under conditions such as nitrogen deficiency, some single-cell microalgae can accumulate a large amount of oil and are the most promising oil-producing organisms. ④ The cultivation of microalgae can utilize CO2 in industrial waste gas to alleviate greenhouse gas emissions, and can also absorb NO in industrial waste gas. x , reducing environmental pollution. ⑤ While producing microalgae biodiesel, a considerable amount of microalgae biomass can also be produced, and high-value products such as protein, polysaccharides, and fatty acids can be further obtained.

[0003] The efficient and large-scale cultivation technology of microalgae, that is, obtaining microalgae biomass efficiently and at low cost by researching and developing new equipment and new processes for large-scale cultivation of microalgae, is one of the core technologies of microalgae biotechnology. Microalgae cultivation is generally carried out in photobioreactors, which can be divided into open photobioreactors and closed photobioreactors. Whether it is a closed photobioreactor or an open raceway pool reactor, in order for the microalgae to grow better under light, it is necessary to drive the movement of the algae liquid. For example, closed photobioreactors generally use compressed air or pumps to drive the movement of the algae liquid, and raceway pool reactors generally use paddle wheels to drive the flow of the algae liquid. However, driving the movement of the algae liquid inevitably leads to an increase in the energy consumption of the culture. Therefore, the energy consumption generated by the drive during large-scale cultivation is very huge, which has become an important reason for the high cost of cultivation. Summary of the invention

[0004] The purpose of the present invention is to overcome the problem of high energy consumption caused by driving during large-scale cultivation in the prior art, and to provide a screening method for floating microalgae suitable for floating cultivation and a floating cultivation method for microalgae. The floating microalgae screened by the screening method basically do not need to drive the movement of algae liquid during the cultivation process, which can greatly reduce the energy consumption during the cultivation process.

[0005] In order to achieve the above-mentioned object, the first aspect of the present invention provides a method for screening floating microalgae suitable for floating culture, the method comprising: inoculating microalgae and culturing them for more than 24 hours, then standing them for 6-12 hours, measuring the proportion of the dry weight of algae cells floating within 1 cm of the liquid surface to the total dry weight of the algae, and screening microalgae with a proportion of more than 25wt% to obtain floating microalgae;

[0006] The culture time is such that the OD of the algae in the algae solution reaches above 0.2;

[0007] Wherein, the static conditions include: light intensity is 20000-40000 lux.

[0008] The second aspect of the present invention provides a floating culture method of microalgae, the method comprising: inoculating the floating microalgae screened by the method as described above into a photobioreactor filled with culture medium for culture;

[0009] The culture process includes a static culture stage and a stirring culture stage, wherein the duration of the stirring culture stage is less than 1 hour per day;

[0010] Wherein, the photobioreactor is at least one layer of culture pool;

[0011] Among them, the depth of the microalgae liquid is 8-20cm.

[0012] The screening method of the present invention is used to screen out microalgae with floating characteristics. The microalgae has a buoyancy characteristic and can float on the surface of a liquid to fully absorb light for photosynthesis, thereby avoiding the problem of light transmission. The microalgae can then be floated and cultured, and the culture cost caused by energy consumption can be reduced during the culture process. The biomass in the floating culture can be increased to maintain a level close to that of the stirring culture. Moreover, under the condition of limited land resources, the microalgae can be cultured with artificial LED supplementary lighting, and the microalgae has the potential for large-scale application, and unstable energy sources such as photovoltaic power generation and wind power generation can be fully utilized. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is the growth curve of static culture of Spirulina with different floating characteristics;

[0014] Figure 2 is the growth curve of the floating culture of Spirulina in Test Example 1;

[0015] Figure 3 This is the growth curve of the agitated culture of Spirulina in Test Example 1. DETAILED DESCRIPTION

[0016] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0017] During the research process, the inventors of the present invention found that under low light intensity conditions, many algae species have floating characteristics when left stationary, but when the light intensity reaches a certain level, the microalgae will settle. This may also be the reason why the yield of microalgae in static culture is generally much lower than that in stirred culture. The inventors have discovered floating microalgae and determined a screening method for them, so that they can be cultivated under static conditions with a cultivation effect close to or even equivalent to that of stirred culture.

[0018] The first aspect of the present invention provides a method for screening floating microalgae suitable for floating culture, the method comprising: inoculating microalgae and culturing for more than 24 hours, then standing for 6-12 hours, measuring the proportion of the dry weight of algae cells floating within 1 cm of the liquid surface to the total dry weight of the algae, and screening microalgae with a proportion of more than 25wt% to obtain floating microalgae;

[0019] The culture time is such that the OD of the algae in the algae solution reaches above 0.2;

[0020] Wherein, the static conditions include: light intensity is 20000-40000 lux.

[0021] In the present invention, the screening method is applicable to all microalgae, such as unicellular algae or multicellular algae, and the microalgae are preferably filamentous algae, preferably one or more selected from Spirulina, Anabaena, Oscillatoria axolotl and Aphanizomenon.

[0022] In a preferred embodiment of the present invention, the microalgae is Spirulina.

[0023] It should be understood that the microalgae is inoculated into a photobioreactor filled with a culture medium, wherein the culture medium can be selected according to the specific microalgae species, such as BG11 culture medium, Zarrouk culture medium, SE culture medium, etc., and Zarrouk culture medium or a culture medium with a similar composition can be used as a Spirulina culture medium for the cultivation of Spirulina. The culture medium and the pH during the cultivation process can be adjusted according to the specific growth requirements of the algae species, for example, the pH of the Spirulina culture medium can be 8.5-10.

[0024] There is no particular limitation on the inoculation amount of the microalgae. In order to shorten the screening time, preferably, the initial OD of the algae solution after the microalgae inoculation is above 0.1, for example, it can be any range consisting of 0.1, 0.15, 0.2, 0.3, 0.4, 0.5 or above, and any two values, more preferably 0.1-0.5.

[0025] It should be understood that, depending on the algae species, the OD value can be measured at different wavelengths. For example, the wavelength for measuring the OD of Spirulina can be 570nm. 570 .

[0026] Preferably, the culture conditions include: a temperature of 20-30°C, for example, 20, 22, 24, 26, 28, 30°C and any range between any two values, and a light intensity of 5000-20000 lux, for example, 5000, 8000, 10000, 12000, 14000, 16000, 18000, 20000 lux and any range between any two values.

[0027] Preferably, the culture time is such that the OD of the algae in the algae liquid reaches 0.2-1, for example, it can be 0.2, 0.4, 0.6, 0.8, 1 or more, and any range between any two values.

[0028] Other conditions in the culture process may be conventional conditions in the art, such as stirring by aeration, where the aerated gas contains CO2, which can provide a carbon source for the growth of microalgae.

[0029] During the static process, the light intensity is controlled to be 20000-40000 lux, for example, it can be 20000, 25000, 30000, 35000, 40000 lux and any range between any two values. The light source during the cultivation and static process can be a natural light source or an artificial light source (such as LED light, etc.).

[0030] Preferably, the standing condition further comprises: a temperature of 20-30° C., for example, 20, 22, 24, 26, 28, 30° C. and any range between any two values.

[0031] In the present invention, after static culture, microalgae accounting for more than 25wt% are screened to obtain floating microalgae, for example, it can be 25, 26, 28, 30, 32, 34, 36, 38, 40, 42, 45, 50, 55wt% and above, and any range between any two values.

[0032] Preferably, the ratio of the dry weight of algal cells floating within 1 cm of the liquid surface to the total dry weight of the algae is determined, and microalgae with a ratio of more than 30 wt % are screened to obtain floating microalgae.

[0033] The screening can be carried out in a conventional photobioreactor in the art. Preferably, the height of the photobioreactor is above 15 cm, more preferably 20-30 cm.

[0034] The second aspect of the present invention provides a floating culture method of microalgae, the method comprising: inoculating the floating microalgae screened by the method as described above into a photobioreactor filled with culture medium for culture;

[0035] The culture process includes a static culture stage and a stirring culture stage, wherein the duration of the stirring culture stage is less than 1 hour per day;

[0036] Wherein, the photobioreactor is at least one layer of culture pool;

[0037] The depth of the microalgae liquid is 8-20 cm, preferably 10-15 cm.

[0038] In the present invention, the time of the stirring culture section is less than 1h / day, and can be even lower, such as less than 30min, less than 20min, less than 10min, or even less than 5min. In one embodiment of the present invention, the algae liquid can be stirred evenly after the illumination ends every day.

[0039] Preferably, the inoculation amount of microalgae is such that the initial OD of the algae solution is 0.1-1, for example, it can be 0.1, 0.15, 0.2, 0.3, 0.4, 0.6, 0.8, 1 or more, and any range between any two values, more preferably 0.1-0.5.

[0040] The culture conditions can be selected or adjusted according to the specific algae species. Preferably, when the microalgae is Spirulina, the culture conditions include: a temperature of 20-30° C.; and a light intensity of 5000-40000 lux.

[0041] Preferably, when the microalgae is Spirulina, the culture conditions further include: a light-dark cycle of 8h-24h:16h-0h.

[0042] Preferably, when the microalgae is Spirulina, the culture conditions further include: pH 8.5-10.

[0043] It should be understood that the light intensity can be gradually increased according to the increase in the concentration of the algae solution, or it can be maintained at a certain level; the culture medium used for culturing microalgae can be a conventional culture medium in the art.

[0044] The culture process may be carried out under natural light or artificial light or both. If artificial light is used, the light-dark cycle may be manually controlled.

[0045] In the present invention, the algae liquid can be evenly mixed in the stirring culture section by various stirring methods, such as ventilation stirring, mechanical stirring, etc.

[0046] Preferably, the photobioreactor is at least one layer of rectangular or circular culture pool, for example, it can be 1 layer, 2 layers, 3 layers, 4 layers or even more layers, as long as it can meet the sufficient light intensity. It should be understood that the shape of the rectangular culture pool can also be similar to a rectangular shape, for example, it can have a certain curvature, and its aspect ratio is not particularly limited.

[0047] The distance between each layer of rectangular culture pools can be selected according to the light intensity. Preferably, the distance between each layer of rectangular culture pools is 10-30 cm. It should be understood that the distance is the distance between the bottom of the upper layer and the upper end of the lower layer.

[0048] Preferably, the height of the culture tank is 20-30 cm.

[0049] Preferably, the distance between the upper liquid surface of the microalgae liquid and the upper edge of the side wall of the culture tank is 5-15 cm.

[0050] In the present invention, unless otherwise specified, static culture and floating culture have the same meaning.

[0051] The present invention will be described in detail below through examples.

[0052] The optical density of Spirulina solution (OD 570 ) was measured using a spectrophotometer, with distilled water used as a control, and the absorbance of the algae solution at a wavelength of 570 nm was measured.

[0053] Unless otherwise specified, all reagents and materials used were commercially available.

[0054] Example 1

[0055] This example is used to illustrate the screening method of floating microalgae suitable for floating culture and the evaluation of the culture performance of microalgae.

[0056] (1) Algal strain screening

[0057] Different strains of Spirulina were inoculated into a rectangular reactor (15 cm in height, 30 cm in length, 20 cm in width, and 10 cm in liquid thickness) filled with Spirulina culture medium, so that the OD of the inoculated algae liquid was 570 The culture was carried out at 25±3℃ and 20,000 lux for 24 hours. After the culture was completed, the mixture was stirred thoroughly and then left to stand for 8 hours. The upper 1 cm algae liquid was taken to determine its dry weight M1, and the total dry weight of the microalgae in the reactor was determined M 总 , calculate the ratio of the two M1 / M 总 , and screened to obtain the following algae strains 1#-3# (as shown in Table 1).

[0058] (2) Performance evaluation

[0059] Spirulina #1-3 were taken and inoculated into a culture pond (height 15 cm, length 100 cm, width 60 cm, liquid thickness 10 cm) for growth performance evaluation.

[0060] Initial inoculum concentration OD 570 =0.22, temperature 25℃±3℃, light intensity 30,000 lux, light-dark cycle 12h:12h, algae liquid thickness 10cm, LED light 20cm away from liquid surface, stir algae liquid evenly after the end of illumination every day, algae cell growth curve is as follows Figure 1 As shown, the OD of Spirulina solution harvested after 8 days of cultivation 570 See Table 1.

[0061] Table 1

[0062] serial number <![CDATA[M1 / M 总 ]]> <![CDATA[Static culture OD 570 > <![CDATA[Stirring culture OD 570 > Spirulina 1# 40％ 18.33 18.14 Spirulina 2# 32％ 17.55 18.02 Spirulina 3# 14％ 14.04 17.95

[0063] Test Example 1

[0064] This test case is a double-layer reactor culture experiment.

[0065] The experiment was conducted using Spirulina 1# screened out in Example 1.

[0066] The photobioreactor is a double-layer rectangular open culture pool (the upper layer is marked as the first layer, and the lower layer is marked as the second layer), with a height of 15 cm, a length of 100 cm, and a width of 60 cm. The distance between the two layers of culture pools is 20 cm, and the light source of the lower culture pool is provided by the LED light source at the bottom of the upper culture pool.

[0067] After mixing the Spirulina culture medium and Spirulina algae, use a submersible pump to pump the Spirulina algae solution into the photobioreactor for cultivation. The initial algae solution concentration OD 570=0.23, algae liquid thickness 10cm, LED lamp 20cm away from liquid surface. During the culture process, the temperature was controlled at 25±3℃, the light intensity was 30,000 lux, and the light-dark cycle was 12h:12h. On the 8th day of culture, the algae liquid was pumped out of the culture tank with a pump and collected with a filter cloth. After the algae cells were collected, the clear liquid was pumped back into the culture tank, and the culture was continued for 15 days, ending the culture.

[0068] Among them, the cultivation methods are divided into floating culture and stirring culture. During the floating culture process, the algae liquid is stirred evenly after the light ends every day. Among them, the growth of algae cells is as follows Figure 2 As shown, a total of 79.18 g of dry weight of Spirulina was obtained after 15 days of cultivation.

[0069] During the stirring culture, the aerator and stirring rod are used to stir the culture medium continuously during the illumination to ensure that the algae liquid is evenly distributed during the culture process. Figure 3 As shown, a total of 79.09 g of dry weight of Spirulina was obtained after 15 days of cultivation.

[0070] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for screening floating microalgae suitable for floating culture, characterized in that: The method comprises: inoculating microalgae and culturing them for more than 24 hours, then standing them for 6-12 hours, determining the proportion of the dry weight of algae cells floating within 1 cm of the liquid surface to the total dry weight of the algae, and screening microalgae with a proportion of more than 25wt% to obtain floating microalgae; The culture time is such that the OD of the algae in the algae solution reaches above 0.2; Wherein, the static conditions include: light intensity is 20000-40000 lux.

2. The method according to claim 1, wherein: The microalgae is a multicellular algae or a unicellular algae, preferably a filamentous algae, more preferably one or more selected from Spirulina, Anabaena, Oscillatoria axolotl and Aphanizomenon; More preferably, the microalgae is Spirulina.

3. The method according to claim 1 or 2, wherein: The initial OD of the algae solution after inoculation with microalgae is above 0.1, preferably 0.1-0.

5.

4. The method according to any one of claims 1 to 3, wherein: The culture conditions include: a temperature of 20-30°C and a light intensity of 5000-20000 lux; and / or The culture time is such that the OD of the algae in the algae solution reaches 0.2-1.

5. The method according to any one of claims 1 to 4, wherein: The static condition also includes: the temperature is 20-30°C.

6. The method according to any one of claims 1 to 5, wherein: The proportion of the dry weight of algal cells floating within 1 cm of the liquid surface to the total dry weight of the algae was determined, and microalgae with a proportion of more than 30 wt% were screened to obtain floating microalgae.

7. A floating culture method for microalgae, characterized in that: The method comprises: inoculating the floating microalgae screened by the method described in any one of claims 1 to 6 into a photobioreactor filled with culture medium for cultivation; The culture process includes a static culture stage and a stirring culture stage, wherein the duration of the stirring culture stage is less than 1 hour per day; Wherein, the photobioreactor is at least one layer of culture pool; The depth of the microalgae liquid is 8-20 cm, preferably 10-15 cm.

8. The method according to claim 7, wherein: The culture conditions include: temperature of 20-30°C; light intensity of 5000-40000 lux.

9. The method according to claim 7 or 8, wherein: The culture conditions also include: a light-dark cycle of 8h-24h:16h-0h.

10. The method according to any one of claims 7 to 9, wherein: In the photobioreactor, the height of the culture tank is 10-30 cm.

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