CN114426928B - Microalgae culture method for inhibiting mixed bacteria - Google Patents
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- 238000012136 culture method Methods 0.000 title claims abstract description 9
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 7
- 238000000034 method Methods 0.000 claims abstract description 26
- 239000001963 growth medium Substances 0.000 claims abstract description 22
- 239000007788 liquid Substances 0.000 claims abstract description 17
- 238000012258 culturing Methods 0.000 claims abstract description 11
- 238000003306 harvesting Methods 0.000 claims abstract description 3
- 239000007789 gas Substances 0.000 claims description 20
- 241000195493 Cryptophyta Species 0.000 claims description 12
- 241000195663 Scenedesmus Species 0.000 claims description 12
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- 238000002360 preparation method Methods 0.000 claims description 5
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 claims description 4
- 239000003546 flue gas Substances 0.000 claims description 4
- 239000002912 waste gas Substances 0.000 claims description 3
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- 230000003698 anagen phase Effects 0.000 claims description 2
- 238000004523 catalytic cracking Methods 0.000 claims description 2
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
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Abstract
The invention relates to a microalgae culture method for inhibiting mixed bacteria, which comprises the steps of adding microalgae seed liquid and microalgae culture medium into an open type photobioreactor, carrying out alternate light-dark culture, and introducing CO during light reaction 2 Introducing CO during dark reaction 2 And NO X The reaction temperature of the gas is 20-35 ℃, and after the alternating culture for a period of time, the dark reaction stops introducing NO X And raising the reaction temperature of the photoreaction to 36-45 ℃, culturing to a stationary phase, and harvesting microalgae cells. The method solves the problem of mixed bacterial pollution in the open culture of the microalgae by a specific culture mode, does not inhibit the growth of the microalgae, reduces the carbon fixation efficiency and reduces the culture cost.
Description
Technical Field
The invention belongs to the technical field of biomass energy, and particularly relates to a microalgae culture method for inhibiting mixed bacteria.
Background
Microalgae is rich in protein, polysaccharide, unsaturated fatty acid and other nutritional components, and can be used in food, medicine and energy sources; can accumulate fatty acid in large quantity, and the content of the fatty acid of the microalgae can account for 30-60% of dry weight. The method for obtaining the grease resource by culturing the microalgae has become a popular research field for developing renewable resources by using solar energy, and has strong market potential and remarkable social value.
At present, microalgae culture modes are closed and open. The closed type refers to a closed type reactor with different structures, such as an airlift type reactor, a stirring type reactor, a tubular reactor and the like, has higher production cost, and can be used for producing products with high added value or used as a seed tank for open culture. Open-cell culture means such as a raceway pond and a round shallow pond are adopted, and the open-cell culture means has the advantages of simple technology and low investment, so that researchers are concerned in recent years. However, open cultures are susceptible to contamination by enemies such as filamentous fungi, rotifers, protozoa, etc., which grow and multiply in the algae culture. When the number of the enemy organisms reaches a certain density, the enemy organisms influence the growth and propagation of the cultured algae, the polluted algae liquid is unfavorable for the expansion of the culture, and the serious people can cause the failure of the culture. Therefore, how to effectively prevent the pollution of enemy organisms is a key problem for the large-scale cultivation of microalgae.
In recent years, many studies have been made on methods for controlling enemies such as microalgae pest and disease damage. The traditional method is to kill and eliminate enemy organisms in algae by adopting a physical method such as a filtering or acidification method; secondly, controlling the pollution of enemy organisms in the open culture of the microalgae by a chemical method; thirdly, a biological control method is adopted for treatment; fourthly, the method of adding plant extracts is adopted for treatment.
CN103773690a discloses a method for culturing microalgae, the microalgae culture adopts conventional culture method and conditions, at the beginning of microalgae culture, plant extract is directly added into microalgae culture medium, and the plant extract is one or more of neem extract, szechwan chinaberry extract and kudzuvine root extract, and the addition amount is 5-80mg/L. The method can solve the problem of pest pollution in the open culture of microalgae, and effectively prevent and treat the pollution of infectious microbe and plant diseases and insect pests in the open culture. However, this method requires the specific preparation of a specific formulation of plant extract, which increases the cost of cultivation.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a microalgae culture method for inhibiting mixed bacteria. The method solves the problem of mixed bacterial pollution in the open culture of the microalgae by a specific culture mode, does not inhibit the growth of the microalgae, reduces the carbon fixation efficiency and reduces the culture cost.
The microalgae culture method for inhibiting the mixed bacteria provided by the invention comprises the following steps:
adding microalgae seed liquid and microalgae culture medium into an open type photobioreactor, performing alternate light and dark culture, and introducing CO during photoreaction 2 Introducing CO during dark reaction 2 And NO X The reaction temperature of the gas is 20-35 ℃, and after the alternating culture for a period of time, the dark reaction stops introducing NO X And raising the reaction temperature of the photoreaction to 36-45 ℃, culturing to a stationary phase, and harvesting microalgae cells.
In the invention, the microalgae can resist NO X And high temperature autotrophic single algae or mixed algae, such as fiber algae SS-B7%Ankistrodesmus sp.) Scenedesmus TMJ-D3Scenedesmus sp.) At least one of the above, preferably Gratelaria TMJ-D3 #Scenedesmus sp.）。
In the invention, the fibrous algae SS-B7 @ isAnkistrodesmus sp.) Has been preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 7478 and the preservation address of the national institute of microbiology, national academy of sciences No. 3, north Chenxi Lu No. 1, the area of Chaoyang in Beijing, as well as in the 15 th 2013. This strain has been disclosed in CN105713836a and submitted for preservation and survival demonstration.
In the invention, the scenedesmus TMJ-D is @ 3 @Scenedesmus sp.) Has been preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 15299 and the preservation address of China academy of China microbiological study (China academy of sciences) with the number 1 of North Chenxi road in the area of Chaoyang in Beijing city in 5 days 2018.
In the present invention, the microalgae culture medium is any one of BG11 culture medium, SE culture medium, BBM culture medium, etc., which are well known to those skilled in the art. Specifically, the preparation of the microalgae culture medium and the microalgae seed liquid is determined according to the types of the microalgae, and the preparation is the same as that of the conventional method.
In the invention, the volume ratio of the microalgae seed liquid to the microalgae culture medium added into the photobioreactor is 1:20-1:5.
In the invention, the preparation method of the microalgae seed liquid comprises the following steps: inoculating microalgae into a microalgae culture medium, and culturing in an oscillating manner until the growth phase of the microalgae seed liquid is logarithmic under the conditions that the pH value is 7-9, the temperature is 20-35 ℃, the illumination period is 24 hours, the light-dark time ratio is 14:10 and the illumination intensity is 2000-20000 Lux, thereby obtaining the microalgae seed liquid.
In the invention, the light and dark alternate culture is carried out, the light and dark period is 24 hours, the light and dark time ratio is 14:10-10:14, and the illumination intensity is 2000-20000 Lux.
In the invention, the CO is introduced by photoreaction 2 In the gas, CO 2 The volume content is 5-40%, preferably 5-20%, and NO is contained X 。
In the invention, the dark reaction is introduced with CO 2 And NO X In the gas, CO 2 The volume content is 5-40%, NO X The volume content is 0.03-0.07%. Said NO X Is NO and/or NO 2 Containing CO 2 And NO X The gas can be self-made, or can be industrial flue gas or waste gas, such as at least one of incineration tail gas and catalytic cracking regeneration tail gas from a sulfur recovery device.
In the present invention, the temperature of the alternate light and dark culture is 20-35 ℃, preferably 25-30 ℃.
In the invention, the NO is stopped to be introduced after the alternate culture for 2 to 5 days X The temperature of the photoreaction is raised to 36-45 ℃, preferably 40-45 ℃, and the culture is carried out until the growth stabilization period is finished.
In the invention, after the culture is finished, the number of the mixed bacteria is detected to be lower than 2.5x10 3 And each mL. Microalgae cells are harvested by centrifugation, sedimentation and other modes, and the dry weight and the grease content of the cells are measured.
Compared with the prior art, the invention has the following beneficial effects:
(1) The inventor of the present application cultivated in an open typeAs found in the research of scenedesmus TMJ-D3, the invention has the advantages that the mixed bacteria number in the finally obtained scenedesmus liquid is lower by the culture mode, and the inventor has NO tolerance on the scenedesmus liquid X Tests on the microalgae SS-B7 and the like show that the bacteria can be inhibited and the normal growth of microalgae is not affected. On the basis, the antibacterial culture method suitable for the microalgae is provided, the problem of mixed bacteria pollution in open culture of the microalgae is solved, the culture cost is reduced, and the method is suitable for large-scale culture.
(2) The culture mode of the invention is suitable for NO X And autotrophic microalgae with certain tolerance at high temperature, which has no toxic or harmful effect on the microalgae, and can maintain the normal growth of the microalgae.
(3) The culture mode is particularly suitable for culturing scenedesmus TMJ-D3, does not influence the normal growth of the scenedesmus, and has better antibacterial effect.
Detailed Description
The invention is illustrated in further detail by the following examples. The embodiments and specific operation procedures are given on the premise of the technical scheme of the invention, but the protection scope of the invention is not limited to the following embodiments.
The experimental methods in the following examples, unless otherwise specified, are all conventional in the art. The experimental materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
In the present invention, the high temperatures involved are those of 20-35℃for microalgae, generally above 36℃and preferably above 40 ℃.
In the invention, CO in the gas 2 、NO X The concentration is detected by a flue gas analyzer.
The preservation number of the fibrous alga SS-B7 is CGMCC No. 7478, which is disclosed in CN105713836A and submitted for preservation and survival certification.
The scenedesmus TMJ-D3 is a new species of algae separated and screened by the inventor and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms; preservation number: CGMCC No. 15299; preservation date: 2.5.2018; preservation address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
The microalgae culture of the invention adopts BG11 culture medium, and the formula is shown in Table 1 and Table 2.
TABLE 1 BG11 Medium
* Table 2 composition of A5+Co solution in Table 1
BG11 liquid media were first prepared according to tables 1 and 2, the pH of the media was adjusted to 8.0, and then microalgae were inoculated into the media, respectively. Culturing in a constant temperature illumination shaking table at 30deg.C for 24 hr with a light-dark time ratio of 14:10 and illumination intensity of 5000Lux at 120rpm to logarithmic phase to obtain microalgae seed liquid.
Example 1
(1) Adding scenedesmus TMJ-D3 seed solution and microalgae culture medium into a 20L open type photoreactor, wherein the adding amount of the seed solution is 800mL, the adding amount of the microalgae culture medium is 8L, performing alternate light and dark culture, the light and dark period is 24h, the light and dark time ratio is 14:10, the illumination intensity is 5000Lux, and introducing CO in gas for photoreaction 2 The volume content is 10%, CO in the gas is introduced in the dark reaction 2 The volume content is 10%, the volume content of NO is 0.05%, and the culture temperature is 25 ℃.
(2) After 4 days of alternate culturing in dark, the gas is introduced into the dark reaction to react with light, NO NO is contained X Raising the culture temperature of photoreaction to 45 ℃, and entering the growth stabilization period after 4 days of culture. After the cultivation, the number of the detected mixed bacteria is less than 1.6X10 3 And each mL.
Microalgae cells are obtained by centrifugation, and the dry weight and the grease content of the cells are measured. And (3) vacuum freeze-drying at-60 ℃ to constant weight, measuring the dry weight of the algae powder, calculating the biomass yield, and measuring the total fat content by adopting an n-hexane-ethyl acetate method. The detection shows that the dry weight of the cells is 10.7g/L, and the grease content is 46.38% of the dry weight of the cells.
Example 2
The difference from example 1 is that: the adding amount of the seed solution is 800mL, the adding amount of the microalgae culture medium is 4L, the light-dark time ratio is 10:14, the illumination intensity is 10000Lux, and after alternate culture for 3 days, gas is introduced to react with light, and NO NO is contained any more X . After the culture is finished, the detection shows that the number of the mixed bacteria is lower than 1.2 multiplied by 10 3 The dry weight of the cells can reach 11.4g/L, and the grease content is 47.12% of the dry weight of the cells.
Example 3
The difference from example 1 is that: the adding amount of the seed solution is 400mL, the adding amount of the microalgae culture medium is 8L, the light-dark time ratio is 12:12, the illumination intensity is 8000Lux, and after 5 days of alternate light-dark culture, gas is introduced to react with light, and NO NO is contained any more X . After the culture is finished, the detection shows that the number of the mixed bacteria is lower than 2.1X10 3 The dry weight of the cells can reach 10.3g/L, and the grease content is 45.72% of the dry weight of the cells.
Example 4
The difference from example 1 is that: the temperature of the light-dark alternate culture is 20 ℃ in the first 4 days, the temperature of the light reaction culture is increased to 40 ℃ after 4 days, and the culture is continued for 4 days. After the culture is finished, the detection shows that the number of the mixed bacteria is lower than 1.8x10 3 The dry weight of the cells can reach 10.5g/L, and the grease content is 45.93% of the dry weight of the cells.
Example 5
The difference from example 1 is that: the temperature of the light and dark alternate culture is 30 ℃ in the first 4 days, the temperature of the light reaction culture is increased to 36 ℃ after 4 days, and the culture is continued for 4 days. After the culture is finished, the detection shows that the number of the mixed bacteria is lower than 2.5X10 3 The dry weight of the cells can reach 10.1g/L, and the grease content is 42.98% of the dry weight of the cells.
Example 6
The difference from example 1 is that: CO 2 The volume content is 5%, and the volume content of NO is increased to 0.07%. After the culture is finished, the detection shows that the number of the mixed bacteria is lower than 1.2 multiplied by 10 3 The dry weight of the cells can reach 11.4g/L per mL, and the grease content is47.21% of the dry weight of the cells.
Example 7
The difference from example 1 is that: CO 2 The volume content was 25% and the volume content of NO was reduced to 0.03%. After the culture is finished, the detection shows that the number of the mixed bacteria is lower than 2.1X10 3 The dry weight of the cells can reach 10.3g/L, and the grease content is 45.66% of the dry weight of the cells.
Example 8
The difference from example 1 is that: the microalgae adopts fibrous algae SS-B7. After the culture is finished, the detection shows that the number of the mixed bacteria is lower than 2.2x10 3 The dry weight of the cells can reach 11.3g/L, and the grease content is 46.41% of the dry weight of the cells.
Comparative example 1
The difference from example 1 is that: photoreaction and darkreaction are carried out by introducing the same mixture containing CO only 2 Is not added with NO in dark reaction X . After the culture is finished, the number of the mixed bacteria is 2.3X10 by detection 5 The dry weight of the cells can reach 8.3g/L, and the grease content is 36.92% of the dry weight of the cells.
Comparative example 2
The difference from example 1 is that: the same light reaction and dark reaction are carried out while containing CO 2 And NO X Gas, CO in gas 2 And NO X The volume content is the same as in example 1. After the culture is finished, the number of the mixed bacteria is 1.5X10 by detection 3 The dry weight of the cells can reach 9.7g/L, and the grease content is 40.69% of the dry weight of the cells.
Comparative example 3
The difference from example 1 is that: the incubation temperature for the photoreaction and dark reaction was always 35 ℃. After the culture is finished, the number of the mixed bacteria is 5.6X10 by detection 5 The dry weight of the cells can reach 9.5g/L, and the grease content is 39.91% of the dry weight of the cells.
Comparative example 4
The difference from example 1 is that: the incubation temperature for the photoreaction and dark reaction was always 25 ℃. After the culture is finished, the number of the mixed bacteria is 3.9X10 by detection 5 The dry weight of the cells can reach 9.4g/L, and the grease content is 40.97% of the dry weight of the cells.
Comparative example 5
The difference from example 1 is that: the microalgae adopts chlorella SF-B1 disclosed in CN 109576158A. After the culture is finished, the detection shows that the number of the mixed bacteria is lower than 5.8x10 3 The dry weight of the cells can reach 7.3g/L, and the grease content is 30.36% of the dry weight of the cells.
Claims (11)
1. A microalgae culture method for inhibiting mixed bacteria is characterized by comprising the following steps: adding microalgae seed liquid and microalgae culture medium into an open type photobioreactor, performing alternate light and dark culture, and introducing CO during photoreaction 2 Introducing CO during dark reaction 2 And NO X The reaction temperature of the gas is 20-35 ℃, and after the alternating culture for a period of time, the dark reaction stops introducing NO X Raising the reaction temperature of the photoreaction to 36-45 ℃, culturing to a stable period, and harvesting microalgae cells;
the microalgae is fibrous algae SS-B7%Ankistrodesmus sp.) Scenedesmus TMJ-D3Scenedesmus sp.) At least one of (a) and (b);
CO-containing photo-reactive feed 2 In the gas, CO 2 The volume content is 5-40%, NO NO is contained X The method comprises the steps of carrying out a first treatment on the surface of the Dark reaction introducing CO 2 And NO X In the gas, CO 2 The volume content is 5-40%, NO X The volume content is 0.03-0.07%.
2. The method according to claim 1, characterized in that: the microalgae is scenedesmus TMJ-D3Scenedesmus sp.）。
3. The method according to claim 1, characterized in that: the microalgae culture medium adopts any one of BG11 culture medium, SE culture medium and BBM culture medium.
4. A method according to claim 1, 2 or 3, characterized in that: the preparation method of the microalgae seed liquid comprises the following steps: inoculating microalgae into a microalgae culture medium, and culturing in an oscillating manner until the growth phase of the microalgae seed liquid is logarithmic under the conditions that the pH value is 7-9, the temperature is 20-35 ℃, the illumination period is 24 hours, the light-dark time ratio is 14:10 and the illumination intensity is 2000-20000 Lux, thereby obtaining the microalgae seed liquid.
5. A method according to claim 1 or 3, characterized in that: the volume ratio of the microalgae seed liquid to the microalgae culture medium added into the photobioreactor is 1:20-1:5.
6. The method according to claim 1, characterized in that: the alternate light and dark cultivation is carried out, the light and dark period is 24 hours, the light and dark time ratio is 14:10-10:14, and the illumination intensity is 2000-20000 Lux.
7. The method according to claim 1 or 6, characterized in that: CO-containing photo-reactive feed 2 In the gas, CO 2 The volume content is 5-20%.
8. The method according to claim 1, characterized in that: said CO-containing 2 And NO X The gas is self-made or industrial flue gas or waste gas; the industrial flue gas or waste gas is derived from at least one of incineration tail gas and catalytic cracking regeneration tail gas of the sulfur recovery device.
9. The method according to claim 1 or 6, characterized in that: the temperature of the alternate light and dark culture is 25-30 ℃.
10. The method according to claim 1, characterized in that: after alternate culturing for 2-5 days, stopping introducing NO X 。
11. The method according to claim 1 or 10, characterized in that: the temperature of the photoreaction is increased to 40-45 ℃.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2011139804A2 (en) * | 2010-04-27 | 2011-11-10 | Sequesco | Use of oxyhydrogen microorganisms for non-photosynthetic carbon capture and conversion of inorganic and/or c1 carbon sources into useful organic compounds |
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