WO2022247225A1 - Symbiotic bacteria composition, preparation method therefor and culture method for microalgae - Google Patents
Symbiotic bacteria composition, preparation method therefor and culture method for microalgae Download PDFInfo
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- WO2022247225A1 WO2022247225A1 PCT/CN2021/137051 CN2021137051W WO2022247225A1 WO 2022247225 A1 WO2022247225 A1 WO 2022247225A1 CN 2021137051 W CN2021137051 W CN 2021137051W WO 2022247225 A1 WO2022247225 A1 WO 2022247225A1
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- microalgae
- liquid
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- 241000894006 Bacteria Species 0.000 title claims abstract description 92
- 239000000203 mixture Substances 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 238000012136 culture method Methods 0.000 title abstract description 3
- 241000195649 Chlorella <Chlorellales> Species 0.000 claims abstract description 39
- 239000013535 sea water Substances 0.000 claims abstract description 37
- 241000193171 Clostridium butyricum Species 0.000 claims abstract description 23
- 241000131970 Rhodospirillaceae Species 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 19
- 239000007788 liquid Substances 0.000 claims description 37
- 239000001963 growth medium Substances 0.000 claims description 20
- 241000223252 Rhodotorula Species 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 238000011081 inoculation Methods 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 8
- 238000012364 cultivation method Methods 0.000 claims description 8
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 7
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- 239000008103 glucose Substances 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 5
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 4
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
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- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 4
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- 239000012362 glacial acetic acid Substances 0.000 claims description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 4
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- 235000007079 manganese sulphate Nutrition 0.000 claims description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 2
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- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 description 2
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- 230000002353 algacidal effect Effects 0.000 description 2
- 230000005791 algae growth Effects 0.000 description 2
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 2
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- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
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- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
Definitions
- the invention relates to the technical field of microalgae cultivation, in particular to a symbiotic bacteria composition, a preparation method thereof and a microalgae cultivation method.
- Seawater chlorella is widely used in the fields of pharmaceutical raw materials, health food and bioenergy because it is rich in protein, lipid, polysaccharide, vitamin, antioxidant and other functional nutrients.
- seawater chlorella culture technology is mostly concentrated in the photoautotrophic system, which has disadvantages such as low production efficiency, large floor area, and high harvest cost, which seriously restricts the development of seawater chlorella industry.
- Increasing the biomass of seawater chlorella through artificial large-scale cultivation technology is the key to the development and utilization of seawater chlorella resources.
- seawater chlorella heterotrophic high cell density culture technology has gradually emerged. This technology can overcome the dependence of light culture system on light, has faster growth rate, can realize pure culture, high cell yield, and can greatly reduce The advantages of downstream post-processing costs and easier automatic control have become a hot spot in the research of seawater chlorella cultivation in recent years.
- algae are also infested by external disease organisms during their growth.
- the main disease organisms of seawater chlorella are viruses, bacteria, fungi and protists. These diseases may seriously affect the growth of algae, especially bring a blow to the production of economical algae, and increase the production cost.
- Bacteria compete with algae for nutrients such as C, N, P, and K, and inhibit the growth of algal cells;
- the bacteria film is formed on the still water surface, which hinders gas exchange and light transmission, and causes algae to die;
- the bacteria enter the algae cells and kill the algae.
- the direct contact of bacteria to kill the algal cells will cause the reduction of chlorophyll a.
- seawater chlorella Commercial large-scale production of seawater chlorella has been hampered by biological contaminants.
- various pollutants will inevitably enter seawater Chlorella culture from water, air or bioreactors.
- Contaminants in seawater chlorella cultures mainly include zooplankton, bacteria, viruses, and other algae.
- Different biological pollutants have different mechanisms of harming the growth of marine chlorella.
- Zooplankton are the main predators in aquaculture systems. Bacteria secrete extracellular compounds with lytic activity on algal cells to inhibit algal growth; viruses infect algal cells, causing culture "collapse"; Resources such as nitrogen, phosphorus, potassium, magnesium, calcium.
- different types of pollutants can cause different degrees of damage to marine chlorella cultures.
- Seawater chlorella is a huge food resource, and the strategic significance of its resource utilization is self-evident.
- the seawater chlorella industry involves many technical fields and has the characteristics of interdisciplinary.
- the disease problem in the cultivation process of seawater chlorella is a practical problem that needs to be solved in seawater chlorella cultivation.
- Mastering the relationship between seawater chlorella and microorganisms in the symbiotic environment is an important basis for promoting the development of my country's seawater chlorella industry and a prerequisite for solving seawater chlorella cultivation diseases. There are still few related studies in our country, and further research investment is needed.
- the present invention provides a symbiotic bacteria composition, a preparation method thereof and a microalgae cultivation method.
- the present invention can effectively suppress the risk of diseases and insect pests in the cultivation process of seawater chlorella through the symbiotic effect of probiotics, reduce its harm, and effectively improve the production capacity and output of industrial cultivation of chlorella.
- the invention provides a symbiotic bacteria composition, which is composed of marine Clostridium butyricum, marine red yeast and marine purple non-sulfur bacteria;
- the ratio of live bacteria of marine Clostridium butyricum, marine rhodotorula and marine purple non-sulfur bacteria was (2-4):(5-7):(1-3).
- Microalgae and bacteria in the symbiotic system of bacteria and algae are both interdependent and inhibit each other under certain conditions, and there is a complex interaction mechanism.
- the most important role played by microalgae in the system is to use carbon dioxide to produce organic matter and release part of it into the environment; while bacteria use the organic matter in sewage to convert it into inorganic matter and continue to be used by microalgae.
- microalgae as photoautotrophs, can produce a large amount of oxygen, which directly increases the dissolved oxygen concentration in the treated sewage, thereby contributing to the catabolism of aerobic bacteria.
- the bacteria in the symbiosis system of bacteria and algae can convert ammonia nitrogen into nitrate, thereby alleviating the inhibition of microalgae by the high ammonia nitrogen environment.
- the oxygen released by the growth of microalgae improves the vitality and function of bacteria, and rapidly secretes all kinds of nutrients needed for algae growth. factors, while inhibiting the growth of harmful bacteria.
- bacterial-algae symbiosis is the most common form of interaction between bacteria and algae. Both endosymbiosis and exosymbiosis are our focus of research.
- algal microenvironment was first proposed, that is, in a certain area where algae live, algal cells diffuse their own extracellular substances into the surrounding environment, forming a special microenvironment around algal cells.
- the flora living in the algae microenvironment are diverse in species, and have unique structures and functions, and can establish special connections with algal cells. They can directly use organic matter derived from algae for their own growth and reproduction.
- the extracellular jelly produced by algae is mostly polysaccharide substances, which can stimulate the growth of bacteria in the environment; in contrast, some compounds produced by bacteria are also specific to the growth of algae.
- the extracellular jelly produced by algae is mostly polysaccharide substances, which can stimulate the growth of bacteria in the environment; on the contrary, some compounds produced by bacteria are also required for the growth of algae.
- the metabolic mechanism of algae is completely different in the process of aseptic culture and bacteria culture.
- the existence of these symbiotic bacteria will mostly benefit the growth of algae, and the bacteria can provide algae with more directly usable small molecules for their growth.
- the exopolysaccharides synthesized and secreted by algae are soluble, and the growing algal cells are completely suspended; but in the process of bacterial culture, most of the exopolysaccharides produced by algae are insoluble and grow
- marine bacteria can catalyze the metabolism of sulfur-containing organic matter dimethyl sulfopropyl ester, and its degradation product dimethyl sulfide is an important component of the marine ecosystem.
- various symbiotic bacteria participating in the physiological metabolism of algae can significantly affect the absorption and transformation of arsenate by Dunaliella salina when the concentration of phosphate in the culture system is 0.112mg/L.
- bacteria can also release some bactericidal substances to inhibit the growth of other harmful bacteria, thereby protecting algae.
- the mode of action of the symbiotic bacteria will change appropriately. They can induce the lysis of algal cells by secreting some special substances, or form a certain effect on the survival of algal cells. environmental pressures in this way to control algae populations.
- algae can also release biological pheromones to the environment and establish species-specific symbiotic relationships with bacteria in the environment.
- some polysaccharides secreted by coccolithophores can act as information molecules to attract specific bacterial groups in the environment, and then establish a symbiotic relationship between the two.
- Some bacteria can produce plant hormones to promote the growth of algae, so as to increase the unit biomass of algae.
- Plant growth-promoting bacteria can promote the accumulation of algae biomass. When algae and plant growth-promoting bacteria are co-cultured, their biomass can be increased by at least 10%, and individual algae can even increase by 70%. If the plant growth-promoting bacteria in the algae microenvironment are removed, the growth rate of algae will be significantly slowed down. This can explain the important role of bacteria in algae culture.
- algicidal bacteria have a certain tendency to algal cells. They fix on the surface of algal cells through flagella, produce algae-dissolving substances, degrade the cell wall of algal cells, and finally lead to the lysis and death of algal cells. Therefore, for this type of algicidal bacteria, they should be avoided as much as possible during the mass cultivation of algae, so that they can be removed in a timely and thorough manner.
- the symbiosis of three probiotics can effectively inhibit the risk of diseases and insect pests in the seawater chlorella culture process, reduce its harm, and effectively improve the quality of pellets. Production capacity of algae industrial culture.
- the present invention also provides a preparation method of the symbiotic bacteria composition, comprising the steps of:
- the bacterium liquid of Clostridium butyricum, the liquid of Rhodotorula marine and the liquid of marine purple non-sulfur bacteria are mixed to obtain a symbiotic bacteria composition.
- the formula of the first culture medium is:
- the formula of the first culture medium is:
- the conditions for the first cultivation are: rotation speed 500-700rpm, temperature 30-35°C, cultivation time 24-30h;
- the conditions for the first cultivation are: rotation speed 600 rpm, temperature 32° C., cultivation time 24 hours.
- the inoculum size of the first culture is 5%-20%;
- the formula of the second culture medium is:
- the formula of the second culture medium is:
- the conditions for the second cultivation are: rotation speed 1000-1500 rpm, temperature 32-35°C, cultivation time 24-30 hours;
- the conditions of the second culture are: the rotation speed is 1000 rpm, the temperature is 35° C., and the culture time is 30 h.
- the inoculum size of the second culture is 5%-20%;
- the formula of the third culture medium is:
- the formula of the third culture medium is:
- the conditions for the third cultivation are: rotation speed 100-150rpm, temperature 28-35°C, cultivation time 48-72h;
- the conditions of the third culture are: the rotation speed is 150 rpm, the temperature is 28° C., and the culture time is 48 hours.
- the inoculation amount of the third culture is 20%-30%.
- the volume ratio of the marine Clostridium butyricum bacteria liquid, the marine Rhodotorula yeast liquid and the marine purple non-sulfur bacteria liquid is (0.2-20): (0.5-50): (0.1-10).
- the volume ratio of the marine Clostridium butyricum bacteria liquid, the marine Rhodotorula yeast liquid and the marine purple non-sulfur bacteria liquid is (0.5-5):(1-10):(0.2-2).
- the volume ratio of the marine Clostridium butyricum bacteria solution, the marine Rhodotorula bacteria solution and the marine purple non-sulfur bacteria solution is 2:5:1.
- the present invention also provides the symbiotic bacteria composition prepared by the above preparation method.
- the invention also provides a mixed symbiotic bacteria liquid, the preparation method of which is: statically culturing the symbiotic bacteria composition to obtain the mixed symbiotic bacteria liquid.
- the conditions for static culture are: light intensity 1000-4000 lux, temperature 25-40°C, light-to-dark ratio 2:1, humidity 10%-30% RH, culture time 20-50h.
- the conditions for static culture are: light intensity 2500 lux, temperature 30° C., light-to-dark ratio 2:1, humidity 12% RH, and culture time 24 hours.
- the invention also provides a microalgae culture method, inoculating the microalgae and the mixed symbiotic bacteria liquid into the microalgae culture liquid for co-cultivation.
- the formula of the microalgae culture solution is: 400-500 g of sodium nitrate, 80-100 g of potassium dihydrogen phosphate, 8-10 g of EDTA-2Na, 40-50 g of hexahydrate ferric chloride, 500-600 mL of glacial acetic acid, clean seawater 1 ton;
- the formula of the microalgae culture solution is: 400g of sodium nitrate, 100g of potassium dihydrogen phosphate, 10g of EDTA-2Na, 40g of hexahydrate ferric chloride, 600mL of glacial acetic acid, and 1 ton of clean seawater.
- the inoculation ratio of microalgae and microalgae culture solution is 1:(500 ⁇ 2000)(V/V);
- the inoculation ratio of the mixed symbiotic bacteria liquid to the microalgae culture liquid is 1: (5000-10000) (V/V).
- the inoculation ratio of microalgae and microalgae culture solution is 1:1000 (V/V);
- the inoculation ratio of the mixed symbiotic bacteria liquid and the microalgae culture liquid is 1:5000 (V/V).
- the co-cultivation conditions are: temperature 25-45°C, illuminance 5000-25000lux, culture time 24-72h;
- the microalgae is one or more of seawater chlorella, diatoms, Nannochloropsis, and oocysts.
- the invention provides a symbiotic bacteria composition, a preparation method thereof and a microalgae cultivation method.
- the symbiotic bacteria composition consists of marine Clostridium butyricum, marine rhodotorula and marine purple non-sulfur bacteria.
- the present invention has following technical effect:
- the present invention effectively suppresses the risk of diseases and insect pests in the cultivation process of seawater chlorella, reduces its harm, and effectively improves the industrialization of chlorella Cultivated capacity yield.
- the invention discloses a symbiotic bacteria composition and a preparation method thereof and a microalgae cultivation method, and those skilled in the art can learn from the contents of this article and appropriately improve the process parameters to realize the method.
- all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.
- the method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.
- the medium or strains used in the present invention can be purchased from the market.
- Example 1 Probiotic culture, preparation of symbiotic bacteria solution and co-cultivation mode
- %RH prepared as a mixed symbiotic bacteria liquid.
- Seawater chlorella was inoculated into the microalgae culture solution according to 1:1000 (V/V, the ratio of the volume of the microalgae species to the volume of the culture solution), and the formula of the culture solution was as follows: 400 grams of sodium nitrate, potassium dihydrogen phosphate 100 grams, 10 grams of EDTA-2Na, 40 grams of Liuheshui ferric chloride, 600 ml of glacial acetic acid, dissolved in 1 ton of clean seawater, that is, prepared into a microalgae culture solution.
- the symbiotic bacteria solution was added to the microalgae culture solution at a ratio of 1:5000 (V/V, the ratio of the volume of the symbiotic bacteria solution to the volume of the microalgae culture solution), and symbiotically cultivated with the microalgae.
- Control group without adding symbiotic bacteria solution, the microalgae culture medium is the same as above.
- Co-culture conditions temperature 30°C, light intensity 12000 lux, culture time 72 hours.
- the cultivation results are as follows:
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Abstract
Provided are a symbiotic bacteria composition, a preparation method therefor, and a culture method for microalgae. The symbiotic bacteria composition is composed of marine Clostridium butyricum, marine red yeast, and marine purple non-sulfur bacteria; by means of the symbiotic effect of the three probiotics: marine Clostridium butyricum, marine red yeast and marine purple non-sulfur bacteria, the risk of diseases and insect pests in the process of chlorella cultivation in seawater can be effectively suppressed, the harm thereof can be reduced, and the production capacity and output of chlorella factory cultivation can be effectively improved.
Description
本申请要求于2021年05月25日提交中国专利局、申请号为202110570827.6、发明名称为“共生菌组合物及其制备方法和微藻的培养方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims the priority of the Chinese patent application submitted to the China Patent Office on May 25, 2021, with the application number 202110570827.6, and the title of the invention is "symbiotic bacteria composition and its preparation method and microalgae cultivation method", the entire content of which Incorporated in this application by reference.
本发明涉及微藻培养技术领域，特别涉及共生菌组合物及其制备方法和微藻的培养方法。The invention relates to the technical field of microalgae cultivation, in particular to a symbiotic bacteria composition, a preparation method thereof and a microalgae cultivation method.
海水小球藻因其富含蛋白质、脂类、多糖、维生素、抗氧化剂和其它功能性营养成分，在医药原料、保健食品及生物能源领域具有广泛的应用。目前，海水小球藻培养技术多集中于光自养体系，该培养体系存在生产效率低、占地面积大、收获成本高等不足，严重制约了海水小球藻产业的发展。通过人工大规模培养技术提高海水小球藻生物量是海水小球藻资源开发利用的关键。近年来，逐渐兴起了海水小球藻异养高细胞密度培养技术，该技术可以克服光培养体系对光的依赖，具有生长速度更快、能实现纯种培养、细胞产率高、可大大降低下游后处理成本，更便于自动化控制等优势，已成为近年来海水小球藻培养研究的热点。Seawater chlorella is widely used in the fields of pharmaceutical raw materials, health food and bioenergy because it is rich in protein, lipid, polysaccharide, vitamin, antioxidant and other functional nutrients. At present, seawater chlorella culture technology is mostly concentrated in the photoautotrophic system, which has disadvantages such as low production efficiency, large floor area, and high harvest cost, which seriously restricts the development of seawater chlorella industry. Increasing the biomass of seawater chlorella through artificial large-scale cultivation technology is the key to the development and utilization of seawater chlorella resources. In recent years, seawater chlorella heterotrophic high cell density culture technology has gradually emerged. This technology can overcome the dependence of light culture system on light, has faster growth rate, can realize pure culture, high cell yield, and can greatly reduce The advantages of downstream post-processing costs and easier automatic control have become a hot spot in the research of seawater chlorella cultivation in recent years.
国内大部分水产育苗企业，在育苗生产中都是自备海水小球藻养殖设施，自行生产各类饵料用海水小球藻。但是一般育苗场都普遍缺乏相应的专业技术力量，只能利用各自的藻池和天然水体粗放培养，在饵料海水小球藻种质、生产技术和应用方法上都各自为政，导致海水小球藻种质混乱、供应不稳定、营养成分不平衡、饵料效价低、缺乏多品种集约化生产应用技术；同时，受限于海水小球藻高密度养殖、采收技术和浓缩液保藏技术的限制，国内几乎没有统一的、专业化的饵料海水小球藻质量标准和集中供应点。所以工厂化育苗需要及时的补充藻种，开口饵料非常重要。Most domestic aquaculture nursery enterprises have their own seawater chlorella culture facilities in seedling production, and produce seawater chlorella for various bait by themselves. However, general nursery farms generally lack corresponding professional and technical strength, and can only use their respective algae pools and natural water bodies for extensive cultivation. They all work independently in terms of bait seawater chlorella germplasm, production technology and application methods, resulting in seawater chlorella species. Quality disorder, unstable supply, unbalanced nutrient composition, low bait titer, and lack of multi-species intensive production and application technology; at the same time, limited by the high-density culture of seawater chlorella, harvesting technology and concentrate storage technology, There are almost no uniform and specialized quality standards and centralized supply points for bait seawater chlorella in China. Therefore, industrial seedlings need to replenish algae species in time, and it is very important to open the bait.
同其他生物一样，藻类在生长过程中也会受到外界病害生物的侵扰。海水小球藻的主要的病害生物有病毒、细菌、真菌和原生生物。这些病害 可能会严重地影响到藻的生长，特别是给经济藻类的生产带来打击，使生产成本提高。Like other organisms, algae are also infested by external disease organisms during their growth. The main disease organisms of seawater chlorella are viruses, bacteria, fungi and protists. These diseases may seriously affect the growth of algae, especially bring a blow to the production of economical algae, and increase the production cost.
在自然界长期的演化过程中，藻类的生长环境里一直生活着不同的细菌，两种类群存在着复杂而独特的相互作用，对于水体环境中物质和能量的循环过程非常关键。藻类与细菌的关系，可以是互利共生的，也可以是相互抑制的，或者没有明显的相互关系。一些细菌可能是藻类的病害物种，可以抑制或裂解藻体。细菌造成藻类病害的原因主要有五大类：During the long-term evolution of nature, different bacteria have always lived in the growth environment of algae, and the two groups have complex and unique interactions, which are critical to the cycle of matter and energy in the water environment. The relationship between algae and bacteria can be mutualistic symbiosis, mutual inhibition, or no obvious mutual relationship. Some bacteria may be pathogenic species of algae, inhibiting or lysing algae. There are five main categories of bacterial causes of algal disease:
(1)通过释放溶藻成份到环境中，从而杀死藻类；(1) Kill algae by releasing algae-dissolving ingredients into the environment;
(2)直接接触藻体，通过释放溶解纤维素的酶而消化藻细胞的细胞壁，而裂解藻细胞；(2) directly contacting the algal body, digesting the cell wall of the algal cell by releasing the enzyme that dissolves the cellulose, and lysing the algal cell;
(3)细菌与藻类竞争C、N、P、K等营养物质，抑制藻细胞生长；(3) Bacteria compete with algae for nutrients such as C, N, P, and K, and inhibit the growth of algal cells;
(4)在静止的水面形成菌胶膜，妨碍气体交换和光线透射，使藻类死亡；(4) The bacteria film is formed on the still water surface, which hinders gas exchange and light transmission, and causes algae to die;
(5)细菌进入藻细胞内杀死藻类。细菌直接接触而杀死藻细胞会造成叶绿素a的降低。(5) The bacteria enter the algae cells and kill the algae. The direct contact of bacteria to kill the algal cells will cause the reduction of chlorophyll a.
海水小球藻的商业化大规模生产已受到生物污染物的阻碍。在大规模培养***中，各种污染物不可避免地会从水、空气或生物反应器进入海水小球藻培养。海水小球藻培养物中的污染物主要包括浮游动物、细菌、病毒和其他藻类。不同的生物污染物具有损害海水小球藻的不同机制生长。浮游动物是养殖***中的主要捕食者。细菌分泌具有对藻类细胞具有裂解活性以抑制藻类生长的能力的细胞外化合物；病毒感染藻细胞，导致培养物“崩溃”；和与所述目标侵入藻类竞争海水小球藻用于限制营养元素的资源，例如，氮、磷、钾、镁、钙。对于海水小球藻，不同类型的污染物会导致海水小球藻培养物受到不同程度的破坏。Commercial large-scale production of seawater chlorella has been hampered by biological contaminants. In large-scale culture systems, various pollutants will inevitably enter seawater Chlorella culture from water, air or bioreactors. Contaminants in seawater chlorella cultures mainly include zooplankton, bacteria, viruses, and other algae. Different biological pollutants have different mechanisms of harming the growth of marine chlorella. Zooplankton are the main predators in aquaculture systems. Bacteria secrete extracellular compounds with lytic activity on algal cells to inhibit algal growth; viruses infect algal cells, causing culture "collapse"; Resources such as nitrogen, phosphorus, potassium, magnesium, calcium. For marine chlorella, different types of pollutants can cause different degrees of damage to marine chlorella cultures.
在海水小球藻开放式养殖过程中，细菌的防控并无太好的方式，在养殖过程中，水质、环境、物料、操作等因素，均会使得藻液中爆发各类细菌。尤其是霉菌，致病杆菌等有害菌，不但竞争性吸收培养液中的营养，抑制藻体生长，破坏藻细胞的完整性，还会残留在藻液中，对下游的产业化应用造成影响。In the open culture process of seawater chlorella, there is no good way to prevent and control bacteria. During the culture process, factors such as water quality, environment, materials, and operation will cause the outbreak of various bacteria in the algae liquid. In particular, harmful bacteria such as molds and pathogenic bacteria not only competitively absorb nutrients in the culture medium, inhibit the growth of algae, destroy the integrity of algal cells, but also remain in the algae liquid, affecting downstream industrial applications.
目前业内常用的抗菌方式是添加各类抗生素来抑制细菌生长，但是抗生素的引入，会带来生物安全性的担忧，同时也会抑制藻体的正常生长。At present, the commonly used antibacterial method in the industry is to add various antibiotics to inhibit the growth of bacteria, but the introduction of antibiotics will bring concerns about biological safety, and will also inhibit the normal growth of algae.
海水小球藻是一个巨大的食品资源，其资源化利用的战略意义不言而喻。海水小球藻产业涉及到多个技术领域，具有学科交叉的特点。目前，海水小球藻培养过程中的病害问题，是海水小球藻养殖需要解决的实际问题。掌握海水小球藻与共生环境中的微生物关系，是推动我国海水小球藻产业发展的重要基础，也是解决海水小球藻养殖病害的前提。我国的相关研究还较少，需要进一步加大研究投入。Seawater chlorella is a huge food resource, and the strategic significance of its resource utilization is self-evident. The seawater chlorella industry involves many technical fields and has the characteristics of interdisciplinary. At present, the disease problem in the cultivation process of seawater chlorella is a practical problem that needs to be solved in seawater chlorella cultivation. Mastering the relationship between seawater chlorella and microorganisms in the symbiotic environment is an important basis for promoting the development of my country's seawater chlorella industry and a prerequisite for solving seawater chlorella cultivation diseases. There are still few related studies in our country, and further research investment is needed.
发明内容Contents of the invention
有鉴于此，本发明提供了共生菌组合物及其制备方法和微藻的培养方法。本发明可通过益生菌的共生作用，有效抑制海水小球藻养殖过程中的病虫害风险，降低其危害，有效提升小球藻工厂化培养的产能产量。In view of this, the present invention provides a symbiotic bacteria composition, a preparation method thereof and a microalgae cultivation method. The present invention can effectively suppress the risk of diseases and insect pests in the cultivation process of seawater chlorella through the symbiotic effect of probiotics, reduce its harm, and effectively improve the production capacity and output of industrial cultivation of chlorella.
为了实现上述发明目的，本发明提供以下技术方案：In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:
本发明提供了一种共生菌组合物，由海洋丁酸梭菌、海洋红酵母和海洋紫色非硫细菌组成；The invention provides a symbiotic bacteria composition, which is composed of marine Clostridium butyricum, marine red yeast and marine purple non-sulfur bacteria;
海洋丁酸梭菌、海洋红酵母与海洋紫色非硫细菌的活菌数比例为(2～4)：(5～7)：(1～3)。The ratio of live bacteria of marine Clostridium butyricum, marine rhodotorula and marine purple non-sulfur bacteria was (2-4):(5-7):(1-3).
菌藻共生***中的微藻与细菌既相互依存又在某些条件下相互抑制，存在复杂的互作机制。***中微藻发挥的最重要作用是利用二氧化碳生产有机物并部分释放入环境；而细菌则利用污水中的有机物转变为无机物继续为微藻所用。在此过程中，微藻作为光合自养生物可产生大量氧气，直接提高处理污水中的溶解氧浓度，从而有助于好氧细菌的分解代谢。菌藻共生***内的细菌可将氨氮转化为硝酸盐，进而缓解高氨氮环境对微藻的抑制，微藻生长释放的氧气提高了细菌的活力和功能，快速分泌藻体生长所需的各类因子，同时抑制有害菌的生长。Microalgae and bacteria in the symbiotic system of bacteria and algae are both interdependent and inhibit each other under certain conditions, and there is a complex interaction mechanism. The most important role played by microalgae in the system is to use carbon dioxide to produce organic matter and release part of it into the environment; while bacteria use the organic matter in sewage to convert it into inorganic matter and continue to be used by microalgae. During this process, microalgae, as photoautotrophs, can produce a large amount of oxygen, which directly increases the dissolved oxygen concentration in the treated sewage, thereby contributing to the catabolism of aerobic bacteria. The bacteria in the symbiosis system of bacteria and algae can convert ammonia nitrogen into nitrate, thereby alleviating the inhibition of microalgae by the high ammonia nitrogen environment. The oxygen released by the growth of microalgae improves the vitality and function of bacteria, and rapidly secretes all kinds of nutrients needed for algae growth. factors, while inhibiting the growth of harmful bacteria.
在自然界中，菌藻共生是细菌和藻类相互作用类型中最为普遍的一种存在形式，无论是内共生还是外共生都是我们重点研究关注的。1972年，首次提出“藻际微环境”的概念，即在藻类生活的一定区域内，藻细胞将自 身产生的胞外物质扩散到周围环境中，使得藻细胞周围形成一个特殊的微环境。生活于藻际微环境中的菌群在种类上呈现多样性，而且具有独特的结构和功能，能够与藻细胞建立特殊的联系。它们可以直接利用源自于藻类的有机物，供自身生长繁殖所需。虽然藻际微环境中细菌种类是呈现多样性，但是其中只有部分细菌能与藻类共生，说明细菌和藻类建立相互联系是具有种属特异性的。藻类产生的胞外胶状物，大多为多聚糖类的物质，这些物质能够刺激环境中细菌的生长；相对的，细菌产生的某些化合物也是藻类生长所需要的特异性的。藻类产生的胞外胶状物，大多为多聚糖类的物质，这些物质能够刺激环境中细菌的生长；相对的，细菌产生的某些化合物也是藻类生长所需要的。In nature, bacterial-algae symbiosis is the most common form of interaction between bacteria and algae. Both endosymbiosis and exosymbiosis are our focus of research. In 1972, the concept of "algal microenvironment" was first proposed, that is, in a certain area where algae live, algal cells diffuse their own extracellular substances into the surrounding environment, forming a special microenvironment around algal cells. The flora living in the algae microenvironment are diverse in species, and have unique structures and functions, and can establish special connections with algal cells. They can directly use organic matter derived from algae for their own growth and reproduction. Although the species of bacteria in the algae microenvironment are diverse, only some of them can symbiose with algae, indicating that the mutual connection between bacteria and algae is species-specific. The extracellular jelly produced by algae is mostly polysaccharide substances, which can stimulate the growth of bacteria in the environment; in contrast, some compounds produced by bacteria are also specific to the growth of algae. The extracellular jelly produced by algae is mostly polysaccharide substances, which can stimulate the growth of bacteria in the environment; on the contrary, some compounds produced by bacteria are also required for the growth of algae.
藻类在无菌培养和有菌培养的过程中，其代谢机制完全不同。这些共生细菌的存在大多会使藻类的生长受益，细菌可以向藻类提供更多可直接利用的小分子物质供其生长所需及。如硅藻在无菌培养时，藻类合成分泌的胞外多糖是可溶性的，生长的藻细胞是完全悬浮的；但在有菌培养过程中，藻类产生的胞外多糖则多是不溶性的，生长也出现贴壁的现象，并与培养体系中的共生细菌一同形成生物膜；海洋细菌能够催化含硫有机物二甲基磺基丙酯的代谢，其降解产物二甲基硫化物是海洋生态***中硫元素的主要来源，参与藻类的各种生理代谢共生细菌在培养体系中磷酸盐的浓度为0.112mg/L时，能够显著影响杜氏盐藻对砷酸盐的吸收和转化。此外，细菌也能释放一些杀菌性物质来抑制其他有害的细菌的生长，进而起到保护藻类的作用。当藻际微环境中的藻类数量超出正常的范围时，共生细菌的作用方式会发生适当的转变，它们可以通过分泌某些特殊的物质诱发藻细胞的裂解，或者对藻细胞的生存形成的一定的环境压力，以这样的方式来控制藻类的数量。另外，藻类也能够向周围中释放生物信息素，与环境中的细菌建立种属特异性的共生关系。如赫氏颗石藻分泌的某些多糖类物质，能够作为信息分子吸引环境中特定的菌群，进而建立起两者之间的共生关系。The metabolic mechanism of algae is completely different in the process of aseptic culture and bacteria culture. The existence of these symbiotic bacteria will mostly benefit the growth of algae, and the bacteria can provide algae with more directly usable small molecules for their growth. For example, during aseptic culture of diatoms, the exopolysaccharides synthesized and secreted by algae are soluble, and the growing algal cells are completely suspended; but in the process of bacterial culture, most of the exopolysaccharides produced by algae are insoluble and grow There is also the phenomenon of sticking to the wall, and forms a biofilm together with the symbiotic bacteria in the culture system; marine bacteria can catalyze the metabolism of sulfur-containing organic matter dimethyl sulfopropyl ester, and its degradation product dimethyl sulfide is an important component of the marine ecosystem. As the main source of sulfur, various symbiotic bacteria participating in the physiological metabolism of algae can significantly affect the absorption and transformation of arsenate by Dunaliella salina when the concentration of phosphate in the culture system is 0.112mg/L. In addition, bacteria can also release some bactericidal substances to inhibit the growth of other harmful bacteria, thereby protecting algae. When the number of algae in the algae microenvironment exceeds the normal range, the mode of action of the symbiotic bacteria will change appropriately. They can induce the lysis of algal cells by secreting some special substances, or form a certain effect on the survival of algal cells. environmental pressures in this way to control algae populations. In addition, algae can also release biological pheromones to the environment and establish species-specific symbiotic relationships with bacteria in the environment. For example, some polysaccharides secreted by coccolithophores can act as information molecules to attract specific bacterial groups in the environment, and then establish a symbiotic relationship between the two.
某些细菌能够产生植物激素促进藻类的生长，以提高藻类的单位生物量。植物促生菌能够促进藻类生物量的积累，当藻类与植物促生菌共同培 养时，其生物量至少可以提高10％，个别的藻类甚至可以提高70％。若去除藻际微环境中的植物促生菌，那么藻类的生长速率将会明显减慢。由此可以说明细菌对藻类培养的重要作用。Some bacteria can produce plant hormones to promote the growth of algae, so as to increase the unit biomass of algae. Plant growth-promoting bacteria can promote the accumulation of algae biomass. When algae and plant growth-promoting bacteria are co-cultured, their biomass can be increased by at least 10%, and individual algae can even increase by 70%. If the plant growth-promoting bacteria in the algae microenvironment are removed, the growth rate of algae will be significantly slowed down. This can explain the important role of bacteria in algae culture.
然而，有些细菌不但对藻类的生长没有促进作用，反而会抑制藻类的生长甚至是直接杀死藻细胞。这种具有杀藻性的细菌对藻细胞具有一定的趋向性，它们通过鞭毛固定在藻细胞表面，产生溶藻性物质，降解藻细胞的细胞壁，最终导致藻细胞的裂解死亡。因此，对于这一类的杀藻性细菌，在藻类的大量培养过程中应尽量回避，能够做到及时且彻底的清除。However, some bacteria not only do not promote the growth of algae, but can inhibit the growth of algae or even directly kill algae cells. This kind of algicidal bacteria has a certain tendency to algal cells. They fix on the surface of algal cells through flagella, produce algae-dissolving substances, degrade the cell wall of algal cells, and finally lead to the lysis and death of algal cells. Therefore, for this type of algicidal bacteria, they should be avoided as much as possible during the mass cultivation of algae, so that they can be removed in a timely and thorough manner.
在本发明中，通过海洋丁酸梭菌、海洋红酵母与海洋紫色非硫细菌三种益生菌的共生作用，有效抑制海水小球藻养殖过程中的病虫害风险，降低其危害，有效提升小球藻工厂化培养的产能产量。In the present invention, through the symbiosis of marine Clostridium butyricum, marine rhodotorula, and marine purple non-sulfur bacteria, the symbiosis of three probiotics can effectively inhibit the risk of diseases and insect pests in the seawater chlorella culture process, reduce its harm, and effectively improve the quality of pellets. Production capacity of algae industrial culture.
本发明还提供了一种共生菌组合物的制备方法，包括如下步骤：The present invention also provides a preparation method of the symbiotic bacteria composition, comprising the steps of:
将海洋丁酸梭菌菌种接种于第一培养基进行第一培养，得到海洋丁酸梭菌菌液；Inoculating the marine Clostridium butyricum species into the first culture medium for the first culture to obtain the marine Clostridium butyricum bacteria liquid;
将海洋红酵母菌种接种于第二培养基进行第二培养，得到海洋红酵母菌菌液；Inoculating the marine rhodotorula strain into the second medium for second culture to obtain the marine rhodotorula strain;
将海洋紫色非硫细菌菌种接种于第三培养基进行第三培养，得到海洋紫色非硫细菌菌液；inoculating the marine purple non-sulfur bacteria into the third culture medium for the third culture to obtain the marine purple non-sulfur bacteria liquid;
将海洋丁酸梭菌菌液、海洋红酵母菌菌液和海洋紫色非硫细菌菌液混合，得到共生菌组合物。The bacterium liquid of Clostridium butyricum, the liquid of Rhodotorula marine and the liquid of marine purple non-sulfur bacteria are mixed to obtain a symbiotic bacteria composition.
作为优选，第一培养基的配方为：As preferably, the formula of the first culture medium is:
在本发明提供的具体实施例中，第一培养基的配方为：In the specific embodiment provided by the invention, the formula of the first culture medium is:
作为优选，第一培养的条件为：转速500～700rpm，温度30～35℃，培养时间24～30h；Preferably, the conditions for the first cultivation are: rotation speed 500-700rpm, temperature 30-35°C, cultivation time 24-30h;
优选地，第一培养的条件为：转速600rpm，温度32℃，培养时间24h。Preferably, the conditions for the first cultivation are: rotation speed 600 rpm, temperature 32° C., cultivation time 24 hours.
作为优选，第一培养的接种量为5％-20％；Preferably, the inoculum size of the first culture is 5%-20%;
作为优选，第二培养基的配方为：As preferably, the formula of the second culture medium is:
在本发明提供的具体实施例中，第二培养基的配方为：In the specific embodiment provided by the invention, the formula of the second culture medium is:
作为优选，第二培养的条件为：转速1000～1500rpm，温度32～35℃，培养时间24～30h；Preferably, the conditions for the second cultivation are: rotation speed 1000-1500 rpm, temperature 32-35°C, cultivation time 24-30 hours;
优选地，第二培养的条件为：转速1000rpm，温度35℃，培养时间30h。Preferably, the conditions of the second culture are: the rotation speed is 1000 rpm, the temperature is 35° C., and the culture time is 30 h.
作为优选，第二培养的接种量为5％-20％；Preferably, the inoculum size of the second culture is 5%-20%;
第三培养基的配方为：The formula of the third culture medium is:
在本发明提供的具体实施例中，第三培养基的配方为：In a specific embodiment provided by the invention, the formula of the third culture medium is:
作为优选，第三培养的条件为：转速100～150rpm，温度28～35℃，培养时间48～72h；Preferably, the conditions for the third cultivation are: rotation speed 100-150rpm, temperature 28-35°C, cultivation time 48-72h;
优选地，第三培养的条件为：转速150rpm，温度28℃，培养时间48h。Preferably, the conditions of the third culture are: the rotation speed is 150 rpm, the temperature is 28° C., and the culture time is 48 hours.
作为优选，第三培养的接种量为20％-30％。Preferably, the inoculation amount of the third culture is 20%-30%.
作为优选，海洋丁酸梭菌菌液、海洋红酵母菌菌液与海洋紫色非硫细菌菌液的体积比例为(0.2～20)：(0.5～50)：(0.1～10)。Preferably, the volume ratio of the marine Clostridium butyricum bacteria liquid, the marine Rhodotorula yeast liquid and the marine purple non-sulfur bacteria liquid is (0.2-20): (0.5-50): (0.1-10).
优选地，海洋丁酸梭菌菌液、海洋红酵母菌菌液与海洋紫色非硫细菌菌液的体积比例为(0.5～5)：(1～10)：(0.2～2)。Preferably, the volume ratio of the marine Clostridium butyricum bacteria liquid, the marine Rhodotorula yeast liquid and the marine purple non-sulfur bacteria liquid is (0.5-5):(1-10):(0.2-2).
更优选地，海洋丁酸梭菌菌液、海洋红酵母菌菌液与海洋紫色非硫细菌菌液的体积比例为2：5：1。More preferably, the volume ratio of the marine Clostridium butyricum bacteria solution, the marine Rhodotorula bacteria solution and the marine purple non-sulfur bacteria solution is 2:5:1.
本发明还提供了由上述制备方法制得的共生菌组合物。The present invention also provides the symbiotic bacteria composition prepared by the above preparation method.
本发明还提供了一种混合共生菌菌液，其制备方法为：静置培养共生菌组合物，得到混合共生菌菌液。The invention also provides a mixed symbiotic bacteria liquid, the preparation method of which is: statically culturing the symbiotic bacteria composition to obtain the mixed symbiotic bacteria liquid.
作为优选，静置培养的条件为：光照强度1000～4000lux，温度25～40℃，光暗比2:1，湿度10％～30％RH，培养时间为20～50h。Preferably, the conditions for static culture are: light intensity 1000-4000 lux, temperature 25-40°C, light-to-dark ratio 2:1, humidity 10%-30% RH, culture time 20-50h.
优选地，静置培养的条件为：光照强度2500lux，温度30℃，光暗比2:1，湿度12％RH，培养时间为24h。Preferably, the conditions for static culture are: light intensity 2500 lux, temperature 30° C., light-to-dark ratio 2:1, humidity 12% RH, and culture time 24 hours.
本发明还提供了一种微藻的培养方法，将微藻、混合共生菌菌液接种于微藻培养液中进行共培养。The invention also provides a microalgae culture method, inoculating the microalgae and the mixed symbiotic bacteria liquid into the microalgae culture liquid for co-cultivation.
作为优选，微藻培养液的配方为：硝酸钠400～500g，磷酸二氢钾80～100g，EDTA-2Na 8～10g，六合水三氯化铁40～50g，冰乙酸500～600mL，清洁海水1吨；Preferably, the formula of the microalgae culture solution is: 400-500 g of sodium nitrate, 80-100 g of potassium dihydrogen phosphate, 8-10 g of EDTA-2Na, 40-50 g of hexahydrate ferric chloride, 500-600 mL of glacial acetic acid, clean seawater 1 ton;
在本发明提供的具体实施例中，微藻培养液的配方为：硝酸钠400g，磷酸二氢钾100g，EDTA-2Na 10g，六合水三氯化铁40g，冰乙酸600mL，清洁海水1吨。In the specific embodiment provided by the present invention, the formula of the microalgae culture solution is: 400g of sodium nitrate, 100g of potassium dihydrogen phosphate, 10g of EDTA-2Na, 40g of hexahydrate ferric chloride, 600mL of glacial acetic acid, and 1 ton of clean seawater.
作为优选，微藻与微藻培养液的接种比例为1:(500～2000)(V/V)；As preferably, the inoculation ratio of microalgae and microalgae culture solution is 1:(500～2000)(V/V);
作为优选，混合共生菌菌液与微藻培养液的接种比例为1:(5000～10000)(V/V)。Preferably, the inoculation ratio of the mixed symbiotic bacteria liquid to the microalgae culture liquid is 1: (5000-10000) (V/V).
在本发明提供的具体实施例中，微藻与微藻培养液的接种比例为1:1000(V/V)；In the specific embodiment provided by the present invention, the inoculation ratio of microalgae and microalgae culture solution is 1:1000 (V/V);
在本发明提供的具体实施例中，混合共生菌菌液与微藻培养液的接种 比例为1:5000(V/V)。In a specific embodiment provided by the invention, the inoculation ratio of the mixed symbiotic bacteria liquid and the microalgae culture liquid is 1:5000 (V/V).
作为优选，共培养的条件为：温度25-45℃，光照度5000-25000lux，培养时间24-72h；Preferably, the co-cultivation conditions are: temperature 25-45°C, illuminance 5000-25000lux, culture time 24-72h;
在本发明中，微藻为海水小球藻、硅藻、微拟球藻、卵囊藻中的一种或几种。In the present invention, the microalgae is one or more of seawater chlorella, diatoms, Nannochloropsis, and oocysts.
本发明提供了共生菌组合物及其制备方法和微藻的培养方法。该共生菌组合物由海洋丁酸梭菌、海洋红酵母和海洋紫色非硫细菌组成。本发明具有如下技术效果：The invention provides a symbiotic bacteria composition, a preparation method thereof and a microalgae cultivation method. The symbiotic bacteria composition consists of marine Clostridium butyricum, marine rhodotorula and marine purple non-sulfur bacteria. The present invention has following technical effect:
本发明通过海洋丁酸梭菌、海洋红酵母与海洋紫色非硫细菌三种益生菌的共生作用，有效抑制海水小球藻养殖过程中的病虫害风险，降低其危害，有效提升小球藻工厂化培养的产能产量。Through the symbiosis of marine Clostridium butyricum, marine rhodotorula and marine purple non-sulfur bacteria, the present invention effectively suppresses the risk of diseases and insect pests in the cultivation process of seawater chlorella, reduces its harm, and effectively improves the industrialization of chlorella Cultivated capacity yield.
本发明公开了共生菌组合物及其制备方法和微藻的培养方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。The invention discloses a symbiotic bacteria composition and a preparation method thereof and a microalgae cultivation method, and those skilled in the art can learn from the contents of this article and appropriately improve the process parameters to realize the method. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.
本发明中所用培养基或菌种均可由市场购得。The medium or strains used in the present invention can be purchased from the market.
下面结合实施例，进一步阐述本发明：Below in conjunction with embodiment, further set forth the present invention:
实施例1 益生菌培养、共生菌液制备及共培养模式Example 1 Probiotic culture, preparation of symbiotic bacteria solution and co-cultivation mode
(一)丁酸梭菌培养(1) Clostridium butyricum culture
1)菌种：海洋丁酸梭菌1) Strain: Clostridium butyricum marine
2)培养基配方(1L)：2) Medium formula (1L):
表1Table 1
| 物料名称Material name | 用量(g/L)Dosage(g/L) |
| 蛋白胨Peptone | 1111 |
| 牛肉浸粉Beef Dip Powder | 1010 |
| 酵母浸粉Yeast Dip Powder | 44 |
| 磷酸氢二钾Dipotassium phosphate | 55 |
| 乙酸钠sodium acetate | 88 |
| 枸橼酸三铵Triammonium citrate | 22 |
| 硫酸镁magnesium sulfate | 0.20.2 |
| 硫酸锰Manganese sulfate | 0.050.05 |
| 吐温80Tween 80 | 11 |
| 葡萄糖glucose | 2525 |
| 碳酸钙calcium carbonate | 55 |
| 氯化钠Sodium chloride | 2020 |
| 纯化水purified water | 1L1L |
3)将上述培养基在三角摇瓶中进行混合，并于121℃灭菌25分钟。3) The above-mentioned culture medium was mixed in an Erlenmeyer shaker flask, and sterilized at 121° C. for 25 minutes.
4)将1毫升海洋丁酸梭菌冻存菌种进行水浴锅复苏，再接入已灭菌好的培养基中，接种量10％，放入摇床中进行培养。4) Resuscitate 1 ml of frozen Clostridium butyricum marine strains in a water bath, then insert them into the sterilized culture medium with an inoculum size of 10%, and put them into a shaker for cultivation.
5)培养条件：转速600rpm，温度32℃，培养时间24h。5) Culture conditions: the rotation speed is 600 rpm, the temperature is 32°C, and the culture time is 24 hours.
(二)海洋红酵母培养(2) Culture of marine red yeast
1)菌种：海洋红酵母1) Strains: marine red yeast
2)培养基配方(1L)：2) Medium formula (1L):
表2Table 2
| 物料名称Material name | 用量(g/L)Dosage(g/L) |
| 蛋白胨Peptone | 55 |
| 麦芽浸粉Malt Dip Powder | 33 |
| 酵母浸粉Yeast Dip Powder | 5.05.0 |
| 葡萄糖glucose | 2525 |
| 氯化钠Sodium chloride | 2020 |
| 纯化水purified water | 1L1L |
3)将上述培养基在三角摇瓶中进行混合，并于121℃灭菌25分钟。3) The above-mentioned culture medium was mixed in an Erlenmeyer shaker flask, and sterilized at 121° C. for 25 minutes.
4)将1毫升海洋红酵母冻存菌种进行水浴锅复苏，再接入已灭菌好的培养基中，接种量10％，放入摇床中进行培养。4) Resuscitate 1 ml of the frozen-stored strain of Rhodotorula marinum in a water bath, then insert it into the sterilized medium with an inoculum size of 10%, and put it into a shaker for cultivation.
5)培养条件：转速1000rpm，温度35℃，培养时间30h。5) Culture conditions: rotation speed 1000rpm, temperature 35°C, culture time 30h.
(三)海洋紫色非硫细菌培养(3) Culture of marine purple non-sulfur bacteria
1)菌种：海洋紫色非硫细菌1) Bacteria: Marine purple non-sulfur bacteria
2)培养基配方(1L)：2) Medium formula (1L):
表3table 3
| 物料名称Material name | 用量(g/L)Dosage(g/L) |
| 碳酸氢钠sodium bicarbonate | 33 |
| 麦芽浸粉Malt Dip Powder | 44 |
| 酵母浸粉Yeast Dip Powder | 22 |
| 葡萄糖glucose | 2020 |
| 氯化钠Sodium chloride | 2020 |
| 纯化水purified water | 1L1L |
3)将上述培养基在三角摇瓶中进行混合，并于121℃灭菌25分钟。3) The above-mentioned culture medium was mixed in an Erlenmeyer shaker flask, and sterilized at 121° C. for 25 minutes.
4)将1毫升海洋红酵母冻存菌种进行水浴锅复苏，再接入已灭菌好的培养基中，接种量25％，放入摇床中进行培养。4) Resuscitate 1 ml of the frozen strain of Rhodotorula marinum in a water bath, then insert it into the sterilized culture medium with an inoculum size of 25%, and put it into a shaker for cultivation.
5)培养条件：转速150rpm，温度28℃，培养时间48h。5) Culture conditions: rotation speed 150 rpm, temperature 28°C, culture time 48 hours.
(四)共生菌液制备(4) Preparation of symbiotic bacteria solution
将培养好的三种细菌按照以下比例在超净台内进行混合，然后置于光照培养箱中静置培养24h，培养条件：光照强度2500lux，温度30℃，光暗比2:1，湿度12％RH，制备成混合共生菌菌液。Mix the three cultured bacteria in the ultra-clean bench according to the following ratio, and then place them in the light incubator for 24 hours. %RH, prepared as a mixed symbiotic bacteria liquid.
表4Table 4
| 细菌bacteria | 比例(V/V)Ratio (V/V) |
| 海洋丁酸梭菌Clostridium butyricum marine | 22 |
| 海洋红酵母marine red yeast | 55 |
| 海洋紫色非硫细菌marine purple nonsulfur bacteria | 11 |
(五)共生菌与微藻共培养模式(5) Co-cultivation model of symbiotic bacteria and microalgae
将海水小球藻按照1:1000(V/V，微藻藻种体积与培养液体积之比)接种至微藻培养液中，所述培养液配方如下：硝酸钠400克，磷酸二氢钾100克，EDTA-2Na 10克，六合水三氯化铁40克，冰乙酸600毫升，溶于1吨清洁海水中，即制备成微藻培养液。Seawater chlorella was inoculated into the microalgae culture solution according to 1:1000 (V/V, the ratio of the volume of the microalgae species to the volume of the culture solution), and the formula of the culture solution was as follows: 400 grams of sodium nitrate, potassium dihydrogen phosphate 100 grams, 10 grams of EDTA-2Na, 40 grams of Liuheshui ferric chloride, 600 ml of glacial acetic acid, dissolved in 1 ton of clean seawater, that is, prepared into a microalgae culture solution.
实验组：将共生菌液按照1:5000(V/V，共生菌液体积与微藻培养液体积之比)比例添加至微藻培养液中，与微藻共生培养。Experimental group: The symbiotic bacteria solution was added to the microalgae culture solution at a ratio of 1:5000 (V/V, the ratio of the volume of the symbiotic bacteria solution to the volume of the microalgae culture solution), and symbiotically cultivated with the microalgae.
对照组：不添加共生菌液，微藻培养基同上。Control group: without adding symbiotic bacteria solution, the microalgae culture medium is the same as above.
共培养条件：温度30℃，光照度12000lux，培养时间72h。Co-culture conditions: temperature 30°C, light intensity 12000 lux, culture time 72 hours.
培养结果如下：The cultivation results are as follows:
表5table 5
通过数据可见，经过72h培养，添加共生菌的海水小球藻，其密度为不添加组的3.87倍。It can be seen from the data that after 72 hours of cultivation, the density of seawater chlorella added with symbiotic bacteria is 3.87 times that of the group without addition.
以上对本发明所提供的共生菌组合物及其制备方法和微藻的培养方法进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。The symbiotic bacteria composition provided by the present invention and its preparation method and the cultivation method of microalgae have been introduced in detail above. This article uses specific examples to illustrate the principle and implementation of the present invention. The description of the above embodiments is only used to help understand the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.
Claims (10)
- 一种共生菌组合物，其特征在于，由海洋丁酸梭菌(Clostridium butyricum)、海洋红酵母(Phaffia rhodozyma)和海洋紫色非硫细菌(Rhodobium marinum)组成；A symbiotic bacteria composition, characterized in that, is composed of Clostridium butyricum, Phaffia rhodozyma and Rhodobium marinum;海洋丁酸梭菌、海洋红酵母与海洋紫色非硫细菌的活菌数比例为(2～4)：(5～7)：(1～3)。The ratio of live bacteria of marine Clostridium butyricum, marine rhodotorula and marine purple non-sulfur bacteria was (2-4):(5-7):(1-3).
- 一种共生菌组合物的制备方法，其特征在于，包括如下步骤：A preparation method of symbiotic bacteria composition, is characterized in that, comprises the steps:将海洋丁酸梭菌菌种接种于第一培养基进行第一培养，得到海洋丁酸梭菌菌液；Inoculating the marine Clostridium butyricum species into the first culture medium for the first culture to obtain the marine Clostridium butyricum bacteria liquid;将海洋红酵母菌种接种于第二培养基进行第二培养，得到海洋红酵母菌菌液；Inoculating the marine rhodotorula strain into the second medium for second culture to obtain the marine rhodotorula strain;将海洋紫色非硫细菌菌种接种于第三培养基进行第三培养，得到海洋紫色非硫细菌菌液；inoculating the marine purple non-sulfur bacteria into the third culture medium for the third culture to obtain the marine purple non-sulfur bacteria liquid;将海洋丁酸梭菌菌液、海洋红酵母菌菌液和海洋紫色非硫细菌菌液混合，得到共生菌组合物。The bacterium liquid of Clostridium butyricum, the liquid of Rhodotorula marine and the liquid of marine purple non-sulfur bacteria are mixed to obtain a symbiotic bacteria composition.
- 根据权利要求2所述的制备方法，其特征在于，所述第一培养基的配方为：The preparation method according to claim 2, wherein the formulation of the first culture medium is:蛋白胨 10.0～15.0g/LPeptone 10.0～15.0g/L牛肉浸粉 10.0～15.0g/LBeef Dip Powder 10.0～15.0g/L酵母浸粉 2.0～5.0g/LYeast extract powder 2.0～5.0g/L磷酸氢二钾 2.0～5.0g/LDipotassium hydrogen phosphate 2.0～5.0g/L乙酸钠 5.0～10.0g/LSodium acetate 5.0～10.0g/L枸橼酸三铵 2.0～3.0g/LTriammonium citrate 2.0～3.0g/L硫酸镁 0.2～0.4g/LMagnesium sulfate 0.2～0.4g/L硫酸锰 0.05～0.1g/LManganese sulfate 0.05～0.1g/L吐温80 1.0～1.5g/LTween 80 1.0～1.5g/L葡萄糖 20.0～35.0g/LGlucose 20.0～35.0g/L碳酸钙 5.0～8.0g/LCalcium carbonate 5.0～8.0g/L氯化钠 20.0～25.0g/LSodium chloride 20.0～25.0g/L水 补足至1L；Water to make up to 1L;第一培养的条件为：转速500～700rpm，温度30～35℃，培养时间24～30h；The conditions for the first cultivation are: rotation speed 500-700rpm, temperature 30-35°C, cultivation time 24-30h;第一培养的接种量为5％-20％；The inoculation amount of the first culture is 5%-20%;所述第二培养基的配方为：The formula of the second culture medium is:蛋白胨 5.0～10.0g/LPeptone 5.0～10.0g/L麦芽浸粉 3.0～5.0g/LMalt powder 3.0～5.0g/L酵母浸粉 2.0～5.0g/LYeast extract powder 2.0～5.0g/L葡萄糖 20.0～35.0g/LGlucose 20.0～35.0g/L氯化钠 20.0～25.0g/LSodium chloride 20.0～25.0g/L水 补足至1L；Water to make up to 1L;第二培养的条件为：转速1000～1500rpm，温度32～35℃，培养时间24～30h；The conditions for the second culture are: rotation speed 1000-1500rpm, temperature 32-35°C, culture time 24-30h;第二培养的接种量为5％-20％；The inoculation amount of the second culture is 5%-20%;所述第三培养基的配方为：The formula of the third culture medium is:碳酸氢钠 2.0～5.0g/LSodium bicarbonate 2.0～5.0g/L麦芽浸粉 3.0～5.0g/LMalt powder 3.0～5.0g/L酵母浸粉 2.0～5.0g/LYeast extract powder 2.0～5.0g/L葡萄糖 20.0～35.0g/LGlucose 20.0～35.0g/L氯化钠 20.0～25.0g/LSodium chloride 20.0～25.0g/L水 补足至1L；Water to make up to 1L;第三培养的条件为：转速100～150rpm，温度28～35℃，培养时间48～72h；The conditions for the third culture are: rotation speed 100-150 rpm, temperature 28-35°C, culture time 48-72 hours;第三培养的接种量为20％-30％。The inoculation amount of the third culture is 20%-30%.
- 根据权利要求2或3所述的制备方法，其特征在于，海洋丁酸梭菌菌液、海洋红酵母菌菌液与海洋紫色非硫细菌菌液的体积比例为(0.2～20)：(0.5～50)：(0.1～10)。According to the preparation method described in claim 2 or 3, it is characterized in that the volume ratio of the marine Clostridium butyricum bacterial liquid, the marine Rhodotorula bacterial liquid and the marine purple non-sulfur bacteria liquid is (0.2～20): (0.5 ~50): (0.1~10).
- 权利要求2至4中任一项所述制备方法制得的共生菌组合物。The symbiotic bacteria composition prepared by the preparation method described in any one of claims 2 to 4.
- 一种混合共生菌菌液，其特征在于，其制备方法为：静置培养权利要求5所述的共生菌组合物，得到混合共生菌菌液。A mixed symbiotic bacteria liquid, characterized in that the preparation method is: statically culturing the symbiotic bacteria composition described in claim 5 to obtain the mixed symbiotic bacteria liquid.
- 根据权利要求6所述的混合共生菌菌液，其特征在于，所述静置培养的条件为：光照强度1000～4000lux，温度25～40℃，光暗比2:1，湿度10％～30％RH，培养时间为20～50h。The mixed symbiotic bacteria liquid according to claim 6, characterized in that, the conditions for the static culture are: light intensity 1000-4000 lux, temperature 25-40°C, light-to-dark ratio 2:1, humidity 10%-30 %RH, the culture time is 20-50h.
- 一种微藻的培养方法，其特征在于，将微藻、权利要求6或7所述混合共生菌菌液接种于微藻培养液中进行共培养。A method for cultivating microalgae, characterized in that the microalgae and the mixed symbiotic bacteria liquid described in claim 6 or 7 are inoculated in the microalgae culture liquid for co-cultivation.
- 根据权利要求8所述的培养方法，其特征在于，所述微藻培养液的配方为：硝酸钠400～500g，磷酸二氢钾80～100g，EDTA-2Na 8～10g，六合水三氯化铁40～50g，冰乙酸500～600mL，清洁海水1吨；The cultivation method according to claim 8, characterized in that, the formula of the microalgae culture solution is: 400-500 g of sodium nitrate, 80-100 g of potassium dihydrogen phosphate, 8-10 g of EDTA-2Na, and Liuhe water trichloride 40-50g of iron, 500-600mL of glacial acetic acid, 1 ton of clean seawater;微藻与微藻培养液的接种比例为1:(500～2000)(V/V)；The inoculation ratio of microalgae and microalgae culture solution is 1:(500～2000)(V/V);混合共生菌菌液与微藻培养液的接种比例为1:(5000～10000)(V/V)。The inoculation ratio of the mixed symbiotic bacteria liquid to the microalgae culture liquid was 1: (5000-10000) (V/V).
- 根据权利要求8或9所述的培养方法，其特征在于，共培养的条件为：温度25-45℃，光照度5000-25000lux，培养时间24-72h；The cultivation method according to claim 8 or 9, characterized in that the co-cultivation conditions are: temperature 25-45°C, illuminance 5000-25000lux, cultivation time 24-72h;所述微藻为海水小球藻、硅藻、微拟球藻、卵囊藻中的一种或几种。The microalgae is one or more of seawater chlorella, diatoms, Nannochloropsis and oocysts.
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