Method for culturing microalgae by using beer waste liquid and application thereof

Abstract

The invention provides a method for culturing microalgae by using beer waste liquid and application of the method, and relates to the technical field of microalgae biological culture. According to the method disclosed by the invention, microalgae, especially chlorella sp. Of chlorophyta, are cultured by adopting the beer wastewater. Moreover, it is found for the first time that the effect of synergistically promoting the growth of the microalgae can be achieved when fermentation tank washing water is adopted as a nutritional ingredient to jointly culture chlorella (Chlorella sp.) FACHB-2900 and Navicula sp. FACHB-1996, and the composite microalgae can keep certain biological activity at a low temperature and also has excellent synergic cold resistance. The invention also provides a preparation method of the composite microalgae related fertilizer, and the microalgae fertilizer can be used for improving the soil performance of saline-alkali soil, and has excellent performances of promoting the growth of beet, increasing the yield and the like.

Description

Technical Field

[0001] This application relates to the technical field of microalgae biological culture, and particularly relates to a method for culturing microalgae using beer waste liquid and its application. Background Art

[0002] In the brewing process of beer, a large amount of wastewater rich in organic matter is generated due to processes such as soaking malt, saccharification, fermentation, and cleaning equipment. The composition of the wastewater mainly includes sugars, proteins, pectins, alcohol acids, and some suspended substances mainly composed of distiller's grains and fibers. Beer wastewater contains a large amount of carbon and nitrogen-containing organic matter, has a high COD / BOD content, and good biodegradability. If directly discharged into the natural environment, it is likely to cause eutrophication pollution of water bodies.

[0003] Microalgae, as a single-celled or multi-celled aquatic organism, has the characteristics of wide distribution and high growth and metabolic rates, has strong environmental adaptability, can survive alone or in groups in environments such as the ocean, desert, atmosphere, and soil, shows characteristics similar to terrestrial microorganisms in living habits, and is regarded as an intermediate species between terrestrial microorganisms and terrestrial plants because its photosynthesis mechanism is similar to that of terrestrial plants. At the same time, because it is an aquatic organism itself, it has the ability to rapidly accumulate biomass synthesis, can perform functions such as nitrogen, phosphorus, and organic matter removal in a water environment rich in nutrients, and thus plays a huge role in polluted water environments. However, there are relatively few related technologies for microalgae to treat beer wastewater. And compared with terrestrial plants, microalgae grow fast, do not occupy arable land, are less affected by seasonal changes in growth, are simple to harvest, have a suitable fatty acid profile, have low by-products, and some microalgae can also absorb CO2 in the atmosphere, such as carbon dioxide in the ecological environment, carbon dioxide in exhaust gases. At the same time, many algae also have rich nutritional components and can be used as good fertilizer materials.

[0004] Lack of soil nutrients is one of the key factors restricting the growth of crops. Microalgae can gradually decompose and release the rich nutrients in cells under the drive of soil microbial metabolism. Compared with chemical fertilizers, microalgae bio-fertilizers can slowly and continuously provide nutrients for crops throughout the planting period. Microalgae has the functions of carbon fixation, nitrogen fixation, and phosphorus solubilization, can improve the chemical and biological properties of the soil, and help restore soil fertility. Microalgae synthesizes organic carbon in vivo through photosynthesis and secretes extracellular polysaccharides to provide a carbon source to the soil, which can effectively prevent farmland degradation problems caused by excessive consumption of soil organic matter. Microalgae also has the ability to convert complex organic phosphorus into inorganic phosphorus and helps dissolve insoluble inorganic phosphorus for plant absorption and utilization. However, the current cost of microalgae cultivation is relatively high, and industrial production is subject to certain restrictions.

[0005] The patent application with the publication number CN105219648A discloses a method for producing Chlorella using beer industrial wastewater and its application. This method combines wastewater treatment with microalgae cultivation, utilizes the heterotrophic characteristics of Chlorella, and addresses the problem of water eutrophication to achieve the resource treatment and utilization of wastewater, with a relatively low content of nutrient elements required to be added. However, in practical applications, this technical solution still has the following technical problems: the content of the added nutrient elements is relatively low and is insufficient to meet the needs of Chlorella at different growth stages, resulting in limited growth of the microalgae biomass. Summary of the Invention

[0006] To solve the above problems, the present application provides a method for cultivating microalgae using beer waste liquid. By using beer waste liquid as an effective component of the culture medium to cultivate microalgae, the reuse of beer waste liquid is achieved, the cultivation cost of microalgae is reduced, and the industrial production process is promoted.

[0007] On the one hand, the present application provides a method for cultivating microalgae using beer waste liquid. The method includes cultivating microalgae using a culture medium; the culture medium contains beer waste liquid; the microalgae are Chlorella sp. and Navicula sp.

[0008] Further, the Chlorella sp. is Chlorella sp. FACHB - 2900, and the Navicula sp. is Navicula sp. FACHB - 1996.

[0009] Further, the beer waste liquid is selected from one or more of distiller's grains wastewater, fermentation tank cooling water, fermentation tank washing water, condenser cooling water, and bottle washing water.

[0010] Further, the beer waste liquid is fermentation tank washing water.

[0011] The fermentation tank washing water contains various nutrient components, such as the remaining beer, yeast, yeast metabolites, hops, incompletely decomposed beer components, various minerals, citric acid, or phosphoric acid in the tank. Compared with other beer waste liquids, it contains more nutrient components and fewer harmful substances such as disinfectants or chemical additives, so it is suitable for cultivating microalgae.

[0012] The present application first proves that using beer waste liquid, especially fermentation tank washing water as a nutrient component to cultivate microalgae helps to promote the growth of microalgae.

[0013] Furthermore, the culture medium comprises 100 - 1000 mg / L of NaNO3, 10 - 50 mg / L of K2HPO4, 10 - 50 mg / L of Na2CO3, 50 - 100 mg / L of MgSO4·7H2O, 10 - 50 mg / L of CaCl2·2H2O, 1 - 5 mg / L of EDTA·Na2, 1 - 5 mg / L of A5 trace elements, 20 - 40 ml / L of soil extract, and 100 - 300 ml / L of beer waste liquid.

[0014] The A5 trace elements include: 1 - 5 g / L of H3BO3, 1 - 5 g / L of MnCl2·4H2O, 0.1 - 0.5 g / L of ZnSO4·7H2O, 0.07 - 0.08 g / L of CuSO4·5H2O, 0.1 - 0.5 g / L of Na2MoO4·2H2O, and 0.04 - 0.05 g / L of Co(NO3)2·6H2O.

[0015] Preferably, the culture medium comprises 500 mg / L of NaNO3, 40 mg / L of K2HPO4, 20 mg / L of Na2CO3, 75 mg / L of MgSO4·7H2O, 36 mg / L of CaCl2·2H2O, 1 mg / L of EDTA·Na2, 1 mg / L of A5 trace elements, 30 ml / L of soil extract, 260 ml / L of fermentation tank washing water, with the balance being water, sterilized at 121°C for 20 min.

[0016] The A5 trace elements include: 2.86 g / L of H3BO3, 1.86 g / L of MnCl2·4H2O, 0.222 g / L of ZnSO4·7H2O, 0.079 g / L of CuSO4·5H2O, 0.390 g / L of Na2MoO4·2H2O, and 0.049 g / L of Co(NO3)2·6H2O.

[0017] Those skilled in the art can understand that the culture medium can be prepared by conventional methods as long as the components in the culture medium are mixed evenly according to the above formula.

[0018] Furthermore, the culture conditions include: the inoculation amount is 1% - 5%, the light intensity is 2000 - 4000 Lux, the light cycle is 12 h:12 h, the culture temperature is 15°C - 25°C, the pH value is 6.5 - 7, and the culture lasts for 10 - 20 days.

[0019] Preferably, the culture conditions include: the inoculation amount is 1%, the light intensity is 3000 Lux, the light cycle is 12 h:12 h, the culture temperature is 20°C, the pH value is 6.5, and the culture lasts for 14 days, wherein only carbon dioxide in the air is used as an inorganic nitrogen source supplement.

[0020] Further, during inoculation, the mass ratio of Chlorella sp. to Navicula sp. is 1:(0.5 - 2); preferably, the OD 680 value of Chlorella sp. is 1 - 2; preferably, the OD 750 value of Navicula sp. is 1 - 2.

[0021] Preferably, the mass ratio of Chlorella sp. to Navicula sp. is 1:1.

[0022] Preferably, the OD 680 value of Chlorella sp. is 1 and / or the OD 750 value of Navicula sp. is 1.

[0023] In a preferred embodiment, a method for culturing microalgae using beer waste liquid, the method comprises the following steps:

[0024] Step 1, prepare a culture medium, the culture medium comprises NaNO3 100 - 1000 mg / L, K2HPO4 10 - 50 mg / L, Na2CO3 10 - 50 mg / L, MgSO4·7H2O 50 - 100 mg / L, CaCl2·2H2O 10 - 50 mg / L, EDTA·Na2 1 - 5 mg / L, A5 trace elements 1 - 5 mg / L, soil extract 20 - 40 ml / L, fermentation tank washing water 100 - 300 ml / L; A5 trace elements include: H3BO3 1 - 5 g / L, MnCl2·4H2O 1 - 5 g / L, ZnSO4·7H2O 0.1 - 0.5 g / L, CuSO4·5H2O 0.07 - 0.08 g / L, Na2MoO4·2H2O 0.1 - 0.5 g / L, Co(NO3)2·6H2O 0.04 - 0.05 g / L;

[0025] Step 2, inoculate Navicula sp. and Chlorella sp. into the culture medium at an inoculation amount of 1% - 5%, the mass ratio of Chlorella sp. to Navicula sp. is 1:(0.5 - 2), the OD 680 value of Chlorella sp. is 1 - 2, the OD of Naviculasp. 750The value is 1 - 2, the light intensity is 2000 - 4000 Lux, the light cycle is 12h:12h, the culture temperature is 15°C - 25°C, the pH value is 6.5 - 7, and only carbon dioxide in the air is used as an inorganic nitrogen source supplement. After culturing for 10 - 20 days, the microalgae grow to the logarithmic growth phase.

[0026] The key technical means of this method include directly using the washing water of the beer fermentation tank as a culture medium, and using its rich nutrients, especially nitrogen, phosphorus, trace elements, etc., to meet the nutritional requirements of microalgae at different growth stages. In this way, the dependence on external chemical additives can be effectively reduced, thereby improving the sustainability and economy of the culture medium.

[0027] This application can achieve the efficient growth of microalgae and solve the technical problems such as low wastewater treatment efficiency and unbalanced nutrient components in the existing microalgae culture medium. Specifically, using the natural nutrients in the washing water of the beer fermentation tank not only improves the growth efficiency of Chlorella but also reduces the production cost to a certain extent and enhances the biological adaptability of microalgae, thus providing a feasible solution for large-scale industrial applications and also providing a new treatment method for beer wastewater.

[0028] On the other hand, this application also provides the use of the described method in the preparation of fertilizer products containing microalgae.

[0029] Preferably, the product includes Chlorella sp. and Navicula sp.

[0030] More preferably, the product includes Chlorella sp. FACHB - 2900 and Navicula sp. FACHB - 1996.

[0031] More preferably, the quantity ratio of Chlorella sp. FACHB - 2900 to Navicula sp. FACHB - 1996 is 16:1.

[0032] It can be understood that auxiliary materials can be added to the fertilizer product of this application, and the auxiliary materials can be appropriate solvents, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, stabilizers, glidants, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, pH value regulators, plasticizers, surfactants, release retardants, etc., which are known auxiliary materials available for fertilizer products.

[0033] It can be understood that a general method can be used to prepare the fertilizer product containing microalgae.

[0034] In a preferred embodiment, a method for preparing a fertilizer product is provided, and the method comprises the following steps:

[0035] Step 1: Prepare a culture medium, which comprises 100 - 1000 mg / L of NaNO3, 10 - 50 mg / L of K2HPO4, 10 - 50 mg / L of Na2CO3, 50 - 100 mg / L of MgSO4·7H2O, 10 - 50 mg / L of CaCl2·2H2O, 1 - 5 mg / L of EDTA·Na2, 1 - 5 mg / L of A5 trace elements, 20 - 40 ml / L of soil extract, and 100 - 300 ml / L of fermenter washing water; the A5 trace elements include: 1 - 5 g / L of H3BO3, 1 - 5 g / L of MnCl2·4H2O, 0.1 - 0.5 g / L of ZnSO4·7H2O, 0.07 - 0.08 g / L of CuSO4·5H2O, 0.1 - 0.5 g / L of Na2MoO4·2H2O, and 0.04 - 0.05 g / L of Co(NO3)2·6H2O;

[0036] Step 2: Inoculate Navicula sp. and Chlorella sp. into the culture medium at an inoculation amount of 1% - 5%, the mass ratio of Chlorella sp. to Navicula sp. is 1:(0.5 - 2), the OD 680 value of Chlorella sp. is 1 - 2, the OD 750 value of Navicula sp. is 1 - 2, the light intensity is 2000 - 4000 Lux, the light cycle is 12h:12h, the culture temperature is 15°C - 25°C, the pH value is 6.5 - 7, and only carbon dioxide in the air is used as an inorganic nitrogen source supplement. Cultivate for 10 - 20 days until the microalgae grow to the logarithmic growth phase to obtain the fertilizer product.

[0037] On the other hand, the present application also provides the application of the described fertilizer product in promoting the growth of sugar beets and / or increasing the sugar content of sugar beets.

[0038] Preferably, the sugar beet planting land is saline-alkali land, and the pH value of the saline-alkali land is 7.5 - 9.

[0039] On the other hand, the present application also provides the application of the described fertilizer product in cold resistance, preventing and controlling sugar beet leaf spot, reducing soil pH and / or saline-alkali land treatment.

[0040] Preferably, the fertilizer product is a cold-resistant fertilizer product, and the application temperature of the fertilizer product is 5°C - 35°C.

[0041] In this application, it was first discovered that the fertilizer product obtained by compounding Chlorella sp. FACHB-2900 and Navicula sp. FACHB-1996 can maintain a certain biological activity at a relatively low temperature, has excellent synergistic cold resistance performance, and can be widely applied to plants that need to grow in cold environments.

[0042] It was also found in this application that the fertilizer product obtained by compounding Chlorella sp. FACHB-2900 and Navicula sp. FACHB-1996 has the effect of preventing and controlling Cercospora beticola, and its control efficiency index can reach 26.33%.

[0043] Preferably, the pH value of the saline-alkali land is 7.5 - 9.

[0044] It can be understood that those skilled in the art can select the application amount of the microalgae fertilizer product according to the actual situation, and this application does not make specific restrictions on this.

[0045] In a preferred embodiment, the application amount of the microalgae fertilizer product is 100 - 1000 mL per mu; preferably, 500 mL per mu.

[0046] The present invention has the following beneficial effects:

[0047] In this application, beer wastewater is used to culture microalgae, especially Chlorella sp. of Chlorophyta. It is proved that the fermentation tank washing water in beer wastewater can replace some nutrient components in the culture medium to cultivate microalgae, which not only reduces the cost in the process of microalgae cultivation, but also solves the problem of beer wastewater treatment, and promotes the industrial production process of microalgae cultivation.

[0048] Moreover, it was first discovered in this application that using the fermentation tank washing water as a nutrient component to co-culture Chlorella sp. FACHB-2900 and Navicula sp. FACHB-1996 can play a role in synergistically promoting the growth of microalgae, and this composite microalgae can maintain a certain biological activity at a relatively low temperature, and also has excellent synergistic cold resistance and the performance of preventing and controlling Cercospora beticola.

[0049] In this application, the formulation of the microalgae culture medium using beer wastewater as a nutrient component and the culture conditions were also improved, and a culture medium and method for efficiently culturing microalgae were obtained.

[0050] This application also provides a preparation method of the fertilizer related to the above composite microalgae, and it is found that this microalgae fertilizer can be used to improve the soil properties of saline-alkali land, and has excellent properties such as promoting the growth of sugar beets and increasing the yield. Detailed implementation mode

[0051] To more clearly illustrate the overall concept of this application, the following will be described in detail by way of examples. In the following description, a large number of specific details are given to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some well-known technical features in the art are not described.

[0052] It should be noted that the following detailed description is illustrative and aims to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0053] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to this application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should also be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0054] Unless otherwise specified, in the following embodiments, reagents or instruments without indicating the manufacturer can be obtained as conventional products through commercial purchase.

[0055] Among them, Chlorella sp. FACHB-2900 belongs to Chlorophyta, strain number: FACHB-2900, collected from Lulang, Tibet. Chlorella sp. FACHB-1997 belongs to Chlorophyta, strain number: FACHB-1997. Navicula sp. belongs to Bacillariophyta, strain number: FACHB-1996, collected from the Niyang River in Tonnyi, Bayi Town. The above-mentioned microalgae were all purchased from the Freshwater Algae Culture Collection of the Chinese Academy of Sciences.

[0056] BG11 medium and CSI medium were purchased from Shanghai Guangyu Biotechnology Co., Ltd.

[0057] BG11 medium formula: NaNO3 1500 mg / L, K2HPO4 40 mg / L, Na2CO3 20 mg / L, MgSO4·7H2O 75 mg / L, CaCl2·2H2O 36 mg / L, EDTA·Na2 1 mg / L, A5 trace elements 1 mg / L, citric acid 6 mg / L, ammonium ferric citrate 6 mg / L, the balance is water, sterilized at 121 °C for 20 min.

[0058] The A5 trace elements include: H3BO3 2.86 g / L, MnCl2·4H2O 1.86 g / L, ZnSO4·7H2O 0.222 g / L, CuSO4·5H2O 0.079 g / L, Na2MoO4·2H2O 0.390 g / L, Co(NO3)2·6H2O 0.049 g / L.

[0059] CSI culture medium formula: Ca(NO3)·4H2O 1 mL / L, KNO3 1 mL / L, MgSO4·7H2O 1 mL / L, β-Na2glycerophosphate·5H2O 1 mL / L, Vitamin B12 0.1 μg / L, Biotin 0.1 μg / L, Thiamine HCl 10 μg / L, PIV* 6 mL / L, HEPES 0.5 g / L, Na2SiO3·9H2O, soil extract 30 mL / L.

[0060] Preparation method of soil extract: Take 200 grams of unfertilized garden soil, place it in a beaker or Erlenmeyer flask, add 1000 milliliters of distilled water, seal the bottle mouth with a breathable plug, heat it in a water bath at boiling water temperature for 3 hours, cool it and let it precipitate for 24 hours. This process is carried out continuously for 3 times, then filter it, take the supernatant, sterilize it in an autoclave and store it in a refrigerator at 4°C for standby.

[0061] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments, rather than limiting the protection scope of the present invention. The test methods without specific conditions noted in the following examples are usually carried out under conventional conditions, or according to the conditions recommended by each manufacturer.

[0062] When the embodiments give numerical ranges, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, equipment, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, equipment, and materials similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to implement the present invention.

[0063] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt the conventional techniques in the fields of microbiology, biochemistry, analytical chemistry, cell culture, and related fields in this technical field.

[0064] In addition, the "water" in the present invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, purified water, etc.

[0065] In the following examples, if there is no other special description, % represents wt%, that is, weight percentage.

[0066] Example 1 Experiment on Cultivating Microalgae with Beer Wastewater

[0067] In this example, beer wastewater at different stages (distillers' grains wastewater, fermenter cooling water, fermenter washing water, condenser cooling water, bottle washing water) was used as nutrient components to cultivate different microalgae (Chlorella sp. FACHB-2900, Chlorella sp. FACHB-1997, Navicula sp. FACHB-1996).

[0068] The specific process is as follows:

[0069] Add beer wastewater at different stages to the culture medium at an addition amount of 300 ml / L (microalgae of Chlorophyta are cultured with BG11 medium, microalgae of Bacillariophyta are cultured with CSI medium, and mixed microalgae of Chlorophyta and Bacillariophyta are cultured with a mixed medium with a volume ratio of BG11 medium to CSI medium of 1:1), sterilize at 121 °C for 20 min, inoculate microalgae at an inoculation amount of 1% (OD of microalgae of Chlorophyta 680 = 1, OD of microalgae of Bacillariophyta 750 = 1), 2000 Lux, light cycle 12 h:12 h, temperature 20 °C, pH = 7, and only use carbon dioxide in the air as an inorganic nitrogen source supplement, culture for 14 days, take samples regularly every 24 h, and measure the OD 680 value of the algal solution to determine the growth of microalgae (record the maximum OD 680 value in 14 days). The specific results are shown in Table 1.

[0070] Table 1

[0071]

[0072] Note: The mass ratio of Chlorella sp. FACHB-2900 or FACHB-1997 to Navicula sp. FACHB-1996 in the mixture is 1:1, and the total inoculation amount is 1%.

[0073] As can be seen from the results in Table 1, beer wastewater can be used to cultivate microalgae. In particular, the washing water of the fermenter can be used as a nutrient component to cultivate microalgae, especially Chlorella sp. of Chlorophyta. Moreover, in this example, it was also found that co-cultivating Chlorella sp. FACHB-2900 and Navicula sp. FACHB-1996 with the washing water of the fermenter as a nutrient component can play a role in synergistically promoting the growth of microalgae. The distribution of microalgae was measured by microscopic counting method, and the maximum OD was reached at 14 days of cultivation. 680 At this time, the number ratio of Chlorella sp. FACHB-2900 to Navicula sp. FACHB-1996 was 16:1.

[0074] In this example, the properties of the washing water of the fermenter were also measured. Its COD (average) content was 521.06 mg / L, total phosphorus (average) content was 10.26 mg / L, and ammonia nitrogen (average) content was 15.71 mg / L.

[0075] Example 2 Medium Formula Optimization Experiment

[0076] On the basis of the preferred medium in Example 1, in order to further improve the cultivation efficiency and cost of microalgae, in this example, part of the organic components in the medium were replaced with the washing water of the fermenter, and the medium components were further optimized.

[0077] Specifically, the medium components in this example include: NaNO3 500 mg / L, K2HPO4 40 mg / L, Na2CO3 20 mg / L, MgSO4·7H2O 75 mg / L, CaCl2·2H2O 36 mg / L, EDTA·Na2 1 mg / L, A5 trace elements 1 mg / L, soil extract 30 ml / L, washing water of the fermenter, and the balance is water. Sterilize at 121 °C for 20 min.

[0078] A5 trace elements include: H3BO3 2.86 g / L, MnCl2·4H2O 1.86 g / L, ZnSO4·7H2O 0.222 g / L, CuSO4·5H2O 0.079 g / L, Na2MoO4·2H2O 0.390 g / L, Co(NO3)2·6H2O 0.049 g / L.

[0079] The microalgae cultivation method and measurement method were the same as in Example 1, and whether there was precipitation was observed. The results are shown in Table 2.

[0080] Table 2

[0081]

[0082]

[0083] As shown in the results of Table 2, when using the fermenter washing water as a medium component to cultivate microalgae, precipitation occurred in the later stage of the medium at an addition amount of 300 ml / L, which inhibited the growth of microalgae. However, there was a problem of insufficient nutrient components at an addition amount of 100 ml / L. Therefore, an addition amount of 260 ml / L had a better effect.

[0084] Moreover, in this example, the microalgae distribution was measured again by the microscopic counting method. At the maximum OD 680 , the ratio of Chlorella vulgaris FACHB-2900 to Navicula sp. FACHB-1996 was about 16:1, proving that this ratio was the optimal ratio for their natural selection.

[0085] Example 3 Optimization Experiment of Culture Conditions

[0086] In this example, an optimization screening experiment was carried out for the culture conditions.

[0087] The specific process is as follows:

[0088] Using the optimized medium in Example 2 (NaNO3 500 mg / L, K2HPO4 40 mg / L, Na2CO3 20 mg / L, MgSO4·7H2O 75 mg / L, CaCl2·2H2O 36 mg / L, EDTA·Na2 1 mg / L, A5 trace elements 1 mg / L, soil extract 30 ml / L, fermenter washing water 260 ml / L, the balance is water, sterilized at 121 °C for 20 min. A5 trace elements include: H3BO3 2.86 g / L, MnCl2·4H2O 1.86 g / L, ZnSO4·7H2O 0.222 g / L, CuSO4·5H2O 0.079 g / L, Na2MoO4·2H2O 0.390 g / L, Co(NO3)2·6H2O 0.049 g / L.), inoculating Chlorella sp. FACHB-2900 and Navicula sp. FACHB-1996 with a mass ratio of 1:1 at an inoculation amount of 1%, the OD of Chlorella sp. FACHB-2900 680 = 1, the OD of Navicula sp. FACHB-1996 750 = 1, 2000 Lux, light cycle 12 h:12 h, at different temperatures, under the condition of pH = 7, only using carbon dioxide in the air as an inorganic nitrogen source supplement, culturing for 14 days, sampling regularly every 24 h, and measuring the OD of the algal solution 680 value to determine the growth situation of microalgae (recording the maximum OD 680 in 14 days), and the specific results are shown in Table 3.

[0089] Table 3

[0090]

[0091] As can be seen from the results in Table 3, in this example, the composite microalgae Chlorella sp. FACHB-2900 and Navicula sp. FACHB-1996 can maintain a certain biological activity at a relatively low temperature, have excellent synergistic cold resistance performance, and their more suitable culture temperature is 15°C - 25°C, with 20°C being the best.

[0092] On the basis of the above preferred conditions, in this example, different inoculation amounts, different light intensities, and different pH conditions were used as single-factor variables for the optimization experiment of the culture conditions. The remaining culture methods were the same as above, and the OD of the algal solution was measured 680 value to determine the growth of microalgae (recording the maximum OD 680 value in 14 days). The specific results are shown in Table 4.

[0093] Table 4

[0094]

[0095] As can be seen from Table 4, the optimal culture conditions for microalgae are as follows: inoculating Chlorella sp. FACHB-2900 and Navicula sp. FACHB-1996 with a mass ratio of 1:1 into the preferred medium at an inoculation amount of 1%, 3000 Lux, a light cycle of 12h:12h, a temperature of 20°C, and a pH = 6.5. Under these culture conditions, the growth of microalgae is the best. Moreover, in this example, the final distribution ratios of Chlorella sp. FACHB-2900 and Navicula sp. FACHB-1996 at different mass ratios were measured, proving that the final ratio has no direct relationship with the mass ratio at the time of inoculation. When the OD 680 reaches the maximum, their ratio is about 16:1.

[0096] Example 4 Preparation method of microalgae fertilizer

[0097] In this example, a preparation method of microalgae fertilizer is provided, which specifically includes the following steps:

[0098] Inoculating Chlorella sp. FACHB-2900 and Navicula sp. FACHB-1996 with a mass ratio of 1:1 into the medium at an inoculation amount of 1%, with the OD of Chlorella 680 = 1 and the OD of Navicula 750= 1, 3000 Lux, light cycle 12 h:12 h, temperature 20 °C, pH = 6.5, using only carbon dioxide in the air as an inorganic nitrogen source supplement, culturing for 14 days to obtain a liquid microalgae fertilizer.

[0099] Among them, the components of the culture medium include: NaNO3 500 mg / L, K2HPO4 40 mg / L, Na2CO3 20 mg / L, MgSO4·7H2O 75 mg / L, CaCl2·2H2O 36 mg / L, EDTA·Na2 1 mg / L, A5 trace elements 1 mg / L, soil extract 30 ml / L, fermentation tank washing water 260 ml / L, with the balance being water, sterilized at 121 °C for 20 min. The A5 trace elements include: H3BO3 2.86 g / L, MnCl2·4H2O 1.86 g / L, ZnSO4·7H2O 0.222 g / L, CuSO4·5H2O 0.079 g / L, Na2MoO4·2H2O 0.390 g / L, Co(NO3)2·6H2O 0.049 g / L.

[0100] Example 5

[0101] The difference between this example and Example 4 is only that Chlorella sp. FACHB-2900 and Navicula sp. FACHB-1996 are cultured separately and mixed in a mass ratio of 16:1.

[0102] Example 6

[0103] The difference between this example and Example 4 is only that Chlorella sp. FACHB-2900 and Navicula sp. FACHB-1996 are cultured separately and mixed in a mass ratio of 1:1.

[0104] Application of the microalgae fertilizer in Example 7

[0105] In this example, the microalgae fertilizer prepared by the methods of Examples 4 - 6 was used as a sample to plant sugar beets in saline-alkali land (pH value 7.5 - 9) in Qingdao, Shandong.

[0106] Using the sugar beet variety Beta468 as the experimental material, it was planted by hill-drop seeding. When the cotyledons grew to 2 - 3 leaves, thinning and final thinning were carried out in a timely manner, and only one sugar beet was retained in each hill. When the sugar beet grew to 6 - 8 leaves, microalgae fertilizer was applied at a rate of 500 mL per mu. An equal amount of commercially available microalgae fertilizer was used as the positive control, and an equal amount of water treatment group was used as the blank control. The remaining field management methods were kept the same. Sowing was carried out in April and harvesting in September, and the sugar beets were harvested after 5 months. The sugar content of the root tuber was obtained by multiplying the soluble solids content of the root tuber measured by a refractometer by 0.83. The results are shown in Table 5.

[0107] The rhizosphere soil before and after planting was air-dried naturally and then passed through a 1 mm sieve. The air-dried soil after sieving was used to prepare a test sample solution according to a soil-water ratio of 5:1. The pH value of the soil was measured using a pH meter for the solution, and all values were averages. The results are shown in Table 6.

[0108] In this example, an investigation was also carried out on the disease situation of sugar beet leaf spot. Using the random sampling method, the field where the overall disease index of sugar beet leaf spot reached 3% was used as the experimental field. Seven days after applying the fertilizer, 50 plants were randomly sampled from each group. The investigation was carried out on a per-plant basis, and the plants were classified according to the severity of the disease occurrence on each plant. Grade 0: The whole plant has no disease spots or only a few leaves have a few disease spots; Grade 1: Most leaves have a few disease spots or a few leaves have many disease spots; Grade 2: Most leaves have a few disease spots, and less than 1 / 4 of the outer leaves are dead due to the disease; Grade 3: Most leaves have many disease spots, and 1 / 4 - 1 / 2 of the outer leaves are dead due to the disease; Grade 4: Most leaves have many disease spots, and 1 / 2 - 3 / 4 of the outer leaves are dead due to the disease; Grade 5: Most leaves are dead due to the disease except for the heart leaves. The disease index and control effect index can be calculated according to the following formula, and the results are shown in Table 7.

[0109]

[0110] Table 5

[0111] Group Sugar content (%) Example 4 20.43 Example 5 18.88 Example 6 16.76 Positive control 14.64 Blank control 10.36

[0112] As can be seen from the results in Table 5, the microalgae fertilizer prepared by the methods of Examples 4 - 6 has a better effect of promoting the growth of sugar beets and promoting the accumulation of dry matter in sugar beets compared with the commercially available fertilizer. Among them, the microalgae fertilizers prepared by the methods of Example 4 and Example 5 have similar promoting effects. However, considering cost savings, it is better to choose the method of Example 4 to prepare the microalgae fertilizer. Since this composite microalgae can maintain a certain biological activity at a lower temperature, even in an environment of about 5°C, compared with other biological fertilizers, it has higher biological activity and thus has a more excellent growth-promoting effect.

[0113] Table 6

[0114] Group pH Soil of Example 4 7.39 Soil of positive control 8.30 Soil of blank control 8.25 Soil before planting 8.26

[0115] As can be seen from the results in Table 6, the microalgae fertilizer prepared by the method of Example 4 has the effect of improving saline-alkali soil.

[0116] Table 7

[0117]

[0118]

[0119] As can be seen from the results in Table 7, the microalgae fertilizer prepared by the method of Example 4 has a certain control effect against Cercospora beticola.

[0120] The above are only the embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the scope of the claims of the present application.

Claims

1. A method for culturing microalgae using beer waste liquid, characterized in that: The method comprises the steps of culturing microalgae by using a culture medium; the culture medium contains beer waste liquid; and the microalgae are Chlorella sp. and Navicula sp.

2. The method according to claim 1, characterized in that The Chlorella sp. is Chlorella sp. FACHB-2900, and the Navicula sp. is Navicula sp. FACHB-1996.

3. The method according to claim 1, characterized in that The beer waste liquid is selected from one or more of lees waste water, fermentation tank cooling water, fermentation tank washing water, condenser cooling water, and bottle washing water.

4. The method according to claim 3, characterized in that The beer waste liquid is fermentation tank washing water.

5. The method according to claim 1, characterized in that: The culture medium comprises NaNO3 100-1000 mg / L, K2HPO4 10-50 mg / L, Na2CO3 10-50 mg / L, MgSO4·7H2O 50-100 mg / L, CaCl2·2H2O 10-50 mg / L, EDTA·Na2 1-5 mg / L, A5 trace elements 1-5 mg / L, soil extract 20-40 ml / L, and beer waste liquid 100-300 ml / L.

6. The method according to claim 1, characterized in that The culture conditions include: inoculation amount of 1%-5%, light intensity of 2000-4000 Lux, photoperiod of 12h:12h, culture temperature of 15°C-25°C, pH value of 6.5-7, and culture for 10-20 days.

7. The method according to claim 6, characterized in that During the inoculation, the mass ratio of Chlorella sp. to Navicula sp. is 1:(0.5-2); preferably, the OD of Chlorella sp. 680 The value is 1-2; preferably, the OD of Navicula sp. 750 The value is 1-2.

8. Use of the method according to any one of claims 1 to 7 in preparing a fertilizer product containing microalgae.

9. Use of the fertilizer product as claimed in claim 8 in promoting the growth of sugar beets and / or increasing the sugar content of sugar beets.

10. Use of the fertilizer product according to claim 8 in cold resistance, prevention and treatment of sugar beet brown spot disease, lowering soil pH and / or saline-alkali land management.

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