Nitrogen resource recycling single-cell protein and preparation method thereof

Abstract

The invention discloses a nitrogen resource recycling single-cell protein and a preparation method thereof, particularly relates to the technical field of biology, and relates to a nitrogen resource recycling single-cell protein and a preparation method thereof. The nitrogen resource recycling single-cell protein is prepared from methanomonas liquid, paracoccus denitrificans liquid, a fermentation culture medium and mixed gas of methane, carbon monoxide and carbon dioxide. According to the invention, methanomonas is adopted to produce the single-cell protein, methane, carbon monoxide and carbon dioxide are used as carbon sources, the gases are gradually converted into cell substances under the metabolism action of microorganisms, a necessary carbon skeleton is provided for synthesis of the single-cell protein, the source is wide, and the cost is low.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to a nitrogen resource recycling single cell protein and a preparation method thereof. Background Art

[0002] With the development of global industrialization, the emission of greenhouse gases such as methane has increased, while the excessive emission of carbon dioxide has led to aggravated environmental problems. The treatment of nitrogen-containing wastewater is also a major problem. If it is directly discharged, it will cause harm such as eutrophication of water bodies. Single-cell protein has high nutritional value. Therefore, it is of great significance to develop a single-cell protein production method that can effectively utilize nitrogen-containing wastewater.

[0003] The single-cell protein preparation in the relevant technology mainly includes starch, ammonium salts, mineral trace elements, water, and vitamins; among them, starch can provide a continuous supply of carbon elements for the growth of microorganisms; ammonium salts are used to synthesize proteins and nucleic acids; mineral trace elements are essential for the growth and metabolism of microorganisms; microorganisms need water to dissolve various nutrients during their growth and participate in biochemical reactions within the cells, and water can also maintain the morphology and structure of the cells; vitamins are organic compounds necessary for the growth of microorganisms, and they participate in various metabolic reactions in the microorganisms and act as coenzymes or cofactors.

[0004] However, there are still some disadvantages in its actual use, such as high cost. Traditional production usually relies on grain crops or food processing waste as raw materials, and a large amount of single-cell protein production is used to increase production costs; low production efficiency. The fermentation cycle of traditional production technology may be long, and the growth and reproduction rate of microorganisms are subject to certain restrictions. In addition, whether it is the pretreatment of raw materials, temperature and pressure control during the fermentation process, or the later product drying and separation, a large amount of energy is required, resulting in high energy consumption; environmental pollution. A large amount of waste is generated in the traditional production process, which will cause serious pollution to the environment. Summary of the invention

[0005] In order to improve the above problems and reduce the high cost, large energy consumption and environmental pollution of single-cell protein, the present invention provides a nitrogen resource recycling single-cell protein and a preparation method thereof to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A nitrogen resource recycling single-cell protein prepared from a methylomonas solution, a denitrifying Paracoccus solution, a fermentation medium, and a mixed gas of methane, carbon monoxide, and carbon dioxide;

[0008] The specific preparation steps are as follows:

[0009] A1. First, a methylomonas solution and a denitrifying Paracoccus solution are mixed, and the mixture is inoculated into a fermenter containing a sterilized fermentation medium at a volume ratio of 5-10%, and the mixture is fermented for 48-72 hours at a temperature of 30-35° C., a stirring speed of 200-300 r / min, and a pH of 7-7.5, during which a mixed gas of methane, carbon monoxide, and carbon dioxide is continuously introduced at a rate of 0.3-0.5 vvm, and nitrogen-containing wastewater is automatically added during the fermentation process to keep the nitrogen source concentration in the fermentation liquid stable, thereby obtaining a fermentation liquid;

[0010] A2. Pour the fermentation liquid obtained in A1 into a centrifuge and centrifuge at a speed of 4000 r / min to obtain the precipitate, then wash the precipitate twice with sterile saline, and then vacuum dry the washed precipitate at a temperature of 60° C. for 12 hours to obtain single-cell protein.

[0011] Preferably, the preparation steps of the Methanogenomyosin solution are as follows:

[0012] C1. In a sterile environment, inoculate the strain of Methanometazobacterium into a shake flask filled with a basal culture medium, and culture for 48 hours at a temperature of 28° C., a rotation speed of 150-200 r / min, and a pH of 6.5-7.5 to obtain an activated Methanometazobacterium liquid;

[0013] C2. Pour nitrogen-containing wastewater containing 500-1000 mg / L of ammonia nitrogen into the activated Methanomethamonas liquid obtained in C1, the wastewater is added at a ratio of 5% of the volume of the culture medium, and at the same time, 0.1-0.3 vvm of a mixed gas containing methane, carbon monoxide and carbon dioxide is introduced, and the culture is carried out under the same conditions as C1; the amount of nitrogen-containing wastewater added is increased by 5-10% every 24 hours until the amount of nitrogen-containing wastewater added reaches 50-70% of the volume of the culture medium, and the addition of nitrogen-containing wastewater is stopped. During this period, the OD600 value of the bacterial liquid is continuously measured. When the OD600 value reaches 0.8-1.2, the culture is stopped to obtain the Methanomethamonas liquid.

[0014] Preferably, the preparation steps of the Paracoccus denitrifying solution are as follows:

[0015] B1. Inoculate the denitrifying Paracoccus strain into the sterilized culture medium at a content of 5% of the volume of the nitrogen-containing culture medium, and culture the nitrogen-containing culture medium in a constant temperature incubator at a temperature of 28-32° C. and a pH of 6.8-7.2 for 1-2 days to obtain a denitrifying Paracoccus liquid.

[0016] Preferably, the fermentation medium consists of a basic medium and nitrogen-containing wastewater in a volume ratio of (1-7): (1-3).

[0017] Preferably, the basic culture medium consists of potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, ferrous sulfate, boric acid, copper sulfate, sodium molybdate, zinc chloride, manganese chloride, and water in a weight ratio of (1.5-2.5): (0.25-0.4): (0.06-0.09): (0.015-0.025): (0.06-0.09): (0.015-0.025); (0.015-0.025): (0.06-0.09): (0.06-0.09): 1000.

[0018] Preferably, the mixed gas of methane, carbon monoxide and carbon dioxide consists of methane, carbon monoxide and carbon dioxide in a volume ratio of (6.5-7.5):(1.2-1.8):(1.3-1.7).

[0019] Preferably, the nitrogen-containing culture medium consists of glucose, ammonium chloride, sodium bicarbonate, magnesium sulfate, and ferrous sulfate in a weight ratio of 5:0.5:0.1:(0-0.2):(0-0.01):1000.

[0020] Technical effects and advantages of the present invention:

[0021] 1. The invention adopts Methylomonas methanotrophicus to produce single-cell protein, which uses methane, carbon monoxide and carbon dioxide as carbon sources, and gradually converts these gases into cellular substances under the metabolism of microorganisms, providing the necessary carbon skeleton for the synthesis of single-cell protein, with a wide source and low cost.

[0022] 2. The present invention utilizes Paracoccus denitrificans to produce single-cell protein, which utilizes oxygen as an electron acceptor, removes nitrogen from the water body by reducing nitrite, converts nitrogen in ammonia-containing wastewater into nitrogen-containing compounds, participates in the metabolic process of cells, and realizes the recycling of nitrogen resources. At the same time, the waste carbon source and nitrogen source are effectively utilized, so that the energy demand in the production process is relatively low;

[0023] 3. The present invention produces single-cell protein by recycling nitrogen resources, which is more in line with the concept of sustainable development and can reduce dependence on limited resources. The waste generated in its production process is relatively small, and resources can be recycled through further processing and utilization. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below in conjunction with the embodiments of the present invention. The raw materials used in the examples and embodiments of the present invention are all commercially available common materials unless otherwise specified below.

[0025] Preparation Example 1

[0026] A Methanometamol liquid is prepared by the following preparation method:

[0027] C1. In a sterile environment, inoculate the strain of Methanometazobacterium into a shake flask filled with a basal culture medium, and culture for 24 hours at a temperature of 28° C., a rotation speed of 150 r / min, and a pH of 6.5 to obtain an activated Methanometazobacterium liquid;

[0028] The basic culture medium is composed of potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, ferrous sulfate, boric acid, copper sulfate, sodium molybdate, zinc chloride, manganese chloride, and water in a weight ratio of 2:0.3:0.08:0.02:0.08:0.02; 0.02:0.08:0.08:1000;

[0029] C2. Pour the activated Methanomethamonas liquid obtained in C1 into nitrogen-containing wastewater containing 500 mg / L ammonia nitrogen, the wastewater being added at a ratio of 5% of the volume of the culture medium, and simultaneously introducing 0.1 vvm of a mixed gas containing methane, carbon monoxide and carbon dioxide, and culturing under the same conditions as C1; increase the amount of nitrogen-containing wastewater added by 5% every 24 hours until the amount of nitrogen-containing wastewater added reaches 50% of the volume of the culture medium, stop adding nitrogen-containing wastewater, continuously measure the OD600 value of the bacterial liquid during this period, and stop culturing when the OD600 value reaches 0.8 to obtain Methanomethamonas liquid.

[0030] The mixed gas of methane, carbon monoxide and carbon dioxide is composed of methane, carbon monoxide and carbon dioxide in a volume ratio of 7:1.5:1.5;

[0031] Preparation Example 2

[0032] A Methanomethoate Methylomonas liquid, which is different from Preparation Example 1 in that the specific preparation steps are as follows:

[0033] C1. In a sterile environment, inoculate the strain of Methanometazobacterium into a shake flask filled with a basal culture medium, and culture for 36 hours at a temperature of 30° C., a rotation speed of 175 r / min, and a pH of 7 to obtain an activated Methanometazobacterium liquid;

[0034] C2. Pour the activated Methanomethamonas liquid obtained in C1 into nitrogen-containing wastewater containing 500 mg / L ammonia nitrogen, the wastewater being added at a ratio of 5% of the volume of the culture medium, and simultaneously introducing 0.1 vvm of a mixed gas containing methane, carbon monoxide and carbon dioxide, and culturing under the same conditions as C1; increase the amount of nitrogen-containing wastewater added by 5% every 24 hours until the amount of nitrogen-containing wastewater added reaches 50% of the volume of the culture medium, stop adding nitrogen-containing wastewater, continuously measure the OD600 value of the bacterial liquid during this period, and stop culturing when the OD600 value reaches 0.8 to obtain an acclimated Methanomethamonas liquid.

[0035] Preparation Example 3

[0036] A Methanomethoate Methylomonas liquid, which is different from Preparation Example 1 in that the specific preparation steps are as follows:

[0037] C1. In a sterile environment, inoculate the strain of Methanometazobacterium into a shake flask filled with a basal culture medium, and culture for 48 hours at a temperature of 32° C., a rotation speed of 200 r / min, and a pH of 7.5 to obtain an activated Methanometazobacterium liquid;

[0038] C2. Pour the activated Methanomethamonas liquid obtained in C1 into nitrogen-containing wastewater containing 500 mg / L ammonia nitrogen, the wastewater being added at a ratio of 5% of the volume of the culture medium, and simultaneously introducing 0.1 vvm of a mixed gas containing methane, carbon monoxide and carbon dioxide, and culturing under the same conditions as C1; increase the amount of nitrogen-containing wastewater added by 5% every 24 hours until the amount of nitrogen-containing wastewater added reaches 50% of the volume of the culture medium, stop adding nitrogen-containing wastewater, continuously measure the OD600 value of the bacterial liquid during this period, and stop culturing when the OD600 value reaches 0.8 to obtain an acclimated Methanomethamonas liquid.

[0039] Preparation Example 4

[0040] A Methanomethoate Methylomonas liquid, which is different from Preparation Example 1 in that the specific preparation steps are as follows:

[0041] C1. In a sterile environment, inoculate the strain of Methanometazobacterium into a shake flask filled with a basal culture medium, and culture for 24 hours at a temperature of 28° C., a rotation speed of 150 r / min, and a pH of 6.5 to obtain an activated Methanometazobacterium liquid;

[0042] C2. Pour the activated Methanomethamonas liquid obtained in C1 into nitrogen-containing wastewater containing 1000 mg / L ammonia nitrogen, the wastewater being added at a ratio of 5% of the volume of the culture medium, and simultaneously introducing 0.1 vvm of a mixed gas containing methane, carbon monoxide and carbon dioxide, and culturing under the same conditions as C1; increase the amount of nitrogen-containing wastewater added by 5% every 24 hours until the amount of nitrogen-containing wastewater added reaches 50% of the volume of the culture medium, stop adding nitrogen-containing wastewater, continuously measure the OD600 value of the bacterial liquid during this period, and stop culturing when the OD600 value reaches 0.8 to obtain an acclimated Methanomethamonas liquid.

[0043] Preparation Example 5

[0044] A Methanomethoate Methylomonas liquid, which is different from Preparation Example 1 in that the specific preparation steps are as follows:

[0045] C1. In a sterile environment, inoculate the strain of Methanometazobacterium into a shake flask filled with a basal culture medium, and culture for 24 hours at a temperature of 28° C., a rotation speed of 15 r / min, and a pH of 6.5 to obtain an activated Methanometazobacterium liquid;

[0046] C2. Pour the activated Methanomethamonas liquid obtained in C1 into nitrogen-containing wastewater containing 500 mg / L ammonia nitrogen, the wastewater being added at a ratio of 5% of the volume of the culture medium, and simultaneously introducing 0.3 vvm of a mixed gas containing methane, carbon monoxide and carbon dioxide, and culturing under the same conditions as C1; increase the amount of nitrogen-containing wastewater added by 5% every 24 hours until the amount of nitrogen-containing wastewater added reaches 50% of the volume of the culture medium, stop adding nitrogen-containing wastewater, continuously measure the OD600 value of the bacterial liquid during this period, and stop culturing when the OD600 value reaches 0.8 to obtain an acclimated Methanomethamonas liquid.

[0047] Preparation Example 6

[0048] A Methanomethoate Methylomonas liquid, which is different from Preparation Example 1 in that the specific preparation steps are as follows:

[0049] C1. In a sterile environment, inoculate the strain of Methanometazobacterium into a shake flask filled with a basal culture medium, and culture for 24 hours at a temperature of 28° C., a rotation speed of 15 r / min, and a pH of 6.5 to obtain an activated Methanometazobacterium liquid;

[0050] C2. Pour the activated Methanomethamonas liquid obtained in C1 into nitrogen-containing wastewater containing 500 mg / L ammonia nitrogen, the wastewater being added at a ratio of 10% of the volume of the culture medium, and simultaneously introducing 0.1 vvm of a mixed gas containing methane, carbon monoxide and carbon dioxide, and culturing under the same conditions as C1; increase the amount of nitrogen-containing wastewater added by 10% every 24 hours until the amount of nitrogen-containing wastewater added reaches 70% of the volume of the culture medium, stop adding nitrogen-containing wastewater, and continuously measure the OD600 value of the bacterial liquid during this period. When the OD600 value reaches 0.8, stop culturing to obtain an acclimated Methanomethamonas liquid.

[0051] Preparation Example 7

[0052] A Methanomethoate Methylomonas liquid, which is different from Preparation Example 1 in that the specific preparation steps are as follows:

[0053] C1. In a sterile environment, inoculate the strain of Methanometazobacterium into a shake flask filled with a basal culture medium, and culture for 24 hours at a temperature of 28° C., a rotation speed of 15 r / min, and a pH of 6.5 to obtain an activated Methanometazobacterium liquid;

[0054] C2. Pour the activated Methanomethamonas liquid obtained in C1 into nitrogen-containing wastewater containing 500 mg / L ammonia nitrogen, the wastewater being added at a ratio of 5% of the volume of the culture medium, and simultaneously introducing 0.1 vvm of a mixed gas containing methane, carbon monoxide and carbon dioxide, and culturing under the same conditions as C1; increase the amount of nitrogen-containing wastewater added by 5% every 24 hours until the amount of nitrogen-containing wastewater added reaches 50% of the volume of the culture medium, stop adding nitrogen-containing wastewater, continuously measure the OD600 value of the bacterial liquid during this period, and stop culturing when the OD600 value reaches 1.2 to obtain an acclimated Methanomethamonas liquid.

[0055] Preparation Example 8

[0056] A Methanemethylomonas solution, which is different from Preparation Example 1 in that the basic culture medium consists of potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, ferrous sulfate, boric acid, copper sulfate, sodium molybdate, zinc chloride, manganese chloride, and water in a weight ratio of 1.5:0.4:0.06:0.015:0.06:0.015; 0.015:0.06:0.06:1000.

[0057] Preparation Example 9

[0058] A Methylomonas solution, which is different from Preparation Example 1 in that the basic culture medium is composed of potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, ferrous sulfate, boric acid, copper sulfate, sodium molybdate, zinc chloride, manganese chloride, and water in a weight ratio of 2.5:0.25:0.09:0.025:0.09:0.025; 0.025:0.09:0.09:1000;

[0059] Preparation Example 10

[0060] A methylomonas solution, which is different from Preparation Example 1 in that the mixed gas of methane, carbon monoxide and carbon dioxide is composed of methane, carbon monoxide and carbon dioxide in a volume ratio of 6.5:1.8:1.7;

[0061] Preparation Example 11

[0062] A methylomonas solution, which is different from Preparation Example 1 in that the mixed gas of methane, carbon monoxide and carbon dioxide is composed of methane, carbon monoxide and carbon dioxide in a volume ratio of 7.5:1.2:1.3;

[0063] Preparation Example 12

[0064] A denitrifying Paracoccus liquid is prepared by the following preparation method:

[0065] B1. Inoculate the denitrifying Paracoccus strain into the sterilized culture medium at a content of 5% of the volume of the nitrogen-containing culture medium, and culture the nitrogen-containing culture medium in a constant temperature incubator at a temperature of 25° C. and a pH of 6.8 for 1 day to obtain a denitrifying Paracoccus liquid.

[0066] The nitrogen-containing medium is composed of glucose, ammonium chloride, sodium bicarbonate, and water in a weight ratio of 5:0.5:0.1:1000;

[0067] Preparation Example 13

[0068] A denitrifying Paracoccus liquid, which is different from Preparation Example 12 in that the preparation method is as follows:

[0069] B1. Inoculate the denitrifying Paracoccus strain into the sterilized culture medium at a content of 5% of the volume of the nitrogen-containing culture medium, and culture the nitrogen-containing culture medium in a constant temperature incubator at a temperature of 30° C. and a pH of 7 for 1.5 days to obtain a denitrifying Paracoccus liquid.

[0070] Preparation Example 14

[0071] A denitrifying Paracoccus liquid, which is different from Preparation Example 12 in that the preparation method is as follows:

[0072] B1. Inoculate the denitrifying Paracoccus strain into the sterilized culture medium at a content of 5% of the volume of the nitrogen-containing culture medium, and culture the nitrogen-containing culture medium in a constant temperature incubator at a temperature of 32° C. and a pH of 7.2 for 2 days to obtain a denitrifying Paracoccus liquid.

[0073] Preparation Example 15

[0074] A denitrifying Paracoccus liquid, which is different from Preparation Example 12 in that the nitrogen-containing culture medium consists of glucose, ammonium chloride, sodium bicarbonate, magnesium sulfate, and ferrous sulfate in a weight ratio of 5:0.5:0.1:0.2:0.01:1000.

[0075] Preparation Examples 16-20

[0076] A nitrogen resource recycling single cell protein, whose components and corresponding volume ratios are shown in the following table, is prepared by the following preparation method:

[0077] A1. Mixing a methylomonas solution and a denitrifying Paracoccus solution, and inoculating the mixture into a fermenter containing a sterilized fermentation medium at a volume ratio of 5%, fermenting the mixture for 48 hours at a temperature of 30° C., a stirring speed of 200 r / min, and a pH of 7, during which a mixed gas of methane, carbon monoxide, and carbon dioxide is continuously introduced at a rate of 0.3 vvm, and nitrogen-containing wastewater is automatically added during the fermentation process to keep the nitrogen source concentration in the fermentation liquid stable, thereby obtaining a fermentation liquid;

[0078] The Methanomethoate monocytogenes solution was prepared as in Preparation Example 1;

[0079] The Paracoccus denitrificans solution was prepared by Preparation Example 12;

[0080] The fermentation medium is composed of basal medium and nitrogen-containing wastewater in a volume ratio of 1:1;

[0081] The basic culture medium and nitrogen-containing wastewater components are the same as those in Preparation Example 1;

[0082] The gas mixture of methane, carbon monoxide and carbon dioxide has the same composition as that in Preparation Example 1;

[0083] A2. Pour the fermentation liquid obtained in A1 into a centrifuge and centrifuge at a speed of 4000 r / min to obtain the precipitate, then wash the precipitate twice with sterile saline, and then vacuum dry the washed precipitate at a temperature of 60° C. for 12 hours to obtain single-cell protein.

[0084] Table: Preparation components and their volume ratios in Preparation Examples 16-20

[0085]

[0086] Preparation Example 21

[0087] A single cell protein for recycling nitrogen resources, which is different from Preparation Example 16 in that the preparation method is as follows:

[0088] A1. Mixing a methylomonas solution and a denitrifying Paracoccus solution, and inoculating the mixture into a fermenter containing a sterilized fermentation medium at a volume ratio of 7.5%, fermenting the mixture for 48 hours at a temperature of 30° C., a stirring speed of 200 r / min, and a pH of 7, during which a mixed gas of methane, carbon monoxide, and carbon dioxide was continuously introduced at a rate of 0.3 vvm, and nitrogen-containing wastewater was automatically added during the fermentation process to keep the nitrogen source concentration in the fermentation liquid stable, to obtain a fermentation liquid;

[0089] A2. Pour the fermentation liquid obtained in A1 into a centrifuge and centrifuge at a speed of 4000 r / min to obtain the precipitate, then wash the precipitate twice with sterile saline, and then vacuum dry the washed precipitate at a temperature of 60° C. for 12 hours to obtain single-cell protein.

[0090] Preparation Example 22

[0091] A single cell protein for recycling nitrogen resources, which is different from Preparation Example 16 in that the preparation method is as follows:

[0092] A1. Mixing a methylomonas solution and a denitrifying Paracoccus solution, and inoculating the mixture into a fermenter containing a sterilized fermentation medium at a volume ratio of 10%, fermenting the mixture for 48 hours at a temperature of 30° C., a stirring speed of 200 r / min, and a pH of 7, during which a mixed gas of methane, carbon monoxide, and carbon dioxide is continuously introduced at a rate of 0.3 vvm, and nitrogen-containing wastewater is automatically added during the fermentation process to keep the nitrogen source concentration in the fermentation liquid stable, thereby obtaining a fermentation liquid;

[0093] A2. Pour the fermentation liquid obtained in A1 into a centrifuge and centrifuge at a speed of 4000 r / min to obtain the precipitate, then wash the precipitate twice with sterile saline, and then vacuum dry the washed precipitate at a temperature of 60° C. for 12 hours to obtain single-cell protein.

[0094] Preparation Example 23

[0095] A single cell protein for recycling nitrogen resources, which is different from Preparation Example 16 in that the preparation method is as follows:

[0096] A1. Mixing a methylomonas solution and a denitrifying Paracoccus solution, and inoculating the mixture into a fermenter containing a sterilized fermentation medium at a volume ratio of 5%, fermenting the mixture for 60 hours at a temperature of 32° C., a stirring speed of 250 r / min, and a pH of 7.2, during which a mixed gas of methane, carbon monoxide, and carbon dioxide was continuously introduced at a rate of 0.3 vvm, and nitrogen-containing wastewater was automatically added during the fermentation process to keep the nitrogen source concentration in the fermentation liquid stable, thereby obtaining a fermentation liquid;

[0097] A2. Pour the fermentation liquid obtained in A1 into a centrifuge and centrifuge at a speed of 4000 r / min to obtain the precipitate, then wash the precipitate twice with sterile saline, and then vacuum dry the washed precipitate at a temperature of 60° C. for 12 hours to obtain single-cell protein.

[0098] Preparation Example 24

[0099] A single cell protein for recycling nitrogen resources, which is different from Preparation Example 16 in that the preparation method is as follows:

[0100] A1. Mixing a methylomonas solution and a denitrifying Paracoccus solution, and inoculating the mixture into a fermenter containing a sterilized fermentation medium at a volume ratio of 5%, fermenting the mixture for 72 hours at a temperature of 35° C., a stirring speed of 300 r / min, and a pH of 7.5, during which a mixed gas of methane, carbon monoxide, and carbon dioxide was continuously introduced at a rate of 0.3 vvm, and nitrogen-containing wastewater was automatically added during the fermentation process to keep the nitrogen source concentration in the fermentation liquid stable, thereby obtaining a fermentation liquid;

[0101] A2. Pour the fermentation liquid obtained in A1 into a centrifuge and centrifuge at a speed of 4000 r / min to obtain the precipitate, then wash the precipitate twice with sterile saline, and then vacuum dry the washed precipitate at a temperature of 60° C. for 12 hours to obtain single-cell protein.

[0102] Preparation Example 25

[0103] A single cell protein for recycling nitrogen resources, which is different from Preparation Example 16 in that the preparation method is as follows:

[0104] A1. Mixing a methylomonas solution and a denitrifying Paracoccus solution, and inoculating the mixture into a fermenter containing a sterilized fermentation medium at a volume ratio of 5%, fermenting the mixture for 48 hours at a temperature of 30° C., a stirring speed of 200 r / min, and a pH of 7, during which a mixed gas of methane, carbon monoxide, and carbon dioxide is continuously introduced at a rate of 0.4 vvm, and nitrogen-containing wastewater is automatically added during the fermentation process to keep the nitrogen source concentration in the fermentation liquid stable, thereby obtaining a fermentation liquid;

[0105] A2. Pour the fermentation liquid obtained in A1 into a centrifuge and centrifuge at a speed of 4000 r / min to obtain the precipitate, then wash the precipitate twice with sterile saline, and then vacuum dry the washed precipitate at a temperature of 60° C. for 12 hours to obtain single-cell protein.

[0106] Preparation Example 26

[0107] A single cell protein for recycling nitrogen resources, which is different from Preparation Example 16 in that the preparation method is as follows:

[0108] A1. Mixing a methylomonas solution and a denitrifying Paracoccus solution, and inoculating the mixture into a fermenter containing a sterilized fermentation medium at a volume ratio of 5%, fermenting the mixture for 48 hours at a temperature of 30° C., a stirring speed of 200 r / min, and a pH of 7, during which a mixed gas of methane, carbon monoxide, and carbon dioxide is continuously introduced at a rate of 0.5 vvm, and nitrogen-containing wastewater is automatically added during the fermentation process to keep the nitrogen source concentration in the fermentation liquid stable, thereby obtaining a fermentation liquid;

[0109] A2. Pour the fermentation liquid obtained in A1 into a centrifuge and centrifuge at a speed of 4000 r / min to obtain the precipitate, then wash the precipitate twice with sterile saline, and then vacuum dry the washed precipitate at a temperature of 60° C. for 12 hours to obtain single-cell protein.

[0110] Preparation Example 27

[0111] A single-cell protein for recycling nitrogen resources, which is different from Preparation Example 16 in that the fermentation medium is composed of a basic medium and nitrogen-containing wastewater in a volume ratio of 3:2;

[0112] The basic culture medium and nitrogen-containing wastewater components are the same as those in Preparation Example 1;

[0113] Preparation Example 28

[0114] A nitrogen resource recycling single cell protein, which is different from Preparation Example 16 in that the fermentation medium is composed of a basic medium and nitrogen-containing wastewater in a volume ratio of 7:3;

[0115] The basic culture medium and nitrogen-containing wastewater components are the same as those in Preparation Example 1;

[0116] Preparation Example 29

[0117] A single-cell protein for recycling nitrogen resources, which is different from Preparation Example 16 in that the fermentation medium is composed of a basic medium and nitrogen-containing wastewater in a volume ratio of 1:1;

[0118] The basic culture medium components are the same as those in Preparation Example 1, and the nitrogen-containing wastewater components are the same as those in Preparation Example 4;

[0119] Preparation Example 30

[0120] A single-cell protein for recycling nitrogen resources, which is different from Preparation Example 16 in that the fermentation medium is composed of a basic medium and nitrogen-containing wastewater in a volume ratio of 1:1;

[0121] The basic culture medium components are the same as those in Preparation Example 8, and the nitrogen-containing wastewater components are the same as those in Preparation Example 1;

[0122] Preparation Example 31

[0123] A single-cell protein for recycling nitrogen resources, which is different from Preparation Example 16 in that the fermentation medium is composed of a basic medium and nitrogen-containing wastewater in a volume ratio of 1:1;

[0124] The basic culture medium components are the same as those in Preparation Example 9, and the nitrogen-containing wastewater components are the same as those in Preparation Example 1;

[0125] Preparation Example 32

[0126] A nitrogen resource recycling single cell protein, which is different from Preparation Example 16 in that the methane, carbon monoxide, and carbon dioxide mixed gas components are the same as those in Preparation Example 10;

[0127] Preparation Example 33

[0128] A nitrogen resource recycling single cell protein, which is different from Preparation Example 16 in that the methane, carbon monoxide, and carbon dioxide mixed gas components are the same as those in Preparation Example 11;

[0129] Preparation Examples 34-43

[0130] A single-cell protein for recycling nitrogen resources, which is different from Preparation Example 16 in that the usage of the Methanogen Methylomonas liquid used in its components is different, and the specific corresponding relationship is shown in the following table.

[0131] Table: Comparison of the use of Methanomethoate solution in Preparation Examples 34-43

[0132]

[0133]

[0134] Preparation Examples 44-46

[0135] A single-cell protein for recycling nitrogen resources, which is different from Preparation Example 16 in that the usage of the denitrifying Paracoccus liquid used in its components is different, and the specific corresponding relationship is shown in the following table.

[0136] Table: Comparison of the use of denitrifying Paracoccus solution in Preparation Examples 44-46

[0137] Group Paracoccus denitrificans liquid Preparation Example 44 Prepared from Preparation Example 13 Preparation Example 45 Prepared from Preparation Example 14 Preparation Example 46 Prepared from Preparation Example 15

[0138] Performance testing

[0139] The nitrogen resource recycling single cell protein prepared in each embodiment was selected for testing, and the test objects were 310 portions of nitrogen resource recycling single cell protein, 10 portions in each group; the protein content and microbial content of the product were tested, and the specific testing steps were as follows:

[0140] Crude protein content:

[0141] First, the nitrogen resource recycling single cell protein prepared in the embodiment is sampled, and the crude protein content of the single cell protein is determined by the Kjeldahl method, and the preparation effect of the nitrogen resource recycling single cell protein is characterized based on this data; the test results and evaluation criteria are as follows:

[0142] Θ: 60 < crude protein content (considered to have significant production effect);

[0143] Ο: 30<crude protein content<60 (considered as general production effect);

[0144] X: Crude protein content value <30 (considered as low production effect).

[0145] Microbial contamination content:

[0146] First, the mixed gas before and after the reaction in the embodiment is sampled, and the changes in the content of methane and carbon dioxide in the biogas before the reaction are detected by the plate counting method. A nutrient agar medium is first prepared, and then the single-cell protein sample is diluted, and the sample solution of different dilutions is spread on the culture medium plate, and the plate is placed at a temperature of 37° C. and cultured for 24 hours. The number of colonies on the plate is counted, and the microbial content in the sample is calculated according to the dilution multiple, and the microbial contamination content in the process of recycling single-cell protein in nitrogen resources is characterized according to this data; the test results and evaluation criteria are as follows:

[0147] Θ: Microbial contamination content <1000 CFU / g (considered to be basically free of contamination);

[0148] Ο: 1000CFU / g<microbial contamination content<5000CFU / g (considered as a normal contamination level);

[0149] X: 5000 CFU / g < microbial contamination content (considered as a high degree of contamination).

[0150] It should be specifically noted that the nitrogen resource recycling single-cell protein obtained above is the nitrogen resource recycling single-cell protein produced in a normal production mode, and the defective nitrogen resource recycling single-cell protein produced has its data discarded.

[0151] Examples 1-5

[0152] A single-cell protein for recycling nitrogen resources and the corresponding relationship between the preparation methods used are shown in the following table.

[0153] Table: Comparison of the use of single cell protein in nitrogen resource recycling in Examples 1-5

[0154] Group Nitrogen resource recycling and utilization by single cell protein Example 1 Prepared from Preparation Example 16 Example 2 Prepared from Preparation Example 17 Example 3 Prepared from Preparation Example 18 Example 4 Prepared from Preparation Example 19 Example 5 Prepared from Preparation Example 20

[0155] The single cell proteins for nitrogen resource recycling in the above Examples 1-5 were extracted, and their crude protein content and microbial contamination content were tested according to the above measurement steps and measurement standards. The average values ​​of the test results were recorded in the following table.

[0156] Table: Performance test results of crude protein content and microbial contamination content of Examples 1-5

[0157]

[0158]

[0159] As can be seen from the above table, the nitrogen resource recycling and single-cell protein preparation processes in Examples 1-7 all have a good effect of improving the nitrogen resource recycling and single-cell protein production effect. By using Methylomonas methanotropha, methane, carbon monoxide, and carbon dioxide are used as carbon sources. These gases are gradually converted into cell substances during the metabolism of microorganisms, providing a carbon skeleton for the synthesis of single-cell proteins; at the same time, Paracoccus denitrifyingi uses oxygen as an electron acceptor to remove nitrogen from the water body by reducing nitrite, converting nitrogen in the ammonia-containing wastewater in the system into nitrogen-containing compounds, which participate in the metabolic process of cells, thereby achieving the purpose of improving nitrogen resource recycling and promoting single-cell protein production;

[0160] Its crude protein content is 63.1-67.4%, which is considered to have significant production effect; its microbial contamination content is 575-592 CFU / g, which is considered to be basically pollution-free;

[0161] It can be seen that when the production raw materials are certain, the production effect of nitrogen resource recycling single-cell protein can be increased by adjusting the preparation ratio. Combined with the data in the above table, it is not difficult to see that when the volume ratio of Methanometha methylmonas and Denitrifying Paracoccus is 2:1, the production effect of nitrogen resource recycling single-cell protein is the highest; the reason for this is that Methanometha methylmonas is dominant at this ratio and can make full use of abundant methane gas as a carbon source; Methanometha methylmonas will produce a variety of intermediate metabolites in the process of methane metabolism, and these products can provide part of the carbon source and energy for Denitrifying Paracoccus. In this process, Denitrifying Paracoccus effectively utilizes ammonia-containing wastewater and integrates the nitrogen source into the cell protein. The two bacteria have a good synergistic effect and promote the growth and protein synthesis of the overall cell. From Examples 1-7, Examples 6-11 are obtained.

[0162] A single-cell protein for recycling nitrogen resources and the corresponding relationship between the preparation methods used are shown in the following table.

[0163] Table: Comparison of the use of single cell protein in nitrogen resource recycling in Examples 6-11

[0164] Group Nitrogen resource recycling and utilization by single cell protein Example 6 Prepared from Preparation Example 21 Example 7 Prepared from Preparation Example 22 Example 8 Prepared from Preparation Example 23 Example 9 Prepared from Preparation Example 24 Example 10 Prepared from Preparation Example 25 Embodiment 11 Prepared from Preparation Example 26

[0165] The single cell proteins for nitrogen resource recycling in the above Examples 6-11 were extracted, and their crude protein content and microbial contamination content were tested according to the above measurement steps and measurement standards. The average values ​​of the test results were recorded in the following table.

[0166] Table: Performance test results of crude protein content and microbial contamination content of Examples 1, 6-11

[0167]

[0168] As can be seen from the above table, the nitrogen resource recycling and single-cell protein preparation processes in Examples 1 and 6-11 all have a good effect of improving the nitrogen resource recycling and single-cell protein production effect. By using Methylomonas methanotropha, methane, carbon monoxide, and carbon dioxide are used as carbon sources. These gases are gradually converted into cell substances during the metabolism of microorganisms, providing a carbon skeleton for the synthesis of single-cell proteins; at the same time, Paracoccus denitrificans uses oxygen as an electron acceptor to remove nitrogen from the water body by reducing nitrite, converting nitrogen in the ammonia-containing wastewater in the system into nitrogen-containing compounds, which participate in the metabolic process of cells, thereby achieving the purpose of improving nitrogen resource recycling and promoting single-cell protein production;

[0169] Its crude protein content is 65.1-67.5%, which is considered to have significant production effect; its microbial contamination content is 512-659 CFU / g, which is considered to be basically pollution-free;

[0170] It can be seen that when the production raw materials are certain, the production conditions can be adjusted to increase the production effect of single-cell protein by recycling nitrogen resources. Combined with the data in the above table, it is not difficult to see that when the inoculation rate is increased, the production effect of single-cell protein by recycling nitrogen resources is improved, but the degree of microbial contamination becomes higher. When the culture temperature, stirring speed, pH, and the content of methane, carbon monoxide, and carbon dioxide mixed gas are increased, the production effect of single-cell protein by recycling nitrogen resources can be effectively improved, and the degree of microbial contamination can be reduced. The reason for this is that more microorganisms can use carbon and nitrogen sources more quickly for metabolic activities and synthesize single-cell proteins, which improves the production effect to a certain extent. However, high inoculation rate liquid will introduce more types of microorganisms, increasing the degree of contamination. Increasing the culture temperature within an appropriate range can accelerate the growth of microorganisms. metabolic rate, promote cell growth and protein synthesis; stirring can make the fermentation liquid mix evenly, ensuring that the microorganisms can fully contact the nutrients; adjusting the pH to an appropriate value can make the metabolic activity of the microorganisms in the best state and maximize the activity of the enzyme; increasing the content of the mixed gas can provide more energy and material basis for the microorganisms; raising the culture temperature; good stirring can reduce local dead corners in the fermentation liquid and reduce the possibility of bacteria breeding; to a certain extent, it can inhibit the growth of some heat-sensitive bacteria; adjusting the pH to a range suitable for the growth of target bacteria but not suitable for the growth of bacteria can effectively inhibit the reproduction of bacteria and reduce the degree of microbial contamination; increasing the content of the introduced gas can make the target bacteria grow rapidly and occupy a dominant position, thereby inhibiting the growth of bacteria, as obtained by Examples 1, 6-11.

[0171] Examples 12-18

[0172] A single-cell protein for recycling nitrogen resources and the corresponding relationship between the preparation methods used are shown in the following table.

[0173] Table: Comparison of the use of single cell protein in nitrogen resource recycling in Examples 12-18

[0174] Group Nitrogen resource recycling and utilization by single cell protein Example 12 Prepared from Preparation Example 27 Embodiment 13 Prepared from Preparation Example 28 Embodiment 14 Prepared from Preparation Example 29 Embodiment 15 Prepared from Preparation Example 30 Example 16 Prepared from Preparation Example 31 Embodiment 17 Prepared from Preparation Example 32 Embodiment 18 Prepared from Preparation Example 33

[0175] The single cell proteins for nitrogen resource recycling in the above Examples 12-18 were extracted, and their crude protein content and microbial contamination content were tested according to the above measurement steps and measurement standards. The average values ​​of the test results were recorded in the following table.

[0176] Table: Performance test results of crude protein content and microbial contamination content in Examples 1, 12-18

[0177]

[0178]

[0179] As can be seen from the above table, the nitrogen resource recycling and single-cell protein preparation processes in Examples 1 and 12-18 all have a good effect of improving the nitrogen resource recycling and single-cell protein production effect. By using Methylomonas methanotropha, methane, carbon monoxide, and carbon dioxide are used as carbon sources. These gases are gradually converted into cell substances during the metabolism of microorganisms, providing a carbon skeleton for the synthesis of single-cell proteins; at the same time, Paracoccus denitrifyingi uses oxygen as an electron acceptor to remove nitrogen from the water body by reducing nitrite, converting nitrogen in the ammonia-containing wastewater in the system into nitrogen-containing compounds, which participate in the metabolic process of cells, thereby achieving the purpose of improving nitrogen resource recycling and promoting single-cell protein production;

[0180] Its crude protein content is 63.1-67.8%, which is considered to have significant production effect; its microbial contamination content is 581-742 CFU / g, which is considered to be basically pollution-free;

[0181] It can be seen that when the production raw materials are certain, the production conditions can be adjusted to increase the production effect of single-cell protein by recycling nitrogen resources. It is not difficult to see from the data in the above table that when cultivating single-cell protein by recycling nitrogen resources, when the fermentation medium is composed of basal culture medium and nitrogen-containing wastewater in a volume ratio of 7:3, the crude protein content of the single-cell protein produced is the highest, but its pollution degree is also the highest. The reason for this is that at this ratio, the basal culture medium is relatively more, which can provide more sufficient nutrients and a suitable growth environment for microorganisms; a certain proportion of nitrogen-containing wastewater also ensures the supply of nitrogen source, and a relatively high proportion of basal culture medium helps to maintain the stability of the fermentation broth, so that the microorganisms can carry out metabolic activities more efficiently, thereby synthesizing more single-cell protein and improving the production effect; a higher proportion of basal culture medium usually contains richer nutrients, which can support the rapid growth and reproduction of the target strain, so that it occupies a dominant position in the fermentation broth, thereby inhibiting the growth of miscellaneous bacteria, as obtained by Examples 1, 12-13;

[0182] When the production raw materials are certain, the production conditions can be adjusted to increase the production effect of single-cell protein by recycling nitrogen resources. It is not difficult to see from the data in the above table that when the amount of ammonia nitrogen in the nitrogen-containing wastewater is increased, the production effect of single-cell protein by recycling nitrogen resources is improved, but the degree of microbial contamination becomes higher. The reason for this is that sufficient nitrogen sources can promote the growth and reproduction of microorganisms and accelerate the synthesis of single-cell protein, thereby improving the production effect; a higher amount of ammonia nitrogen not only provides more nitrogen sources for the target production strains, but also creates favorable conditions for the growth of other miscellaneous bacteria, as shown in Examples 1 and 14;

[0183] When the production raw materials are certain, the production conditions can be adjusted to increase the production effect of single-cell protein by recycling nitrogen resources. It is not difficult to see from the data in the above table that when the basic culture medium is composed of potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, ferrous sulfate, boric acid, copper sulfate, sodium molybdate, zinc chloride, manganese chloride, and water in a weight ratio of 2.5: 0.25: 0.09: 0.025: 0.09: 0.025; 0.025: 0.09: 0.09: 1000, the crude protein content of the produced single-cell protein is the highest. The reason for this is that the content of potassium dihydrogen phosphate is relatively high, which can provide sufficient phosphorus source for microbial growth and promote cell metabolism and protein synthesis, which is obtained from Examples 1 and 15-16;

[0184] When the production raw materials are certain, the production conditions can be adjusted to increase the production effect of single-cell protein by recycling nitrogen resources. It is not difficult to see from the data in the above table that when the mixed gas of methane, carbon monoxide and carbon dioxide is composed of methane, carbon monoxide and carbon dioxide in a volume ratio of 7:1.5:1.5, the crude protein content of the produced single-cell protein is the highest. The reason for this is that the ratio is relatively balanced, and methane accounts for a high proportion as the main carbon source, which meets the carbon source requirements of the strain. At the same time, the ratio of carbon monoxide and carbon dioxide is moderate, which can provide the strain with a diverse carbon source structure, which helps the strain to maintain a good growth and protein synthesis state during the metabolic process, as obtained from Examples 1 and 17-18.

[0185] Examples 19-24

[0186] A single-cell protein for recycling nitrogen resources and the corresponding relationship between the preparation methods used are shown in the following table.

[0187] Table: Comparison of the use of single cell protein in nitrogen resource recycling in Examples 19-24

[0188]

[0189]

[0190] The single-cell proteins for nitrogen resource recycling in the above Examples 19-24 were extracted, and their crude protein content and microbial contamination content were tested according to the above measurement steps and measurement standards. The average values ​​of the test results were recorded in the following table.

[0191] Table: Performance test results of crude protein content and microbial contamination content of Examples 1, 19-24

[0192]

[0193] As can be seen from the above table, the nitrogen resource recycling single-cell protein preparation process in Examples 1 and 19-24 has a good effect of improving the nitrogen resource recycling single-cell protein production effect. By using Methylomonas methanotrophicola, methane, carbon monoxide, and carbon dioxide are used as carbon sources. These gases are gradually converted into cell substances during the metabolism of microorganisms, providing a carbon skeleton for the synthesis of single-cell proteins; at the same time, Paracoccus denitrifyingi uses oxygen as an electron acceptor to remove nitrogen from the water body by reducing nitrite, converting nitrogen in the ammonia-containing wastewater in the system into nitrogen-containing compounds, and participating in the metabolic process of cells, thereby achieving the purpose of improving nitrogen resource recycling and promoting single-cell protein production;

[0194] Its crude protein content is 62.1-67.4%, which is considered to have significant production effect; its microbial contamination content is 511-764 CFU / g, which is considered to be basically pollution-free;

[0195] It can be seen that when the production raw materials are certain, the preparation conditions of the methylmonas methanotrophic bacteria solution can be adjusted to increase the production effect of the nitrogen resource recycling single-cell protein. Combined with the data in the above table, it is not difficult to see that when the temperature, rotation speed, pH and culture time when activating methylmonas are increased, the production effect of the nitrogen resource recycling single-cell protein can be effectively improved and the degree of microbial contamination can be reduced. The reason is that appropriately increasing the temperature can accelerate the metabolic rate of microorganisms; higher temperatures can inhibit the growth of heat-sensitive bacteria; increasing the rotation speed can fully mix the culture solution to ensure that the microorganisms are evenly exposed to nutrients; good stirring can reduce local dead corners and reduce the possibility of bacteria breeding; adjusting the pH to a range suitable for the growth of methylmonas can keep the metabolic activity of the microorganisms in the best state and maximize the activity of the enzyme; it can also selectively promote the growth of methylmonas and inhibit the reproduction of bacteria, thereby reducing the degree of microbial contamination; appropriately extending the culture time can allow the microorganisms to have more sufficient time to grow and metabolize; as the culture time increases, the target strain methylmonas may gradually occupy a dominant position, and inhibit the growth of bacteria by competing for nutrients and living space, as shown in Examples 1, 19-20;

[0196] It can be seen that when the production raw materials are certain, the preparation conditions of the Methane Methylomonas liquid can be adjusted to increase the production effect of the single-cell protein by recycling nitrogen resources. It is not difficult to see from the data in the above table that when the amount of ammonia nitrogen in the nitrogen-containing wastewater is increased, the production effect of the single-cell protein by recycling nitrogen resources is improved, but the degree of microbial contamination becomes higher. The reason for this is that sufficient nitrogen sources can promote the growth and reproduction of microorganisms and accelerate the synthesis of single-cell proteins, thereby improving the production effect; a higher amount of ammonia nitrogen not only provides more nitrogen sources for the target production strains, but also creates favorable conditions for the growth of other miscellaneous bacteria, as obtained from Examples 1 and 21;

[0197] It can be seen that when the production raw materials are constant, the production effect of single-cell protein recycled by nitrogen resources can be increased by adjusting the preparation conditions of the Methane Methylomonas liquid. It is not difficult to see from the data in the above table that when the content of the mixed gas of methane, carbon monoxide and carbon dioxide is increased, the production effect of single-cell protein recycled by nitrogen resources can be effectively improved. The reason for this is that increasing the content of the mixed gas can provide more energy and material basis for microorganisms, which is obtained from Examples 1 and 22;

[0198] It can be seen that when the production raw materials are certain, the preparation conditions of the Methane Methylomonas liquid can be adjusted to increase the production effect of the single-cell protein by recycling nitrogen resources. It is not difficult to see from the data in the above table that when the amount of nitrogen wastewater added is increased, the production effect decreases and the degree of pollution increases. The reason is that although the nitrogen-containing wastewater provides a nitrogen source, if the amount added is too much, the proportion of the culture medium is reduced, which will break the balance of the carbon-nitrogen ratio in the fermentation medium. The addition of high-nitrogen wastewater will also introduce more miscellaneous bacteria, which is obtained from Examples 1 and 23;

[0199] It can be seen that when the production raw materials are certain, the preparation conditions of the Methane Methylomonas solution can be adjusted to increase the production effect of single-cell protein by recycling nitrogen resources. Combined with the data in the above table, it is not difficult to see that when the OD600 value of the fixed bacterial solution is increased, the production effect of single-cell protein by recycling nitrogen resources can be effectively improved, but the degree of microbial contamination will increase. The reason for this is that when the OD600 value is increased, it indicates that the number of microbial cells in the bacterial solution increases, and more microorganisms are involved in the process of utilizing the carbon source, so that metabolic activities can be carried out more efficiently and single-cell protein can be synthesized, which is obtained from Examples 1 and 24.

[0200] Examples 25-28

[0201] A single-cell protein for recycling nitrogen resources and the corresponding relationship between the preparation methods used are shown in the following table.

[0202] Table: Comparison of the use of single cell protein in nitrogen resource recycling in Examples 25-28

[0203]

[0204]

[0205] The single-cell proteins for nitrogen resource recycling in the above Examples 25-28 were extracted, and their crude protein content and microbial contamination content were tested according to the above measurement steps and measurement standards. The average values ​​of the test results were recorded in the following table.

[0206] Table: Performance test results of crude protein content and microbial contamination content of Examples 1, 25-28

[0207]

[0208] As can be seen from the above table, the nitrogen resource recycling single-cell protein preparation process in Examples 1 and 25-28 has a good effect of improving the nitrogen resource recycling single-cell protein production effect. By using Methylomonas methanotrophicola, methane, carbon monoxide, and carbon dioxide are used as carbon sources. These gases are gradually converted into cell substances during the metabolism of microorganisms, providing a carbon skeleton for the synthesis of single-cell proteins; at the same time, Paracoccus denitrifyingi uses oxygen as an electron acceptor to remove nitrogen from the water body by reducing nitrite, converting nitrogen in the ammonia-containing wastewater in the system into nitrogen-containing compounds, and participating in the metabolic process of cells, thereby achieving the purpose of improving nitrogen resource recycling and promoting single-cell protein production;

[0209] Its crude protein content is 62.1-66.1%, which is considered to have significant production effect; its microbial contamination content is 581-586 CFU / g, which is considered to be basically pollution-free;

[0210] It can be seen that when the production raw materials are certain, the production effect of single-cell protein by recycling nitrogen resources can be increased by adjusting the preparation conditions of the Methanogenomyosin liquid. It is not difficult to see from the data in the above table that when the basic culture medium is composed of potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, ferrous sulfate, boric acid, copper sulfate, sodium molybdate, zinc chloride, manganese chloride, and water in a weight ratio of 2.5: 0.25: 0.09: 0.025: 0.09: 0.025; 0.025: 0.09: 0.09: 1000, the crude protein content of the produced single-cell protein is the highest, which is obtained from Examples 1, 25-27;

[0211] It can be seen that when the production raw materials are certain, the preparation conditions of the Methylomonas methylotrophic bacteria liquid can be adjusted to increase the production effect of single-cell protein by recycling nitrogen resources. Combined with the data in the above table, it is not difficult to see that when the mixed gas of methane, carbon monoxide and carbon dioxide is composed of methane, carbon monoxide and carbon dioxide in a volume ratio of 7:1.5:1.5, the crude protein content of the produced single-cell protein is the highest, which is obtained from Examples 1 and 28.

[0212] Examples 29-31

[0213] A single-cell protein for recycling nitrogen resources and the corresponding relationship between the preparation methods used are shown in the following table.

[0214] Table: Comparison of the use of single cell protein in nitrogen resource recycling in Examples 29-31

[0215] Group Nitrogen resource recycling and utilization by single cell protein Embodiment 29 Prepared from Preparation Example 44 Embodiment 30 Prepared from Preparation Example 45 Embodiment 31 Prepared from Preparation Example 46

[0216] The single-cell proteins for nitrogen resource recycling in the above Examples 29-31 were extracted, and their crude protein content and microbial contamination content were tested according to the above measurement steps and measurement standards. The average values ​​of the test results were recorded in the following table.

[0217] Table: Performance test results of crude protein content and microbial contamination content of Examples 1, 29-31

[0218]

[0219]

[0220] As can be seen from the above table, the nitrogen resource recycling single-cell protein preparation process in Examples 1 and 29-31 has a good effect of improving the nitrogen resource recycling single-cell protein production effect. By using Methylomonas methanotrophicola, methane, carbon monoxide, and carbon dioxide are used as carbon sources. These gases are gradually converted into cell substances during the metabolism of microorganisms, providing a carbon skeleton for the synthesis of single-cell proteins; at the same time, Paracoccus denitrifyingi uses oxygen as an electron acceptor to remove nitrogen from the water body by reducing nitrite, converting nitrogen in the ammonia-containing wastewater in the system into nitrogen-containing compounds, and participating in the metabolic process of cells, thereby achieving the purpose of improving nitrogen resource recycling and promoting single-cell protein production;

[0221] Its crude protein content is 65.1-67.2%, which is considered to have significant production effect; its microbial contamination content is 579-594 CFU / g, which is considered to be basically pollution-free;

[0222] It can be seen that when the production raw materials are certain, the production effect of single-cell protein by recycling nitrogen resources can be increased by adjusting the preparation conditions of denitrifying Paracoccus liquid. It is not difficult to see from the data in the above table that when the culture conditions are 30°C and PH7 for 1.5 days, the crude protein content of the produced single-cell protein is the highest, which is obtained from Examples 1 and 29-30;

[0223] It can be seen that when the production raw materials are certain, the preparation conditions of denitrifying Paracoccus liquid can be adjusted to increase the production effect of single-cell protein by recycling nitrogen resources. Combined with the data in the above table, it is not difficult to see that when the nitrogen-containing culture medium contains magnesium sulfate and ferrous sulfate, the produced single-cell protein has a higher crude protein content. The reason for this is that in the single-cell protein production process, magnesium ions can activate enzymes related to nitrogen metabolism and carbon metabolism, and ferrous ions play a role in the electron transport chain in the process of cellular respiration, which is obtained from Examples 1 and 31.

[0224] This specific embodiment is only an explanation of the present invention, not a limitation of the present invention. After reading this specification, those skilled in the art can make non-creative modifications to this embodiment as needed, but as long as it is within the scope of the claims of the present invention, it is protected by the patent law.

Claims

1. A single cell protein formula for recycling nitrogen resources, characterized in that: The mixture of Methylomonas and Paracoccus denitrificans is inoculated into a fermentation medium for fermentation, during which a mixed gas of methane, carbon monoxide and carbon dioxide is introduced; The components and volume ratios of the single-cell protein formula for recycling nitrogen resources are as follows: 1-3 parts of Methylomonas methanotrophicola solution and 1-3 parts of Paracoccus denitrificans solution; The fermentation medium is composed of a basic medium and nitrogen-containing wastewater in a volume ratio of (1-7): (1-3); The methane, carbon monoxide and carbon dioxide mixed gas is composed of methane, carbon monoxide and carbon dioxide in a volume ratio of (6.5-7.5):(1.2-1.8):(1.3-1.7).

2. A single cell protein formula for recycling nitrogen resources according to claim 1, characterized in that: The method for preparing single-cell protein for recycling nitrogen resources comprises the following steps: A1. First, a methylomonas solution and a denitrifying Paracoccus solution are mixed, and the mixture is inoculated into a fermenter containing a sterilized fermentation medium at a volume ratio of 5-10%, and the mixture is fermented for 48-72 hours at a temperature of 30-35° C., a stirring speed of 200-300 r / min, and a pH of 7-7.5, during which a mixed gas of methane, carbon monoxide, and carbon dioxide is continuously introduced at a rate of 0.3-0.5 vvm, and nitrogen-containing wastewater is automatically added during the fermentation process to keep the nitrogen source concentration in the fermentation liquid stable, thereby obtaining a fermentation liquid; A2. Pour the fermentation liquid obtained in A1 into a centrifuge and centrifuge at a speed of 4000 r / min to obtain the precipitate, then wash the precipitate twice with sterile saline, and then vacuum dry the washed precipitate at a temperature of 60° C. for 12 hours to obtain single-cell protein.

3. A single cell protein formula for recycling nitrogen resources according to claim 1, characterized in that: The preparation steps of the Methylomonas solution are as follows: C1. In a sterile environment, inoculate the strain of Methanometazobacterium into a shake flask filled with a basal culture medium, and culture for 48 hours at a temperature of 28° C., a rotation speed of 150-200 r / min, and a pH of 6.5-7.5 to obtain an activated Methanometazobacterium liquid; C2. Pour nitrogen-containing wastewater containing 500-1000 mg / L of ammonia nitrogen into the activated Methanomethamonas liquid obtained in C1, the wastewater is added at a ratio of 5% of the volume of the culture medium, and at the same time, 0.1-0.3 vvm of a mixed gas containing methane, carbon monoxide and carbon dioxide is introduced, and the culture is carried out under the same conditions as C1; the amount of nitrogen-containing wastewater added is increased by 5-10% every 24 hours until the amount of nitrogen-containing wastewater added reaches 50-70% of the volume of the culture medium, and the addition of nitrogen-containing wastewater is stopped. During this period, the OD600 value of the bacterial liquid is continuously measured. When the OD600 value reaches 0.8-1.2, the culture is stopped to obtain the Methanomethamonas liquid.

4. A single cell protein formula for recycling nitrogen resources according to claim 1, characterized in that: The preparation steps of the Paracoccus denitrificans liquid are as follows: B1. Inoculate the denitrifying Paracoccus strain into the sterilized culture medium at a content of 5% of the volume of the nitrogen-containing culture medium, and culture the nitrogen-containing culture medium in a constant temperature incubator at a temperature of 28-32° C. and a pH of 6.8-7.2 for 1-2 days to obtain a denitrifying Paracoccus liquid.

5. A single cell protein formula for recycling nitrogen resources according to claim 2, characterized in that: The basic culture medium is composed of potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, ferrous sulfate, boric acid, copper sulfate, sodium molybdate, zinc chloride, manganese chloride and water in a weight ratio of (1.5-2.5): (0.25-0.4): (0.06-0.09): (0.015-0.025): (0.06-0.09): (0.015-0.025); (0.015-0.025): (0.06-0.09): (0.06-0.09): 1000.

6. A single cell protein formula for recycling nitrogen resources according to claim 4, characterized in that: The nitrogen-containing culture medium is composed of glucose, ammonium chloride, sodium bicarbonate, magnesium sulfate and ferrous sulfate in a weight ratio of 5:0.5:0.1:(0-0.2):(0-0.01):1000.

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