阅读说明：本技术 一种微生物发酵生产单细胞蛋白的方法 (Method for producing single-cell protein by microbial fermentation ) 是由 王雯 牛子津 杨紫怡 于 2021-01-11 设计创作，主要内容包括：本发明公开了一种微生物发酵生产单细胞蛋白的方法,包括如下步骤：向装有驯化培养基的反应器中通入混合气,得到驯化体系,用于对接种物进行培养和驯化；向装有发酵培养基的反应器中通入混合气,得到混合发酵体系,用于对驯化微生物进行发酵培养；将厌氧微生物和好氧微生物接种到驯化体系中,进行培养和驯化,得到驯化微生物；将驯化微生物接种到混合发酵体系中,进行发酵培养；将发酵产物进行处理,得到单细胞蛋白。本发明相较于单一底物发酵体系,同时供应混合气和有机碳源的双底物发酵体系更有助于微生物转化氨氮,生产单细胞蛋白。(The invention discloses a method for producing single-cell protein by microbial fermentation, which comprises the following steps: introducing mixed gas into a reactor filled with a domestication culture medium to obtain a domestication system for culturing and domesticating the inoculum; introducing mixed gas into a reactor filled with a fermentation medium to obtain a mixed fermentation system for carrying out fermentation culture on the domesticated microorganisms; inoculating anaerobic microorganisms and aerobic microorganisms into a domestication system, and culturing and domesticating to obtain domesticated microorganisms; inoculating the domesticated microorganism into a mixed fermentation system for fermentation culture; and (4) processing the fermentation product to obtain the single-cell protein. Compared with a single substrate fermentation system, the double-substrate fermentation system for simultaneously supplying the mixed gas and the organic carbon source is more beneficial to the conversion of ammonia nitrogen by microorganisms and the production of single-cell protein.)

1. A method for producing single-cell protein by microbial fermentation is characterized by comprising the following steps:

s1, introducing mixed gas into the reactor filled with the domestication culture medium to obtain a domestication system for culturing and domesticating the inoculum;

s2, introducing mixed gas into the reactor filled with the fermentation medium to obtain a mixed fermentation system for carrying out fermentation culture on the domesticated microorganisms;

s3, inoculating the anaerobic microorganisms and the aerobic microorganisms into a domestication system, and culturing and domesticating to obtain domesticated microorganisms;

s4, inoculating the domesticated microorganisms into a mixed fermentation system for fermentation culture;

and S5, processing the fermentation product to obtain the single cell protein.

2. The method for producing single-cell protein by microbial fermentation according to claim 1, wherein: in step S1, the preparation of the acclimatization medium includes the following steps:

s1-1, preparing a basic culture medium

The basic culture medium comprises the following substances in per liter of solution: 2.3g of monopotassium phosphate, 2.9g of disodium hydrogen phosphate, 1g of ammonium chloride, 0.5g of magnesium sulfate, 0.5g of sodium carbonate, 0.01g of calcium chloride, 0.05g of ferric ammonium citrate, 0.6mg of boric acid, 0.4mg of cobalt chloride, 0.2mg of zinc sulfate, 0.06mg of manganese chloride, 0.06mg of sodium molybdate, 0.04mg of nickel chloride and 0.02mg of copper sulfate;

s1-2, sterilizing the basic culture medium at the temperature of 120-125 ℃ for 15-30min, cooling to room temperature, and adding 15g/L agar to obtain the acclimatized culture medium.

3. The method for producing single-cell protein by microbial fermentation according to claim 1, wherein: in step S1, H in the mixed gas₂And O₂The components are variable, and the volume composition of the mixed gas is H₂：O₂：CO₂＝50:35:15～70:15:15。

4. The method for producing single-cell protein by microbial fermentation according to claim 1, wherein: in step S2, the preparation of the fermentation medium includes the following steps:

s2-1, preparing a basic culture medium

The basic culture medium comprises the following substances in per liter of solution: 2.3g of monopotassium phosphate, 2.9g of disodium hydrogen phosphate, 1g of ammonium chloride, 0.5g of magnesium sulfate, 0.5g of sodium carbonate, 0.01g of calcium chloride, 0.05g of ferric ammonium citrate, 0.6mg of boric acid, 0.4mg of cobalt chloride, 0.2mg of zinc sulfate, 0.06mg of manganese chloride, 0.06mg of sodium molybdate, 0.04mg of nickel chloride and 0.02mg of copper sulfate;

s2-2, sterilizing the basic culture medium at the temperature of 120-125 ℃ for 15-30min, cooling to room temperature, adding an exogenous organic carbon source and a nitrogen source, and uniformly mixing to obtain the fermentation culture medium.

5. The method for producing single-cell protein by microbial fermentation according to claim 4, wherein: in the step S2-2, the exogenous organic carbon source is glucose, and the addition amount of the glucose is 1-100 g/L;

preferably, in step S2-2, the nitrogen source is NH₄Cl, addition of NH₄Cl the initial nitrogen content of the medium was controlled at 100-10000 mg/L.

6. The method for producing single-cell protein by microbial fermentation according to claim 1, wherein: in step S2, H in the mixed gas₂And O₂The components are variable, and the volume composition of the mixed gas is H₂：O₂：CO₂＝50:35:15～70:15:15。

7. The method for producing single-cell protein by microbial fermentation according to claim 1, wherein: in step S3, the culturing and acclimating are performed in a rocker reactor under conditions of a temperature of 25 to 35 ℃, a pH of 6.0 to 8.0, and an oscillation rate of 150 ± 50 rpm;

preferably, the volume ratio of the liquid-phase matrix to the headspace of the rocking bed reactor in the rocking bed reactor is 200: 415-300: 315;

preferably, in step S3, the volume ratio of the inoculum (anaerobic microorganism and aerobic microorganism) to the usage amount of the acclimatization medium is 50: 250-50: 150;

preferably, in step S3, the acclimatization system is supplemented with mixed gas to 0.1-5atm every 12-24 hours;

preferably, in step S3, the volume ratio of the anaerobic microorganism and the aerobic microorganism inoculation amount is 25: 25;

preferably, in step S3, the conditions for determining the completion of acclimatization are as follows: the daily consumption of the mixed gas in the acclimatization system is more than 60%.

8. The method for producing single-cell protein by microbial fermentation according to claim 1, wherein: in step S4, the fermentation culture is performed in a rocker reactor under the conditions of a temperature of 25-35 ℃, a pH of 6.0-8.0, and an oscillation rate of 150 ± 50 rpm;

preferably, the volume ratio of the liquid phase matrix to the reactor headspace in the swing bed reactor is 100: 515-250: 365;

preferably, in step S4, the volume ratio of the domesticated microorganism to the amount of the fermentation medium in the mixed fermentation system is 40: 210-40: 60;

preferably, in step S4, the mixed gas is supplemented to the mixed fermentation system to 0.1-5atm every 12-24 hours.

9. The method for producing single-cell protein by microbial fermentation according to claim 1, wherein: in step S5, the process includes the steps of:

s5-1, centrifuging the fermentation product at 9000-11000rpm for 10-15min, and discarding the supernatant;

s5-2, washing the centrifuged fermentation product with deionized water, centrifuging again, and repeating for 1-3 times;

s5-3, baking the fermentation product after multiple times of centrifugal cleaning at 103-108 ℃ for 20-28 hours to obtain the single cell protein.

10. The method for producing single-cell protein by microbial fermentation according to claim 1, wherein: in step S5, the standard for single-cell protein formation is one or more of the following methods: 1) the conversion amount of ammonia nitrogen in the culture medium is more than 40 percent; 2) the crude protein content in the product is more than 30%.

Technical Field

The invention relates to a method for producing single-cell protein. More particularly, it relates to a method for producing single-cell protein by microbial fermentation.

Background

In 2050, the world population is expected to increase to 90-100 billion, and the food demand will increase by 30-60% at that time, and with the improvement of living standard, people will have more and more additional demands for high-protein food. Nitrogen in nature is mainly N₂In the form of (1), in the existing nitrogen cycle, N₂Mainly converted into active nitrogen by the ways of thunder, microorganism fixation, Haber Bosch process and the likeAnd then the active nitrogen is further fixed by crop planting. Part of nitrogen fixed by crop planting is processed into plant protein products required by people; the other part is converted into animal protein through livestock breeding, and then the animal protein is subjected to a series of processing to finally become an animal protein product required by people. Recent studies predict that the efficiency of existing nitrogen cycles is only 16%. The huge loss is due to the defects of the traditional planting method on one hand; on the other hand, high ammonia nitrogen waste is generated in each nitrogen circulation process, and active nitrogen in the waste is finally converted into N in the conventional treatment mode₂Is returned to the atmosphere, causing a substantial loss of active nitrogen. Today, the protein supply centered on crop planting is saturated and it is crucial to find a new protein supply.

The single-cell protein refers to stem cells of microorganisms such as yeast, mold, bacteria and algae, which can be used as food and animal feed for human beings. The single cell protein has high crude protein content, rich amino acid types, higher nutritive value than plant protein, and is similar to animal protein. Compared with the existing protein supply chain, the protein supply chain taking the microorganism as the core has wider development prospect, which is specifically shown as follows: 1) the raw material source is wide: industrial sewage, beer waste liquid, waste beet pulp, pomace, corn straw and other wastes can be used as raw materials for producing single-cell protein, the environment burden can be reduced by using the wastes as substrates, the high-efficiency recovery of resources can be realized, and new value is created; 2) high production efficiency and large yield: the large-scale production can be realized by utilizing the existing fermentation technology, and the microorganism is fast to propagate, the production efficiency is high and the yield is stable; 3) the suitable condition range is wide: most of microorganisms capable of synthesizing protein can grow and propagate at normal temperature, are less influenced by climate and seasons, and can realize all-weather production all the year round; 4) the floor area is small, and the equipment is simple and easy to control: the production of single cell protein is carried out in a completely controllable and closed automatic bioreactor, which not only gets rid of the dependence on land and climate, but also the production process is easier to control, and the utilization rate of active nitrogen can almost reach 100%.

In view of the foregoing, it would be desirable to provide a method for efficiently producing single-cell proteins by microbial fermentation, which utilizes the fermentation of microorganisms to convert active nitrogen into high-protein products.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for efficiently producing single-cell protein by microbial fermentation. The method couples the mixed gas with a specific culture medium to form a specific acclimation system and a mixed fermentation system. The domestication system can culture and domesticate the inoculum to obtain a mixed flora with the ability of producing the protein; the mixed fermentation system is used for carrying out fermentation culture on domesticated microorganisms, and active nitrogen in the system is converted into a high value-added protein product by utilizing the fermentation effect of the microorganisms.

In order to solve the technical problems, the invention adopts the following technical scheme:

a method for producing single-cell protein by microbial fermentation comprises the following steps:

s1, introducing mixed gas into the reactor filled with the domestication culture medium to obtain a domestication system for culturing and domesticating the inoculum;

s2, introducing mixed gas into the reactor filled with the fermentation medium to obtain a mixed fermentation system for carrying out fermentation culture on the domesticated microorganisms;

s3, inoculating the anaerobic microorganisms and the aerobic microorganisms into a domestication system, and culturing and domesticating to obtain domesticated microorganisms;

s4, inoculating the domesticated microorganisms into a mixed fermentation system for fermentation culture;

and S5, processing the fermentation product to obtain the single cell protein.

In the present invention, the term "anaerobic microorganism" refers to a mixed microorganism capable of normally performing anaerobic fermentation and producing biogas. The microorganism is mainly from the discharge of the anaerobic fermentation tank, and the substrate of the fermentation tank is mainly livestock and poultry manure.

In the present invention, the term "aerobic microorganism" means a mixed microorganism capable of normally performing aerobic fermentation. The source of the microorganisms is mainly sludge from municipal sewage treatment plants.

In the present invention, the term "acclimation system" refers to a system capable of culturing and acclimating anaerobic microorganisms and aerobic microorganisms to finally obtain microorganisms capable of producing single-cell proteins, and specifically, a reactor filled with an acclimation medium and filled with mixed gas. The domesticated microorganism has functions including: 1) growing by using mixed gas or an organic carbon source as a substrate; 2) converting the active nitrogen into single-cell protein.

In the present invention, the term "mixed fermentation system" refers to a system capable of maintaining the growth of domesticated microorganisms and converting active nitrogen in the system into single cell protein, and specifically to a reactor filled with a fermentation medium and a mixed gas.

As a further improvement of the technical solution, in step S1, the preparation of the acclimatization medium comprises the following steps:

s1-1, preparing a basic culture medium

The basic culture medium comprises the following substances in per liter of solution: 2.3g of monopotassium phosphate, 2.9g of disodium hydrogen phosphate, 1g of ammonium chloride, 0.5g of magnesium sulfate, 0.5g of sodium carbonate, 0.01g of calcium chloride, 0.05g of ferric ammonium citrate, 0.6mg of boric acid, 0.4mg of cobalt chloride, 0.2mg of zinc sulfate, 0.06mg of manganese chloride, 0.06mg of sodium molybdate, 0.04mg of nickel chloride and 0.02mg of copper sulfate;

s1-2, sterilizing the basic culture medium at the temperature of 120-125 ℃ for 15-30min, cooling to room temperature, and adding 15g/L of agar to obtain the acclimatization culture medium.

Preferably, in step S1, H in the mixed gas₂And O₂The components are variable, and the volume composition of the mixed gas is H₂： O₂：CO₂＝50:35:15～70:15:15。

As a further improvement of the technical scheme, in step S2, the preparation of the fermentation medium comprises the following steps:

s2-1, preparing a basic culture medium

The basic culture medium comprises the following substances in per liter of solution: 2.3g of monopotassium phosphate, 2.9g of disodium hydrogen phosphate, 1g of ammonium chloride, 0.5g of magnesium sulfate, 0.5g of sodium carbonate, 0.01g of calcium chloride, 0.05g of ammonium ferric citrate, 0.6mg of boric acid, 0.4mg of cobalt chloride, 0.2mg of zinc sulfate, 0.06mg of manganese chloride, 0.06mg of sodium molybdate, 0.04mg of nickel chloride and 0.02mg of copper sulfate;

s2-2, sterilizing the basic culture medium at the temperature of 120-125 ℃ for 15-30min, cooling to room temperature, adding an exogenous organic carbon source and a nitrogen source, and uniformly mixing to obtain the fermentation culture medium.

Preferably, in step S2-2, the exogenous organic carbon source is glucose, and the addition amount of glucose is 1-100 g/L;

preferably, in step S2-2, the nitrogen source is NH₄Cl, addition of NH₄Cl the initial nitrogen content of the medium was controlled at 100-10000 mg/L.

Preferably, in step S2, H in the mixed gas₂And O₂The components are variable, and the volume composition of the mixed gas is H₂： O₂：CO₂＝50:35:15～70:15:15。

In a further improvement of the technical solution, in step S3, the culturing and acclimating are carried out in a shaking bed reactor under the reaction conditions of a temperature of 25 to 35 ℃, a pH of 6.0 to 8.0, and a shaking rate of 150 ± 50 rpm.

Preferably, the volume ratio of the liquid phase matrix in the rocker reactor to the headspace of the rocker reactor is 200: 415-300: 315.

Preferably, in step S3, the volume ratio of the inoculum (anaerobic microorganism and aerobic microorganism) to the acclimatization medium usage is 50:250 to 50: 150.

Preferably, in step S3, the acclimatization system is supplemented with mixed gas to 0.1-5atm every 12-24 hours.

Preferably, in step S3, the volume ratio of the anaerobic microorganism and the aerobic microorganism inoculum is 25: 25.

Preferably, in step S3, the conditions for determining the completion of acclimatization are as follows: the daily consumption of the mixed gas in the acclimatization system is more than 60%.

In a further improvement of the technical scheme, in step S4, the fermentation culture is carried out in a shaking bed reactor, the reaction conditions are that the temperature is 25-35 ℃, the pH is 6.0-8.0, and the shaking rate of the shaking bed reactor is 150 +/-50 rpm.

Preferably, the volume ratio of the liquid phase matrix to the reactor headspace in the swing bed reactor is 100: 515-250: 365.

Preferably, in step S4, the volume ratio of the domesticated microorganism to the amount of the fermentation medium in the mixed fermentation system is 40:210 to 40: 60.

Preferably, in step S4, the mixed gas is supplemented to the mixed fermentation system to 0.1-5atm every 12-24 hours.

As a further improvement of the technical solution, in step S5, the processing includes the steps of:

s5-1, centrifuging the fermentation product at 9000-11000rpm for 10-15min, and discarding the supernatant;

s5-2, washing the centrifuged fermentation product with deionized water, centrifuging again, and repeating for 1-3 times;

s5-3, baking the fermentation product after multiple times of centrifugal cleaning at 103-108 ℃ for 20-28 hours to obtain the single cell protein.

Preferably, in step S5, the standard for single-cell protein formation is one or more of the following methods: 1) the conversion amount of ammonia nitrogen in the culture medium is more than 40 percent; 2) the crude protein content in the product is more than 30%.

Any range recited herein is intended to include the endpoints and any number between the endpoints and any subrange subsumed therein or defined therein.

Unless otherwise specified, the raw materials of the present invention are commercially available, and the apparatus used in the present invention may be any apparatus conventionally used in the art or may be any apparatus known in the art.

Compared with the prior art, the invention has the following beneficial effects:

1) the invention designs an acclimatization system for culturing and acclimatizing microorganisms and a mixed fermentation system for fermenting and culturing the acclimatized microorganisms. Wherein, the domestication system is used for culturing and domesticating the inoculum to obtain a mixed flora with the capability of producing protein; the mixed fermentation system is used for carrying out fermentation culture on the domesticated microorganisms, and active nitrogen in the system is converted into a high-protein product by utilizing the fermentation action of the microorganisms.

2) In the study of single-cell protein-producing bacteria, the metabolic characteristics of the bacteria were mostly studied using pure bacteria as a target. However, the aseptic culture using pure bacteria as a core is difficult to be applied to the treatment of actual wastewater due to the reasons of complicated operation, difficulty in maintaining an aseptic environment, difficulty in enlarging the culture scale, and the like. Compared with pure bacteria, the mixed bacteria has stronger buffering capacity to the external environment change, and the operation required by culture is simple, so that large-scale culture is easy to realize. The invention designs a domestication system which can quickly enrich mixed flora with the capability of producing single cell protein.

3) At present, the methods for treating active nitrogen in water body include nitrification-denitrification, anaerobic ammonia oxidation and the like, and the result is that the active nitrogen is converted into N₂And finally returns to the atmosphere, causing a large degree of nitrogen loss. On one hand, the mixed fermentation system designed by the invention can obviously reduce the active nitrogen level in the system and reduce the environmental pollution caused by nitrogen; on the other hand, the microorganisms in the system can convert active nitrogen into high value-added protein products, thereby realizing resource recovery.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

As one aspect of the invention, the invention provides a method for producing single-cell protein by microbial fermentation, which comprises the following steps:

s1, introducing mixed gas into the reactor filled with the domestication culture medium to obtain a domestication system for culturing and domesticating the inoculum;

s2, introducing mixed gas into the reactor filled with the fermentation medium to obtain a mixed fermentation system for carrying out fermentation culture on the domesticated microorganisms;

s3, inoculating the anaerobic microorganisms and the aerobic microorganisms into a domestication system, and culturing and domesticating to obtain domesticated microorganisms;

s4, inoculating the domesticated microorganisms into a mixed fermentation system for fermentation culture;

and S5, processing the fermentation product to obtain the single cell protein.

According to some embodiments of the invention, in step S1, the preparation of the acclimatization medium comprises the following steps:

s1-1, preparing a basic culture medium

The basic culture medium comprises the following substances in per liter of solution: 2.3g of monopotassium phosphate, 2.9g of disodium hydrogen phosphate, 1g of ammonium chloride, 0.5g of magnesium sulfate, 0.5g of sodium carbonate, 0.01g of calcium chloride, 0.05g of ammonium ferric citrate, 0.6mg of boric acid, 0.4mg of cobalt chloride, 0.2mg of zinc sulfate, 0.06mg of manganese chloride, 0.06mg of sodium molybdate, 0.04mg of nickel chloride and 0.02mg of copper sulfate;

s1-2, sterilizing the basic culture medium at the temperature of 120-125 ℃ for 15-30min, cooling to room temperature, and adding 15g/L of agar to obtain the acclimatization culture medium.

According to some embodiments of the invention, in step S1, H in the mixture is₂And O₂The composition is variable, and the volume composition of the mixed gas is H₂：O₂：CO₂50:35:15 to 70:15: 15.

According to certain embodiments of the invention, in step S2, the preparation of the fermentation medium comprises the steps of:

s2-1, preparing a basic culture medium

The basic culture medium comprises the following substances in per liter of solution: 2.3g of monopotassium phosphate, 2.9g of disodium hydrogen phosphate, 1g of ammonium chloride, 0.5g of magnesium sulfate, 0.5g of sodium carbonate, 0.01g of calcium chloride, 0.05g of ammonium ferric citrate, 0.6mg of boric acid, 0.4mg of cobalt chloride, 0.2mg of zinc sulfate, 0.06mg of manganese chloride, 0.06mg of sodium molybdate, 0.04mg of nickel chloride and 0.02mg of copper sulfate;

s2-2, sterilizing the basic culture medium at the temperature of 120-125 ℃ for 15-30min, cooling to room temperature, adding an exogenous organic carbon source and a nitrogen source, and uniformly mixing to obtain the fermentation culture medium.

Further, in step S2-2, the exogenous organic carbon source is glucose, and the amount of glucose added is 1-100 g/L. The low addition of the organic carbon source does not greatly promote the growth of microorganisms, so that the yield is reduced; the addition amount is too high, so that the crude protein content in the product is reduced.

Further, in step S2-2, the nitrogen source is NH₄Cl, addition of NH₄Cl the initial nitrogen content of the medium was controlled at 100-10000 mg/L. If the content of the nitrogen source in the mixed fermentation system is too low, the mixed flora can stop the production of the single-cell protein, and then the excessive carbon source is synthesized into other polymers; too high a nitrogen source content can affect the nutrient balance of the system and further affect the synthesis of single-cell proteins.

According to some embodiments of the invention, in step S2, H in the mixture is₂And O₂The composition is variable, and the volume composition of the mixed gas is H₂：O₂：CO₂50:35:15 to 70:15: 15. .

According to certain embodiments of the present invention, in step S3, the culturing and acclimating are performed in a rocker reactor under reaction conditions of a temperature of 25-35 ℃, a pH of 6.0-8.0, and a shaking rate of 150 ± 50 rpm.

Further, the volume ratio of the liquid-phase matrix to the headspace of the rocker reactor in the rocker reactor is from 200:415 to 300: 315. Too low an effective volume can affect reactor utilization; if the concentration is too high, the mixed gas required for the growth of the microorganisms cannot be supplied, and the progress of the fermentation is influenced.

Further, in step S3, the volume ratio of the inoculum (anaerobic microorganism and aerobic microorganism) to the acclimatization medium usage is 50:250 to 50: 150. Too large or too small inoculation amount can affect fermentation, and too large can cause insufficient supply of mixed gas and affect product synthesis; too small will prolong the cultivation time and reduce the productivity of the reactor.

Further, in step S3, the acclimatization system is supplemented with mixed gas to 0.1 to 5atm every 12 to 24 hours. The mixed gas is consumed for the growth of the microorganisms, and the fermentation activity of the microorganisms and the fermentation process are influenced by the too little supply of the mixed gas; it is difficult to maintain the internal pressure by supplying too many reactors.

Further, in step S3, the volume ratio of the anaerobic microorganism and the aerobic microorganism inoculum is 25: 25.

Further, in step S3, the conditions for determination of the completion of acclimatization are as follows: the daily consumption of the mixed gas in the acclimatization system is more than 60%.

According to certain embodiments of the invention, in step S4, the fermentation culture is performed in a rocker reactor under conditions of a temperature of 25-35 ℃, a pH of 6.0-8.0, and a shaking rate of 150 ± 50 rpm.

Further, the volume ratio of the liquid phase matrix to the reactor headspace in the rocker reactor is from 100:515 to 250: 365. Too low an effective volume can affect reactor utilization; if the concentration is too high, the mixed gas required for the growth of the microorganisms cannot be supplied, and the progress of the fermentation is influenced.

Further, in step S4, the ratio of the domesticated microorganism to the amount of fermentation medium used in the mixed fermentation system is 40:210 to 40:60 by volume. Too large or too small inoculation amount can affect fermentation, and too large can cause insufficient supply of mixed gas and affect product synthesis; too small a size will prolong the culture time and reduce the productivity of the reactor.

Further, in step S4, the mixed gas is supplemented to the mixed fermentation system to 0.1-5atm every 12-24 hours. The mixed gas is consumed for the growth of the microorganisms, and the fermentation activity of the microorganisms and the fermentation progress are influenced by the too little supply of the mixed gas; it is difficult to maintain the internal pressure by supplying too many reactors.

According to some embodiments of the invention, in step S5, the processing includes the steps of:

s5-1, centrifuging the fermentation product at 9000-11000rpm for 10-15min, and discarding the supernatant;

s5-2, washing the centrifuged fermentation product with deionized water, centrifuging again, and repeating for 1-3 times;

s5-3, baking the fermentation product after multiple times of centrifugal cleaning at 103-108 ℃ for 20-28 hours to obtain the single cell protein.

Further, in step S5, the standard for single-cell protein formation is one or more of the following methods: 1) The conversion amount of ammonia nitrogen in the culture medium is more than 40 percent; 2) the crude protein content in the product is more than 30%.

Practice ofExample 1

A method for domesticating microorganisms capable of producing single-cell protein comprises the following steps:

1) preparing a basic culture medium, sterilizing, and adding 15g/L agar; the basic culture medium comprises the following substances in per liter of solution: 2.3g of monopotassium phosphate, 2.9g of disodium hydrogen phosphate, 1g of ammonium chloride, 0.5g of magnesium sulfate, 0.5g of sodium carbonate, 0.01g of calcium chloride, 0.05g of ferric ammonium citrate, 0.6mg of boric acid, 0.4mg of cobalt chloride, 0.2mg of zinc sulfate, 0.06mg of manganese chloride, 0.06mg of sodium molybdate, 0.04mg of nickel chloride and 0.02mg of copper sulfate;

2) inoculating anaerobic microorganisms and aerobic microorganisms;

3) adjusting the temperature and pH of the acclimatization system, and introducing the mixture into the acclimatization system according to the volume ratio of H₂：O₂：CO₂60: 25: 15, reacting in a shaking bed reactor;

4) supplementing mixed gas into the reaction system to about 1atm every 12-24 hours;

5) when the daily consumption of the gas mixture in the headspace of the reactor is greater than 60%, it is determined that acclimation is complete and a microorganism useful for the production of single-cell protein is obtained.

In this example, the temperature was controlled to 30 ℃, the oscillating speed of the rocker reactor was 150rpm, the initial pH was set to 7.0, and the pH was controlled within the range of 7.0. + -. 0.2 throughout the process.

The consumption of the mixed gas by the microorganisms in the acclimation system of the embodiment is shown in the following table 1:

table 1: daily consumption of mixed gas of microorganisms in acclimatization process

In this example, mixed gas was supplied to the acclimatization system to 1atm every 24 hours for the first 4 days in the acclimatization process; after 4 days, the acclimatization system was supplemented with mixed gas to 1atm every 12 hours. The acclimatization process is completed quickly, the consumption of the mixed gas of the acclimatized microorganisms is over 60 percent after 5 days, and the microorganisms can be kept stable.

Example 2

A method for efficiently producing single-cell protein by microbial fermentation comprises the following specific steps:

1) preparing basic culture medium, sterilizing, and adding NH₄Cl until the initial nitrogen content in the system is 1000 mgN/L; the basic culture medium comprises the following substances in per liter of solution: 2.3g of monopotassium phosphate, 2.9g of disodium hydrogen phosphate, 1g of ammonium chloride, 0.5g of magnesium sulfate, 0.5g of sodium carbonate, 0.01g of calcium chloride, 0.05g of ammonium ferric citrate, 0.6mg of boric acid, 0.4mg of cobalt chloride, 0.2mg of zinc sulfate, 0.06mg of manganese chloride, 0.06mg of sodium molybdate, 0.04mg of nickel chloride and 0.02mg of copper sulfate;

2) adding glucose into the culture medium, wherein the addition amount of the glucose is 8.25 g/L;

3) adjusting the temperature and the pH value, and introducing the mixture into the reactor according to the volume ratio of H₂：O₂：CO₂60: 25: 15, reacting in a shaking table reactor;

4) supplementing mixed gas into the reaction system to about 1atm every 12 hours;

5) after culturing in a fermentation system for 12 days, stopping the reaction;

6) centrifuging the fermentation product at 10000rpm for 10min, and removing the supernatant; washing the centrifuged fermentation product with deionized water, centrifuging again, and repeating for 2 times;

7) and baking the fermentation product after 3 times of centrifugation at 105 ℃ for 24 hours to obtain the single cell protein.

In this example, the temperature was controlled to 30 ℃, the oscillating speed of the rocker reactor was 150rpm, the initial pH was set to 7.0, and the pH was controlled within the range of 7.0. + -. 0.2 throughout the process.

The results of the ammonia nitrogen conversion amount of the microorganism and the yield analysis of the single cell protein in the fermentation system of this example are shown in table 2. As can be seen from Table 2, the ammonia nitrogen conversion rate in the fermentation system of the present example is higher than 40%, the crude protein content in the product is higher than 30%, and the formation conditions of single cell protein are met.

Example 3

Example 2 was repeated with the only difference that: in the step 2), the adding amount of the glucose is 16.50 g/L.

The ammonia nitrogen conversion amount and the single cell protein yield of the microorganism in the fermentation system of this example are shown in Table 2. As can be seen from Table 2, the ammonia nitrogen conversion amount and the single-cell protein yield of the fermentation system are higher than those of the fermentation system in the embodiment 2, and the crude protein content of the product is close to that of the fermentation system in the embodiment 2.

Example 4

Example 2 was repeated with the only difference that: in the step 2), the adding amount of the glucose is 24.75 g/L.

The results of the ammonia nitrogen conversion amount of the microorganism and the yield analysis of the single cell protein in the fermentation system of this example are shown in table 2. As can be seen from Table 2, the ammonia nitrogen conversion amount, the single cell protein yield and the crude protein content in the product in the fermentation system of the present example are all higher than those in example 3.

Example 5

Example 2 was repeated with the only difference that: in the step 2), the addition amount of the glucose is 33.00 g/L.

The results of the ammonia nitrogen conversion amount of the microorganism and the yield analysis of the single cell protein in the fermentation system of this example are shown in table 2. As can be seen from Table 2, the ammonia nitrogen conversion amount and the single-cell protein yield of the fermentation system in the present example are higher than those of the fermentation system in the example 4, but the crude protein content of the product is reduced.

Example 6

Example 2 was repeated with the only difference that: in the step 2), the adding amount of the glucose is 41.25 g/L.

The results of the ammonia nitrogen conversion amount of the microorganism and the yield analysis of the single cell protein in the fermentation system of this example are shown in table 2. As can be seen from Table 2, the ammonia nitrogen conversion amount and the single-cell protein yield of the fermentation system in the present example are higher than those of the fermentation system in the example 5, and the crude protein content of the product is close to that of the fermentation system in the example 5.

Table 2: fermentation conditions of each fermentation system under different organic carbon source adding amounts

Comparative example 1

A method for efficiently producing single-cell protein by microbial fermentation comprises the following specific steps:

1) preparing basic culture medium, sterilizing, and adding NH₄Cl until the initial nitrogen content in the system is 1000 mgN/L;

2) adjusting the temperature and the pH value, and introducing the mixture into the reactor according to the volume ratio of H₂：O₂：CO₂60: 25: 15, reacting the mixed gas in a shaking table reactor;

3) supplementing mixed gas into the reaction system to about 1atm every 12 hours;

4) after culturing in a fermentation system for 12 days, stopping the reaction;

5) centrifuging the fermentation product at 10000rpm for 10min, and removing the supernatant; washing the centrifuged fermentation product with deionized water, centrifuging again, and repeating for 2 times;

6) and baking the fermentation product after 3 times of centrifugation at 105 ℃ for 24 hours to obtain the single cell protein.

In this comparative example, the temperature was controlled to 30 ℃, the oscillating speed of the rocker reactor was 150rpm, the initial pH was set to 7.0, and the pH was controlled within the range of 7.0. + -. 0.2 throughout the process.

The results of the analysis of the ammonia nitrogen conversion amount and the single cell protein yield of the microorganism in the fermentation system of this comparative example are shown in Table 3 below. As can be seen from Table 3, the ammonia nitrogen conversion in the fermentation system of this comparative example was higher than 40%, but the crude protein content in the product was lower than 30%, and the standard for formation of the single-cell protein identified in the present invention was not met in terms of the crude protein content in the product.

Comparative example 2

A method for efficiently producing single-cell protein by microbial fermentation comprises the following specific steps:

1) preparing basic culture medium, sterilizing, and adding NH₄Cl until the initial nitrogen content in the system is 1000 mgN/L;

2) adding glucose into the culture medium, wherein the adding amount of the glucose is 16.50 g/L;

3) adjusting the temperature and the pH value, and stopping the reaction after culturing for 12 days in a fermentation system;

4) centrifuging the fermentation product at 10000rpm for 10min, and removing the supernatant; washing the centrifuged fermentation product with deionized water, centrifuging again, and repeating for 2 times;

5) and baking the fermentation product after 3 times of centrifugation at 105 ℃ for 24 hours to obtain the single cell protein.

In this comparative example, the temperature was controlled to 30 ℃, the oscillating speed of the rocker reactor was 150rpm, the initial pH was set to 7.0, and the pH was controlled within the range of 7.0. + -. 0.2 throughout the process.

The results of the analysis of the ammonia nitrogen conversion amount and the single cell protein yield of the microorganism in the fermentation system of this comparative example are shown in Table 3 below. As can be seen from Table 3, the conversion of ammonia nitrogen in the fermentation system of the comparative example was less than 40%, and the crude protein content in the product was less than 30%, which did not meet the standard for the formation of single-cell protein identified in the present invention.

Table 3: fermentation conditions of various fermentation systems under different substrates

From the results of comparative examples 1, 2 and example 3, it can be seen that the acclimated microorganism in example 1 can convert ammonia nitrogen to produce single-cell protein using one or both of a mixed gas or an organic carbon source as a substrate. However, as shown in table 3, in the single substrate fermentation system, the ammonia nitrogen conversion amount and the crude protein content in the product are both lower than those of the double substrate fermentation system; and in terms of crude protein content in the product, a single substrate fermentation system cannot efficiently produce single-cell protein. Therefore, compared with a single-substrate fermentation system, the double-substrate fermentation system which simultaneously supplies the mixed gas and the organic carbon source is more beneficial to the microbial conversion of ammonia nitrogen and the production of single-cell protein.

It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. Not all embodiments are exhaustive. All obvious changes and modifications which are obvious to the technical scheme of the invention are covered by the protection scope of the invention.