CN116676233A

CN116676233A - Method for producing single-cell protein by methane oxidizing bacteria through methane

Info

Publication number: CN116676233A
Application number: CN202310760889.2A
Authority: CN
Inventors: 杨紫怡; 王雯; 李旭天; 陈进
Original assignee: Beijing Deliuanyuan Environmental Technology Co ltd; Beijing University of Chemical Technology
Current assignee: Beijing Deliuanyuan Environmental Technology Co ltd; Beijing University of Chemical Technology
Priority date: 2023-06-26
Filing date: 2023-06-26
Publication date: 2023-09-01

Abstract

The invention provides a method for producing single-cell protein by using methane oxidizing bacteria, which belongs to the field of fermentation engineering, and particularly relates to a method for producing single-cell protein by using methane oxidizing bacteria, wherein a basic culture medium is prepared, and a nitrogen source is added; inoculating methane oxidizing bacteria into basic culture medium, culturing at constant temperature, transferring culture medium into reactor, introducing mixed gas containing CH ₄ And O ₂ The reactor is fed with water every day, the reaction system is aerated, fermented and separated to obtain single cell protein. The method of the invention can fix the greenhouse gas CH ₄ Improves the utilization of methane and converts the methane into single-cell protein.

Description

Method for producing single-cell protein by methane oxidizing bacteria through methane

Technical Field

The invention belongs to the field of fermentation engineering, and particularly relates to a method for producing single-cell proteins by using methane oxidizing bacteria.

Background

Since the 20 th century, with the increasing use of fossil fuels, a great deal of greenhouse gas emissions have resulted in global warming, which is a result ofThis raises a series of environmental crises. In the greenhouse gas, CH ₄ Although the discharge amount of (C) is not as high as that of CO ₂ But CH ₄ For example: exploitation processes of fossil energy sources such as oil fields, gas fields, coal mines and the like, processes in sewage treatment and the like. And CH (CH) ₄ The potential of the greenhouse effect of (C) is CO ₂ And thus reducing greenhouse gas emissions and low carbon development has become a global consensus.

With the continuous development of technology, the technology of synthesizing single cell proteins by microbial fermentation is receiving continuous attention from scientific researchers. The greenhouse gas is used as a carbon source for producing chemicals, fuels and other products, so that the production cost can be reduced, the grain problem can be solved, the pressure of greenhouse gas emission can be reduced, and the single-cell protein produced by microbial fermentation has the advantages of reducing the pressure on cultivated land and synthesizing high-quality protein.

Single cell proteins, which may also be referred to as microbial proteins, can be produced by fungi, bacteria, algae and bacteria, are a high nutritional value product, and are currently used mostly in the feed industry. The single cell proteins produced by methane-oxidizing bacteria have nutritional values comparable to, or even slightly higher than, fish meal, soybean meal, etc. Methane-oxidizing bacteria are the only carbon and energy sources that use methane to directly convert methane into bacterial biomass, while assimilating mineral nitrogen (i.e., ammonium) into quality proteins. The end product of this single cell protein production technology has been approved as a protein-rich feed additive with an amino acid profile approaching that of high quality animal proteins.

Disclosure of Invention

The invention aims to provide a method for producing single-cell proteins by using methane-oxidizing bacteria, which is characterized in that a specific culture medium is prepared, and mixed gas containing methane is introduced to promote the methane-oxidizing bacteria to produce the single-cell proteins by using the methane, so that methane and carbon dioxide can be fixed higher, and the yield of the single-cell proteins is improved.

In the present invention, the term "inoculation ratio" means the ratio of the volume of fermentation broth inoculated with methane-oxidizing bacteria subjected to preculture to the working volume in the final reaction system.

In the invention, the term "purging the headspace" means that in the microbial fermentation process, gas is introduced into the fermentation tank, the gas in the fermentation tank in the previous day is purged, and the gas prepared according to a certain proportion is introduced, so that the gas in the fermentation tank can react according to the same proportion every day.

In the present invention, the term "headspace volume" refers to the volume of gas at the top of the reactor after the fermentation system is loaded into the reactor.

In the present invention, the term "methane-oxidizing bacteria" refers to microorganisms capable of utilizing methane as a carbon source.

In the present invention, the term "single cell protein" is also called mycoprotein and microbial protein.

In the present invention, the term "crude protein" refers to the protein component of the fermentation product.

In the present invention, the term "crude protein content" refers to the crude protein content as determined by a Kjeldahl apparatus.

In the present invention, the term "water inlet and outlet" means adding a culture medium and discharging a fermentation broth.

In order to achieve the above object, the present invention provides a method for producing single cell proteins by methane oxidizing bacteria using methane, comprising the steps of:

s1, preparing a basic culture medium, and adding a nitrogen source;

s2, inoculating methane-oxidizing bacteria into a basic culture medium, and culturing at a constant temperature;

s3, transferring the culture medium in the step S2 into a reactor, and introducing mixed gas, wherein the mixed gas contains CH ₄ And O ₂ ；

S4, water enters and exits the reactor every day, and aeration is carried out on a reaction system;

s5, fermenting and separating to obtain single-cell protein.

In some embodiments, the basal medium in step S1 is formulated to contain the following per liter of solution: 0.37g-3.7g of dipotassium hydrogen phosphate, 0.17g-1.7g of monopotassium hydrogen phosphate, 0.085g-0.85g of magnesium chloride hexahydrate, 0.015g-0.015g of calcium chloride, 2.5mg-25mg of sodium iron ethylenediamine tetraacetate, 0.25mg-25mg of disodium ethylenediamine tetraacetate, 0.75mg-7.5mg of ferrous chloride tetrahydrate, 0.015mg-0.15mg of boric acid, 0.01mg-0.1mg of cobalt chloride, 0.0025mg-0.025mg of zinc chloride, 0.0012mg-0.012mg of manganese sulfate, 0.0015mg-0.015mg of potassium molybdate, 0.001mg-0.01mg of nickel chloride, and 0.0085mg-0.085mg of copper chloride dihydrate.

In some embodiments, the basal medium in step S1 is formulated to contain the following per liter of solution: 0.74g of dipotassium hydrogen phosphate, 0.34g of potassium dihydrogen phosphate, 0.17g of magnesium chloride hexahydrate, 0.03g of calcium chloride, 5mg of sodium iron ethylenediamine tetraacetate, 0.5mg of disodium ethylenediamine tetraacetate, 0.15mg of ferrous chloride tetrahydrate, 0.03mg of boric acid, 0.02mg of cobalt chloride, 0.005mg of zinc chloride, 0.0024mg of manganese sulfate, 0.003mg of potassium molybdate, 0.002mg of nickel chloride, 0.017mg of cupric chloride dihydrate

In some embodiments, the nitrogen source in step S1 is selected from one or more of amino acids, ammonium salts, peptones, amino sugars, polypeptides, cyclic nitrides.

In some embodiments, the nitrogen source is ammonium chloride.

In some embodiments, the ammonium chloride concentration is 0.11g/L to 1.91g/L, and the ammonia nitrogen concentration is controlled to be: 28mg N/L to 500mg N/L.

In some embodiments, in step S3, the volume of microbial fermentation medium within the reactor: headspace volume= (150-200): (415-465).

In some embodiments, the volume of microbial fermentation medium within the reactor: headspace volume = 200:415.

in some embodiments, in step S2, the methane-oxidizing bacteria are selected from the group consisting of: methylocystis, methylosinus, methylomonas, methylobacter, methylocapsa, methylosarina, methylovulumd.

In some embodiments, the methane-oxidizing bacteria are methane-oxidizing bacteria, the bacteria comprising the following relative abundance of bacteria: 55% -60% of methyl peptides, 20% -25% of methyl peptides and 15% -25% of methyl peptides.

In some embodiments, the methane-oxidizing bacteria are methane-oxidizing bacteria, the bacteria comprising the following relative abundance of bacteria: methyldynamics sp.49242 (55% -60%), methylotenera versatilis BAA-2224 (20% -25%), methylovulum sp TSD-255 (15% -25%).

In some embodiments, the methane-oxidizing bacteria are methane-oxidizing bacteria, the bacteria comprising the following relative abundance of bacteria: methylcysts sp.49242.57%, methylotenera versatilis BAA-2224 23%, methylovulum sp TSD-255%.

In some embodiments, in step S2, the inoculation ratio is 20%; in the step S2, fermentation culture is carried out in a constant temperature shaking incubator, the reaction temperature is 26 ℃, the pH of the reactor is 7.0+/-0.2, and the shaking speed of the constant temperature shaking incubator is 150rpm.

In some embodiments, in step S3, CH ₄ And O ₂ The proportion is (1-3): 3.

in some embodiments, in step S3, CH ₄ And O ₂ The proportion is 2:3.

in some embodiments, in step S3, the mixture further contains CO ₂ ，N ₂ Wherein CH is ₄ 、O ₂ 、CO ₂ And N ₂ The mixing proportion is (10-190): (15-45): (30-80): (250-300).

In some embodiments, in step S4, the reactor inlet/outlet water is 40mL-50mL per day, and the hydraulic retention time is 3d-6d.

In some embodiments, the reactor has 50mL of water in and out per day, with a hydraulic residence time of 4d;

in some embodiments, in step S4, the reactor is aerated once every 12-36 hours for 2-3 minutes for each purge of the headspace.

In some embodiments, the reactor was aerated once every 24 hours for 2.5 minutes for each purge of the headspace.

In some embodiments, the conditions for completion of the fermentation of step S5 are: the ammonia nitrogen conversion rate is more than 5mg N/L d, the methane fixation efficiency is more than 23%, the flora grows to the stationary phase and OD ₆₀₀ The stability is achieved;

step S5 separation includes the steps of:

s6-1, centrifuging the fermentation product at 10000rpm for 10min, and pouring out supernatant;

s6-2, cleaning the centrifuged product with deionized water, centrifuging again, and repeating for 1-3 times;

s6-3, baking the fermentation product subjected to centrifugal washing for many times at 103-110 ℃ for 20-30 hours to obtain the microbial protein.

The inventors found that methane oxidizing bacteria fix methane volumes of > 180mL and single cell protein content of > 40% by adding specific medium and introducing a mixture gas containing methane.

The beneficial effects are that:

through the technical scheme, the greenhouse gas CH is fixed ₄ The methane fixation efficiency is improved, and the ammonia nitrogen concentration in the wastewater can be determined optimally by simulating the ammonia nitrogen in the wastewater by adding ammonium chloride, so that the ammonia nitrogen in the wastewater is further treated and converted into a high added value product.

Not only solves the problem of the influence of ammonia nitrogen in natural environment on the environment and the loss of nitrogen in nitrogen circulation, but also contributes to the solution of greenhouse effect. Solves the problem of insufficient grain in the prior art and can reduce the pressure of cultivated land.

Detailed Description

Embodiments of the present disclosure are described in further detail below in conjunction with examples. The following detailed description of the embodiments is provided to illustrate the principles of the present disclosure and not to limit the scope of the disclosure, which may be embodied in many different forms and is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

The present disclosure provides these embodiments in order to make the present disclosure thorough and complete, and fully convey the scope of the disclosure to those skilled in the art. It should be noted that: the relative arrangement of parts and steps, the composition of materials, numerical expressions and numerical values set forth in these embodiments should be construed as exemplary only and not limiting unless otherwise specifically stated.

In the description of the present disclosure, unless otherwise indicated, the meaning of "plurality" is greater than or equal to two; the terms "upper," "lower," "left," "right," "inner," "outer," and the like indicate an orientation or positional relationship merely for convenience of describing the present disclosure and simplifying the description, and do not indicate or imply that the devices or elements being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus are not to be construed as limiting the present disclosure. When the absolute position of the object to be described is changed, the relative positional relationship may be changed accordingly.

All terms used in the present disclosure have the same meaning as understood by one of ordinary skill in the art to which the present disclosure pertains, unless specifically defined otherwise. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

Techniques, methods, and apparatus known to one of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, the techniques, methods, and apparatus should be considered part of the specification.

In order to more clearly illustrate the present invention, the present invention will be further described with reference to preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.

Example 1

S1, preparing a basic culture medium

The basic culture medium in the step S1 comprises the following substances in each liter of solution: 0.74g of dipotassium hydrogen phosphate, 0.34g of potassium dihydrogen phosphate, 0.17g of magnesium chloride hexahydrate, 0.03g of calcium chloride, 5mg of sodium iron ethylenediamine tetraacetate, 0.5mg of disodium ethylenediamine tetraacetate, 0.15mg of ferrous chloride tetrahydrate, 0.03mg of boric acid, 0.02mg of cobalt chloride, 0.005mg of zinc chloride, 0.0024mg of manganese sulfate, 0.003mg of potassium molybdate, 0.002mg of nickel chloride, and 0.017mg of copper chloride dihydrate;

sterilizing the basic culture medium at 121-125deg.C for 20min, cooling to room temperature, and adding external nitrogen source.

The external nitrogen source is ammonium chloride, the concentration of the added ammonium chloride is 0.11g/L, and the concentration of ammonia nitrogen is controlled to be 28mg N/L, so as to obtain the fermentation culture medium.

S2, inoculating methane-oxidizing bacteria (comprising bacteria with the relative abundance of methyl stissp.49557%, methylotenera versatilis BAA-2224 23%, methylovulum sp TSD-255%) into a fermentation culture medium, wherein the inoculation ratio is 20%.

In the step S2, fermentation culture is carried out in a constant temperature shaking incubator, the reaction temperature is 26 ℃, the pH of the reactor is 7.0+/-0.2, and the shaking speed of the constant temperature shaking incubator is 150rpm.

S3, transferring the culture medium in the step S3 into a reactor, and introducing mixed gas, wherein the mixed gas comprises CH ₄ And O ₂ The ratio of the mixed gas is 200:300.

In step S3, the volume of the microbial fermentation medium in the reactor: headspace volume = 200:415.

s4, water enters and exits the reactor every day, and aeration is carried out on the reaction system.

In step S4, the reactor had 50mL of water fed and discharged each day, wherein "in" means adding 50mL of medium, and "out" means discharging 50mL of fermentation broth, and the hydraulic retention time was 4d.

In the step S4, the reactor is used for carrying out aeration on the reaction system every 24 hours, and the time for purging the headspace every time is 2.5min.

S5, fermenting and separating to obtain the microbial protein. Conditions for completion of fermentation: ammonia nitrogen conversion rate is more than 6mg N/L d, methane fixation efficiency is more than 60%, bacterial colony grows to stationary phase, OD ₆₀₀ And the stability is achieved.

The separation of step S5 comprises the steps of:

s5-1, centrifuging the fermentation product at 10000rpm for 10min, and pouring out supernatant;

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

s5-3, baking the fermentation product subjected to centrifugal washing for many times at 103-110 ℃ for 20-30 hours to obtain single cell protein.

Example 2

Example 1 was repeated, with the only differences that: in step 1), NH is added ₄ The initial ammonia nitrogen content in the Cl-to-system is 100mg N/L.

Example 3

Example 1 was repeated, with the only differences that: in step 1), NH is added ₄ The initial ammonia nitrogen content in the Cl-to-system is 200mg N/L.

Example 4

Example 1 was repeated, with the only differences that: in step 1), NH is added ₄ The initial ammonia nitrogen content in the Cl-to-system is 400mg N/L.

Example 5

Example 1 was repeated, with the only differences that: in step 1), NH is added ₄ The initial ammonia nitrogen content in the Cl-to-system is 500mg N/L.

Example 6

S1, preparing a basic culture medium

The external nitrogen source is ammonium chloride, and the initial ammonia nitrogen content is controlled to be 200mg N/L, so that the fermentation medium is obtained.

S3, transferring the culture medium in the step S3 into a reactor, and introducing mixed gas, wherein the volume of the mixed gas is CH ₄ 、CO ₂ 、O ₂ And N ₂ The mixing ratio of the components is 100:50:45:300.

in step S3, the volume of the microbial fermentation medium in the reactor: headspace volume=150:465.

In step S4, the reactor had 40mL of water fed and discharged each day, wherein "in" means adding 40mL of medium, and "out" means discharging 40mL of fermentation broth, and the hydraulic retention time was 4d.

S5, fermenting and separating to obtain the microbial protein.

In step S5, conditions for completion of fermentation: the ammonia nitrogen conversion rate is more than 5mg N/L d, the methane fixation efficiency is more than 23%, the flora grows to the stationary phase and OD ₆₀₀ And the stability is achieved.

The separation of step S5 comprises the steps of:

Example 7

Example 6 was repeated, with the only difference that: in step 4), the gas mixture has a volume composition of CH ₄ 、CO ₂ 、O ₂ And N ₂ The mixing ratio of the components is 190:80:25:300.

example 8

Example 6 was repeated, with the only difference that: in step 4), the gas mixture has a volume composition of CH ₄ 、CO ₂ 、O ₂ And N ₂ The mixing ratio of the components is 10:30：15：250。

example 9

Example 3 was repeated, with the only difference that: in the step 1), the formula of the culture medium is as follows:

3.7g of dipotassium hydrogen phosphate, 1.7g of potassium dihydrogen phosphate, 0.85g of magnesium chloride hexahydrate, 0.015g of calcium chloride, 25mg of sodium iron ethylenediamine tetraacetate, 25mg of disodium ethylenediamine tetraacetate, 7.5mg of ferrous chloride tetrahydrate, 0.15mg of boric acid, 0.1mg of cobalt chloride, 0.025mg of zinc chloride, 0.012mg of manganese sulfate, 0.015mg of potassium molybdate, 0.01mg of nickel chloride, and 0.085mg of copper chloride dihydrate.

Example 10

The procedure of example 3 was repeated, with the only differences that: in the step 1), the formula of the culture medium is as follows:

0.37g of dipotassium hydrogen phosphate, 0.17g of potassium dihydrogen phosphate, 0.085g of magnesium chloride hexahydrate, 0.015g of calcium chloride, 2.5mg of sodium iron ethylenediamine tetraacetate, 0.25mg of disodium ethylenediamine tetraacetate, 0.75mg of ferrous chloride tetrahydrate, 0.015mg of boric acid, 0.01mg of cobalt chloride, 0.0025mg of zinc chloride, 0.0012mg of manganese sulfate, 0.0015mg of potassium molybdate, 0.001mg of nickel chloride and 0.0085mg of cupric chloride dihydrate.

Example 11

S1, preparing a basic culture medium

The additional nitrogen source is ammonium chloride, and the ammonia nitrogen concentration is controlled to be 200mg N/L, so that the fermentation medium is obtained.

S3, transferring the culture medium in the step S3 into a reactor, and introducing mixed gas, wherein the mixed gas comprises CH ₄ And O ₂ The ratio of the mixed gas is 300:300.

The separation of step S5 comprises the steps of:

The results of the yield analysis of the fermentation system of this comparative example are shown in the following table. As can be seen from the table, the ammonia nitrogen conversion rate in the fermentation system of this example is greater than 6mg N/L d, and the crude protein content in the product is lower than 44%, and the methane fixation efficiency is greater than 23%.

Example 12

S1, preparing a basic culture medium

S3, transferring the culture medium in the step S3 into a reactor, and introducing mixed gas, wherein the mixed gas comprises CH ₄ And O ₂ The ratio of the mixed gas is 100:300.

The separation of step S5 comprises the steps of:

The results of the yield analysis of the fermentation system of this comparative example are shown in the following table. As can be seen from the table, although the ammonia nitrogen conversion rate in the fermentation system is more than 6mg N/L d, the methane fixation efficiency is more than 60%, and the crude protein content in the product is lower than 44%.

Example 13

Example 3 was repeated, with the only difference that: in step 2), the methane-oxidizing bacteria consist of bacteria of the following relative abundance: methylcysts sp.49242, methylotenera versatilis BAA-2224 20%, methylovulum sp TSD-255%.

Example 14

Example 3 was repeated, with the only difference that: in step 2), the methane-oxidizing bacteria consist of bacteria of the following relative abundance: methylcysts sp.49242, methylotenera versatilis BAA-2224 25%, methylovulum sp TSD-255%.

Comparative example 1

S1, preparing a basic culture medium

The basic culture medium in the step S1 comprises the following substances in each liter of solution: 0.74g of dipotassium hydrogen phosphate, 0.34g of potassium dihydrogen phosphate, 0.17g of magnesium chloride hexahydrate, 5mg of sodium iron ethylenediamine tetraacetate, 0.5mg of disodium ethylenediamine tetraacetate, 0.15mg of ferrous chloride tetrahydrate, 0.03mg of boric acid, 0.02mg of cobalt chloride, 0.005mg of zinc chloride, 0.0024mg of manganese sulfate, 0.003mg of potassium molybdate, 0.002mg of nickel chloride and 0.017mg of cupric chloride dihydrate;

S5, fermenting and separating to obtain the microbial protein. The flora grows to a stable period and OD ₆₀₀ And the stability is achieved.

The separation of step S5 comprises the steps of:

The results of the yield analysis of the fermentation system of this comparative example are shown in the following table. As can be seen from the table, the ammonia nitrogen conversion rate in the fermentation system of the comparative example is less than 6mg N/L d, the crude protein content in the product is less than 44%, and the methane fixation efficiency is less than 23%, which is not up to the single cell protein formation standard identified in the invention.

Comparative example 2

S1, preparing a basic culture medium

The basic culture medium in the step S1 comprises the following substances in each liter of solution: 0.74g of dipotassium hydrogen phosphate, 0.34g of potassium dihydrogen phosphate, 0.17g of magnesium chloride hexahydrate, 0.03g of calcium chloride, 2.5mg of sodium iron ethylenediamine tetraacetate, 0.5mg of disodium ethylenediamine tetraacetate, 0.15mg of ferrous chloride tetrahydrate, 0.03mg of boric acid, 0.02mg of cobalt chloride, 0.005mg of zinc chloride, 0.0024mg of manganese sulfate, 0.003mg of potassium molybdate, 0.002mg of nickel chloride, and 0.017mg of cupric chloride dihydrate;

S3, transferring the culture medium in the step S3 into a reactor, and introducing mixed gas, wherein the volume of the mixed gas is CH ₄ 、CO ₂ 、O ₂ And N ₂ The mixing ratio of the components is 190:80:25:300.

5) The reactor had a daily water inlet/outlet of 40mL and a hydraulic residence time of 4d.

6) The reactor was aerated once every 24 hours, and the time for purging the headspace was 2.5min each time.

S5, fermenting and separating to obtain the protein.

The separation of step S5 comprises the steps of:

The results of the yield analysis of the fermentation system of this comparative example are shown in the following table. As can be seen from the table, the ammonia nitrogen conversion rate in the fermentation system of the comparative example is lower than 5mg N/L d, the crude protein content in the product is lower than 30%, and the methane fixation efficiency is less than 23%, so that the formation standard of the single cell protein identified in the invention is not met.

Comparative example 3

Example 3 was repeated, with the only difference that: in step 2), the methane-oxidizing bacteria consist of bacteria of the following relative abundance: methylcysts sp.49242, methylotenera versatilis BAA-2224 23%, methylovulum sp TSD-255% by weight.

Comparative example 4

The parameters of the fermentation process of examples 1-10, comparative examples 1-2, and the single cell protein yield and crude protein content of the product were tested by the following test methods:

1. ammonia nitrogen conversion rate test:

wherein:

V _NH4 ⁺ _-N ammonia nitrogen conversion rate (mg N/L x d);

NH ₄ ⁺ -N _i -initial ammonia nitrogen content (mg N/L) of the reactor;

NH ₄ ⁺ -N _r -initial ammonia nitrogen content (mg N/L) of the reactor;

t-cultivation time (d)

2. Methane consumption test

Methane consumption = Σ (V _{Initial i} -V _{Last i} )

3. Single cell protein yield test

4. Crude protein content assay

Crude protein content measurement was performed using a Kjeldahl nitrogen determination instrument

5. Methane fixation efficiency test

The test results are shown in Table 1

TABLE 1 fermentation conditions in each reaction System

As is clear from Table 1, in examples 1 to 5, the crude protein content was gradually increased with increasing ammonia nitrogen concentration, the single-cell protein yield was decreased, and in example 3, ammonia nitrogen concentration was 200mg N/L, and methane fixation efficiency was highest, reaching 82.5%.

Examples 6 to 8 employ a gas of CH ₄ 、CO ₂ 、O ₂ And N ₂ Ammonia of example 3The nitrogen conversion rate and methane fixation efficiency are better than those of examples 6-8, however, the crude protein content of example 6 is higher than that of example 3, and the ratio of the basic culture medium is different between examples 9-10 and example 3, and the ammonia nitrogen conversion rate and methane fixation efficiency and crude protein content of example 3 are better than those of examples 9-10.

In example 3, compared with examples 13-14, the bacteria ratio in the methane-oxidizing bacteria is different, the ammonia nitrogen conversion rate and the methane fixation efficiency and the crude protein content of example 3 are better than those of examples 13-14, and the bacteria ratio of the methane-oxidizing bacteria in examples 3-4 is outside the protection range of the invention, and the ammonia nitrogen conversion rate and the methane fixation efficiency and the crude protein content are lower than those of examples 3, 13 and 14.

Comparative example 1 As compared with example 3, caCl was not added to the basal medium of comparative example 1 ₂ The single cell protein yield, crude protein content and methane fixation efficiency were lower than in example 3.

Comparative example 2 the basic medium of comparative example 2 had a lower iron content of sodium ethylenediamine tetraacetate than example 6, and the same applies to the single cell protein production of comparative example 2, and the crude protein content and methane fixation efficiency were lower than those of example 6.

In summary, the composition and proportion of bacteria in the methane-oxidizing bacteria flora, the formula of the culture medium, and the composition and proportion of methane and other gases all affect the single-cell protein yield, the crude protein content and the methane fixation efficiency.

Thus, various embodiments of the present invention have been described in detail. In order to avoid obscuring the concepts of the invention, some details known in the art have not been described. How to implement the inventive solutions herein will be fully apparent to those skilled in the art from the above description.

While certain specific embodiments of the invention have been described in detail by way of example, it will be appreciated by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the invention. It will be understood by those skilled in the art that the foregoing embodiments may be modified and equivalents substituted for elements thereof without departing from the scope and spirit of the invention. In particular, the technical features mentioned in the respective embodiments may be combined in any manner as long as there is no structural conflict.

Claims

1. A method for producing single-cell protein by using methane by methane oxidizing bacteria comprises the following steps:

s1, preparing a basic culture medium, and adding a nitrogen source;

s5, fermenting and separating to obtain single-cell protein.

2. The method of claim 1, wherein the basal medium in step S1 is formulated to contain the following per liter of solution: 0.37g-3.7g of dipotassium hydrogen phosphate, 0.17g-1.7g of monopotassium hydrogen phosphate, 0.085g-0.85g of magnesium chloride hexahydrate, 0.015g-0.015g of calcium chloride, 2.5mg-25mg of sodium iron ethylenediamine tetraacetate, 0.25mg-25mg of disodium ethylenediamine tetraacetate, 0.75mg-7.5mg of ferrous chloride tetrahydrate, 0.015mg-0.15mg of boric acid, 0.01mg-0.1mg of cobalt chloride, 0.0025mg-0.025mg of zinc chloride, 0.0012mg-0.012mg of manganese sulfate, 0.0015mg-0.015mg of potassium molybdate, 0.001mg-0.01mg of nickel chloride, and 0.0085mg-0.085mg of copper chloride dihydrate.

3. The method according to claim 1, wherein the nitrogen source in the step S1 is one or more selected from the group consisting of amino acids, ammonium salts, peptones, amino sugars, polypeptides, and cyclic nitrides, and the nitrogen source is ammonium chloride, and the ammonia nitrogen concentration is controlled to be 28mg N/L to 500mg N/L.

4. The method of claim 1, wherein in step S3, the volume of the microbial fermentation medium in the reactor is: headspace volume= (150-200): (415-465).

5. The method of claim 1, wherein in step S2, the methane-oxidizing bacteria are selected from the group consisting of: methylocystis, methylosinus, methylomonas, methanobacterium, methylobacter, methylocapsa, methylosarina, methylovulumd, said methane-oxidizing bacteria being a methane-oxidizing flora comprising bacteria in relative abundance of: methyldynamics (55% -60%), methyltenera (20% -25%), methylcaldium (15% -25%).

6. The method according to claim 1, wherein in step S2, the inoculation ratio is 20%; the fermentation culture in the step S2 is carried out in a constant temperature shaking incubator, the reaction temperature is 26 ℃, the pH of the reactor is 7.0+/-0.2, and the shaking speed of the constant temperature shaking incubator is 150rpm.

7. The method of claim 1, wherein in step S3, CH ₄ And O ₂ The proportion is (1-3): 3.

8. the method of claim 1, wherein in step S3, the mixture further comprises CO ₂ ，N ₂ Wherein CH is ₄ 、O ₂ 、CO ₂ And N ₂ The mixing proportion is (10-190): (15-45): (30-80): (250-300).

9. The method according to claim 1, wherein in the step S4, the water inlet and outlet of the reactor is 40-50 mL each day, and the hydraulic retention time is 3-6 d; in the step S4, the reactor is used for aerating the reaction system every 12-36 h, and the time for purging the headspace every time is 2-3min.

10. The method of claim 1, wherein the fermentation of step S5 is accomplished under the following conditions: the ammonia nitrogen conversion rate is more than 5mg N/L d, the methane fixation efficiency is more than 23%, and the flora growsLong to stationary phase, OD ₆₀₀ The stability is achieved;

the separation of step S5 includes the steps of: