Method for preparing isobutanol by converting methane through microorganisms and application thereof

Abstract

The invention provides a method for preparing isobutanol by converting methane through microorganisms and application of the method. The method comprises the following steps: mixing lactococcus lactis and methanophilic bacteria which reach a stable growth period according to a volume ratio of (5-30): 100, and then converting methane to prepare the isobutanol, the conditions for preparing the isobutanol by converting the methane through the microorganisms are as follows: the methane concentration is 10-30%, the conversion temperature is 25-35 DEG C, and the conversion period is 2-7 days. The invention provides a method for catalyzing conversion of methane into isobutanol through mixed culture of two microorganisms, and a channel for preparing isobutanol by using methane as a raw material is opened in a simple mode.

Description

Technical Field

[0001] The invention relates to the technical field of producing alcohol compounds through methane bioconversion, and in particular to a method for preparing isobutanol by utilizing microorganisms to convert methane and application thereof. Background Art

[0002] How to achieve effective recycling of carbon resources has become one of the most serious problems facing human society. As a carbon greenhouse gas, methane (CH 4 ) has a wide range of sources and is also an important carbon source for microorganisms. Its bioconversion to produce chemicals can not only help reduce greenhouse gas emissions, but also broaden the source of raw materials for biomanufacturing. As an important type of chemical in petroleum refining, alcohol compounds can be widely used in energy, materials, medicine and other industries. In order to get rid of dependence on traditional petrochemical resources, the use of microorganisms to synthesize alcohol compounds has become a very promising biomanufacturing route and plays an important role in promoting the green economy. Isobutanol is an important organic chemical raw material with high energy density, low volatility and weak water absorption. It is widely used in many fields, such as in the manufacture of petroleum additives, isobutyl acetate, diisobutyl phthalate plasticizers and artificial musks, etc. It is a potential liquid fuel.

[0003] Methanophiles are a branch of methyl-oxidizing bacteria, also known as methanotrophs. Methanophiles mainly include type I and type II, which belong to γ-Proteobacteria and α-Proteobacteria respectively. Type I belongs to γ-Proteobacteria, such as Methylomonas, Methylococcus, Methylocaldum, Methylobacter, Methylomicrobium and Methylothermus, and type II belongs to α-Proteobacteria, such as Methylosinus, Methylocella, Methylocapsa and Methylocystis. Among them, some species of bacteria in the genus such as Methylomicrobium belong to facultative methanotrophs. Methanophiles contain key enzymes that can catalyze the conversion of methane into methanol, pyruvate, ketoisovalerate and various amino acids, and are the most important group of bacteria in the global methane cycle. However, there is no ketoisovalerate decarboxylase in the cells of methanogens, and ketoisovalerate cannot be catalyzed to produce isobutyraldehyde. Therefore, it is impossible to directly convert methane into isobutanol using only methanogens.

[0004] At present, some scientific researchers have tried to construct an isobutanol synthesis pathway in methanophiles. For example, patent application CN113151130A discloses a genetically engineered bacterium and its application in the bioconversion of methane to produce isobutanol. The bacterium introduces a 2-ketoisovalerate decarboxylase gene and an alcohol dehydrogenase gene into an alkaliphilic methanobacterium, or introduces a 2-ketoisovalerate decarboxylase gene into an alkaliphilic methanobacterium, and integrates an exogenous DNA molecule having the 2-ketoisovalerate decarboxylase gene into the genomic DNA of the alkaliphilic methanobacterium to obtain a recombinant bacterium that can express both the 2-ketoisovalerate decarboxylase gene and the alcohol dehydrogenase gene, and the recombinant bacterium is used to convert methane to produce isobutanol. However, the method of converting methane to produce isobutanol requires that genetic engineering be used to obtain recombinant bacteria that can simultaneously express 2-ketoisovalerate decarboxylase genes and ethanol dehydrogenase genes. This method requires high technical requirements and is difficult to reconstruct. In addition, the yield of isobutanol produced by converting methane using the recombinant bacteria is low. In addition, this pathway has not been established in Methylomicrobium species such as Methylomicrobium buryatense 5GB1 and Methylomicrobium alcaliphilum 20Z.

[0005] Therefore, there is an urgent need for a method for efficiently converting methane to prepare isobutanol. Lactococcus lactis (L. lactis) is a prokaryotic microorganism that can produce ketoisovalerate decarboxylase to catalyze ketoisovalerate to produce isobutyraldehyde. So far, there has been no research on the combined catalytic conversion of methane by methanophilic bacteria and Lactococcus lactis. Summary of the invention

[0006] In view of the problems that the existing technology uses a single methanotrophic bacterium to transform methane, the types of methane products are few, and the use of recombinant bacteria to transform methane has the problems of great recombination difficulty, high cost, low isobutanol yield and the inability to open up the transformation pathway of some methanotrophic bacteria, the present invention provides a method for preparing isobutanol by transforming methane using microorganisms and its application. By mixing Lactococcus lactis and methanotrophic bacteria that have reached a stable growth period and then using them to transform methane to prepare isobutanol, a channel for preparing isobutanol using methane as a raw material is opened in a simple way.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing isobutanol by converting methane using microorganisms, the method comprising mixing Lactococcus lactis and methanophilic bacteria that have reached a stable growth phase in a volume ratio of (5-30):100 and then using the mixture to convert methane to prepare isobutanol.

[0009] In some embodiments of the present invention, the volume ratio of Lactococcus lactis that has reached a stable growth phase to the methanophilic bacteria that has reached a stable growth phase is (15-30):100, preferably 20:100.

[0010] According to the method provided by the present invention, the conditions for preparing isobutanol by converting methane using microorganisms are: methane concentration is 10-30%, conversion temperature is 25-35°C, and conversion cycle is 2-7 days.

[0011] Preferably, the methane concentration is 10-20%, more preferably, the methane concentration is 15%.

[0012] Preferably, the conversion period is 3-5 days.

[0013] In some embodiments of the present invention, the pH value of the culture medium for the mixed culture of Lactococcus lactis and methanophilic bacteria is controlled to be 7-10, and the rotation speed of the mixed culture is 50-300 rpm.

[0014] According to the method provided by the present invention, the lactococcus lactis is selected from one or more species of bacteria capable of producing ketoisovalerate decarboxylase, preferably, selected from two or more species of Lactococcus lactis, Lactococcus lactis subsp. Lactis, and Lactococcus lactis subsp. Cremoris.

[0015] In some embodiments of the present invention, the method for culturing Lactococcus lactis that reaches the stable growth phase is: inoculating Lactococcus lactis in the culture medium at an inoculation amount of 8-12%, and then culturing at a culture temperature of 32-38°C, a culture speed of 150-500rpm, and a pH value of 5.5-7.5.

[0016] According to the method provided by the present invention, the methanotrophic bacteria are selected from at least one species of the genus Methylomicrobium; preferably, at least two species of the methanotrophic bacteria are selected from Methylomicrobium alcaliphilum, Methylomicrobium albus, Methylomicrobium buryatense, Methylomicrobium albumstrain, and Methylomicrobiumsp.

[0017] In some embodiments of the present invention, the cultivation method of methanogens that have reached a stable growth phase is as follows: inoculating methanogens in a culture medium at an inoculation rate of 8-12%, and then cultivating at a culture temperature of 25-35°C, a culture speed of 150-500rpm, a pH value of 8.5-9.5, and a methane concentration of 15-25%.

[0018] In some embodiments of the present invention, the carbon source of the above culture medium is selected from one or more of methane, methanol and glucose.

[0019] In some embodiments of the present invention, the nitrogen source of the above culture medium is selected from one or more of potassium nitrate, peptone and beef extract.

[0020] In some embodiments of the present invention, the culture medium formula for inoculating the lactococcus lactis strains as described above refers to the culture medium formula for culturing lactococcus lactis used in existing public documents, preferably a culture medium formula that enables lactococcus lactis to produce high ketoisovalerate decarboxylase, such as MRS solid culture medium or MRS liquid culture medium. The culture medium formula for inoculating the methanophilic bacteria strains as described above refers to the culture medium formula for culturing methanophilic bacteria used in existing public documents, preferably a culture medium formula that enables methanophilic bacteria to produce high ethanol dehydrogenase, such as NMS solid culture medium or NMS liquid culture medium.

[0021] In some embodiments of the present invention, Lactococcus lactis and methanophilus are controlled to reach a stable growth phase at the same time.

[0022] In a second aspect, the present invention provides application of the method described in the first aspect in a biofuel production process.

[0023] In some embodiments of the invention, the biofuel comprises isobutanol.

[0024] In some embodiments of the present invention, the biofuel may also include n-butanol, n-propanol, and the like.

[0025] The beneficial effects of the present invention are as follows:

[0026] The present invention proposes for the first time a method for converting methane to prepare isobutanol by mixing Lactococcus lactis and methanophiles that have reached a stable growth period. The method does not require the construction of a recombinant microorganism, the strain is relatively stable, and the methane conversion rate and isobutanol yield are high. Lactococcus lactis and methanophiles are first cultured separately, and strains in a stable growth period can be quickly cultured, and Lactococcus lactis and methanophiles can also be controlled to reach a stable growth period at the same time according to demand. The strain culture method in the stable growth period is simple and easy to culture in large quantities, and it is beneficial to ensure that the mixed strain has a higher alcohol dehydrogenase activity and ketoisovalerate decarboxylase activity. The method of the present invention opens up a channel for preparing isobutanol using methane as a raw material in a simple manner, and the method for preparing isobutanol has good prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the growth curve of methanophilic bacteria and the curve of alcohol dehydrogenase activity changing with culture time (h);

[0028] Figure 2 is the growth curve of Lactococcus lactis and the curve of ketoisovalerate decarboxylase activity changing with culture time (h);

[0029] Figure 3 It is a growth curve of microorganisms in a mixed solution containing methanophilic bacteria and Lactococcus lactis (volume ratio is 15:100) under a methane concentration of 15% and a curve of isobutanol production changing with conversion time (h). DETAILED DESCRIPTION

[0030] The following examples further illustrate the technology of the present invention. These examples are for illustration and example of the present invention and are not intended to limit the scope of the present invention in any form.

[0031] Example 1

[0032] A method for preparing isobutanol by converting methane using microorganisms comprises the following steps:

[0033] (1) Cultivating Lactococcus lactis in the stable growth phase: Inoculate Lactococcus lactis seed solution in MRS liquid culture medium at an inoculation rate of 10%, and then culture for 48 hours at a culture temperature of 35° C., a culture rotation speed of 300 rpm, and a pH value of 5.5-7.5.

[0034] (2) Cultivation of methanogens in the stable growth phase: Inoculate the NMS liquid medium with a 10% inoculum of methanogens (Methylomicrobium alcaliphilum) seed solution, and then culture for 48 h at a temperature of 30° C., a rotation speed of 300 rpm, a pH of 8.5-9.5, and a methane concentration of 20%.

[0035] (3) The culture solution containing Lactococcus lactis in the stable growth phase obtained by the above step (1) and the culture solution containing methanogens in the stable growth phase obtained by the step (2) were mixed, and 7 groups of experiments were set up. In each group of experiments, the volume ratio of the culture solution containing Lactococcus lactis in the stable growth phase: the culture solution containing methanogens in the stable growth phase was 0:100, 5:100, 10:100, 15:100, 20:100, 25:100, and 30:100. The pH value of the mixed solution containing Lactococcus lactis and methanogens was controlled to be 7-10, and the rotation speed of the mixed culture was 180 rpm.

[0036] (4) methane is introduced into the mixed solution containing Lactococcus lactis and methanogens, and the methane concentration in the reactor is controlled to be 15% and the conversion temperature is 30° C. The conversion is carried out for 4 days, so that methane is converted into isobutanol under the catalytic action of the biological enzymes produced by Lactococcus lactis and methanogens.

[0037] The final isobutanol yields of different mixed systems are shown in Table 1. It can be seen from Table 1 that when only the culture solution containing methanogens in the stable growth period is used to convert methane, isobutanol cannot be obtained; when the volume ratio of the culture solution containing Lactococcus lactis in the stable growth period: the culture solution containing methanogens in the stable growth period is 20:100, the isobutanol yield is the highest.

[0038] Table 1 Final isobutanol yields of different mixing systems

[0039] Volume ratio 0:100 5:100 10:100 15:100 20:100 25:100 30:100 Isobutanol (mg / L) 0 3.16 18.21 54.21 62.19 51.37 55.98

[0040] Example 2

[0041] A method for preparing isobutanol by converting methane using microorganisms comprises the following steps:

[0042] (1) Cultivating Lactococcus lactis in the stable phase: Inoculate Lactococcus lactis seed solution in an inoculum of 10% in MRS medium, and then culture for 48 hours at a culture temperature of 35° C., a culture rotation speed of 300 rpm, and a pH value of 5.5-7.5.

[0043] (2) Cultivation of methanogens in the stable growth phase: Inoculate the NMS medium with a 10% inoculum of methanogens (Methylomicrobium alcaliphilum) seed solution, and then culture for 48 h at a temperature of 30° C., a rotation speed of 300 rpm, a pH of 8.5-9.5, and a methane concentration of 20%.

[0044] (3) The culture solution containing Lactococcus lactis in the stable growth phase obtained in step (1) above and the culture solution containing methanophilic bacteria in the stable growth phase obtained in step (2) were mixed at a volume ratio of 20:100, the pH value of the mixed solution containing Lactococcus lactis and methanophilic bacteria was controlled to be 7-10, and the rotation speed of the mixed culture was 180 rpm. The composition and content of the nutrients in the mixed solution are shown in Table 2 below.

[0045] Table 2 Composition and content of nutrients in the mixed solution (100mL)

[0046]

[0047]

[0048] (4) methane was introduced into the mixed solution containing Lactococcus lactis and methanogens, and 6 experimental groups were set up. The methane concentration in the reactor was 5%, 10%, 15%, 20%, 25%, and 30%. The conversion temperature in the reactor was controlled to 30° C. and the conversion was carried out for 4 days. Methane was converted into isobutanol under the catalytic action of the biological enzymes produced by Lactococcus lactis and methanogens.

[0049] The final isobutanol production of the conversion system at different methane concentrations is shown in Table 3. When the methane concentration is 5%, very little isobutanol is produced and it is almost undetectable. When the methane concentration is 15%, the isobutanol production is the highest.

[0050] Table 3 Final isobutanol production of conversion systems with different methane concentrations

[0051] Methane concentration (%) 5 10 15 20 25 30 Isobutanol (mg / L) - 55.34 62.19 58.60 49.12 45.74

[0052] It should be noted that the above-described embodiments are only used to explain the present invention and do not constitute any limitation of the present invention. The embodiments describe the present invention and it should be understood that the words used therein are descriptive and explanatory words, rather than limiting words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention.

Claims

1. A method for preparing isobutanol by converting methane using microorganisms, characterized in that: The method comprises mixing lactococci that have reached a stable growth phase with methanophilic bacteria in a volume ratio of (5-30):100 and then using the mixture to convert methane to prepare isobutanol.

2. The method according to claim 1, characterized in that The conditions for preparing isobutanol by converting methane using microorganisms are: methane concentration is 10-30%, conversion temperature is 25-35° C., and conversion period is 2-7 days.

3. The method according to claim 2, characterized in that In the process of converting methane to prepare isobutanol, the pH value of the culture medium for the mixed culture of Lactococcus lactis and methanophilic bacteria is controlled to be 7-10, and the rotation speed of the mixed culture is 50-300 rpm.

4. The method according to any one of claims 1 to 3, characterized in that The lactococcus lactis is selected from one or more bacteria that can produce ketoisovalerate decarboxylase.

5. The method according to claim 4, characterized in that The method for culturing Lactococcus lactis that reaches the stable growth phase is as follows: inoculating Lactococcus lactis in a culture medium at an inoculation amount of 8-12%, and then culturing at a culture temperature of 32-38° C., a culture speed of 150-500 rpm, and a pH value of 5.5-7.

5.

6. The method according to any one of claims 1 to 5, characterized in that The methanophilic bacteria are selected from at least one species of the genus Methylomicrobium.

7. The method according to claim 6, characterized in that The method for culturing methanogens that have reached a stable growth phase is as follows: inoculating methanogens in a culture medium at an inoculation rate of 8-12%, and then culturing at a culture temperature of 25-35°C, a culture speed of 150-500rpm, a pH value of 8.5-9.5, and a methane concentration of 15-25%.

8. The method according to claim 5 or 7, characterized in that: The carbon source of the culture medium is selected from at least one of methane, methanol and glucose; and / or the nitrogen source of the culture medium is selected from at least one of potassium nitrate, peptone and beef extract.

9. The method according to any one of claims 1 to 8, characterized in that Control Lactococcus lactis and methanogens to reach the stable growth phase at the same time.

10. Use of the method according to any one of claims 1 to 9 in a biofuel production process.

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