A methanogenic archaea that metabolizes bicarbonate, culture medium and culture method

Abstract

The present invention relates to the field of microbial culture technology, and provides a methanogenic archaeon that metabolizes bicarbonate, a culture medium and a culture method. The methanogenic archaeon that metabolizes bicarbonate is named MZD130B, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Committee on May 14, 2024. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 41193 and a classification name of Methanobacterium loongson This strain is a new type of methanogenic archaea that metabolizes bicarbonate. The present invention provides a new cultivation scheme by using different carbon sources and hydrogen environments to promote the growth of Methanobacterium longzhizi and methane production, thereby providing an efficient and environmentally friendly technical solution for methane bioproduction.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a methanogenic archaea capable of metabolizing bicarbonate, a culture medium and a culture method. Background Art

[0002] As a class of strictly anaerobic archaea, most methanogenic archaea require an anaerobic, low-sulfur environment to grow, and their growth rate is extremely slow, which makes it extremely difficult to obtain their pure culture.

[0003] Methanobacterium truncatum based on a novel hydrogenotrophic metabolic pathway Methanobacterium loongson It grows very slowly and is difficult to culture pure bacteria. It is necessary to maintain a high concentration of carbon dioxide and a neutral environment in the culture medium. It is difficult to produce methane in acidic and alkaline environments or in an environment without bicarbonate. Summary of the invention

[0004] In view of the problems existing in the prior art, the purpose of the present invention is to provide a methanogenic archaeon that metabolizes bicarbonate, a culture medium and a cultivation method. The strain is a new type of methanogenic archaeon that metabolizes bicarbonate. The present invention greatly improves its growth rate by optimizing its cultivation scheme.

[0005] In order to achieve the above-mentioned invention object, the present invention adopts the following technical scheme:

[0006] The present invention provides a methanogenic archaeon that metabolizes bicarbonate, named MZD130B, which has been deposited in the General Microbiological Center of China Microbiological Culture Collection Committee on May 14, 2024, with the deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No. 41193, and the classification name is Methanobacterium loongson .

[0007] Furthermore, the nucleotide sequence of the methanogenic archaea 16S rDNA is shown in SEQ ID No.1.

[0008] The present invention also provides a culture medium for methanogenic archaea that metabolizes bicarbonate, including: 1.0 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.5 g / L ammonium chloride, 0.2 g / L calcium chloride dihydrate, 5 g / L sodium bicarbonate; 1 ‰ v / v of 0.1% resazurin solution, 1 ‰ v / v of vitamin solution, and 1 ‰ v / v of trace element solution. Wherein, "v / v" represents the volume ratio of each solution in the culture medium. The culture medium is a basic culture medium and can be used for the purification and cultivation of the methanogenic archaea MZD130B.

[0009] Preferably, the culture medium is additionally supplemented with sodium bicarbonate, and cysteine ​​salts are added after nitrogen deoxygenation. The culture medium is a specific culture medium and can be used to determine the optimal methanogenic conditions of the methanogenic archaeon MZD130B.

[0010] More preferably, the molar concentration of the additional sodium bicarbonate added in the culture medium is 0.3 mol / L; the amount of cysteine ​​salt added in the culture medium is 0.3-0.4 g / L (the maximum recommended amount is 0.4 g / L).

[0011] The present invention further provides a method for culturing methanogenic archaea that metabolizes bicarbonate, comprising: inoculating the methanogenic archaea into the above culture medium, and culturing at 35-45° C. for 10 days under anaerobic conditions, more preferably culturing at 40° C. for 10 days under anaerobic conditions.

[0012] Preferably, the method further comprises adding a methanogenic stimulator to the culture medium, wherein the methanogenic stimulator comprises yeast powder, sodium acetate and coenzyme M.

[0013] More preferably, the methanogenic metabolism temperature is 35-55 °C, and the methanogenic metabolism pH is 6.5-10.

[0014] More preferably, the molar concentrations of sodium acetate and coenzyme M in the culture medium are both 0.1 mol / L, and the amount of yeast powder added to the culture medium is 1 g / L.

[0015] More preferably, the methanogenic metabolism temperature is 40°C and the methanogenic metabolism pH is 7.5.

[0016] The beneficial effects of the present invention are:

[0017] The present invention provides a new species of hydrogenotrophic methanogenic archaea capable of utilizing substances other than carbon dioxide, named MZD130B. The species is a new type of methanogenic archaea that metabolizes bicarbonate, and the culture conditions of methanogenic archaea are optimized through different carbon sources and hydrogen environments, thereby increasing methane production. The present invention is environmentally friendly and efficient, and is suitable for large-scale methane bioproduction. Furthermore, it can contribute to China's biological resources, environment and energy undertakings, and fill the gap in the utilization of bicarbonate by methanogenic archaea in the world. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 strains Methanobacterium loongson Evolutionary tree of MZD130B.

[0019] Figure 2 strains Methanobacterium loongson Carbon source selection experiment of MZD130B.

[0020] Figure 3 strains Methanobacterium loongsonExperiments on the optimal pH, optimal temperature and methanogenic stimulating factors of MZD130B.

[0021] Figure 4 strains Methanobacterium loongson Growth curve of MZD130B before and after optimization. DETAILED DESCRIPTION

[0022] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0023] The methanogenic archaea of ​​the present invention was isolated from the food waste biogas station in Huzhou, Zhejiang Province, named MZD130B, and was deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Committee on May 14, 2024. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No. 41193, and the classification name is Methanobacterium loongson .

[0024] Example

[0025] 1. Methanogenic Archaea Methanobacterium loongson Species identification and substrate selection experiments of MZD130B

[0026] A pure culture of a methanogenic microorganism was obtained by the Hungate tube method and named MZD130B. The 16S rRNA gene was amplified and sequenced using Met86F / Met1340R as primers. After successful sequencing, BLAST species comparison was performed on NCBI and EzBiocloud. The final comparison results were consistent with the currently discovered species. Methanobacterium kanagiense and Methanobacterium petrolearium The highest similarity is less than 98.7% (16S rRNA similarity of 98.7% is used as the critical value for species identification), Methanobacterium The similarity of species under the genus Methanobacterium is between 95% and 97.6%, such as Figure 1 Therefore, MZD130B belongs to a new species of Methanobacterium and is named Methanobacterium loongson MZD130B.

[0027] Prepare basal culture medium: (1) Weigh 1.0 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.5 g ammonium chloride, 0.2 g calcium chloride dihydrate, and 5 g sodium bicarbonate; (2) Dissolve the above ingredients in 1000 mL pure water; (3) Add 1 mL of 0.1% resazurin solution, 1 mL of vitamins from DSMZ medium 141, and 1 mL of trace element solution from DSMZ medium 320; (4) After mixing well, dispense the culture medium into 1 L flasks.

[0028] Preparation of specific culture medium: (1) Add different substrates (sodium formate, methanol, carbon dioxide, sodium bicarbonate, sodium carbonate, sodium acetate, ethanol and ethylene glycol) to 100 mL of basal culture medium; (2) After deoxygenation by nitrogen flow for 20 minutes, add 0.04 g of cysteine ​​salt; (3) Prepare culture medium with different carbon sources (C sources) according to the following formula:

[0029] Medium A1: Use C source containing only sodium formate, hydrogen (after filling the test tubes, extract 5 mL of the test tube headspace, inject 5 mL of hydrogen, and deoxygenate the syringe in the anaerobic bottle first), and divide into 5 test tubes;

[0030] Medium A2: Use C source containing only methanol, and the rest is the same as above;

[0031] Medium B1: Use C source containing only carbon dioxide, and the rest is the same as above;

[0032] Medium B2: Use C source containing only sodium bicarbonate, and the rest is the same as above;

[0033] Medium B3: Use C source containing only sodium carbonate, and the rest is the same as above;

[0034] Medium C1: Use C source containing only sodium acetate, and the rest is the same as above;

[0035] Medium C2: Use C source containing only anhydrous ethanol, and the rest is the same as above;

[0036] Culture medium C3: Use C source containing only ethylene glycol, and the rest is the same as above.

[0037] Methanobacterium truncatum MZD130B was inoculated (inoculation amount was 10%) into the above culture medium and cultured at 40°C for 10 days under anaerobic conditions to measure the methane production in its headspace.

[0038] The amount of the above carbon source substances is 0.3 mol / L, and the final results are as follows Figure 2 Among these eight substrates, Methanobacterium spp. MZD130B only utilized sodium bicarbonate for methanogenic metabolism.

[0039] 2. Methanogenic Archaea Methanobacterium loongsonAcid-base tolerance test, temperature tolerance test and methanogenic stimulating factor test of MZD130B

[0040] Preparation of specific culture medium: (1) Add different methanogenic stimulants (sodium acetate, sodium butyrate, yeast powder and coenzyme M) to 100 mL of the basal culture medium prepared in step 1; or add an acid-base regulator to prepare a culture medium with different pH gradients; (2) After deoxygenation by nitrogen flow for 20 minutes, add 0.04 g of cysteine ​​salt; (3) Prepare a culture medium containing methanogenic stimulants (the carbon source used only contains sodium bicarbonate, and the amount of carbon source material is 0.3 mol / L) according to the following formula:

[0041] Sodium acetate medium: The stimulus is sodium acetate, measure its pH and titrate it to pH 7.5, hydrogen (after filling the test tubes, extract 5 mL of the test tube headspace and inject 5 mL of hydrogen. The syringe is first deoxygenated in the anaerobic bottle), and fill it into 5 test tubes;

[0042] Sodium butyrate medium: the stimulus is sodium butyrate, and the rest is the same as above;

[0043] Yeast powder culture medium: the stimulator is yeast powder, and the rest is the same as above;

[0044] Coenzyme M culture medium: The stimulator is coenzyme M, and the rest is the same as above.

[0045] Methanobacterium truncatum MZD130B was inoculated (with the same inoculum size) into the above culture medium and cultured at 40°C for 10 days under anaerobic conditions to measure the methane production in the headspace.

[0046] In the different temperature experiment, the methane production in the headspace was measured by cultivating the plants at different gradient temperatures of 30-55℃ for 10 days; in the different pH experiment, the methane production in the headspace was measured by cultivating the plants at different gradient pH of 5.0-11.0 for 10 days.

[0047] The amount of the above stimulant substances is 0.1 mol / L (the amount of yeast powder added is 1 g / L), and the final results are as follows Figure 3 As shown in the figure, yeast extract, acetic acid and coenzyme M can all be used as growth stimulators for the bacterium, among which yeast extract has the strongest promoting effect. Among these 8 substrates, Methanobacterium longzhizi MZD130B only utilizes sodium bicarbonate ( Figure 2 Methanobacterium longzhizi MZD130B is a mesohydrogenotrophic methanogenic archaeon of the genus Methanobacterium with an optimum methanogenic temperature of 40°C and an optimum methanogenic pH of 7.5.

[0048] 3. Methanogenic Archaea Methanobacterium loongson MZD130B cultivation optimization experiment.

[0049] Combining steps one and two, we end up with Methanobacterium loongsonThe optimization plan for cultivating MZD130B is shown in Table 1.

[0050] Table 1 Methanobacterium loongson MZD130B cultivation optimization plan

[0051]

[0052] Before and after optimization Methanobacterium loongson The growth curve of MZD130B is as follows Figure 4 As shown in Table 1. Methanobacterium loongson The growth rate of MZD130B increased by an average of 2.26 times.

[0053] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A methanogenic archaeon that metabolizes bicarbonate, characterized in that The name is MZD130B, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on May 14, 2024. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is: CGMCC No. 41193, and the classification name is Methanobacterium loongson The nucleotide sequence of the methanogenic archaea 16S rDNA is shown in SEQ ID No.1.

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