Alkali-resistant and high-temperature-resistant methanogenic archaea, culture medium and screening method

Abstract

The present invention provides an alkali-resistant and high-temperature-resistant methanogenic archaeon, a culture medium and a screening method. The alkali-resistant and high-temperature-resistant methanogenic archaeon is named MZD129, which has been deposited in the General Microbiological Center of 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. 41192 and a classification name of Methanoculleus bourgensis The bacteria has the characteristics of high methane production, alkali resistance, high temperature resistance and can grow in a simple synthetic culture medium. The invention effectively overcomes the shortcomings of traditional methanogenic archaea that are difficult to cultivate and have harsh environmental conditions, while realizing the resource utilization of waste and solving the problem of the difficulty in cultivating methanogenic archaea.

Description

Technical Field

[0001] The invention belongs to the field of biological environment technology, and specifically relates to an alkali-resistant and high-temperature-resistant methanogenic archaeon, a culture medium and a screening method. Background Art

[0002] Methanogenic archaea are a class of single-celled archaeons that are often considered to be one of the early forms of life because they have similar characteristics to prokaryotes and eukaryotes. Due to their evolutionary importance and their research value on the origin of life and genome evolution, researchers are interested in the cultivation and study of archaea. However, isolating and culturing pure methanogenic archaea from nature is a challenging task for the following reasons:

[0003] Extreme living environment: Many archaea live in extreme environments, such as high temperature, high salinity, high pressure, etc. These environments may be fatal to the cultivation of bacteria and fungi, thus increasing the difficulty of isolating and culturing archaea from nature.

[0004] Lack of appropriate culture conditions: Archaea may have different culture requirements than those we typically use to culture bacteria and fungi. For example, some archaea may require specific nutrients, atmospheric conditions, or media components to grow.

[0005] Contamination risk: When archaea are isolated in nature, they are often contaminated by other microorganisms. These contaminants may interfere with the cultivation of archaea, making it difficult to purify and identify them.

[0006] Lack of effective isolation methods: Archaea may have low population densities in nature, making it more difficult to isolate archaea from environmental samples. Summary of the invention

[0007] In view of the problems existing in the prior art, the purpose of the present invention is to provide an alkali-resistant and high-temperature resistant methanogenic archaeon, a culture medium and a screening method. The strain can produce methane under the conditions of pH 10 and 50 °C, making great contributions to my country's energy, biology, environmental undertakings and national strategic significance.

[0008] In order to achieve the above-mentioned invention object, the present invention adopts the following technical scheme:

[0009] The present invention provides an alkali-resistant and high-temperature-resistant methanogenic archaeon, named MZD129, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration (abbreviated as: CGMCC) 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. 41192, and the classification name is Methanoculleus bourgensis .

[0010] Furthermore, the nucleotide sequence of 16S rDNA of the alkali-resistant and thermostable methanogenic archaea is shown in SEQ ID No.1.

[0011] Furthermore, the methanogenic metabolism temperature of the alkali-resistant and high-temperature-resistant methanogenic archaea is 35~50 °C, and the methanogenic metabolism pH is 5.5~10.

[0012] The present invention also provides a screening culture medium for the above-mentioned alkali-resistant and high-temperature-resistant methanogenic archaea, wherein the screening culture medium comprises: 0.2 g NH4Cl, 1 g sodium formate, 1 g sodium acetate, 0.1 g MgSO4•7H2O, 0.1 g K2HPO4•3H2O, 1 g NaHCO3, 0.05 g CaCO3, 0.01 g FeSO4•7H2O, 5 mL DSM medium320 TRACE, 0.2 g sodium sulfide nonahydrate, 495 mL H2O, and a pH value of 7.0-8.0.

[0013] The present invention also provides an MZD129 culture medium for culturing the above-mentioned alkali-resistant and thermostable methanogenic archaea, wherein the MZD129 culture medium comprises: 2 mL of methanol, 1 g of sodium acetate, 1 g of NaCl, 2 g of sodium formate, 0.2 g of cysteine, 1 g of sodium bicarbonate, 498 mL of distilled water, 10 mL of vitamins, 0.25 mL of anaerobic indicator and 1 mL of trace metal solution.

[0014] The present invention also provides a method for preparing the above-mentioned MZD129 culture medium, comprising:

[0015] a) Add methanol, sodium acetate, NaCl, sodium formate, sodium bicarbonate, distilled water, vitamins, anaerobic indicator and trace metal solution into a beaker in sequence;

[0016] b) Blow high-purity nitrogen gas into the MZD129 culture medium for 30 minutes to replace the dissolved oxygen inside, and then add cysteine;

[0017] c) Dispense the MZD129 culture medium into individual anaerobic test tubes, blow nitrogen gas into the test tubes for 10 minutes, and then sterilize them in an autoclave at 121°C for 20 minutes.

[0018] Preferably, the anaerobic indicator is a 0.1% resazurin solution; the vitamin is a vitamin solution of DSMZ Medium 141; and the trace metal solution is a trace metal solution of DSMZ Medium 320.

[0019] The preparation method of the MZD129 culture medium can prepare a completely anaerobic liquid test tube culture medium (0.1% resazurin is used as an anaerobic indicator, red means trace oxygen is contained, and colorless means no oxygen), and the anaerobic liquid culture medium test tubes are all colorless. The preparation method improves the deoxygenation effect of the culture medium, can effectively improve the efficiency of anaerobic culture medium preparation, improve the survival rate of methanogenic archaea, and can also reduce the influence of trace oxygen on the methanogenic metabolism of methanogenic archaea.

[0020] The present invention further provides a method for screening the above-mentioned alkali-resistant and high-temperature-resistant methanogenic archaea, comprising:

[0021] (1) First, take the supernatant of fresh food waste sample and add it to MZD129 medium, culture at 38 °C for 7-10 days; MZD129 medium contains: 2 mL of methanol, 1 g of sodium acetate, 1 g of NaCl, 2 g of sodium formate, 0.2 g of cysteine, 1 g of sodium bicarbonate, 498 mL of distilled water, 10 mL of vitamins, 0.25 mL of anaerobic indicator and 1 mL of trace metal solution;

[0022] (2) Inoculate the culture medium enriched for 7-10 days into a new screening medium at a 10% inoculum size and culture at 38°C for another 7-10 days; the screening medium contains: 0.2 g NH4Cl, 1 g sodium formate, 1 g sodium acetate, 0.1 g MgSO4•7H2O, 0.1 g K2HPO4•3H2O, 1 g NaHCO3, 0.05 g CaCO3, 0.01 g FeSO4•7H2O, 5 mL DSM medium 320TRACE, 0.2 g sodium sulfide nonahydrate, 495 mL H2O, pH 7.0-8.0;

[0023] (3) The culture medium after the initial enrichment screening was diluted and spread on the solid culture medium inside the Hungate roller tube, and cultured in a 38 °C incubator. After 21 days of culture, the dominant colonies were selected and placed in test tubes containing 10 mL of liquid screening culture medium. Deoxygenated ampicillin and streptomycin were added to each test tube to ensure that the final antibiotic concentration in each test tube was 50 μg / mL. After 7 days of enrichment culture, the tubes were rolled and enriched again until the pure strain MZD129 was obtained. The solid culture medium of the Hungate roller tube was obtained by adding 15-20 g / L of agar to the screening culture medium and sterilizing it.

[0024] Preferably, the volume ratio of fresh food waste sample supernatant to MZD129 culture medium is 1:9.

[0025] Preferably, the anaerobic indicator is a 0.1% resazurin solution; the vitamin is a vitamin solution of DSMZ Medium 141; and the trace metal solution is a trace metal solution of DSMZ Medium 320.

[0026] The beneficial effects of the present invention are:

[0027] The present invention isolates a new methanogenic archaea from a restaurant kitchen waste biogas station in Huzhou, Zhejiang. The bacterium has the characteristics of high methane production, alkali resistance, high temperature resistance and can grow in a simple synthetic culture medium. The present invention effectively overcomes the shortcomings of traditional methanogenic archaea that are difficult to cultivate and demanding on environmental conditions, while realizing the resource utilization of waste and solving the problem of the difficulty in cultivating methanogenic archaea. It has five strategic benefits for my country:

[0028] Energy development: The discovery of new methanogenic archaea can help the country develop more methane resources and improve the country's energy supply security;

[0029] Environmental protection: Methane is a greenhouse gas that has a significant impact on global climate change. By studying new strains of methanogenic archaea, more effective methods can be found to control and utilize methane emissions, which is conducive to reducing greenhouse gas emissions and protecting the environment;

[0030] Scientific research: The study of new species of methanogenic archaea can promote the country's scientific research in the fields of microbiology and biotechnology, and improve the country's scientific and technological level and competitiveness in related fields;

[0031] Economic development: The discovery of new methanogenic archaea can drive the development of related industries, such as bioenergy industry, biotechnology industry, etc., and promote the development and transformation and upgrading of the national economy;

[0032] International impact: Through the research and application of new methanogenic archaea, the country's international scientific and technological strength and influence will be enhanced, which will help the country occupy a more advantageous position in the global scientific and technological competition. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 strain Methanoculleus bourgensis Fluorescence image (left) and crystal violet staining image (right) of MZD129 colonies.

[0034] Figure 2 strain Methanoculleus bourgensis Phylogenetic tree of MZD129.

[0035] Figure 3 strain Methanoculleus bourgensis Carbon source selection for MZD129.

[0036] Figure 4 strainMethanoculleus bourgensis Antibiotic susceptibility testing of MZD129.

[0037] Figure 5 strain Methanoculleus bourgensis High temperature tolerance test of MZD129.

[0038] Figure 6 strain Methanoculleus bourgensis Acidity and alkalinity tolerance test of MZD129.

[0039] Figure 7 strain Methanoculleus bourgensis Osmosensitivity test of MZD129. DETAILED DESCRIPTION

[0040] 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.

[0041] The methanogenic archaea of ​​the present invention was isolated from the food waste biogas station in Huzhou, Zhejiang Province, named MZD129, 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. 41192, and the classification name is Methanoculleus bourgensis .

[0042] Example

[0043] 1. Methanogenic Archaea Methanoculleus bourgensis Isolation and screening of MZD129

[0044] 1. First, take a fresh sample of kitchen waste slurry and shake it in physiological saline. Take 10 mL of the supernatant and add it to 90 mL of MZD129 culture medium and culture it at 38 °C for 7 days.

[0045] Wherein, the preparation method of MZD129 culture medium comprises:

[0046] a) Add 2 mL of methanol, 1 g of sodium acetate, 1 g of NaCl, 2 g of sodium formate, 1 g of sodium bicarbonate, 498 mL of distilled water, 10 mL of DSMZ Medium 141 vitamin solution (specific specifications can be found at https: / / www.dsmz.de / ), 0.25 mL of 0.1% resazurin solution, and 1 mL of DSMZ Medium 320 trace metal solution (specific specifications can be found at https: / / www.dsmz.de / ) into a 500 mL beaker.

[0047] b) Blow high-purity nitrogen (purity 99.999%) into the MZD129 culture medium for 30 minutes to replace the dissolved oxygen inside, and then add 0.2 g of cysteine;

[0048] c) The MZD129 culture medium was dispensed into individual anaerobic test tubes. The test tubes were blown with high-purity nitrogen for 10 minutes and then sterilized in an autoclave at 121 °C for 20 minutes.

[0049] The preparation method of the MZD129 culture medium can prepare a completely anaerobic liquid test tube culture medium (0.1% resazurin is used as an anaerobic indicator, red means containing trace oxygen, and colorless means no oxygen), and the anaerobic liquid culture medium test tubes are all in a colorless state.

[0050] 2. Inoculate 10% of the culture medium enriched for 7 days into a new 100 mL screening culture medium with sodium acetate and sodium formate as the sole carbon source, and culture again at 38 °C for 7 days.

[0051] Specifically, the screening culture medium comprises: 0.2 g NH4Cl, 1 g sodium formate, 1 g sodium acetate, 0.1 g MgSO4•7H2O, 0.1 g K2HPO4•3H2O, 1 g NaHCO3, 0.05 g CaCO3, 0.01 g FeSO4•7H2O, 5 mL DSM medium320 TRACE, 0.2 g sodium sulfide nonahydrate, 495 mL H2O, and a pH value of 7.0-8.0; 15-20 g / L agar is added to the solid culture medium in the Hungate roller tube (roller tube volume 25 mL, culture medium 5 mL) and sterilized at 121 °C for 20 min.

[0052] 3. Use a disposable syringe to inject the culture medium after the enrichment and initial screening into the Hungate roller tube solid culture medium for roller tube rolling. After the culture medium solidifies, culture it in a 38 °C incubator. After 21 days of culture, select the dominant colonies and put them into test tubes containing 10 mL of liquid screening culture medium. Add deoxygenated ampicillin and streptomycin to each test tube to ensure that the final concentration of antibiotics in each test tube is 50 μg / mL. After 7 days of enrichment culture, roll the tube again and enrich until the pure strain MZD129 is obtained.

[0053] 2. Methanogenic Archaea Methanoculleus bourgensis Identification of MZD129

[0054] Methanoculleus bourgensisMZD129 is a methanogenic archaeon that grows well in a medium containing acetate and formate as the sole carbon source. It takes 21 days for this strain to grow yellow rhombus-shaped colonies in a Hungate roller tube. Since it contains the F420 coenzyme, it can be determined using a fluorescence microscope ( Figure 1 , left); MZD129 is an extremely anaerobic archaeon, which is smaller than common bacteria and is pseudo-spherical ( Figure 1 ,right).

[0055] 1. Different substrate selectivity test: Hydrogen / carbon dioxide, formate, methanol, acetate, and ethylene glycol were used as the sole carbon source, and the substrate concentration was set to 0.1 mol / L. The other components in the culture medium were consistent with those in MZD129 culture medium. Methanoculleus bourgensis MZD129 pure culture was then cultured in an anaerobic incubator at 38 °C for 14 days. Methanoculleus bourgensis Methanogenic characteristics of MZD129.

[0056] The results show that Methanoculleus bourgensis The MZD129 strain can effectively utilize at least four carbon sources, including formate, methanol, acetate, and bicarbonate ( Figure 3 ).

[0057] 2. Experiment on inhibition of methanogenesis by different antibiotics: 50 µg / mL of chlortetracycline, streptomycin, vancomycin, chloramphenicol, penicillin, sulfamethoxazole, and tetracycline inhibitor were added to MZD129 medium, and the isolated Methanoculleus bourgensis MZD129 was cultured in an anaerobic incubator at 38°C for 14 days. Methanoculleus bourgensis Methanogenic characteristics of MZD129.

[0058] The results show that Methanoculleus bourgensis The MZD129 strain was severely sensitive to one antibiotic (chloramphenicol) and moderately sensitive to two antibiotics (vancomycin and sulfamethoxazole). Figure 4 .

[0059] strain Methanoculleus bourgensis The statistical results of substrate selectivity and antibiotic tolerance of MZD129 are shown in Table 1.

[0060] Table 1 Strains Methanoculleus bourgensis Substrate selectivity and antibiotic tolerance of MZD129

[0061]

[0062] 3. Temperature Methanoculleus bourgensisEffect of temperature gradients on the methanogenic performance of MZD129: The liquid culture medium of MZD129 was set at 7 temperatures, namely 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ and 60 ℃, and the isolated Methanoculleus bourgensis MZD129 pure culture was then cultured in an anaerobic incubator at the corresponding temperature for 14 days. Methanoculleus bourgensis Methanogenic characteristics of MZD129.

[0063] The results show that Methanoculleus bourgensis The strain MZD129 can still produce a lot of methane at around 50°C, and the optimal temperature for methane production is around 45°C ( Figure 5 ).

[0064] 4. pH value Methanoculleus bourgensis Effect of MZD129 on methanogenesis: Hydrochloric acid / sodium hydroxide was used to adjust the pH value of the medium at different gradients, and a total of 17 gradients of MZD129 liquid culture medium with pH values ​​of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, and 11.0 were set. Methanoculleus bourgensis MZD129 pure culture was then cultured in an anaerobic incubator at 38 °C for 14 days. Methanoculleus bourgensis Methanogenic characteristics of MZD129.

[0065] The results show that Methanoculleus bourgensis The lowest pH that the MZD129 strain can tolerate is 5.5, and the highest pH that it can tolerate is 10.5 ( Figure 6 ).

[0066] 5. Methane production test at different salt concentrations: MZD129 medium was adjusted with NaCl and KCl to have a total of 10 salinity gradients of 0.5%, 0.7%, 1%, 1.2%, 2%, 2.4%, 3%, 3.5%, 5% and 10%, and the isolated Methanoculleus bourgensis MZD129 pure culture was then cultured in an anaerobic incubator at 38 °C for 14 days. Methanoculleus bourgensis Methanogenic characteristics of MZD129.

[0067] The results showed that the methanogenic performance of Methanoculleus bourgensis MZD129 strain was significantly inhibited when the salinity reached 3%, and completely inhibited when it reached 10% ( Figure 7 ).

[0068] strain Methanoculleus bourgensis The results of the physiological and biochemical characteristics of MZD129 are shown in Table 2:

[0069] Table 2 Strains Methanoculleus bourgensis Physiological and biochemical characteristics of MZD129

[0070]

[0071] The results of physiological and biochemical identification of strain LYC were closest to the characteristics of M. brevis. The phylogenetic tree constructed by the maximum likelihood method was as follows: Figure 2 shown.

[0072] The methanogenic archaea strain Methanoculleus bourgensis Identification method of MZD129 at the molecular level: The 16S rDNA partial sequence (SEQ ID No.1) of strain MZD129 was amplified using universal primers Met-86F (5′- GCTCAGTAACACGTGG-3′, as shown in SEQ ID No.2) and Met-1340R (5′-CGGTGTGTGCAAGGAG-3′, as shown in SEQ ID No.3). The obtained fragment was sequenced, and the 16S rDNA sequence was compared with the NCBI database. At the molecular level, strain MZD129 was identified as belonging to the species Methanobacterium breisi.

[0073] 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. An alkali-resistant and thermotolerant methanogenic archaeon, named MZD129, was deposited in the General Microbiological Center of the 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. 41192, and the classification name is Methanoculleus bourgensis .

2. The alkali-resistant and thermostable methanogenic archaea according to claim 1, characterized in that: The nucleotide sequence of 16S rDNA of the alkali-resistant and thermostable methanogenic archaea is shown in SEQ ID No.

1.

3. The alkali-resistant and thermostable methanogenic archaea according to claim 1, characterized in that: The methanogenic metabolism temperature of the alkali-resistant and high-temperature-resistant methanogenic archaea is 35-50° C., and the methanogenic metabolism pH is 5.5-10.

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