A method for in situ enrichment of sulfate-dependent anaerobic methane-oxidizing archaea

Abstract

The present invention belongs to the field of environmental protection technology, and specifically relates to a method for in situ enrichment of sulfate-dependent anaerobic methane-oxidizing archaea. The method mainly comprises the following steps: placing a specific adsorption material in an in situ sulfate-methane transition zone sediment environment for a period of time and then washing it to obtain enriched sulfate-dependent anaerobic methane-oxidizing archaea. The in situ enrichment effect of the above method was further revealed by labeled isotope method and DNA sequencing. This method overcomes the non-directionality of in situ culture, and also overcomes the shortcomings of the traditional enrichment culture of anaerobic methane-oxidizing archaea, such as the long cycle and low success rate. It can provide a new reference approach for the study of methane emission hotspot ecosystems, and is conducive to promoting the development of methane emission reduction technology in related ecosystems.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental protection, and more specifically, relates to a method for in-situ enrichment of sulfate-dependent anaerobic methane-oxidizing archaea. Background Art

[0002] Continued emissions of greenhouse gases have led to a range of environmental problems, including global warming, changes in precipitation patterns, changes in groundwater levels, ocean acidification, changes in soil conditions, and a series of extreme climate events. 4 ) is second only to carbon dioxide (CO 2 ) is the second largest greenhouse gas in terms of global warming potential. 2 28 times of that of the atmosphere, contributing 20% ​​to global warming, and its emissions are increasing year by year, and are expected to play an important role in future climate change. Microorganisms that perform metabolic functions in nature mainly include methanogenic archaea, aerobic methanotrophs (methanophiles), anaerobic methanotrophs (ANME) and bacteria (NC10). Among them, ANME has not yet been cultured in pure form, and the mechanism of anaerobic methane oxidation (AOM) and the microorganisms involved are still unclear. The AOM process driven by ANME is an important methane sink. It is estimated that more than half of the methane produced globally is oxidized to carbon dioxide by aerobic and anaerobic methane-oxidizing microorganisms before reaching the atmosphere, and in the deep sea, AOM coupled with sulfate reduction processes may consume up to 90% of the methane released from sediments.

[0003] The AOM pathway lacks pure culture of key microorganisms, and the AOM process is mainly explored based on enrichments. AOM activity experiments estimated that the doubling time of ANME is 2 to 7 months, revealing that ANME utilizes CH 4 Slow growth. The coupling of AOM and sulfate reduction was first discovered in marine sediments. The main limitations of ANME cultivation that mediates AOM-coupled sulfate reduction (SR) are as follows: (1) Among the AOM with known electron acceptors, the energy generated by AOM-coupled SR under standard thermodynamic conditions is the lowest; (2) ANME grows very slowly; (3) the solubility concentration of the substrate methane at 1 atmosphere is limited (1.4 mmol / L); (4) the SR product sulfide inhibits the growth of ANME. These have brought challenges to the cultivation and isolation of ANME.

[0004] At present, there are two main ANME enrichment methods: reactor enrichment and culture bottle enrichment. Both of them increase the number of target archaea by introducing nutrients or environmental conditions for the growth of specific archaea, and belong to non-in situ directional enrichment methods based on original sediments. The advantage of the above method is high selectivity, and the main disadvantage is that the cycle is long, often measured in years, and changes in environmental conditions may cause the enrichment of target microorganisms to fail. For example, China's patent application CN103275868A discloses a co-cultivation method of a highly efficient denitrification anaerobic methane oxidation and anaerobic ammonia oxidation mixed microbial system, which uses an SMBR reactor for three-stage enrichment for a total of 480 days, which takes a long time. In situ cultivation is also a solution for cultivating difficult-to-cultivate microorganisms. The so-called in situ cultivation refers to the use of diffusion chambers to allow uncultured microorganisms to grow in a natural environment. The biggest advantage of in situ cultivation is that it removes the culture medium component restrictions of traditional solid separation cultivation, and its disadvantages are also obvious: lack of selectivity, and it is difficult to directly separate the target microorganisms from the environment. Therefore, there is an urgent need for a method for effectively and quickly enriching anaerobic methane oxidizing archaea. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing ANME enrichment methods, which are mainly non-in situ directional enrichment methods based on original sediments, and have long cycles and are easily affected by the environment, while the in situ culture lacks selectivity and it is difficult to directly separate the target microorganisms from the environment. A method for in situ enrichment of sulfate-dependent anaerobic methane-oxidizing archaea is provided.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] The present invention provides a method for in-situ enrichment of sulfate-dependent anaerobic methane-oxidizing archaea (ANME), comprising the following steps: placing an adsorption material in a sulfate-methane transition zone sediment environment for full contact, collecting the adsorption material, washing it, and collecting the washed adsorption material;

[0008] wherein the sulfate-dependent anaerobic methane-oxidizing archaea is attached to the washed adsorption material;

[0009] The sulfate-dependent anaerobic methane-oxidizing archaea is ANME-1b; the specific type of archaea is determined by three-generation full-length 16S rDNA sequencing;

[0010] The adsorption material is a material having the functions of releasing sulfate radicals and adsorbing microorganisms.

[0011] In order to solve the problem that in-situ culture lacks selectivity and is difficult to directly separate the target microorganisms from the environment, the present invention creatively selects materials that can release sulfate and have microbial adsorption function as ideal materials for in-situ enrichment of ANME. Such materials have a layered structure and interlayer ions, and are preferably sulfate anion type magnesium aluminum layered double metal hydroxide (hydrotalcite). Taking hydrotalcite as an example, in the layered structure of hydrotalcite, Mg(OH) 2 Mg 2+ Can be Al within a certain range 3+ The substitution makes the layers positively charged, the interlayer sulfate ions are balanced with the positive charges on the layers, and the interlayer ions of the hydrotalcite are exchangeable. For example, the sulfate between the layers of the sulfate anion type magnesium aluminum hydrotalcite can be replaced by carbonate, thereby achieving the purpose of slow release of sulfate. In the wetland environment, carbonate is almost everywhere, and the positive charge of the layers and the negative charge of the microbial membrane attract each other and have adsorption. Therefore, the hydrotalcite material can achieve the functions of slow release of sulfate and microbial adsorption. Placing it in the sediment is equivalent to artificially creating an environmental hotspot to enrich sulfate-preferring microorganisms in a targeted manner.

[0012] Furthermore, the washing is to remove impurities attached to the surface of the hydrotalcite.

[0013] Furthermore, the adsorption material is in full contact with the sulfate-methane transition zone sediment environment through a carrier. The specific process is: placing the adsorption material in a mesh bag with an opening at the top, and fixing the mesh bag in the carrier, burying the carrier containing the adsorption material in the sulfate-methane transition zone sediment environment, and the burial depth is ≥50cm, so that most of the sediment can penetrate into the carrier and can be exchanged with the surrounding environment medium.

[0014] Furthermore, the carrier is a container with holes on the side and an opening on the top, preferably an acrylic cylindrical container with holes on the side and an opening on the top.

[0015] Furthermore, the appearance characteristics of the acrylic cylindrical container are as follows: bottom diameter 12 cm, height 13 cm, side holes with a hole diameter of 3 cm, evenly distributed, a total of 3 rows and 9 columns of 27 holes, the bottom is sealed, and the top is open.

[0016] Furthermore, the method further comprises anaerobic culture, wherein the washed adsorption material is placed in a culture solution and cultured in a dark environment under the condition of methane. The anaerobic culture can further enhance the effect of in-situ enrichment of sulfate-dependent anaerobic methane-oxidizing archaea.

[0017] Preferably, the adsorbent material is hydrotalcite containing sulfate.

[0018] Furthermore, the preparation method of the hydrotalcite comprises the following steps: mixing a metal salt solution containing magnesium ions and aluminum ions with an alkaline reagent, allowing the mixture to react fully under protective atmosphere conditions, filtering after the reaction is complete, washing, drying and sieving the precipitate, and obtaining the hydrotalcite.

[0019] Preferably, the particle size of the hydrotalcite is ≥0.15 mm.

[0020] Furthermore, the magnesium ions are from a magnesium salt or a hydrate thereof, and the magnesium salt is selected from magnesium sulfate.

[0021] Furthermore, the aluminum ions are from aluminum salts or their hydrates, and the aluminum salts are selected from aluminum sulfate.

[0022] Preferably, the molar ratio of the magnesium ions to the aluminum ions is (2-3):1.

[0023] Preferably, the alkaline agent is sodium hydroxide or potassium hydroxide.

[0024] Furthermore, the gas of the protective atmosphere condition is selected from nitrogen, argon, neon or helium, preferably nitrogen.

[0025] Furthermore, the sufficient contact time is 60 to 90 days, preferably 70 to 80 days, and more preferably 74 days.

[0026] Preferably, the temperature of the anaerobic culture is 20-25°C.

[0027] Preferably, the anaerobic culture time is ≥ 7 days.

[0028] Preferably, the volume percentage of methane in the condition in which methane exists is 3-5%, more preferably 4%.

[0029] Furthermore, the culture solution is a mineral culture solution, and the components of the mineral culture solution include artificial seawater, alkaline trace elements, acidic trace elements, vitamins, resazurin and sulfate.

[0030] Furthermore, the sulfate radical comes from sodium sulfate.

[0031] Furthermore, the salinity of the culture solution is 1 to 3 ppt, and the salinity is adjusted according to the sediment environment of the sulfate-methane transition zone, preferably 2 ppt.

[0032] Furthermore, the artificial seawater contains NaC, KCl, MgCl 2 6H 2 O、NH 4 Cl, CaCl 2 ·2H 2 O.KH 2 PO 4 .

[0033] Furthermore, the trace elements in the alkaline trace elements are selected from SeO 2 、Na 2 WO 4 ·2H 2 O、Na 2 MoO 4 ·2H 2 O; the alkalinity of the alkaline trace elements is provided by NaOH.

[0034] Furthermore, the trace elements in the acidic trace elements are selected from FeCl 2 ·4H 2 O、ZnCl 2 , H 3 BO 3 ; The acidity of the acidic trace elements is provided by HCl.

[0035] Preferably, the mass volume ratio of the washed adsorption material to the culture solution is 1:2-4 g / mL.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The present invention discloses a method for in-situ enrichment of sulfate-dependent anaerobic methane-oxidizing archaea. The method mainly comprises the following steps: placing a specific adsorption material in an in-situ sulfate-methane transition zone sediment environment for a period of time, and then washing to obtain enriched sulfate-dependent anaerobic methane-oxidizing archaea. The in-situ enrichment effect of the above method was further revealed by labeled isotope method and DNA sequencing. This method overcomes the non-directionality of in-situ culture, and also overcomes the shortcomings of traditional enrichment and culture of sulfate-dependent anaerobic methane-oxidizing archaea, such as long cycle and low success rate. It can provide a new reference approach for the study of methane emission hotspot ecosystems, and is conducive to promoting the development of methane emission reduction technology in related ecosystems. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Statistical chart of the physical and chemical indicators of sediments in the experimental group and the control group.

[0039] Figure 2 Statistical graph of the methane oxidation activity of the in situ sediment community in the control group and the hydrotalcite-attached microbial community in the experimental group.

[0040] Figure 3 Statistical diagram of the relative abundance of the in situ sediment community in the control group and the sulfate-dependent ANME archaeal community attached to hydrotalcite in the experimental group.

[0041] Figure 4Statistical diagram of the relative abundance of ANME-1b in the in situ sediment community in the control group and hydrotalcite-attached ANME-1b in the experimental group. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0043] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0044] Example 1 A method for in situ enrichment of sulfate-dependent anaerobic methane-oxidizing archaea

[0045] A method for in situ enrichment of sulfate-dependent anaerobic methane-oxidizing archaea comprises the following steps:

[0046] S1. Synthesize a material that releases sulfate slowly and has microbial adsorption function. The material is sulfate anion type magnesium aluminum layered double metal hydroxide, referred to as hydrotalcite (LDH). The synthesis method is coprecipitation. Ultrapure water is used as the solvent to prepare 14mM MgSO 4 and 3 mM Al 2 (SO 4 ) 3 200 mL of mixed salt solution was added to obtain a salt solution with a total cation concentration of 20 mM (Mg 2 + :Al 3+ The ion concentration ratio is about 2:1), 800mL of 0.2M NaOH is prepared as the alkaline solution, and 1L of ultrapure water is prepared additionally. The containers all use conical flasks to reduce the contact surface with the air. All three liquids are nitrogen-blown for 30 minutes. Under high-speed stirring, the salt solution is quickly added to the alkaline solution and reacted for 30 minutes, during which all liquids are kept nitrogen-blown. Use a medium-speed qualitative filter paper with a diameter of 150cm for filtration, wash the precipitated product with ultrapure water purged with nitrogen, and stir with a glass rod to clean the alkali solution (2-3 times). Use a vacuum heating drying method to dry the precipitate at 80°C, scrape it off the filter paper, and pass it through a 100-mesh (pore size 0.15mm) sieve, and select sulfate anion-type magnesium aluminum hydrotalcite with a particle size greater than 0.15mm for subsequent experiments.

[0047] S2. Select the methane emission hotspot ecosystem to be studied, place the hydrotalcite in the sulfate reduction area in the in-situ sediment for about two months, and collect in-situ sediment samples, as well as sediments near the LDH and attached samples. The selected methane emission hotspot ecosystem is a coastal mangrove ecosystem, located in the Sonneratia apetala mangrove forest in Hualong Town, Panyu District, Guangzhou. The sulfate content of mangrove sediments decreases with increasing depth in the range of 0-50cm, and fluctuates steadily below 50cm. Therefore, the burial depth of 50cm was selected.

[0048] Specifically, an acrylic cylindrical container with a side hole and an open top was used as a carrier. The bottom diameter was 12 cm, the height was 13 cm, and the side was perforated with a hole diameter of 3 cm, which was evenly distributed. There were 27 holes in 3 rows and 9 columns, the bottom was sealed, and the top was open. The particle size of mangrove sediments is generally less than 250 μm, so a 60-mesh (pore size 250 μm) top-opened nylon cylindrical mesh bag was placed inside the carrier and fixed to the carrier with a nylon mesh line, so that most of the sediments can penetrate and exchange with the surrounding environment medium. The control group only placed the carrier and the mesh bag to obtain in-situ sediments; the experimental group added 40g of hydrotalcite wrapped in a 100-mesh nylon mesh, and the mesh was tied with a nylon mesh line to prevent the hydrotalcite from overflowing. Each pair of experimental groups and control groups was separated by about 1m, and each repeated group was separated by more than 5m, with a total of five repeated groups. The samples were taken out after 74 days of burial, and the mesh bag sediments and hydrotalcite were placed in self-sealing bags and immediately placed in 7L anaerobic tanks for storage. In the anaerobic operation box, take out the hydrotalcite wrapped in nylon mesh and wash it with a sterilized beaker and ultrapure water until the water is clear. A portion of the washed LDH is stored at -80°C for DNA extraction, and a portion is still stored at room temperature in the anaerobic tank for isotope experiments. All physical and chemical index measurements are based on the sediments in the mesh bags of the experimental group and the control group. The isotope experiment is based on the washed hydrotalcite (experimental group) and the in situ sediments (control group). All samples used for DNA extraction are stored at -80°C.

[0049] S3. After 74 days of in-situ enrichment, the washed hydrotalcite-attached microorganisms are the initially enriched anaerobic methane-oxidizing archaea, which can be used for subsequent laboratory reactor enrichment or culture bottle enrichment.

[0050] Example 2 Determination of physical and chemical indicators

[0051] 1. Experimental methods

[0052] The sediments in the mesh bags of the experimental group and the control group obtained in Example 1 were measured by conventional methods for physical and chemical indicators, including pH, salinity, dissolved organic carbon, total carbon, total nitrogen, total sulfur and sulfate content. The precipitate was dried at 60°C to constant weight and the moisture content of each sediment was measured. After centrifuging the sediment, the supernatant was filtered through a 0.22 μm filter membrane, and the pH, salinity, and dissolved organic carbon content of the supernatant were measured by a pH meter, a salinity meter, and a TOC meter; the sulfate content of the supernatant was measured by an ion chromatograph; and the total carbon, total sulfur, and total nitrogen content of the dry sediment were analyzed by an elemental analyzer. The water content was converted into the content of each physical and chemical index of the sediment.

[0053] 2. Experimental results

[0054] The present invention selects mangroves as a typical methane emission hotspot ecosystem case, located in the Sonneratia apetala mangroves in Hualong Town, Panyu District, Guangzhou City, and the material is buried at a depth of 50 cm. After 74 days of burial, physical and chemical tests were carried out. The specific physical and chemical indicators measured included pH, salinity, dissolved organic carbon (DOC), total carbon (TC), total nitrogen (TN), total sulfur (TS) and sulfate (SO 4 2- ) content. The results are as follows Figure 1 As shown, there was a significant difference between the experimental group (Treat) and the control group (Control) only in the sulfate content, and the sulfate content of the experimental group was higher than that of the control group, indicating that adding hydrotalcite to the environment during the experimental period can significantly increase the sulfate content in the sediment.

[0055] Example 3 Isotope Activity Test

[0056] 1. Experimental methods

[0057] Build a mud mixing system and add 13 CH 4 Isotope activity experiments were conducted to determine the rate of anaerobic methane oxidation (AOM) processes. The following mother solutions were prepared: artificial seawater, alkaline trace elements, acidic trace elements, vitamins, and resazurin. Each liter of artificial seawater contains: NaCl (26 g), KCl (0.5 g), MgCl 2 6H 2 O(5g),NH 4 Cl(0.3g),CaCl 2 ·2H 2 O (1.4 g), KH 2 PO 4 (0.1g). Each liter (1000×) of acidic trace element solution contains: HCl 100mmol, FeCl 2 ·4H 2 O 1.49 g, ZnCl2 32.2mg, H 3 BO 3 14mg, CoCl 2 6H 2 O 120 mg, MnCl 2 ·4H 2 O 500mg, CuCl 2 270mg, NiCl 2 6H 2 O95mg; each liter (1000×) of alkaline trace elements contains: NaOH 10mmol, SeO 2 67mg, Na 2 WO 4 ·2H 2 O 50 mg, Na 2 MoO 4 ·2H 2 O 242mg; Each liter (1000×) vitamin solution (pH 7.0) contains: vitamin B 12 1mg, D-(+)-biotin 20mg, folic acid 20mg, nicotinic acid 50mg, p-aminobenzoic acid 50mg, calcium D-(1)-pantothenate 50mg, pyridoxine HCl 100mg, riboflavin50mg, thiamine50mg, thioctic acid 50mg. (1000×) Resazurin stock solution concentration is 1g / L. According to the in-situ salinity data, the salinity of the isotope experiment culture medium is set at 2ppt, and the salinity of artificial seawater is about 35ppt. The amount of artificial seawater added is adjusted according to the salinity. Each liter of MM (inorganic mineral) culture medium is prepared as follows: 57mL artificial seawater, 1mL acidic trace element stock solution, 1mL alkaline trace element stock solution, 1mL vitamin stock solution, 1mL resazurin stock solution, add ultrapure water to make up to 1L.

[0058] 18 g of the in-situ sediment sample of the control group in Example 1 and 18 g of the hydrotalcite sample of the experimental group were transferred to a 100 ml sterile conical flask, and 36 ml of sterile MM culture medium was added thereto. 2 / CO 2 The mixed slurry was purged with mixed gas (volume ratio 80:20) for 60 min to replace the residual sulfate in the hydrotalcite, and 0.2 M BaCl 20.5mL of solution was added to precipitate sulfate in the homogenate. Continue to stir at high speed and blow helium for 20 minutes to remove oxygen in the sample. The mixed homogenate was placed in a 100mL serum bottle, aerated for 20 minutes, immediately sealed with a rubber stopper, and then pressed with an aluminum cap. The serum bottle was then placed horizontally in a constant temperature shaker at 25°C and 150rpm / min for pre-incubation for 7 days to consume various electron acceptors and residual oxygen remaining in the soil sample. After pre-incubation, sulfate reduction coupled anaerobic methane oxidation (SR-AOM) activity experiments were performed. Under high-speed stirring, 5 20mL anaerobic serum bottles were allocated to each sample, corresponding to 5 time points (0, 7, 14, 21, and 28 days). 4mL of homogenate was added to each serum bottle while passing helium, and aeration was continued for 20 minutes. The electron acceptor solution (600uL 50mM Na 2 SO 4 ) to make the final sulfate concentration of the homogenate about 7.5 mM, and then replace it with 0.6 mL of 13 CH 4 ,make 13 CH 4 All serum bottles were placed in a 25°C constant temperature shaker in the dark and cultured at a speed of 150 rpm / min. 150 μL of 7M zinc chloride solution blown with helium for 15 minutes was added to the samples at 0, 7, 14, 21, and 28 days to terminate the microbial activity. Then 2 mL of 3M HCl deoxygenated with helium was added to make the liquid phase 13 CO 2 The samples were then shaken and left to stand for 2 hours before being stored at -20°C. 13 CO 2 The mixture in the serum bottle was dried and the dry weight was recorded.

[0059] The same dry weight of sediment was oxidized at each time point. 13 CH 4 The total amount of methane is obtained by the following calculation formula:

[0060] n(t)=c(t)v / m(t)

[0061] c(t)(mmol / L) is the headspace temperature on day t 3 CO 2 concentration, v is the headspace volume of the serum bottle (16 mL), and m(t) is the dry weight of the sample on day t (g).

[0062] 2. Experimental results

[0063] The methane oxidation activity of the in situ sediment community (control group) and the hydrotalcite-attached microbial community was compared by isotope activity experiments. Figure 2 As shown, in the same time and at the same dry weight, the amount of anaerobic methane oxidation by the microbial community attached to the hydrotalcite in the experimental group was higher than that by the in situ sediment microbial community in the control group.

[0064] Example 4 Determination of ANME microbial changes

[0065] 1. Experimental methods

[0066] The initial extraction of DNA in the sample (LDH after cleaning obtained in Example 1) was performed by sodium dodecyl sulfate SDS lysis method (Zhou et al., 1996. DNA recovery from soils of diverse composition. Applied and Environmental Microbiology). After obtaining crude DNA, the DNA was further purified using the DNeasy PowersoilPro Kit purification kit. DNA was extracted using more than 5g of original sediment or cleaned hydrotalcite. It is worth noting that there is very little DNA in the cleaned hydrotalcite, and it is difficult to ensure successful extraction by traditional kit extraction, so it is necessary to cooperate with sodium dodecyl sulfate SDS lysis method for extraction.

[0067] The extracted DNA was PCR amplified, and the universal archaeal primer pair 20F_1492R was used to sequence the full-length 16S rDNA using the PacBio third-generation sequencing platform. DADA2 was used to analyze the amplicon data, including filtering (removing sequences <1000bp and >1600bp, and read loss <2% for each sample), deduplication (threshold: similarity 100%), learning errors, checking errors, noise reduction, etc., and abundance calculation and species annotation were performed, among which the species annotation relied on the silva 138.1 database. The relevant analysis methods refer to the DADA2 analysis of the PacBio full-length 16S rDNA high-throughput amplicon sequencing process, and the code and methods can be obtained on github (https: / / github.com / benjjneb / LRASManuscript).

[0068] 2. Experimental results

[0069] The universal archaeal primer pair 20F_1492R was used to sequence the full-length 16S rDNA using the PacBio third-generation sequencing platform. Amplicon data were analyzed using DADA2. The results are shown in Figures 3-4As shown, the relative abundance of the ANME-1b group in the hydrotalcite-attached archaeal community in the experimental group was significantly higher than that in the in situ sediment archaeal community in the control group, and the abundance of ANME-1b in the experimental group was about 7 times that of the control group, indicating that the in situ addition of hydrotalcite can significantly increase the abundance of specific sulfate-dependent ANME-1b in the in situ environment. There was no significant difference between the experimental group and the control group for other sulfate-dependent ANMEs such as ANME-2b, ANME-1a, and ANME-2a-2b. It should be noted that the relative abundance of archaea in the microbial community is originally very low, so it is extremely challenging to achieve a significant (P < 0.05) improvement after enrichment.

[0070] Among them, the full-length 16S rDNA sequence information and species annotation result table of the sulfate-dependent anaerobic methane-oxidizing archaea described in the present invention can be downloaded on the open source platform gitee (https: / / gitee.com / djj9012 / zhuanli_-sr_-anme_seq / blob / master / SR_ANME_seq.xlsx). The DNA sequence of the sulfate-dependent anaerobic methane-oxidizing archaea ANME-2b described in the present invention is shown in the sequence "ASV1446" in the file "SR_ANME_seq" on the above-mentioned open source platform gitee. The strain belongs to the Archaea kingdom, the Halobacterota phylum, the Methanosarcina class, the Methanosarciniales order, the ANME-2a-2b family, and the ANME-2b genus. At present, it can only be identified to the genus classification level. The strains with the above DNA sequence are all named ANME-2b.

[0071] The DNA sequence of the sulfate-dependent anaerobic methane-oxidizing archaeon ANME-2a-2b described in the present invention is shown in the sequence "ASV1447", sequence "ASV3114", sequence "ASV3215", sequence "ASV3348", sequence "ASV3820", sequence "ASV3951", and sequence "ASV5013" in the file "SR_ANME_seq" on the above-mentioned open source platform gitee. Such strains belong to the Archaea kingdom, Halobacterota phylum, Methanosarcina class, Methanosarcinales order, ANME-2a-2b family, and can only be identified to the family classification level at present. The strains with the above-mentioned DNA sequences are all named ANME-2a-2b.

[0072] The DNA sequence of the sulfate-dependent anaerobic methane-oxidizing archaeon ANME-1a described in the present invention is shown in the sequence "ASV1129", sequence "ASV1130", sequence "ASV1131", sequence "ASV1653", sequence "ASV2695", sequence "ASV3010", sequence "ASV3322", sequence "ASV4108", sequence "ASV4732", and sequence "ASV5158" in the file "SR_ANME_seq" on the above-mentioned open source platform gitee. Such strains belong to the Archaea kingdom, Halobacterota phylum, ANME-1 class, ANME-1a order, and can only be identified to the order classification level at present. Strains with the above-mentioned DNA sequences are all named ANME-1a.

[0073] The DNA sequence of the sulfate-dependent anaerobic methane-oxidizing archaeon ANME-1b described in the present invention is shown in the sequence "ASV1126", sequence "ASV1127", sequence "ASV1128", sequence "ASV1139", sequence "ASV1861", sequence "ASV2414", sequence "ASV3020", sequence "ASV3330", sequence "ASV3333", and sequence "ASV4784" in the file "SR_ANME_seq" on the above-mentioned open source platform gitee. Such strains belong to the Archaea kingdom, Halobacterota phylum, ANME-1 class, ANME-1b order, and can currently only be identified to the order classification level. Strains with the above-mentioned DNA sequences are all named ANME-1b.

[0074] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for in situ enrichment of sulfate-dependent anaerobic methane-oxidizing archaea, characterized in that: The method comprises the following steps: placing the adsorbent material in a sulfate-methane transition zone sediment environment for full contact, collecting the adsorbent material, washing it, and collecting the washed adsorbent material; wherein the sulfate-dependent anaerobic methane-oxidizing archaea is attached to the washed adsorption material; The sulfate-dependent anaerobic methane-oxidizing archaea is ANME-1b; The adsorbent material is hydrotalcite containing sulfate.

2. The method according to claim 1, characterized in that: The method further comprises anaerobic culture, wherein the anaerobic culture is to place the washed adsorption material in a culture solution and perform anaerobic culture in the absence of light under the condition of the presence of methane.

3. The method according to claim 1, characterized in that: The preparation method of the hydrotalcite comprises the following steps: mixing a metal salt solution containing magnesium ions and aluminum ions with an alkaline reagent, reacting them fully under protective atmosphere conditions, filtering after the reaction is complete, washing, drying and sieving the precipitate to obtain the hydrotalcite.

4. The method according to claim 1, characterized in that The particle size of the hydrotalcite is ≥0.15 mm.

5. The method according to claim 1, characterized in that The sufficient contact time is 60 to 90 days.

6. The method according to claim 2, characterized in that: The temperature of the anaerobic culture is 20-25°C.

7. The method according to claim 2, characterized in that: The anaerobic culture time is ≥7 days.

8. The method according to claim 2, characterized in that: The volume percentage content of methane in the presence of methane is 3-5%.

9. The method according to claim 2, characterized in that: The culture solution is a mineral culture solution, and the components of the mineral culture solution include artificial seawater, alkaline trace elements, acidic trace elements, vitamins, resazurin and sulfate.

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