Carbon sequestration bacterium and application thereof

Abstract

The invention discloses a carbon sequestration bacterium and application thereof, and belongs to the technical field of biological carbon sequestration. The carbon sequestration bacterium is Stutzeremonasfrequens and is named as strainWS-9, and the preservation number of the carbon sequestration bacterium is CGMCC (China General Microbiological Culture Collection Center) No. 31181. The stterimonas frequently disclosed by the invention has remarkable carbon dioxide (CO2) immobilization capability, and shows the capability of promoting the microalgae to immobilize CO2 in a mixed culture system of the stterimonas frequently and the microalgae, and the CO2 immobilization capability is further improved through the cooperation of the stterimonas frequently and the microalgae. Metabonomics analysis shows that the concentrations of lactic acid, oxalic acid, malic acid and pyruvic acid in a mixed system are reduced, and the activity of acetyl coenzyme A is increased at the same time, which indicates that complex metabolic interaction exists between microalgae and bacteria, and the synergistic effect is attributed to metabolite exchange, metabolic pathway change, oxidation resistance and other environmental adaptability.

Description

Technical Field

[0001] The present application belongs to the technical field of biological carbon fixation, and specifically relates to a carbon-fixing bacterium and its application. Background Art

[0002] CO 2 As the most important greenhouse gas, it has caused a series of environmental problems. Carbon fixation refers to measures to increase the carbon content of carbon pools outside the atmosphere, including physical carbon fixation and biological carbon fixation. Physical carbon fixation is the long-term storage of carbon dioxide in exploited oil and gas wells, coal seams and deep seas. Biological carbon fixation is the conversion of inorganic carbon, that is, carbon dioxide in the atmosphere, into organic carbon, that is, carbohydrates, and fixes it in plants or soil. There are two common methods of carbon fixation: photosynthesis, such as various green plants and photoautotrophic microorganisms (such as cyanobacteria); chemosynthesis, such as nitrifying bacteria using oxidized ammonia to synthesize organic matter. Biological carbon fixation increases the carbon absorption and storage capacity of the ecosystem and reduces the concentration of carbon dioxide in the atmosphere.

[0003] In the process of biological carbon fixation using microorganisms, combining carbon fixation bacteria with microalgae may further increase the overall CO 2 Carbon fixation efficiency. Carbon fixation bacteria refer to those bacteria that can fix or reduce atmospheric carbon dioxide (CO 2 ) microorganisms. These microorganisms convert CO 2 It is converted into organic carbon, thus participating in the global carbon cycle and playing an important role in mitigating climate change. Summary of the invention

[0004] 1. Purpose of the Invention

[0005] The purpose of the present invention is to provide a carbon-fixing bacterium screened from an environmental sample and having the ability to fix carbon. The strain is identified as Stutzerimonasfrequens and named as strain WS-9. The strain is deposited in the General Microbiological Center of China Microbiological Culture Collection Committee, with the deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.31181. The frequent Stutzerimonas has significant CO 2 The fixation capacity of CO2 was shown to promote the fixation of CO2 by microalgae in mixed culture system with microalgae. 2 The two work together to further improve the CO 2 Fixed capacity.

[0006] 2. Technical solution

[0007] In order to solve the above problems, the technical solutions adopted in this application are as follows:

[0008] The present application provides a frequently occurring Stutzerimonas frequents strain WS-9, which is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with a deposit date of July 5, 2024, a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit number of CGMCC No. 31181.

[0009] The present application also provides the above-mentioned frequent Stutzerimonas frequens strain WS-9 in fixed CO 2 Application in.

[0010] Furthermore, the above-mentioned microalgae include one or more of Chlorella sp., Tetradesmus obliquus, Scenedesmus quadricauda, ​​Chlamydomonas reinhardtii, Chlorella sorokiniana and Desmodesmus sp.

[0011] Further, the microalgae include Chlorella. Further, the Chlorella include FACHB-5 and / or FACHB-31. Further, the Chlorella include FACHB-5.

[0012] Furthermore, the above-mentioned Tetraselmisoleum obliquus includes FACHB-12.

[0013] Furthermore, the above-mentioned Scenedesmus quadricauda includes FACHB-44.

[0014] Furthermore, the above-mentioned Chlamydomonas reinhardtii includes FACHB-265.

[0015] Furthermore, the above-mentioned Chlorella sorokinensis includes FACHB-1068.

[0016] Furthermore, the above-mentioned Desmodesmus algae includes FACHB-2042.

[0017] The above-mentioned FACHB refers to the Freshwater Algae Seed Bank of the National Aquatic Biological Germplasm Resources Bank of the Institute of Hydrobiology, Chinese Academy of Sciences.

[0018] The present application also provides a microbial agent, which includes the above-mentioned Stutzerimonas frequents strain WS-9.

[0019] Furthermore, the above-mentioned microbial agent also includes a microorganism capable of fixing CO 2 Capacity of microalgae.

[0020] The present application also provides the above-mentioned frequent Stutzerimonas frequens strain WS-9 in promoting microalgae to fix CO 2 In the application of this bacteria, the frequently-occurring Sterzerimonas can cooperate with microalgae to improve the fixation of CO 2 ability.

[0021] Furthermore, the above-mentioned microalgae include one or more of Chlorella sp., Tetradesmus obliquus, Scenedesmus quadricauda, ​​Chlamydomonas reinhardtii, Chlorella sorokiniana and Desmodesmus sp.

[0022] Further, the microalgae include Chlorella. Further, the Chlorella include FACHB-5 and / or FACHB-31. Further, the Chlorella include FACHB-5.

[0023] Furthermore, the above-mentioned Tetraselmisoleum obliquus includes FACHB-12.

[0024] Furthermore, the above-mentioned Scenedesmus quadricauda includes FACHB-44.

[0025] Furthermore, the above-mentioned Chlamydomonas reinhardtii includes FACHB-265.

[0026] Furthermore, the above-mentioned Chlorella sorokinensis includes FACHB-1068.

[0027] Furthermore, the above-mentioned Desmodesmus algae includes FACHB-2042.

[0028] The above-mentioned FACHB refers to the Freshwater Algae Seed Bank of the National Aquatic Biological Germplasm Resources Bank of the Institute of Hydrobiology, Chinese Academy of Sciences.

[0029] The present application also provides the above-mentioned microbial agent for fixing CO 2 Application in.

[0030] 3. Beneficial effects

[0031] (1) The frequently occurring Stutzerimonas (Stutzerimonas frequents) strain WS-9 and its application provided in the present application, the frequently occurring Stutzerimonas is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, the deposit date is July 5, 2024, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC No. 31181, and it is a carbon-fixing bacterium with carbon fixation ability.

[0032] (2) The present application provides a frequent Stutzerimonas frequens strain WS-9 and its application, wherein the frequent Stutzerimonas frequens has significant CO 2 The fixation capacity of CO2 was shown to promote the fixation of CO2 by microalgae in mixed culture system with microalgae. 2 The two work together to further improve the CO 2 Fixed capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 These are the colony morphology characteristics and scanning electron microscope observation images of strain WS-9.

[0034] Figure 2 This is the phylogenetic tree of strain WS-9 based on the 16S rRNA gene sequence.

[0035] Figure 3 It is the change of pH during pure culture of microalgae and co-culture of microalgae with strain WS-9.

[0036] Figure 4 It is the change of chlorophyll concentration during pure culture of microalgae and co-culture of microalgae with strain WS-9.

[0037] Figure 5 It is the change of lactic acid concentration during pure culture of microalgae and co-culture of microalgae with strain WS-9.

[0038] Figure 6 It is the change of gallic acid concentration during pure culture of microalgae and co-culture of microalgae with strain WS-9.

[0039] Figure 7 It is the change of oxalic acid concentration during pure culture of microalgae and co-culture of microalgae with strain WS-9.

[0040] Figure 8 It is the change of malic acid concentration during pure culture of microalgae and co-culture of microalgae with strain WS-9.

[0041] Fig. 9 It is the change of pyruvate concentration during pure culture of microalgae and co-culture of microalgae with strain WS-9.

[0042] Fig.10 It is the change of benzoic acid concentration during pure culture of microalgae and co-culture of microalgae with strain WS-9.

[0043] Fig.11 It is the change of reactive oxygen activity level during pure culture of microalgae and co-culture of microalgae with strain WS-9.

[0044] Fig.12 It is the change of acetyl-CoA activity during pure culture of microalgae and co-culture of microalgae with strain WS-9.

[0045] Fig.13 This is an electron microscope image of the microalgae and the strain WS-9 observed by electron microscope scanning during the co-culture of the microalgae and the strain WS-9.

[0046] Fig.14 The principal component analysis (PCA) of pure culture of microalgae and co-culture of microalgae with strain WS-9 was performed.

[0047] Fig.15 These are the results of differential metabolite analysis of pure culture of microalgae and co-culture of microalgae with strain WS-9.

[0048] Fig.16 It is the result of changes in metabolic pathways and metabolites in pure culture of microalgae and co-culture of microalgae with strain WS-9.

[0049] Fig.17 This is the KEGG enrichment map of differential metabolism between pure culture of microalgae and co-culture of microalgae with strain WS-9.

[0050] Fig.18 These are the results of the metabolite interaction analysis of pure culture of microalgae and co-culture of microalgae with strain WS-9.

[0051] Fig.19 It is the changing trend of different metabolite clusters in pure culture of microalgae and co-culture of microalgae with strain WS-9. DETAILED DESCRIPTION

[0052] The present application is further described below in conjunction with specific embodiments.

[0053] It should be noted that the terms such as "upper", "lower", "left", "right", "middle", etc. cited in this specification are only for the convenience of description and are not used to limit the scope of implementation. Changes or adjustments to their relative relationships should be regarded as the scope of implementation of this application without substantially changing the technical content.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0055] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0056] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement or value. The degree of flexibility for a particular variable can be easily determined by one skilled in the art.

[0057] As used herein, the term "at least one of" is intended to be synonymous with "one or more of." For example, "at least one of A, B, and C" expressly includes only A, only B, only C, and combinations of each thereof.

[0058] Concentration, amount and other numerical data can be presented in range format herein.It should be understood that such range format is only used for convenience and simplicity, and should be flexibly interpreted as not only including the numerical value clearly described as range limit, but also including all single numerical values ​​or sub-ranges contained in the range, just as each numerical value and sub-range are clearly described.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly described, but also including single numerals (such as 2,3,4) and sub-ranges (such as 1 to 3,2 to 4, etc.).The same principle is applicable to the scope of only narrating a numerical value, such as "less than about 4.5", which should be interpreted as including all the above-mentioned values ​​and ranges.In addition, no matter how the breadth of the described range or feature is, this explanation should be applicable.

[0059] In this application, some of the culture media and their components used are as follows:

[0060] Minimal Salt Medium (MSM) medium: Na 2 HPO 4 12H 2 O 4.5g / L, K 2 HPO 41.0g / L, NH 4 SO 4 1.0g / L, MgSO 4 7H 2 O 0.2g / L, CaCl 2 ·2H 2 O 0.02g / L, trace element stock solution 1mL / L, vitamin stock solution 1mL / L, agar powder 15-20g / L (solid culture medium only), including:

[0061] Trace element stock solution refers to ATCC Trace Mineral Supplement: MnSO 4 ·H 2 O 0.10g / L, Na 2 MoO 4 ·2H 2 O 0.02g / L, H 3 BO 3 0.01g / L, FeSO 4 7H 2 O 1.00g / L, ZnSO 4 7H 2 O 0.10g / L, CuSO 4 ·5H 2 O 0.02g / L, CoCl 2 6H 2 O 0.02 g / L;

[0062] The vitamin stock solution refers to ATCC Vitamin Supplement: folic acid 2.00g / L, pyridoxine hydrochloride 10.00g / L, riboflavin 5.00g / L, biotin 2.00g / L, thiamine 5.00g / L, niacin 5.00g / L, calcium pantothenate 5.00g / L, vitamin B12 0.10g / L, p-aminobenzoic acid 5.00g / L, lipoic acid 5.00g / L.

[0063] LB medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar powder 15-20 g / L (solid medium only).

[0064] Microalgae C-free BG11 medium: NaNO 3 1.5g / L, K 2 HPO 4 0.04g / L, MgSO 4 7H 2 O 0.075 g / L, CaCl 2·2H 2 O 0.036g / L, citric acid 0.006g / L, ammonium ferric citrate 0.006g / L, EDTA: 0.001g / L.

[0065] In this application, the main reagents used are as follows:

[0066] PBS and Tris-HCl buffer were purchased from Fisher;

[0067] Bradford protein concentration assay kit was purchased from Beijing Solebow Technology Co., Ltd.;

[0068] The bacterial DNA extraction kit was purchased from Omega;

[0069] Other reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0070] In this application, the main instruments used are as follows:

[0071] Agilent 8860 gas chromatography system, Agilent Technologies, USA;

[0072] UV spectrophotometer, LabTech Co., Ltd., USA;

[0073] 5810R high-speed refrigerated centrifuge, Eppendorf, Germany;

[0074] Clean bench, Suzhou Antai Air Technology Co., Ltd.;

[0075] Nanodrop One spectrophotometer, Qbuit 3.0 fluorometer, Thermo Fisher Scientific, USA;

[0076] PromethION sequencer, Oxford Nanopore Technologies, UK;

[0077] NovaSeq6000 PE150 platform, Illumina, USA.

[0078] In the present application, the microalgae is Chlorella vulgaris, which was purchased from the Freshwater Algae Seed Bank (FACHB) of the National Aquatic Biological Germplasm Resource Bank of the Institute of Hydrobiology, Chinese Academy of Sciences, and is numbered FACHB-5.

[0079] Example 1

[0080] This example provides screening and identification of carbon-fixing bacteria.

[0081] (1) Sample

[0082] Water samples were collected from the vicinity of a factory in Nanjing, placed in sterile containers, stored at 4°C and transported to the laboratory for immediate enrichment and culture experiments of the strains.

[0083] (2) Screening

[0084] Take 5 mL of water sample and place it in a sterile conical flask containing 100 mL of basal salt medium (MSM medium) and add 10 mg / L HCO 3 - The enrichment culture was carried out at 30°C and 150 rpm as the sole carbon source; 5% of the enrichment culture medium was transferred to fresh medium containing 100 mg / L HCO every other week. 3 - After six months of enrichment culture, the CO2-fixing 2 of bacterial liquid.

[0085] 1 mL of bacterial solution was diluted with sterile PBS buffer to obtain 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 and 10 -8 times diluted bacterial solution; the diluted bacterial solution was spread on the surface of the plate containing 100 mg / L NaHCO 3 agar solid plates and placed in a 30°C constant temperature incubator for culture; after 5 days of culture, single colonies with good growth were selected and streaked on the plates to obtain CO 2 The fixed strain (carbon-fixing bacteria) was named strainWS-9.

[0086] (3) Identification

[0087] (a) Morphological observation of carbon-fixing bacteria

[0088] The screened CO 2 The fixed strain WS-9 was streaked on LB agar plates and cultured at 30°C for 2-3 days. Then, single colonies with good growth were picked and cultured in LB liquid medium. When the culture reached the logarithmic phase, the bacterial liquid was collected and centrifuged at 5000r / min at 4°C for 3min. The supernatant was discarded and the plates were washed twice with PBS buffer. After retaining the bacterial precipitate, paraformaldehyde-glutaraldehyde fixative (2% / 2.5%) was slowly added for scanning electron microscopy observation.

[0089] result:

[0090] CO 2 The morphological characteristics of single colonies formed by the fixed strain WS-9 on LB agar plates are as follows Figure 1As shown, the colonies are round with a diameter of about 1 to 2 mm, with a smooth surface and neat edges; the colonies are opaque and white. Scanning electron microscopy observed that the cells of strain WS-9 were rod-shaped, with a cell length of about 3-5 μm and a diameter of about 1 μm.

[0091] (b) 16S rDNA identification and phylogenetic analysis of carbon-fixing bacteria

[0092] Collect the strain WS-9 cells cultured to the logarithmic phase, and extract DNA according to the manual of the bacterial DNA extraction kit. Using the extracted DNA as a template, the 16S rRNA gene sequence was amplified using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2). TransStartFastpfu DNA Polymerase was used in the PCR reaction system (50μL). PCR reaction conditions: 95℃ pre-denaturation for 5min; 30 cycles (95℃ denaturation for 30s, 54℃ annealing for 30s, 72℃ extension for 45s); 72℃ extension for 10min, 10℃ until the end of use.

[0093] The purified PCR products were detected by 2% agarose gel electrophoresis and sequenced by Shanghai Lingen Biotechnology Co., Ltd. The obtained sequences were uploaded to the NCBI database (https: / / www.ncbi.nlm.nih.gov) for BLAST analysis, and the genetic distances were calculated using MEGA 11.0 software, and the phylogenetic tree was constructed by the neighbor-joining method (NJ).

[0094] Result analysis:

[0095] The 16S rRNA gene sequence (SEQ ID NO.3) of strain WS-9 obtained by sequencing was compared in the GenBank database. Phylogenetic tree ( Figure 2) shows the relationship between strain WS-9 and other related strains. The numbers on the branch points are the developmental distances (%), and the numbers in brackets correspond to the GenBank accession numbers of the strain 16S rRNA gene sequences. The similarity between strain WS-9 and Stutzerimonasfrequens strain DNSP21 (MZ 276326.1) is 93%. The branch where strain WS-9 is located is closely related to Stutzerimonas stutzeri ATCC 17588=LMG 11199 (MT 027239.1) and Pseudomonas phenolilytica strain RBPA9 (NR 181536.1), showing 93% and 96% similarity, respectively. Further upward branches show more Stutzerimonas and Pseudomonas strains. These data indicate that strain WS-9 is phylogenetically closely related to Stutzerimonasfrequens, supporting the classification of strain WS-9 as part of the genus Stutzerimonas. It is therefore classified and named Stutzerimonasfrequensstrain WS-9 and deposited in the General Microbiology Center of China Microorganism Culture Collection, with a deposit date of July 5, 2024 at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and a deposit number of CGMCC No.31181.

[0096] (4)Save

[0097] Carbon-fixing bacteria suspension: single colony strain WS-9 was selected and inoculated into LB liquid medium to amplify and culture to the logarithmic phase, and the cells were collected by centrifugation at 8000r / min for 3min at 4°C, the supernatant was discarded, and the cells were washed 2-3 times with PBS buffer solution; finally, the cells were resuspended with PBS buffer solution to obtain the carbon-fixing bacteria suspension. The cell density of the bacterial suspension was OD 600 Not less than 0.05, the bacterial suspension is stored at 4°C for short-term use (0 to 14 days).

[0098] Long-term storage of StrainWS-9: Mix the carbon-fixing bacteria suspension with 50% glycerol at a ratio of 1:1 under sterile conditions and store in a -80°C refrigerator.

[0099] Example 2

[0100] This example provides analysis of the carbon fixation capacity, extracellular carbonic anhydrase activity and extracellular polymer content of Stutzerimonas frequency strain WS-9.

[0101] Strain WS-9 carbon fixation capacity test:

[0102] Inoculate Stutzerimonas frequency strain WS-9 in a medium containing 100 mM NaHCO 3 The cells were cultured in MSM medium for 24 h, and their carbon fixation capacity was verified by biomass concentration.

[0103] Strain WS-9 extracellular carbonic anhydrase activity assay:

[0104] The extracellular carbonic anhydrase (CA) activity of the strain was determined by the Wilbur-Anderson assay. 9 mL of ice-cold Tris-HCl (20 mmol / L, pH 8.0) buffer solution was added to a 25 mL beaker, and 200 μL of the culture medium (OD 600 =1.0) strain WS-9 bacterial solution. After the pH stabilized, add 6 mL of saturated CO 2 At the same time, quickly press the stopwatch to record the time T required for the pH to drop from 8.3 to 6.3. Replace 200 μL of bacterial solution with H 2 The repeated measurement process is recorded as T 0 The temperature of the whole process is between 0 and 4°C. The carbonic anhydrase (CA) activity WAU is calculated by formula (1):

[0105] WAU=(T 0 -T) / T……Formula (1)

[0106] Strain WS-9 Extracellular Polymer Content Determination:

[0107] The activities and contents of ribulose-1,5-bisphosphate carboxylase (RuBisCO), acetyl-coenzyme A (Acetyl-CoA) and extracellular polymeric polysaccharide (EPS, ng / mL) were determined using enzyme-linked immunosorbent assay (ELISA) kits produced by Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., China.

[0108] Result analysis:

[0109] Table 1 Extracellular enzyme activity and extracellular polymer content of carbon-fixing bacteria strain WS-9 after 24 hours of culture

[0110]

[0111] In the presence of 100 mM NaHCO 3 After culturing in MSM medium for 24 h, the biomass concentration of strain WS-9 reached 0.463 g / L, demonstrating its carbon fixation ability.

[0112] Table 1 shows the extracellular enzyme activity and extracellular polymer content of carbon-fixing bacteria strain WS-9 after 24 hours of culture:

[0113] The carbonic anhydrase (WAU / mL) activity was 0.39 WAU / mL, indicating that strain WS-9 could fix CO 2 The process has a certain carbonic anhydrase activity, which can catalyze CO 2 The hydration reaction helps to increase CO 2 Utilization efficiency;

[0114] The activity of ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO, U / L) was 91.18 U / L. As the most important carbon fixation enzyme in plants and some bacteria, strain WS-9 showed a high RuBisCO activity, indicating that it played an important role in the carbon fixation process.

[0115] The activity of acetyl-CoA (U / mL) was 107.75U / mL. Acetyl-CoA is a key intermediate in cell metabolism. Its high activity indicates that strain WS-9 has a strong function in the metabolism of organic matter and energy conversion.

[0116] The content of extracellular polymer polysaccharide (EPS, ng / mL) was 11.05 ng / mL. EPS plays an important role in bacterial biofilm formation, cell adhesion and protection. Strain WS-9 produces a certain amount of EPS, which helps it to grow stably and function in the environment.

[0117] In general, strain WS-9 showed significant activity in multiple extracellular enzymes related to carbon fixation, indicating its ability to fix CO 2 and potential in metabolic transformation.

[0118] Example 3

[0119] This example provides a method for promoting microalgae to fix CO 2 Application in.

[0120] Experimental group: microalgae and strain WS-9 co-culture system (FACHB-5+WS-9), activated carbon-fixing bacteria strain WS-9 (OD 600 =1) according to 2mL and microalgae (OD 670 =1) 2 mL was inoculated into 150 mL of C-free BG11 medium and 20% CO was introduced. 2As a carbon source, the cells were incubated in a 25°C 5000 Lux white LED light incubator and samples were taken at 0 to 10 days.

[0121] Control group: microalgae culture system (FACHB-5), i.e. pure microalgae culture. Note: strain WS-9 cannot survive in C-free BG11 medium.

[0122] Each experiment was repeated three times. The pH, chlorophyll content, organic acid content, key enzyme activity, reactive oxygen level and other parameters were measured to investigate the effect of strain WS-9 on CO fixation in microalgae. 2 The parameters are determined as follows:

[0123] (1) pH measurement

[0124] Use a pH meter to measure the pH value of the microalgae solution. Stir the microalgae solution evenly with a glass rod, then submerge the pH meter probe in the solution, and record the data after the reading stabilizes.

[0125] (2) Determination of chlorophyll content

[0126] Take 1 mL of microalgae solution, centrifuge at 10,000 rpm for 5 min, resuspend in 1 mL of methanol, stand at 45°C in the dark for 30 min, centrifuge and take the supernatant in a 96-well plate, and measure OD 649 OD 665 and OD 750 , Calculate chlorophyll (Chla) according to formula (2), where: A 649 , A 665 and A 750 OD 649 OD 665 and OD 750 The value of .

[0127] Chla (mg / mL) = 5.19 × (A 649 -A 750 )+13.36×(A 665 -A 750 )……Formula (2)

[0128] (3) Determination of organic acid content

[0129] 100 mg of microalgae liquid was ground with liquid nitrogen, and the homogenate was resuspended in pre-cooled 80% methanol and 0.1% formic acid, incubated on ice for 5 min, and then centrifuged at 15,000g for 20 min at 4°C. The supernatant was diluted with LC-MS grade water to a final concentration of 53% methanol. The sample was then transferred to a new Eppendorf tube and centrifuged at 15,000g for 20 min at 4°C. Finally, the supernatant was injected into the LC-MS / MS system for analysis. Ultra-high performance liquid chromatography-mass spectrometry / mass spectrometry analysis was performed by a Vanquish ultra-high performance liquid chromatography system (ThermoFisher, Germany) and an Orbitrap Q ExactiveTMHF-X mass spectrometer (ThermoFisher, Germany) from Shanghai Lingen Biotechnology Co., Ltd. The sample was injected through a Hypesil Gold column (100×2.1 mm, 1.9 μm) with a flow rate of 0.2 mL / min and a linear gradient of 17 min. Eluents A (0.1% FA water) and B (methanol) were used in the positive polarity mode, and eluents A (5 m ammonium acetate, pH 9.0) and B (methanol) were used in the negative polarity mode. The solvent gradient was set as follows: 2% B, 1.5 min; m / z = 1, 2-100% B, 12.0 min; 100% B, 14.0 min; 100-2% B, 14.1 min; 2% B, 17 min. The Q ExactiveTMHF-X mass spectrometer was operated in positive / negative polarity mode, with a spray voltage of 3.2 kV, a capillary temperature of 320 ° C, a sheath gas flow rate of 40 arb, and an auxiliary gas flow rate of 10 arb.

[0130] (4) Key enzyme activities and reactive oxygen levels

[0131] During the co-culture process, the activities of extracellular carbonic anhydrase (CA), ribulose-1,5-bisphosphate carboxylase (RuBisCO) and acetyl-coenzyme A (Acetyl-CoA) were measured according to the method described in Example 2.

[0132] The level of ROS in algae cells was detected using a cellular reactive oxygen species (ROS) detection kit (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.), and the specific operation was carried out according to the instructions.

[0133] (5) Biological electron microscopy

[0134] After the co-culture of strain WS-9 and microalgae (FACHB-5) was completed, 5 mL of bacterial solution was collected and centrifuged at 8000 r / min for 5 min at 4°C. The supernatant was discarded and washed three times with PBS buffer. After retaining the bacterial precipitate, paraformaldehyde-glutaraldehyde fixative (2% / 2.5%) was slowly added for scanning electron microscopy observation.

[0135] (6) Difference Analysis

[0136] GraphPad Prism 8.0.1 software was used to perform an unpaired t-test to assess the significant differences between the groups. When the P value was less than 0.05 (P<0.05), the difference was considered statistically significant.

[0137] Result analysis:

[0138] (1) Changes in pH during pure culture of microalgae and co-culture of microalgae with strain WS-9 Figure 3 As shown. During the period from 0 to 10 days, the pH values ​​of both groups gradually increased. In the initial stage (0 days), the pH values ​​of both groups were close to 6. By the 10th day, the pH values ​​of both groups rose to close to 12. Overall, the pH value of the FACHB-5+WS-9 group was slightly higher than that of the pure culture of FACHB-5, but the trend was similar. This shows that the addition of the carbon-fixing bacteria strain WS-9 accelerated the alkalinization of the culture medium, reflecting that co-culture (synergistic effect) has a significant effect on metabolic activities during carbon fixation.

[0139] (2) The concentration of chlorophyll a (Chla) changes during pure culture of microalgae and co-culture of microalgae with strain WS-9. Figure 4 As shown. During the period from 0 to 4 days, the chlorophyll a concentration of both groups gradually increased. During the period from 4 to 6 days, the chlorophyll a concentration of the FACHB-5 group decreased, while the chlorophyll a concentration of the FACHB-5+WS-9 group continued to increase, reaching a peak of about 14 mg / mL on the 8th day and then decreased, but still maintained a high level. This study shows that the addition of carbon-fixing bacteria strain WS-9 significantly promoted the photosynthetic capacity of microalgae, thereby increasing the content of chlorophyll a, showing a synergistic effect on the photosynthetic activity during carbon fixation.

[0140] (3) During the pure culture of microalgae and the co-culture of microalgae with strain WS-9, the concentration of organic acids on the 10th day was Figures 5 to 10As shown. During the synergistic carbon fixation process between microalgae and carbon-fixing bacteria, lactic acid, oxalic acid, malic acid and pyruvic acid decreased significantly. Specifically, the lactic acid concentration of the FACHB-5 group was 3068 μg / L, while the lactic acid concentration of the FACHB-5+WS-9 group was significantly reduced to 158 μg / L. The oxalic acid concentration of the FACHB-5 group was 6258 μg / L, while the oxalic acid concentration of the FACHB-5+WS-9 group was significantly reduced to 2053 μg / L. The pyruvic acid concentration of the FACHB-5 group was 625 μg / L, while the lactic acid concentration of the FACHB-5+WS-9 group was significantly reduced to 31 μg / L. The concentrations of gallic acid and benzoic acid decreased during co-culture but not significantly. The reasons for the decrease in organic acid concentrations may include the following aspects: The metabolic activity of carbon-fixing bacteria may utilize the organic acids produced by microalgae metabolism as carbon sources and energy sources. In addition, there may be a co-metabolic relationship between microalgae and carbon-fixing bacteria. The organic acids produced by microalgae are used by bacteria, and the bacterial metabolic activities may produce an environment or substances that are beneficial to microalgae and promote the growth of microalgae. Therefore, the organic acids in the co-culture system are consumed faster.

[0141] (4) Changes in reactive oxygen levels and acetyl-CoA activity during pure culture of microalgae and co-culture of microalgae with strain WS-9 Figure 11-12 As shown. The reactive oxygen level concentration in the FACHB-5 group was 193pg / mL, while the reactive oxygen level concentration in the FACHB-5+WS-9 group was reduced to 179pg / mL. The acetyl-CoA activity in the FACHB-5 group was 75.24U / mL, while the activity in the FACHB-5+WS-9 group increased significantly to 118.78U / mL. The synergistic effect between microalgae and carbon-fixing bacteria strain WS-9 may involve multiple mechanisms in reducing reactive oxygen generation and increasing acetyl-CoA production. First, they may reduce oxidative stress through a symbiotic relationship, with the microalgae releasing oxygen to provide a favorable oxygen environment for carbon-fixing bacteria strain WS-9 and reduce the generation of reactive oxygen. Second, they may increase acetyl-CoA production through the optimization of common metabolic pathways, such as microalgae using photosynthesis to produce organic matter to provide substrates for carbon-fixing bacteria strain WS-9. In addition, they may jointly participate in the antioxidant enzyme system and key enzyme systems in the carbon metabolic pathway, such as superoxide dismutase and acetaldehyde dehydrogenase, to regulate the generation of reactive oxygen species and the production of acetyl-CoA. Finally, microalgae and carbon-fixing bacteria strain WS-9 may interact through signal transduction and regulatory networks to regulate metabolic pathways and enzyme activities to adapt to symbiotic growth conditions. Taken together, these mechanisms work together to enable microalgae and carbon-fixing bacteria strain WS-9 to participate in the carbon cycle process more effectively.

[0142] (5) Scanning electron microscopy (SEM) images of pure culture of microalgae and co-culture of microalgae with strain WS-9 Fig.13 As shown in the figure, the larger algae cells on the surface and the smaller carbon-fixing bacteria strain WS-9 cells attached to them can be clearly seen. The carbon-fixing bacteria strain WS-9 exists in the form of aggregates, which may be attached to the surface of the microalgae cells or to each other. Fig.13 The structural features of the microalgae cell surface, such as wrinkles and protrusions, are shown, which may provide attachment sites for the carbon-fixing bacteria strain WS-9. Under high magnification, the distribution of carbon-fixing bacteria strain WS-9 on the microalgae cell surface can be seen, and the bacteria are tightly attached to the wrinkles and depressions on the microalgae surface in aggregates or individually. There may also be aggregation of carbon-fixing bacteria strain WS-9 between microalgae cells, forming a tight network. In summary, this set of SEM images shows the attachment and distribution of carbon-fixing bacteria strain WS-9 on the microalgae cell surface during the synergistic carbon fixation process between carbon-fixing bacteria strain WS-9 and microalgae. These results indicate that carbon-fixing bacteria strain WS-9 may promote the carbon fixation efficiency of microalgae by interacting with the surface of microalgae. This synergistic effect is of great significance in the research of microbial carbon fixation technology, and provides new perspectives and ideas for further optimization and application of this technology.

[0143] Example 4

[0144] This example provides metabolomic analysis of pure culture of microalgae and co-culture of microalgae with strain WS-9.

[0145] After the pure culture and co-culture experiments, the microalgae cells were collected by centrifugation and washed with PBS buffer. The precipitated samples were placed in centrifuge tubes, grinding beads and extracts were added, and then processed by freezing tissue grinder and low-temperature ultrasonic treatment, and the supernatant was drawn into the injection bottle for analysis. The samples were analyzed by LC-MS / MS using an ultra-high performance liquid chromatography-Fourier transform mass spectrometry system (UHPLC-Q ExactiveHF-X, Shanghai Meiji Biopharmaceutical Technology Co., Ltd.). The raw data were preprocessed and corrected by the metabolomics processing software Progenesis QI, and then matched and analyzed with the metabolic public database HMDB (http: / / www.hmdb.ca / ) and Metlin (https: / / metlin.scripps.edu / ) to obtain metabolite information. The experimental procedures included: sample pretreatment, LC-MS analysis, data preprocessing, pattern recognition (multivariate statistical analysis), differential metabolite analysis, and change trend analysis of different metabolite clusters.

[0146] (1) Principal component analysis

[0147] The principal component analysis (PCA) of pure culture of microalgae and co-culture of microalgae with strain WS-9 was as follows Fig.14 As shown. Fig.14 It can be seen that the scores of pure culture of microalgae (CK group, red triangle) and co-culture of microalgae with strain WS-9 (WS-9 group, green plus sign) on principal component 1 (PC1) and principal component 2 (PC2) are clearly separated, indicating that there are significant differences between the two groups. The scores of samples in the CK group on PC1 and PC2 are relatively concentrated, indicating that they have high consistency and stability, and are mainly distributed on the left side of the figure. The samples in the WS-9 group scored higher on PC1 and clustered on the right side of the figure, showing obvious separation, indicating that the microalgae and carbon-fixing bacteria strain WS-9 in the co-culture system have significant changes in metabolites, gene expression or other biological characteristics. PC1 and PC2 explain a total of 81.2% of the variation, which can well show the differences between different treatment groups. Therefore, Fig.14 The results showed that the microalgae exhibited significantly different metabolic or biological characteristics under monoculture and co-culture conditions with the carbon-fixing bacteria strain WS-9. The co-culture system may have caused significant changes in the metabolic pathways, material exchange and growth environment adaptation of the microalgae.

[0148] (2) Metabolite difference analysis

[0149] The results of metabolite difference analysis after pure culture of microalgae and co-culture of microalgae with strain WS-9 are shown in Fig.15 As shown. The horizontal axis is Log 2 FC (logarithmic fold change) indicates the relative abundance change of metabolites under co-culture conditions and mono-culture conditions; the vertical axis lists the names of specific metabolites. Fig.15 Circles of different colors and sizes are used to represent the significance and VIP (variable importance projection) values ​​of metabolites. The color changes from yellow to red to represent the change of p-value. The redder the color, the higher the significance. The larger the circle, the higher the VIP value. Fig.15It can be seen that L-Methionine, D-Glucose 6-phosphate, Tartaric acid, Ergothioneine, Demethylnobiletin and Glutamic acid were significantly downregulated under co-culture conditions, while Geniposidic acid, Orotic acid, 5-Sulfosalicylic acid, 3,4,5-Trimethoxycinnamic acid and 2-{[(4,5-dimethoxy-2-nitrophenethyl)imino]methyl}phenol were significantly upregulated. Fig.15 The results showed that the metabolite composition of microalgae changed significantly under the conditions of monoculture and co-culture with carbon-fixing bacteria strain WS-9, reflecting the influence and regulation of the co-culture system on the metabolic pathways of microalgae.

[0150] (3) Analysis of changes in metabolic pathways and metabolites

[0151] The results of changes in metabolic pathways and metabolites after pure culture of microalgae and co-culture of microalgae with strain WS-9 are shown in Fig.16 The nodes represent different metabolites and metabolic pathways, where orange nodes represent metabolic pathways, red nodes represent up-regulated metabolites, and blue nodes represent down-regulated metabolites. Fig.16 The up-regulated and down-regulated metabolites were widely distributed in the metabolic network, indicating that multiple metabolic pathways were significantly affected in the co-culture system, indicating that the co-culture environment of microalgae and carbon-fixing bacteria strainWS-9 has a significant regulatory effect on metabolic activities.

[0152] (4) Analysis of metabolic pathways

[0153] The results of differential analysis of metabolic pathways in pure culture of microalgae and co-culture of microalgae with strain WS-9 are shown in Fig.17 As shown. The vertical axis lists the specific metabolic pathway name, and the horizontal axis is Rich factor, which indicates the proportion of differential metabolites in the pathway to the total number. The color of the circle indicates the p-value, and the color from blue to red indicates the p-value from high to low, that is, the significance from low to high; the size of the circle indicates Diff_number, that is, the number of differential metabolites in the pathway. The larger the circle, the more differential metabolites. Fig.17It can be seen that metabolic pathways with high enrichment factors and significant p-values ​​are mainly concentrated in riboflavin, purine and amino acid metabolism. For example, Riboflavin metabolism, Purine metabolism, Alanine, aspartate and glutamate metabolism, and Glutathione metabolism reflect the extensive regulation of microalgae metabolic pathways by the co-culture system. The enrichment of these metabolic pathways may be due to the following reasons: First, under co-culture conditions, the metabolic activities of carbon-fixing bacteria strain WS-9 and microalgae are enhanced, and the demand for coenzymes (such as riboflavin) increases. Carbon-fixing bacteria strain WS-9 may secrete riboflavin or its derivatives to promote the growth and metabolism of microalgae, thereby enhancing riboflavin metabolism in mixed culture. Second, co-culture may promote cell proliferation and division, increase the demand for nucleic acid synthesis, and lead to the enhancement of purine metabolic pathways. Microalgae and carbon-fixing bacteria strain WS-9 may maintain balance through metabolite exchange, and purine metabolites play an important role in this interaction. Third, there may be a complementary relationship in nitrogen metabolism between the carbon-fixing bacteria strain WS-9 and the microalgae, which enhances the synthesis and decomposition of amino acids to meet the cell's demand for amino acids. Fourth, there may be higher oxidative stress in the co-culture environment, causing cells to require more glutathione to maintain redox balance. The carbon-fixing bacteria strain WS-9 and the microalgae may protect each other from oxidative damage by enhancing glutathione metabolism, thereby better surviving and growing in the co-culture environment. In short, the activity of these metabolic pathways not only helps maintain cell growth and division, but also enhances antioxidant capacity and metabolic efficiency, thereby optimizing the overall metabolic state of the co-culture system.

[0154] (5) Analysis of metabolite interactions

[0155] The results of differential analysis of metabolic pathways in pure culture of microalgae and co-culture of microalgae with strain WS-9 are shown in Fig.18As shown in the figure. The figure contains multiple metabolite categories, which are classified and color-coded according to different categories (Class_I). The specific category identifiers from top to bottom are: Alkaloids and derivatives, Benzenoids, Lipids and lipid-like molecules, Nucleosides, nucleotides, and analogues, Organic acids and derivatives, Organic nitrogen compounds, Organic oxygen compounds, Organic heterocyclic compounds, Other, and Phenylpropanoids and polyketides. Each color represents a metabolite category. The chord connections in the chord diagram represent the interactions between different metabolites, where red chords represent positive interactions and blue chords represent negative interactions. The interactions between metabolites in the co-culture system are more complex, with far more positive correlations than negative correlations, and there are close relationships between multiple metabolites.

[0156] (6) Changing trends of different metabolite clusters

[0157] The changing trends of different metabolite clusters in pure culture of microalgae and co-culture of microalgae with strain WS-9 are shown in Figure 2. Fig.19 As shown. Each small figure in the figure represents a metabolite cluster, and the black line represents the average change trend of the metabolites in the cluster. The metabolites of Cluster-3, 4, 5, 8, and 10 increased significantly in the co-culture system, and the metabolites of Cluster-1, 2, 6, and 7 decreased overall. These change trends indicate that different metabolites respond differently in the co-culture system, and some metabolites are significantly regulated in the co-culture environment.

Claims

1. A Stutzerimonas frequents strain WS-9, characterized in that: It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit date is July 5, 2024, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.31181.

2. Use of the recurrent Sterzerimonas described in claim 1 in fixing CO2.

3. Use of the recurrent Sterzerimonas described in claim 1 in promoting CO2 fixation in microalgae.

4. The use according to claim 3, characterized in that: The microalgae include one or more of Chlorella sp., Tetradesmus obliquus, Scenedesmus quadricauda, ​​Chlamydomonas reinhardtii, Chlorella sorokiniana and Desmodesmus sp.

5. The use according to claim 4, characterized in that: Said Chlorella includes FACHB-5 and / or FACHB-31; Tetraselmis obliquus includes FACHB-12; Scenedesmus quadricauda includes FACHB-44; Chlamydomonas reinhardtii includes FACHB-265; Sorokin Chlorella includes FACHB-1068; Desmodesmus includes FACHB-2042.

6. The use according to claim 4 or 5, characterized in that: The Chlorella includes FACHB-5.

7. A microbial agent, characterized in that: The microbial agent includes the frequently occurring Sterzerimonas described in claim 1.

8. A microbial agent according to claim 7, characterized in that: The microbial agent also includes microalgae capable of fixing CO2.

9. A microbial agent according to claim 8, characterized in that: The microalgae include one or more of Chlorella sp., Tetradesmus obliquus, Scenedesmus quadricauda, ​​Chlamydomonas reinhardtii, Chlorella sorokiniana and Desmodesmus sp.

10. Use of the microbial agent according to any one of claims 7 to 9 in fixing CO2.

Images