Arthrobacter GD-20 and application thereof in carbon sequestration and phosphorus solubilization

Abstract

The invention relates to the technical field of microbial carbon sequestration, in particular to arthrobacter GD-20 and application thereof in carbon sequestration and phosphorus solubilization. The invention provides an arthrobacter sp. GD-20 strain with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.31727. The arthrobacter sp. GD-20 strain is provided by the invention. The arthrobacter GD-20 provided by the invention is an arthrobacter separated from saline-alkali photovoltaic field corn field soil, has carbon sequestration capacity and phosphate solubilizing capacity, can tolerate acidic conditions, has wide adaptability to oligotrophic conditions, provides a strain resource for improving the carbon sequestration and emission reduction capacity of saline-alkali soil, and has a wide application prospect. Technical support is provided for deeply knowing the microorganism regulation process of the saline-alkali soil nutrient substance occurrence form, a foundation is laid for providing a photovoltaic field soil microorganism improvement technology, and high-quality development of the saline-alkali fragile area photovoltaic industry can be promoted.

Description

Technical Field

[0001] The invention relates to the technical field of microbial carbon fixation, in particular to a strain of Arthrobacter GD-20 and application thereof in carbon fixation and phosphorus dissolution. Background Art

[0002] The development of new energy has become the core way to deal with climate change. However, more than 80% of my country's new energy is distributed in fragile areas (such as saline-alkali land). It is urgent to explore new models of organic integration of energy, ecological restoration, and characteristic industrial development. Studies have shown that functional microorganisms such as nitrogen-fixing bacteria and cellulose-degrading bacteria play an important role in improving saline-alkali land, restoring soil physical and chemical properties, and ecological environment. Improving the potential of soil carbon sequestration and carbon sink is an urgent need to restore the ecological function of saline-alkali land and mitigate climate change. However, there is still a lack of research on the isolation and screening of soil carbon-fixing microorganisms and their carbon sequestration ability. There is even less research on the precise identification and screening of soil carbon-fixing microorganisms in photovoltaic fields in saline-alkali land. To this end, the separation and screening of efficient carbon-fixing indigenous functional microorganisms in photovoltaic fields in saline-alkali land has important potential application value for stimulating and enhancing the synergistic benefits of indigenous microorganisms in improving soil and fixing carbon and increasing carbon sinks. Summary of the invention

[0003] In order to solve the above problems, the present invention provides a strain of Arthrobacter GD-20 and its application in carbon fixation and phosphorus solubilization. The Arthrobacter GD-20 provided by the present invention has carbon fixation and phosphorus solubilization capabilities, can tolerate acidic conditions, has a wide range of adaptability to oligotrophic conditions, and has good application value for improving the soil carbon fixation and sink potential, promoting the high-quality development of the photovoltaic industry in salinization-vulnerable areas, and alleviating global warming.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The invention provides an Arthrobacter sp. GD-20, whose preservation number is CGMCC No.31727.

[0006] The present invention provides a carbon-fixing bacterial agent, comprising the Arthrobacter GD-20 described in the above technical solution.

[0007] Preferably, the OD of Arthrobacter GD-20 in the carbon-fixing bacteria agent is 600 Value ≥ 0.6.

[0008] The present invention provides a method for preparing the carbon-fixing bacteria agent described in the above technical solution, comprising the following steps:

[0009] The Arthrobacter GD-20 described in the above technical solution is inoculated into a culture medium for cultivation to obtain the carbon-fixing bacterial agent; the culture medium is a culture medium containing a carbon source or a culture medium without a carbon source; and the pH value of the culture medium is 5.5-7.4.

[0010] Preferably, the culture medium containing no carbon source comprises components with the following concentrations: Na2HPO4 0.5g / L, KH2PO4 0.5g / L, MgSO4 0.3g / L, CaCl2 0.2g / L, NH4Cl 0.5g / L, NaNO3 0.25g / L, NaCl 0.4g / L, and trace element solution 10mL / L; the trace element solution comprises components with the following concentrations: FeCl2 0.3g / L, FeSO4·7H2O 0.3g / L, MnSO4·H2O 0.15g / L, ZnSO4·7H2O 0.25g / L, and CoCl2 0.2g / L.

[0011] Preferably, the carbon source of the culture medium containing a carbon source comprises one or more of sodium carbonate, sodium bicarbonate, glucose, oxalic acid and sodium carboxymethyl cellulose.

[0012] Preferably, the culture medium containing a carbon source comprises a first culture medium or an LB culture medium; the first culture medium is prepared by adding a carbon source to the culture medium not containing a carbon source.

[0013] The present invention provides the use of the Arthrobacter GD-20 described in the above technical scheme or the carbon-fixing bacteria agent described in the above technical scheme or the carbon-fixing bacteria agent prepared by the preparation method described in the above technical scheme in carbon fixation and / or dissolution of insoluble inorganic phosphorus, and the carbon source for carbon fixation includes an inorganic carbon source.

[0014] Preferably, the inorganic carbon source includes one or more of carbon dioxide, sodium bicarbonate and sodium carbonate; and the sparingly soluble inorganic phosphorus includes calcium phosphate.

[0015] Preferably, during the application, the pH value of the area to be fixed with carbon and / or to be dissolved with sparingly soluble inorganic phosphorus is 5.5 to 7.4.

[0016] Beneficial effects:

[0017] The present invention provides a strain of Arthrobacter GD-20, with a deposit number of CGMCC No. 31727. The Arthrobacter GD-20 provided by the present invention is an Arthrobacter isolated from corn field soil in a salinized photovoltaic field area, has carbon fixation and phosphorus solubilization capabilities, can tolerate acidic conditions, and has a wide range of adaptability to oligotrophic conditions. It provides strain resources for improving the carbon fixation and emission reduction capabilities of salinized soils, provides technical support for in-depth understanding of the microbial regulation process of the occurrence form of nutrients in salinized soils, lays the foundation for proposing soil microbial improvement technology in photovoltaic fields, and can promote the high-quality development of the photovoltaic industry in salinized vulnerable areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0019] Figure 1 This is the bacterial morphology of Arthrobacter GD-20;

[0020] Figure 2 This is the colony characteristic diagram of Arthrobacter GD-20;

[0021] Figure 3 is the phylogenetic tree of the 16S rRNA gene of Arthrobacter GD-20;

[0022] Figure 4 The results of the growth and carbon fixation ability of Arthrobacter GD-20 under different conditions;

[0023] Figure 5 The results are the growth test results of Arthrobacter GD-20 under the addition of different types of carbon sources;

[0024] Figure 6 It is the carbon fixation pathway of Arthrobacter GD-20;

[0025] Figure 7 The results are for determining the effective phosphorus content of Arthrobacter GD-20 after culturing in a liquid medium containing insoluble phosphate salts.

[0026] Biological Deposit Description

[0027] Arthrobacter GD-20, classified and named Arthrobacter sp., was deposited in the China General Microbiological Culture Collection (CGMCC) on August 23, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.31727. DETAILED DESCRIPTION

[0028] The present invention provides a strain of Arthrobacter GD-20, with a deposit number of CGMCC No. 31727. The Arthrobacter GD-20 of the present invention is an Arthrobacter isolated from corn field soil in a salinized photovoltaic field area, and the bacterial body morphology is rod-shaped to oval ( Figure 1 ), the colonies produced on solid culture medium are small, round, yellow, opaque, smooth and flat with neat edges ( Figure 2), can tolerate acidic conditions, still has the ability to fix carbon in a low-nutrient environment, and has a wide range of carbon source utilization capabilities and the ability to dissolve insoluble inorganic phosphorus. The results of the examples show that Arthrobacter GD-20 can maintain a good growth rate and carbon fixation capacity under acidic conditions, in extremely low-nutrient salt culture media, and in the presence of electron donors NaNO2 and Na2S2O3, and its carbon fixation capacity is significantly improved (more than five times) under the condition of adding only inorganic carbon source NaHCO3.

[0029] Based on the above advantages, the present invention provides a carbon-fixing bacterial agent, including the Arthrobacter GD-20 described in the above technical solution.

[0030] As an embodiment, the OD of Arthrobacter GD-20 in the carbon-fixing bacteria agent is 600 Value ≥ 0.6.

[0031] The present invention provides a method for preparing the carbon-fixing bacteria agent described in the above technical solution, comprising the following steps:

[0032] The Arthrobacter GD-20 described in the above technical solution is inoculated into a culture medium for cultivation to obtain the carbon-fixing bacterial agent; the culture medium is a culture medium containing a carbon source or a culture medium without a carbon source; and the pH value of the culture medium is 5.5-7.4.

[0033] As an embodiment, the culture medium without carbon source includes components of the following concentrations: Na2HPO4 0.5g / L, KH2PO4 0.5g / L, MgSO4 0.3g / L, CaCl2 0.2g / L, NH4Cl 0.5g / L, NaNO3 0.25g / L, NaCl0.4g / L, trace element solution 10mL / L; the trace element solution includes components of the following concentrations: FeCl2 0.3g / L, FeSO4·7H2O 0.3g / L, MnSO4·H2O 0.15g / L, ZnSO4·7H2O 0.25g / L, CoCl2 0.2g / L. The Arthrobacter GD-20 of the present invention can utilize carbon dioxide in the atmosphere, so that it can reproduce and fix carbon dioxide in the atmosphere in a culture medium without a carbon source.

[0034] As an embodiment, the carbon source of the culture medium containing a carbon source comprises: one or more of sodium carbonate, sodium bicarbonate, glucose, oxalic acid and sodium carboxymethyl cellulose. As another embodiment, the carbon source of the culture medium containing a carbon source is sodium bicarbonate.

[0035] As an embodiment, the culture medium containing a carbon source includes a first culture medium or an LB culture medium; the first culture medium is prepared by adding a carbon source to the culture medium not containing a carbon source.

[0036] As an embodiment, the culture time is 5 to 7 days; the culture temperature is 28°C.

[0037] Based on the above advantages, the present invention provides the use of the Arthrobacter GD-20 described in the above technical scheme or the carbon-fixing bacteria agent described in the above technical scheme or the carbon-fixing bacteria agent prepared by the preparation method described in the above technical scheme in carbon fixation and / or dissolution of insoluble inorganic phosphorus, and the carbon source for carbon fixation includes an inorganic carbon source.

[0038] As an embodiment, the inorganic carbon source includes one or more of carbon dioxide, sodium bicarbonate and sodium carbonate. The Arthrobacter GD-20 provided by the present invention has a higher carbon fixation potential in a soil environment with a high carbonate (especially bicarbonate) content, which helps to develop the application of the strain in special environments (such as soda saline-alkali land or acidic soil).

[0039] As an embodiment, the poorly soluble inorganic phosphorus may be calcium phosphate. The Arthrobacter GD-20 provided by the present invention can dissolve the poorly soluble inorganic phosphorus into soluble phosphate.

[0040] As an embodiment, during the application, the pH value of the area to be fixed carbon and / or to be dissolved insoluble inorganic phosphorus is 5.5 to 7.4. The Arthrobacter GD-20 provided by the present invention can tolerate acidic conditions, has a wide range of carbon source utilization capabilities and the ability to dissolve insoluble inorganic phosphorus, and can be used for carbon fixation and / or dissolution of insoluble inorganic phosphorus in an acidic environment.

[0041] In order to further illustrate the present invention, a strain of Arthrobacter GD-20 provided by the present invention and its application in carbon fixation and phosphorus dissolution are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1 Isolation and Identification of Carbon-Fixing Bacteria Arthrobacter sp. GD-20

[0043] The corn field soil in the salinized photovoltaic field area of ​​Daqing was used as the target soil, and the specific screening scheme was as follows:

[0044] 1. Collect soil from a depth of 0 to 20 cm, take 10.0 g of the collected soil, add it to 100 mL of sterilized water, and place it on a shaker for shaking (180 rpm, shaking for 30 min) to make a soil suspension. Then dilute the soil suspension in a 10-fold gradient to 10 -4times, take 0.2mL of soil dilution, apply it to a solid culture medium without an organic carbon source, and culture it at 28°C for 7 days. The formula of the solid culture medium is: Na2HPO4 0.5g / L, KH2PO4 0.5g / L, MgSO4 0.3g / L, CaCl20.2g / L, NaHCO3 0.5g / L, NH4Cl 0.5g / L, NaNO3 0.25g / L, NaCl 0.4g / L, trace element solution 10mL / L, agar 20g / L; the pH value of the solid culture medium is 7.0. The formula of the trace element solution is: FeCl20.3g / L, FeSO4·7H2O 0.3g / L, MnSO4·H2O 0.15g / L, ZnSO4·7H2O 0.25g / L, CoCl20.2g / L.

[0045] 2. Pick different colonies with better morphology, better growth potential and faster growth rate from the solid culture medium without organic carbon source, and purify them multiple times on a new solid culture medium without organic carbon source by inoculation loop plate streak method until pure culture. Preliminary separation and purification of 25 strains of carbon-fixing bacteria were carried out by dilution plate method, and the dominant bacteria were selected for carbon fixation function rescreening (refer to Example 2), and the one with the best effect was selected as the research object, recorded as GD-20. Scanning electron microscopy was used to determine that the bacterial morphology of the strain was rod-shaped to oval ( Figure 1 ), the colonies produced on solid culture medium are small, round, yellow, opaque, smooth and flat with neat edges ( Figure 2 ).

[0046] 3. The strain was further subjected to 16S rRNA gene sequencing: the genomic DNA of the strain was amplified by PCR using universal primers 27F (SEQ ID NO.1) and 1492R (SEQ ID NO.2), and the amplified product was sequenced. The sequencing result is shown in SEQ ID NO.3, which is as follows:

[0047] SEQ ID NO.1: 5'-AGTTTGATCMTGGCTCAG-3';

[0048] SEQ ID NO.2: 5'-GGTTACCTTGTTACGACTT-3';

[0049] SEQ ID NO.3:5'-TGCAAGTCGAACGATGATCCGGTGCTTGCACCGGGGATTAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCTTAACTCTGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACTCCTCATCGCATGGTGGGGGGTGGAAAGCTTTTGTGGTTTTGGATGGACTCGCGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGTAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGCCGTGAAAGTCCGGGGCTCAACTCCGGATCTGCGGTGGGTACGGGCAGACTAGAGTGATGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTCTCTGGGCATTAACTGACGCTGAGGAGCGAAAGCATGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGTTGGGCACTAGGTGTGGGGGACATTCCACGTTTTCCGCGCCGTAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATGGACCGGACCGCCGCAGAAATGTGGTTTCCCCTTTGGGGCCGGTTCACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCCATGTTGCCAGCGCGTAATGGCGGGGACTCATGGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAAAGGGTTGCGATACTGTGAGGTGGAGCTAATCCC AAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCACGAAAGTTGGTAACACCCGAAGCCGGTGGCCTAACCCCTTGTGGGAGGGA-3'.

[0050] Then, BLAST analysis was performed based on the strain 16S rRNA gene sequence using the NCBI database. The results showed that the strain had 100% homology with the 16S rRNA gene sequence of Arthrobacter globiformis 1TCS.8 (Accession No. PP301853.1), 99.71% homology with the 16S rRNA gene sequence of Arthrobacter humicola ICMP 22236 (Accession No. MH392660.1), and 99.57% homology with the 16S rRNA gene sequences of Arthrobacter pascens MR-26 (Accession No. KY753225.1) and Arthrobacter ramosus (Accession No. X80742.1). Figure 3As shown in the figure, 16S rRNA gene phylogenetic analysis showed that the strain had the closest phylogenetic relationship with Arthrobacter globiformis. Combined with its bacterial morphology and colony characteristics, the strain was identified as Arthrobacter sp. However, the accurate classification of the strain cannot be determined by 16S rRNA gene sequencing analysis alone. Further, the whole genome sequencing was performed on it: the extracted whole genome was sent to Shanghai Paisono Biotechnology Co., Ltd. for second-generation sequencing, and the obtained sequence results were compared with the whole genome sequences of the closely related species reference strains Arthrobacter globiformis NBRC12137, Arthrobacter humicola 53, Arthrobacter pascens CC187 and Arthrobacterramosus ZX_2022b in the NCBI database for average nucleotide identity (ANI) analysis. When the ANI value is greater than 95%, the two can be considered to be the same species. The ANI results were 82.43%, 79.60%, 94.53% and 77.45%, respectively, indicating that the strain was not the same species as the closest related species mentioned above and was a potential new strain. Arthrobacter GD-20 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with the deposit number CGMCC No.31727.

[0051] Example 2 Study on the growth ability and carbon fixation ability of Arthrobacter GD-20 under different conditions

[0052] 1. Materials and methods

[0053] 1.1 Test strain: Arthrobacter GD-20, isolated and identified in Example 1.

[0054] Carbon-free liquid culture medium: Na2HPO4 0.5g / L, KH2PO4 0.5g / L, MgSO4 0.3g / L, CaCl20.2g / L, NH4Cl 0.5g / L, NaNO3 0.25g / L, NaCl 0.4g / L, trace element solution 10mL / L; pH value is 7.0. The formula of the trace element solution is: FeCl2 0.3g / L, FeSO4·7H2O 0.3g / L, MnSO4·H2O 0.15g / L, ZnSO4·7H2O0.25g / L, CoCl2 0.2g / L.

[0055] Na2CO3 liquid culture medium: Add 0.63g / L Na2CO3 to the carbon-free liquid culture medium, and the pH value is 7.0.

[0056] NaHCO3 liquid culture medium: Add 0.5g / L NaHCO3 to the carbon-free liquid culture medium and adjust the pH to 5.5, 6.2 and 7.0 respectively.

[0057] NaNO2 liquid culture medium: NaNO2 4.6g / L, Na2HPO4 0.5g / L, KH2PO4 0.5g / L, MgSO4 0.3g / L, CaCl2 0.2g / L, NaHCO3 0.5g / L, NH4Cl 0.5g / L, NaNO3 0.25g / L, NaCl 0.4g / L; pH value is 7.0.

[0058] Na2S2O3 liquid culture medium: Replace NaNO2 4.6g / L in NaNO2 liquid culture medium with Na2S2O35g / L, pH value is 7.0.

[0059] 1.2 Study on the growth and carbon fixation ability of Arthrobacter GD-20 under different pH, different inorganic carbon sources and different electron donors

[0060] The GD-20 colony purified in Example 1 was picked and inoculated into 5 mL of carbon-free liquid medium, and cultured at 28°C, 160 r / min for 5 days. 1 mL of the bacterial solution at the end of the culture was taken, and after the strain abundance was determined by flow cytometry, 400 μL was inoculated into 5 mL of carbon-free liquid medium (denoted as CO2), Na2CO3 liquid medium, NaHCO3 liquid medium with a pH value of 5.5 (denoted as pH5.5), NaHCO3 liquid medium with a pH value of 6.2 (denoted as pH6.2), NaHCO3 liquid medium with a pH value of 7.0 (denoted as NaHCO3), NaNO2 liquid medium and Na2S2O3 liquid medium, and 3 parallels were made. After culturing at 28°C for 5 days, the strain abundance in the bacterial solution was detected again. The specific measurement method of microbial abundance is as follows: SYBR-Green (10000×, Solebol) was diluted 100 times and mixed with propidium iodide (PI, Solebol) at a ratio of 50:1 (the volume of SYBR-Green and PI was 50:1) to obtain the fluorescent dye. After 1 mL of bacterial solution was passed through a 300-mesh nylon sieve to remove large particles of impurities, 10 μL of fluorescent dye was added to stain in the dark for 15 minutes, and the number of viable bacteria was determined after vortexing.

[0061] 1.3 Data processing

[0062] The experimental data were analyzed in CytExpert SRT software and then preliminarily sorted using Microsoft ExcelOffice 2016. The experimental results were plotted using Graphpad Pism 9.5.

[0063] 2. Results and Analysis

[0064] Since the only carbon sources available to the strain during the culture process are atmospheric carbon dioxide and inorganic carbon sources in the culture medium, the increase in the strain biomass comes from carbon fixation. The experimental results of the growth and carbon fixation ability of Arthrobacter GD-20 under different conditions are shown in Figure 4 , where different letters indicate significant differences between different groups (p < 0.05). The results showed that the strain could tolerate acidic conditions as low as pH 5.5, and grow and fix carbon well under acidic conditions, with an average daily growth rate of 3923.11 Events / μL. In the case of atmospheric carbon dioxide alone, the concentration of the strain can increase by 4352.07 Events / μL per day, indicating that it has a good fixation ability for atmospheric carbon dioxide. When the inorganic carbon source NaHCO3 is added, its carbon fixation ability is significantly improved (about five times), and the average daily growth rate of the strain can reach 22116.68 Events / μL, which may be the result of carbon fixation by the strain through other pathways. When an electron donor is added, the carbon fixation ability of the strain is not further improved, but will be weakened to varying degrees. When Na2S2O3 is added, its average daily growth rate is 3897.28 Events / μL, while when NaNO2 is added, its average daily growth rate is 2792.12 Events / μL. The results showed that the strain can maintain a high growth rate under all conditions and has a wide range of environmental adaptability. It can not only tolerate acidic conditions, but also grow in extremely low-nutrient salt medium without relying on electron donors. At the same time, the optimal growth conditions for the strain are 28°C, pH 7.0, and the addition of inorganic carbon source NaHCO3. This phenomenon shows that the strain has a higher carbon fixation potential in soil environments with high carbonate content, which is helpful for the development of strain applications in special environments (such as soda saline-alkali land).

[0065] Example 3 Carbon source utilization ability of carbon-fixing bacterium Arthrobacter GD-20

[0066] 1. Materials and methods

[0067] 1.1 Test strain: Arthrobacter GD-20, isolated and identified in Example 1.

[0068] Liquid LB medium: yeast extract 5 g / L, tryptone 10 g / L, NaCl 5 g / L; pH value was 7.0.

[0069] 1.2 Growth of carbon-fixing bacteria Arthrobacter GD-20 under different carbon sources

[0070] The GD-20 colony purified in Example 1 was picked up and inoculated into 100 mL LB medium and cultured until OD 600The value is 0.8, and the obtained bacterial precipitate after centrifugation is washed and resuspended using the carbon-free liquid culture medium in Example 2, one part of which is sterilized by high pressure (121°C, 30 minutes) as microbial residue (MNC), and the other part is directly inoculated into 70mL carbon-free liquid culture medium (recorded as NaHCO3-Free) and NaHCO3 liquid culture medium, and different types of organic carbon sources are added to each culture medium. Soluble organic matter (oxalic acid (OA), glucose (GLU), sodium carboxymethyl cellulose (CMC)) and microbial residues present in the soil are selected as exogenous additives according to the structure from simple to complex, and various carbon sources are added to the two culture media according to the total organic carbon content of 2mg / L. At the same time, a control group (CK) without adding a carbon source is set, and each treatment is repeated 3 times, and cultured for 7 days at 28°C and 160r / min. Take the bacterial liquid at the end of the culture, and use flow cytometry to determine the abundance of the strain, and its specific measurement method is referenced to Example 2.

[0071] 1.3 Data processing

[0072] The experimental data were preliminarily sorted using Microsoft Excel Office 2016 software. The experimental results were plotted using Graphpad Pism 9.5. Figure 5 and Table 1, where **** means p<0.0001.

[0073] 2. Results and Analysis

[0074] Carbon sources are essential for the growth and metabolism of microorganisms. Figure 5 Table 1 shows that the strain concentration under the addition of various carbon sources is higher than that of the control group, indicating that the growth ability of strain GD-20 has been improved to varying degrees, among which the promotion effect of microbial residue carbon on the growth activity of the strain is better than other carbon sources, because microbial residues can provide a variety of nutrients for the strain. Glucose can also significantly stimulate the growth of the strain, but oxalic acid and cellulose, a difficult-to-degrade substance, only have a weak growth promotion on the strain, indicating that the strain has low degradation activity for both. In addition, the addition of NaHCO3 will lead to differential responses to the growth of the strain under various treatments. The growth ability of the strain in the control group is improved, while the growth of the strain is inhibited in the treatments with the addition of different carbon sources. This phenomenon shows that the effect of NaHCO3 on the growth of the strain in oligotrophic and eutrophic environments is completely opposite, proving that the promotion effect of NaHCO3 on the carbon fixation potential of the strain can be better achieved under oligotrophic conditions.

[0075] Table 1 Strain concentrations under different carbon source additions (Events / μL)

[0076] Group CK OA GLU CMC MNC <![CDATA[NaHCO3-Free]]> 19204.33 23875.67 39889.33 25141.67 50204.00 <![CDATA[NaHCO3]]> 23415.67 18216.67 38583.67 21342.33 47701.67

[0077] Example 4 Analysis of carbon fixation pathways of carbon-fixing bacteria Arthrobacter GD-20

[0078] 1. Materials and methods

[0079] The Arthrobacter GD-20 isolated and identified in Example 1 was sent to Shanghai Paisono Biotechnology Co., Ltd. for second-generation sequencing using the Illumina Novaseq platform, and the whole genome framework map was assembled and spliced. The bacteria were annotated and functionally identified using bioinformatics software and databases, and the carbon fixation pathway of strain GD-20 was inferred through the KEGG database to provide a basis for studying the carbon fixation ability of the strain. The results are shown in Figure 6 , where the letters represent the key enzymes in the carbon fixation pathway, pps—phosphoenolpyruvate carboxylase; ppc—phosphoenolpyruvate carboxylase; pycA—pyruvate carboxylase; aspC—aspartate aminotransferase; ItaE—threonine aldolase; glyA—glycine hydroxymethyltransferase; SDS—serine dehydrogenase.

[0080] 2. Results and Analysis

[0081] Based on the KEGG database, the CO2 fixation pathway of strain GD-20 was determined. First, CO2 was fixed by phosphoenolpyruvate to generate oxaloacetate, which was then converted to L-aspartate by aspartate aminotransferase, and then converted to threonine. Threonine was cleaved into glycine by threonine aldolase, and glycine was then converted to serine by glycine hydroxymethyltransferase, and finally converted to pyruvate by serine dehydrogenase to form a carbon fixation cycle ( Figure 6 ). The first stable product produced when this pathway fixes CO2 is the four-carbon compound oxaloacetate, which is then converted into phosphoenolpyruvate through the conversion of various amino acids. The synthesis and degradation of threonine are the core processes in this cycle. In addition, biological carbon fixation is usually completed under the catalysis of a series of enzymes. Strain GD-20 can also achieve the conversion of acetyl-CoA with HCO3 under the catalysis of acetyl-CoA carboxylase (ACC), an enzyme with high carbon fixation efficiency. - The reaction produces malonyl-CoA, which is the first step in the synthesis of fatty acids and other substances. This also explains to a certain extent that the addition of inorganic carbon source NaHCO3 significantly improves the carbon fixation capacity of this strain.

[0082] Example 5 Study on the Phosphate Solubilization Capacity of Carbon-Fixing Bacteria Arthrobacter GD-20

[0083] 1. Materials and methods

[0084] 1.1 Test strain: Arthrobacter GD-20, isolated and identified in Example 1.

[0085] Pikovskaya liquid medium: yeast extract 0.5 g / L, glucose 10 g / L, Ca3(PO4)2 5 g / L, (NH4)2SO4 0.5 g / L, KCl 0.2 g / L, Mg3(PO4)2 0.1 g / L, MgSO4 0.0001 g / L, FeSO4 0.0001 g / L; pH 7.0.

[0086] 1.2 Phosphate solubilization ability test of carbon-fixing bacteria Arthrobacter GD-20

[0087] The 5g / L KH2PO4 standard solution was diluted to 0, 2, 2.5, 250, 500, and 1000mg / L concentrations, and the absorbance was determined by the molybdenum antimony anti-colorimetric method. The standard curve equation was y=0.0028x+0.048, R 2 =0.9996.

[0088] Take the colony purified from the solid culture medium without organic carbon source in Example 1, inoculate it into 10mL Pikovskaya liquid culture medium, culture it at 28℃, 160r / min, for 5 days, set up a control group without inoculation of strains, centrifuge the bacterial solution at the end of the culture, add 1mL sterile water to the bacterial precipitate, grind it with a tissue crusher, mix it with the supernatant, add NaHCO3, shake and filter it, and determine its organic phosphorus content by molybdenum antimony colorimetric method. The results of the determination are shown in Figure 7 , wherein 1 to 4 are the other 4 carbon-fixing bacteria isolated and purified in Example 1.

[0089] 1.3 Data processing

[0090] The experimental data were preliminarily sorted using Microsoft Excel Office 2016. The experimental results were plotted using Graphpad Pism 9.5.

[0091] 2. Results and Analysis

[0092] Using the ability to dissolve tricalcium phosphate as an indicator, it was determined that the Arthrobacter GD-20 had the ability to dissolve phosphate and achieved the utilization of insoluble phosphates. Its phosphate dissolving capacity was 409.11 mg / L, while the phosphate dissolving capacity of the other four carbon-fixing bacteria was as low as 115.18 mg / L and as high as 301.25 mg / L, all lower than that of the carbon-fixing bacteria.

[0093] In summary, the Arthrobacter GD-20 isolated by the present invention has the ability of carbon fixation and phosphorus solubilization, can tolerate acidic conditions, and has a wide range of adaptability to oligotrophic conditions. It provides bacterial resources for improving the carbon fixation and emission reduction capacity of saline-alkali soils, provides technical support for in-depth understanding of the microbial regulation process of the storage form of nutrients in saline-alkali soils, lays the foundation for proposing soil microbial improvement technology in photovoltaic fields, and can promote the high-quality development of the photovoltaic industry in saline-alkali vulnerable areas.

[0094] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Arthrobacter sp. GD-20, with a deposit number of CGMCC No.31727.

2. A carbon-fixing bacterial agent, characterized in that: The invention comprises the Arthrobacter GD-20 described in claim 1.

3. The carbon-fixing bacterial agent according to claim 2, characterized in that The OD of Arthrobacter GD-20 in the carbon-fixing bacteria agent 600 Value ≥ 0.

6.

4. The method for preparing the carbon-fixing bacteria agent according to claim 2 or 3, characterized in that: The following steps are involved: The Arthrobacter GD-20 described in claim 1 is inoculated into a culture medium for cultivation to obtain the carbon-fixing bacterial agent; the culture medium is a culture medium containing a carbon source or a culture medium without a carbon source; and the pH value of the culture medium is 5.5 to 7.

4.

5. The preparation method according to claim 4, characterized in that: The culture medium without carbon source includes components with the following concentrations: Na2HPO4 0.5g / L, KH2PO4 0.5g / L, MgSO4 0.3g / L, CaCl20.2g / L, NH4Cl 0.5g / L, NaNO3 0.25g / L, NaCl 0.4g / L, and trace element solution 10mL / L; the trace element solution includes components with the following concentrations: FeCl2 0.3g / L, FeSO4·7H2O 0.3g / L, MnSO4·H2O 0.15g / L, ZnSO4·7H2O 0.25g / L, and CoCl20.2g / L.

6. The preparation method according to claim 4, characterized in that: The carbon source of the culture medium containing a carbon source comprises one or more of sodium carbonate, sodium bicarbonate, glucose, oxalic acid and sodium carboxymethyl cellulose.

7. The preparation method according to claim 4 or 6, characterized in that: The culture medium containing a carbon source includes a first culture medium or an LB culture medium; the first culture medium is prepared by adding a carbon source to the culture medium not containing a carbon source.

8. Use of the Arthrobacter GD-20 described in claim 1, the carbon-fixing bacteria agent described in claim 2 or 3, or the carbon-fixing bacteria agent prepared by the preparation method described in any one of claims 4 to 7 in carbon fixation and / or dissolution of insoluble inorganic phosphorus, wherein the carbon source for carbon fixation includes an inorganic carbon source.

9. The use according to claim 8, characterized in that: The inorganic carbon source includes one or more of carbon dioxide, sodium bicarbonate and sodium carbonate; and the sparingly soluble inorganic phosphorus includes calcium phosphate.

10. The use according to claim 8, characterized in that: During the application, the pH value of the area where carbon is to be fixed and / or where sparingly soluble inorganic phosphorus is to be dissolved is 5.5 to 7.4.

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