Arthrobacter sp. GD-20 and its application in carbon fixation and phosphorus solubilization

Abstract

The present invention relates to the technical field of microbial carbon fixation, in particular to an Arthrobacter sp. GD-20 and its application in carbon fixation and phosphorus solubilization. The present invention provides an Arthrobacter sp. GD-20 with a preservation number of CGMCC No. 31727. The Arthrobacter GD-20 provided by the present invention is an Arthrobacter isolated from the soil of a corn field in a salinized photovoltaic field area, which has the ability of carbon fixation and phosphorus solubilization, can tolerate acidic conditions, and has a wide adaptability to oligotrophic conditions. It provides strain resources for improving the carbon fixation and emission reduction ability of salinized soil, provides technical support for in-depth understanding of the microbial regulation process of the occurrence form of nutrients in salinized soil, lays a foundation for proposing soil microbial improvement technologies in photovoltaic field areas, and can promote the high-quality development of the photovoltaic industry in fragile salinized areas.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial carbon fixation, and particularly relates to an Arthrobacter sp. GD-20 and its application in carbon fixation and phosphorus solubilization. Background Art

[0002] The development of new energy has become the core approach to address climate change. However, more than 80% of new energy in China is distributed in fragile areas (such as saline-alkali lands), and there is an urgent need to explore a new model for the organic integration of energy, ecological restoration, and the development of characteristic industries. Existing studies have shown that functional microorganisms such as nitrogen-fixing bacteria and cellulose-degrading bacteria play an important role in improving saline-alkali lands, restoring soil physical and chemical properties, and ecological environment. Improving the potential of soil carbon sequestration and sink enhancement is an urgent need to restore the ecological function of saline-alkali lands 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-fixing abilities, and there is even less research on the precise identification and screening of soil carbon-fixing microorganisms in photovoltaic fields of saline-alkali lands. Therefore, isolating and screening efficient indigenous carbon-fixing functional microorganisms in photovoltaic fields of saline-alkali lands has important potential application value for stimulating and enhancing the synergistic benefits of indigenous microorganisms in soil improvement and carbon sequestration and sink enhancement. Summary of the Invention

[0003] To solve the above problems, the present invention provides an Arthrobacter sp. GD-20 and its application in carbon fixation and phosphorus solubilization. The Arthrobacter sp. GD-20 provided by the present invention has the ability of carbon fixation and phosphorus solubilization, can tolerate acidic conditions, has a wide adaptability to oligotrophic conditions, and has good application value for enhancing the potential of soil carbon sequestration and sink, promoting the high-quality development of the photovoltaic industry in fragile saline-alkali areas, and alleviating global warming.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides an Arthrobacter sp. ( Arthrobacter sp.) GD-20, with the preservation number of CGMCC No. 31727.

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

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

[0008] The present invention provides a preparation method of the carbon-fixing bacterium agent described in the above technical solution, including the following steps:

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

[0010] Preferably, the carbon source-free medium comprises components with the following concentrations: 0.5 g / L of Na2HPO4, 0.5 g / L of KH2PO4, 0.3 g / L of MgSO4, 0.2 g / L of CaCl2, 0.5 g / L of NH4Cl, 0.25 g / L of NaNO3, 0.4 g / L of NaCl, and 10 mL / L of trace element solution; the trace element solution comprises components with the following concentrations: 0.3 g / L of FeCl2, 0.3 g / L of FeSO4·7H2O, 0.15 g / L of MnSO4·H2O, 0.25 g / L of ZnSO4·7H2O, and 0.2 g / L of CoCl2.

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

[0012] Preferably, the carbon source-containing medium includes the first medium or LB medium; the first medium is prepared by adding a carbon source to the carbon source-free medium.

[0013] The present invention provides the application of Arthrobacter GD-20 described in the above technical solution, or the carbon sequestration agent described in the above technical solution, or the carbon sequestration agent prepared by using the preparation method described in the above technical solution in carbon sequestration and / or dissolving insoluble inorganic phosphorus, and the carbon source for carbon sequestration includes inorganic carbon sources.

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

[0015] Preferably, during the application, the pH value of the area to be carbon-sequestered and / or the area where insoluble inorganic phosphorus is to be dissolved is 5.5 - 7.4.

[0016] Beneficial effects:

[0017] The present invention provides an Arthrobacter GD-20 with a preservation number of CGMCC No. 31727. The Arthrobacter GD-20 provided by the present invention is an Arthrobacter isolated from the soil of a corn field in a salinized photovoltaic field area, which has the ability of carbon sequestration and phosphorus solubilization, can tolerate acidic conditions, and has a wide adaptability to oligotrophic conditions, providing strain resources for improving the carbon sequestration and emission reduction ability of salinized soil, providing technical support for in-depth understanding of the microbial regulation process of the occurrence form of nutrients in salinized soil, laying a foundation for proposing soil microbial improvement technologies in photovoltaic field areas, and promoting the high-quality development of the photovoltaic industry in salinized vulnerable areas. Description of the Drawings

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0019] Figure 1 It is a cell morphology diagram of Arthrobacter sp. GD-20;

[0020] Figure 2 It is a colony characteristic diagram of Arthrobacter sp. GD-20;

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

[0022] Figure 4 It is the determination result of the growth and carbon fixation ability of Arthrobacter sp. GD-20 under different conditions;

[0023] Figure 5 It is the determination result of the growth of Arthrobacter sp. GD-20 under the addition of different types of carbon sources;

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

[0025] Figure 7 It is the determination result of the available phosphorus content after culturing Arthrobacter sp. GD-20 in a liquid medium containing insoluble phosphate salts.

[0026] Biological deposit description

[0027] Arthrobacter sp. GD-20, taxonomically named Arthrobacter sp., was deposited at the China General Microbiological Culture Collection Center (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. The deposit number is CGMCC No. 31727. Detailed implementation manners

[0028] The present invention provides a strain of Arthrobacter sp. GD-20 with a deposit number of CGMCC No. 31727. The Arthrobacter sp. GD-20 described in the present invention is a strain of Arthrobacter isolated from the soil of a cornfield in a salinized photovoltaic field. The cell morphology is rod-shaped to oval-shaped ( Figure 1 ), and the colonies it produces on the solid medium are small, circular, yellow, opaque, smooth and flat, with neat edges ( Figure 2) It 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 good growth rates and carbon fixation capabilities under acidic conditions, in a very low-nutrient salt medium, and in the presence of electron donors NaNO2 and Na2S2O3. And under the condition of only adding inorganic carbon source NaHCO3, its carbon fixation ability is significantly improved (more than five times).

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

[0030] As an implementation manner, the OD of Arthrobacter GD-20 in the carbon-fixing bacterium agent 600 value ≥ 0.6.

[0031] The present invention provides a preparation method of the carbon-fixing bacterium agent described in the above technical solution, including the following steps:

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

[0033] As an implementation manner, the medium without a carbon source includes the following components in the following concentrations: Na2HPO4 0.5 g / L, KH2PO4 0.5 g / L, MgSO4 0.3 g / L, CaCl2 0.2 g / L, NH4Cl 0.5 g / L, NaNO3 0.25 g / L, NaCl 0.4 g / L, trace element solution 10 mL / L; the trace element solution includes the following components in the following concentrations: FeCl2 0.3 g / L, FeSO4·7H2O 0.3 g / L, MnSO4·H2O 0.15 g / L, ZnSO4·7H2O 0.25 g / L, CoCl2 0.2 g / L. Arthrobacter GD-20 of the present invention can utilize carbon dioxide in the atmosphere, so it can reproduce in a medium without a carbon source and fix carbon dioxide in the atmosphere.

[0034] As an implementation manner, the carbon source of the medium containing a carbon source includes: one or more of sodium carbonate, sodium bicarbonate, glucose, oxalic acid, and sodium carboxymethylcellulose. As another implementation manner, the carbon source of the medium containing a carbon source is sodium bicarbonate.

[0035] As an implementation manner, the medium containing a carbon source includes the first medium or LB medium; the first medium is prepared by adding a carbon source to the medium without a carbon source.

[0036] As an implementation manner, 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 application of Arthrobacter sp. GD-20 described in the above technical solution, or the carbon-fixing bacterial agent described in the above technical solution, or the carbon-fixing bacterial agent prepared by the preparation method described in the above technical solution in carbon fixation and / or dissolution of insoluble inorganic phosphorus, and the carbon source for carbon fixation includes inorganic carbon sources.

[0038] As an implementation manner, the inorganic carbon source includes one or more of carbon dioxide, sodium bicarbonate, and sodium carbonate. Arthrobacter sp. GD-20 provided by the present invention has higher carbon fixation potential in soil environments with higher 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 implementation manner, the insoluble inorganic phosphorus may be calcium phosphate. Arthrobacter sp. GD-20 provided by the present invention can dissolve insoluble inorganic phosphorus into soluble phosphate.

[0040] As an implementation manner, during the application, the pH value of the area to be carbon-fixed and / or the area where insoluble inorganic phosphorus is to be dissolved is 5.5 to 7.4. Arthrobacter sp. 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 acidic environments.

[0041] To further illustrate the present invention, the following describes in detail an Arthrobacter sp. GD-20 provided by the present invention and its application in carbon fixation and phosphorus dissolution in combination with the drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

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

[0043] Taking the cornfield soil in the Daqing salinized photovoltaic field area as the target soil, the specific screening scheme is as follows:

[0044] 1. Collect the soil at a depth of 0 to 20 cm, take 10.0 g of the collected soil, add it to 100 mL of sterilized water, place it on a shaker and shake (180 revolutions per minute, shake for 30 min) to make a soil suspension. Then dilute the soil suspension by a factor of 10 in a gradient to 10 -4times, take 0.2 mL of the soil dilution and spread it on a solid medium without an organic carbon source, and culture it at 28 °C for 7 days. The formula of the solid medium is: 0.5 g / L of Na2HPO4, 0.5 g / L of KH2PO4, 0.3 g / L of MgSO4, 0.2 g / L of CaCl2, 0.5 g / L of NaHCO3, 0.5 g / L of NH4Cl, 0.25 g / L of NaNO3, 0.4 g / L of NaCl, 10 mL / L of trace element solution, and 20 g / L of agar; the pH value of the solid medium is 7.0. The formula of the trace element solution is: 0.3 g / L of FeCl2, 0.3 g / L of FeSO4·7H2O, 0.15 g / L of MnSO4·H2O, 0.25 g / L of ZnSO4·7H2O, and 0.2 g / L of CoCl2.

[0045] 2. Pick different colonies with good morphology, growth vigor and fast growth rate on the solid medium without an organic carbon source, and use the inoculation loop to purify them on a new solid medium without an organic carbon source by the streak plate method until pure culture. Through the dilution plate method, 25 carbon-fixing bacteria were initially isolated and purified. Select the dominant bacteria for the secondary screening of carbon-fixing function (refer to Example 2), and pick the best-performing 1 strain as the research object, denoted as GD-20. Use a scanning electron microscope to determine that the cell morphology of this strain is rod-shaped to oval-shaped ( Figure 1 ), and the colonies it produces on the solid medium are small, round, yellow, opaque, smooth and flat, with neat edges ( Figure 2 ).

[0046] 3. Further sequence the 16S rRNA gene of this strain: Use the universal primers 27F (SEQ ID NO.1) and 1492R (SEQ ID NO.2) to perform PCR amplification on the genomic DNA of the strain, and then sequence the amplification product. The sequencing result is shown in SEQ ID NO.3, as follows:

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

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

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

[0050] Then, using the NCBI database, BLAST analysis was performed based on the 16S rRNA gene sequence of the strain. The results showed that the strain had 100% homology with the 16S rRNA gene sequence of Arthrobacter globiformis 1TCS.8 (accession number PP301853.1), 99.71% homology with the 16S rRNA gene sequence of Arthrobacter humicola ICMP 22236 (accession number MH392660.1), 99.57% homology with the 16S rRNA gene sequences of Arthrobacter pascens MR-26 (accession number KY753225.1) and Arthrobacter ramosus (accession number X80742.1). As Figure 3 shown, the phylogenetic analysis of the 16S rRNA gene indicated that the strain had the closest phylogenetic relationship with Arthrobacter globiformis Combining its cell morphology and colony characteristics, the strain was identified as Arthrobacter sp. However, the accurate classification of the strain could not be determined solely by 16S rRNA gene sequencing analysis. Further, whole-genome sequencing was performed on it: the extracted whole genome was sent to Shanghai Personal Biotechnology Co., Ltd. for second-generation sequencing, and the obtained sequence results were compared with the reference strains of related species in the NCBI database Arthrobacter sp.). However, the accurate classification of this strain could not be determined solely by 16S rRNA gene sequencing analysis. Further, its whole genome was sequenced: the extracted whole genome was sent to Shanghai Personal Biotechnology Co., Ltd. for second-generation sequencing, and the obtained sequence results were compared with the reference strains of related species in the NCBI database Arthrobacter globiformis NBRC12137, Arthrobacter humicola 53, Arthrobacter pascens CC187 and Arthrobacter ramosusThe average nucleotide identity (ANI) analysis was performed on the whole-genome sequence of ZX_2022b. When the ANI value is greater than 95%, the two can be considered to be of the same bacterial species. The ANI results were shown as 82.43%, 79.60%, 94.53% and 77.45% respectively, indicating that this strain is not of the same species as the above-mentioned closest related species and is a potential new strain. Arthrobacter GD-20 was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms with the deposit number of 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 strains: Arthrobacter GD-20, isolated and identified in Example 1.

[0054] Carbon-free liquid medium: Na2HPO4 0.5 g / L, KH2PO4 0.5 g / L, MgSO4 0.3 g / L, CaCl2 0.2 g / L, NH4Cl 0.5 g / L, NaNO3 0.25 g / L, NaCl 0.4 g / L, trace element solution 10 mL / L; pH value is 7.0. The formula of the trace element solution is: FeCl2 0.3 g / L, FeSO4·7H2O 0.3 g / L, MnSO4·H2O 0.15 g / L, ZnSO4·7H2O 0.25 g / L, CoCl2 0.2 g / L.

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

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

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

[0058] Na2S2O3 liquid medium: Replace NaNO2 4.6 g / L in the NaNO2 liquid medium with Na2S2O3 5 g / L, and the pH value is 7.0.

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

[0060] Pick the purified GD-20 colonies in Example 1 and inoculate them into 5 mL of carbon-free liquid medium. Culture at 28 °C and 160 r / min for 5 days. Take 1 mL of the bacterial liquid at the end of the culture. After measuring the strain abundance using a flow cytometer, take 400 μL and inoculate them 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 respectively, and make 3 parallels. After culturing at 28 °C for 5 days, detect the strain abundance in the bacterial liquid again. The specific measurement method of microbial abundance is as follows: Dilute SYBR-Green (10000×, Solarbio) 100 times and mix it with propidium iodide (PI, Solarbio) at a ratio of 50:1 (the volume ratio of SYBR-Green and PI is 50:1) to obtain a fluorescent dye. Pass 1 mL of the bacterial liquid through a 300-mesh nylon sieve to remove large particle impurities, add 10 μL of the fluorescent dye, stain it in the dark for 15 minutes, and vortex it before detection to determine the number of viable bacteria.

[0061] 1.3 Data processing

[0062] The data of this experiment were analyzed in CytExpert SRT software and then preliminarily sorted using Microsoft Excel Office 2016 software. The experimental results were plotted using Graphpad Prism 9.5.

[0063] 2. Results and analysis

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

[0065] Example 3 Carbon source utilization ability of the 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: 5 g / L yeast extract, 10 g / L tryptone, 5 g / L NaCl; pH value is 7.0.

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

[0070] Pick the purified GD-20 colonies in Example 1 and inoculate them into 100 mL of LB medium, and culture until OD 600The value is 0.8. The obtained bacterial cell precipitate after centrifugation was washed and resuspended with the carbon-free liquid medium in Example 2. One part of it was autoclaved (121 °C, 30 minutes) to serve as microbial necromass (MNC), and the other part was directly inoculated into 70 mL of carbon-free liquid medium (denoted as NaHCO3-Free) and NaHCO3 liquid medium, and different types of organic carbon sources were added to each medium. Soluble organic matters present in the soil (oxalic acid (OA), glucose (GLU), sodium carboxymethyl cellulose (CMC)) and microbial necromass were selected as exogenous additives according to the order from simple to complex structure. Various carbon sources were added to the two media at a total organic carbon content of 2 mg / L. At the same time, a control group without adding carbon source (CK) was set, and each treatment was repeated 3 times and cultured at 28 °C and 160 r / min for 7 days. The bacterial liquid at the end of the culture was taken, and the abundance of the strain was measured by flow cytometry. The specific measurement method refers to Example 2.

[0071] 1.3 Data processing

[0072] The data of this experiment were preliminarily sorted out using Microsoft Excel Office 2016 software. The experimental results were plotted using Graphpad Prism 9.5. The results are shown in Figure 5 and Table 1, where **** represents p < 0.0001.

[0073] 2. Results and analysis

[0074] Carbon sources are crucial for the growth and metabolism of microorganisms. Figure 5 Table 1 shows that the strain concentrations under the addition of various carbon sources are all higher than those of the control group, indicating that the growth ability of strain GD-20 has been improved to varying degrees. Among them, the promotion effect of microbial necromass carbon on the growth activity of the strain is better than that of other carbon source substances because microbial necromass can provide various nutrients for the strain. Glucose can also significantly stimulate the growth of the strain. However, oxalic acid and the refractory substance cellulose only have a weak growth promotion effect on the strain, indicating that the degradation activity of the strain towards the two is relatively low. In addition, the addition of NaHCO3 will lead to different responses in the growth of the strain under various treatments. The growth ability of the strain in the control group has been improved, while the growth of the strain in the treatments with different carbon sources added is inhibited. This phenomenon indicates that the role of NaHCO3 in the growth of this strain in oligotrophic and eutrophic environments is exactly the opposite, proving that the promotion effect of NaHCO3 on the carbon fixation potential of this strain can be better realized under oligotrophic conditions.

[0075] Table 1 Strain concentrations (Events / μL) under the addition of different carbon sources

[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] Analysis of the Carbon Fixation Pathway of the Carbon-Fixing Bacterium Arthrobacter GD-20 in Example 4

[0078] 1. Materials and Methods

[0079] The Arthrobacter GD-20 isolated and identified in Example 1 was sent to Shanghai Personal Biotechnology Co., Ltd. for second-generation sequencing using the Illumina Novaseq platform. A whole-genome framework map was assembled and spliced, and bioinformatics software and databases were used to annotate and functionally identify the bacteria. 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 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 converted to L-aspartate under the action of aspartate aminotransferase, and then further converted to threonine. Threonine was cleaved by threonine aldolase into glycine, and glycine was then converted to serine by glycine hydroxymethyltransferase. Finally, it was converted to pyruvate under the action of serine dehydrogenase to form a carbon fixation cycle( Figure 6 ). The first stable product generated when this pathway fixes CO2 is the four-carbon compound oxaloacetate. Through the conversion of various amino acids, the renewal of phosphoenolpyruvate is finally achieved, and 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 reaction of acetyl-CoA with HCO3 - to generate malonyl-CoA under the catalysis of the enzyme with higher carbon fixation efficiency - acetyl-CoA carboxylase (ACC). This step of reaction is the first step in fatty acid synthesis and the synthesis of other substances, which also explains to a certain extent the phenomenon that the addition of the inorganic carbon source NaHCO3 significantly improves the carbon fixation ability of this strain.

[0082] Study on the Phosphorus-Solubilizing Ability of the Carbon-Fixing Bacterium Arthrobacter GD-20 in Example 5

[0083] 1. Materials and Methods

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

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

[0086] 1.2 Detection of phosphate-solubilizing ability of the carbon-fixing bacterium Arthrobacter sp. GD-20

[0087] Dilute the 5 g / L KH2PO4 standard solution to concentrations of 0, 2, 2.5, 250, 500, and 1000 mg / L. Measure the absorbance using the molybdenum antimony anti-colorimetric method. The standard curve equation obtained through measurement is y = 0.0028x + 0.048, R 2 = 0.9996.

[0088] Take the colonies purified from the solid medium without organic carbon source in Example 1, inoculate them into 10 mL of Pikovskaya liquid medium, culture at 28 °C and 160 r / min for 5 days. Set a control group without inoculating the strain. At the end of the culture, centrifuge the bacterial liquid, add 1 mL of sterile water to the bacterial cell precipitate, grind it using a tissue disruptor and mix it with the supernatant, add NaHCO3 and then shake and filter. Measure its organic phosphorus content using the molybdenum antimony anti-colorimetric method. The measurement results are shown in Figure 7 , where 1 - 4 are the other 4 carbon-fixing bacteria isolated and purified in Example 1.

[0089] 1.3 Data processing

[0090] The data of this experiment was preliminarily sorted using Microsoft Excel Office 2016 software. The experimental results were plotted using Graphpad Prism 9.5.

[0091] 2. Results and analysis

[0092] Taking the ability to dissolve tricalcium phosphate as an index, it was determined that the Arthrobacter sp. GD-20 has phosphate-solubilizing ability and realizes the utilization of insoluble phosphate. Its phosphate-solubilizing ability is 409.11 mg / L, while the phosphate-solubilizing abilities of the other 4 carbon-fixing bacteria are as low as 115.18 mg / L at the lowest and only 301.25 mg / L at the highest, all lower than that of this carbon-fixing bacterium.

[0093] In summary, the Arthrobacter sp. GD-20 isolated in the present invention has the abilities of carbon fixation and phosphorus solubilization, can tolerate acidic conditions, and has a wide adaptability to oligotrophic conditions, providing strain resources for enhancing the carbon sequestration and emission reduction capacity of saline-alkali soil, providing technical support for in-depth understanding of the microbial regulation process of nutrient occurrence forms in saline-alkali soil, laying a foundation for proposing soil microbial improvement technologies in photovoltaic fields, and promoting the high-quality development of the photovoltaic industry in fragile saline-alkali areas.

[0094] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An Arthrobacter sp. GD-20, with the deposit number of CGMCC No. 31727. Arthrobacter ​ 2. A carbon-fixing bacterial agent, characterized in that, including Arthrobacter GD-20 described in claim 1.

3. The carbon-fixing microbial inoculum according to claim 2, wherein The OD of Arthrobacter sp. GD-20 in the carbon fixation bacterial agent 600 value ≥ 0.

6.

4. The preparation method of the carbon-fixing bacterial agent according to claim 2 or 3, characterized in that, comprising the following steps: Inoculating Arthrobacter GD-20 described in claim 1 into a culture medium for culturing to obtain the carbon-fixing microbial agent; the culture medium is a culture medium containing a carbon source or a culture medium without a carbon source; 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 a carbon source comprises components with the following concentrations: 0.5 g / L of Na2HPO4, 0.5 g / L of KH2PO4, 0.3 g / L of MgSO4, 0.2 g / L of CaCl2, 0.5 g / L of NH4Cl, 0.25 g / L of NaNO3, 0.4 g / L of NaCl, 10 mL / L of trace element solution; the trace element solution comprises components with the following concentrations: 0.3 g / L of FeCl2, 0.3 g / L of FeSO4·7H2O, 0.15 g / L of MnSO4·H2O, 0.25 g / L of ZnSO4·7H2O, 0.2 g / L of CoCl2.

6. The preparation method according to claim 4, wherein The carbon source of the culture medium containing a carbon source includes one or more of sodium carbonate, sodium bicarbonate, glucose, oxalic acid, and sodium carboxymethylcellulose.

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 without a carbon source.

8. Use of Arthrobacter GD-20 described in claim 1 or the carbon-fixing microbial agent described in claim 2 or 3 or the carbon-fixing microbial agent prepared by using 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 application according to claim 8, characterized in that, The inorganic carbon source includes one or more of carbon dioxide, sodium bicarbonate, and sodium carbonate; the insoluble inorganic phosphorus includes calcium phosphate.

10. The application according to claim 8, characterized in that, When the application is carried out, the pH value of the area to be carbon-fixed and / or the area where insoluble inorganic phosphorus is to be dissolved is 5.5 to 7.4.

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