Pseudomonas GD-9 with carbon fixation ability and its application

Abstract

The present invention provides a Pseudomonas GD-9 with carbon fixation ability and its application, which relate to the field of microbial technology. The Pseudomonas described in the present invention is classified and named as Pseudomonas Pseudomonas sp., and the deposit number is CGMCC No. 31725. The Pseudomonas described in the present invention is isolated from the soil in the rocky desertification photovoltaic field area of ​​Shilin County, Yunnan Province. Experiments have shown that the Pseudomonas can maintain good growth activity and carbon fixation ability in an extremely low-nutrient salt culture medium, and its carbon fixation ability is significantly improved under the condition of only adding an inorganic carbon source NaHCO3. The Pseudomonas isolated and obtained by the present invention has important scientific significance for improving the soil carbon fixation and sink potential and promoting the high-quality development of the photovoltaic industry in rocky desertification vulnerable areas.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and specifically relates to Pseudomonas GD-9 with carbon fixation ability and application thereof. Background Art

[0002] New energy development has become a core approach to implementing the "dual carbon" strategy and addressing climate change. However, over 80% of new energy resources are located in vulnerable areas, such as rocky desertification. These areas, characterized by shallow, calcium-rich, and alkaline soils, present a fragile ecosystem, severely hindering regional economic and social development. There is an urgent need to explore new models that organically integrate energy with ecological restoration and the development of specialized industries.

[0003] Currently, research on key technologies for eco-photovoltaic systems in rocky desertification-prone areas primarily focuses on monitoring and evaluating meteorological effects within photovoltaic sites and the impact of soil heterogeneity. However, there is a lack of research on the precise identification and screening of functional soil microorganisms within photovoltaic sites. There is an urgent need to analyze the carbon conversion pathways and strategies of key microorganisms and isolate and screen highly efficient carbon-sequestering indigenous functional microorganisms within photovoltaic sites. This has significant potential for stimulating and enhancing the synergistic benefits of indigenous microorganisms in soil improvement and carbon sequestration. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a Pseudomonas GD-9 with carbon fixation ability and its application. The Pseudomonas GD-9 has the ability to fix inorganic carbon sources, especially the ability to efficiently fix bicarbonate, and has strong adaptability under oligotrophic conditions.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a Pseudomonas GD-9. The Pseudomonas GD-9 is classified as Pseudomonas sp. and has a preservation number of CGMCC No. 31725.

[0007] Preferably, the nucleotide sequence of Pseudomonas sp. GD-9 is shown as SEQ ID NO.1.

[0008] The present invention also provides a soil conditioner, which comprises the Pseudomonas GD-9.

[0009] The present invention also provides a use of the Pseudomonas GD-9 or the soil conditioner in improving soil carbon sequestration capacity.

[0010] Preferably, the soil is karst soil.

[0011] The present invention also provides an application of the Pseudomonas GD-9 in fixing inorganic carbon.

[0012] Preferably, the inorganic carbon comprises one or more of carbon dioxide, sodium carbonate and sodium bicarbonate.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention provides a strain of Pseudomonas sp. GD-9, which can efficiently fix bicarbonate and has strong adaptability under oligotrophic conditions. Experiments have shown that the present invention inoculates Pseudomonas GD-9 in a culture medium containing different inorganic carbon sources, and the bacteria can fix the carbon in the culture medium; by adding an inorganic carbon source, especially sodium bicarbonate, its carbon fixation capacity can be significantly improved. The present invention discloses for the first time that Pseudomonas has a strong adaptability to oligotrophic conditions, has the ability to fix inorganic carbon sources, and is particularly capable of efficiently fixing bicarbonate, providing bacterial resources for improving the carbon fixation and emission reduction capacity of rocky desertification soils, providing a theoretical basis for in-depth understanding of the microbial regulation process of the occurrence form of nutrients in rocky desertification soils, laying the foundation for proposing soil microbial improvement technology in photovoltaic areas, and promoting the high-quality development of the photovoltaic industry in rocky desertification-vulnerable areas.

[0015] Biological deposit information

[0016] The Pseudomonas GD-9 described in the present invention is classified and named as Pseudomonas sp., and the depository unit is the General Microbiology Center of China Culture Collection Administration; the depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No. 31725; and the deposit date is August 23, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a bacterial morphology diagram of Pseudomonas GD-9 of the present invention.

[0018] Figure 2 This is a colony characteristic diagram of Pseudomonas GD-9 of the present invention.

[0019] Figure 3 This is a phylogenetic tree of the 16S rRNA gene of Pseudomonas sp. GD-9 of the present invention.

[0020] Figure 4 1 is the RubisCO enzyme activity of Pseudomonas GD-9 of the present invention. In the figure, GD-9 is the Pseudomonas of the present invention, and 1 to 4 are other 4 carbon-fixing bacteria. In the figure, compared with Pseudomonas GD-9 of the present invention, * indicates significant difference at the p<0.05 level.

[0021] Figure 5Figure 2 is the carbon fixation ability of Pseudomonas sp. GD-9 under different conditions. Different letters in a, b, and c in the figure indicate significant differences at the p<0.05 level.

[0022] Figure 6 This is the carbon fixation pathway of the Pseudomonas GD-9 of the present invention. DETAILED DESCRIPTION

[0023] The present invention provides Pseudomonas sp. GD-9, which is classified as Pseudomonas sp. and has a deposit number of CGMCC No. 31725. Pseudomonas GD-9 was isolated from soil in a rocky desertification photovoltaic field in Shilin County, Yunnan Province. Scanning electron microscopy confirmed that the strain has a rod-shaped morphology. The colonies produced on solid culture medium are small, round, yellow, and opaque, with a smooth, viscous surface and irregular edges.

[0024]

[0025] The present invention also provides a soil conditioner comprising the aforementioned Pseudomonas GD-9. In one embodiment, the active ingredient of the soil conditioner comprises Pseudomonas GD-9; the soil conditioner may further comprise acceptable excipients. In another embodiment, the sole ingredient of the soil conditioner is Pseudomonas GD-9.

[0026] The present invention also provides the use of the Pseudomonas GD-9 or soil conditioner described herein for improving soil carbon sequestration capacity. In the present invention, the soil is preferably karst soil. Experiments have shown that the Pseudomonas GD-9 described herein can grow in extremely low-nutrient salt media without relying on electron donors, and has a higher carbon sequestration potential in soil environments with high carbonate content. This facilitates the development of strains for use in specialized environments, such as karst soils.

[0027] The present invention also provides a use of the Pseudomonas GD-9 in fixing inorganic carbon. The inorganic carbon preferably includes one or more of carbon dioxide, sodium carbonate, and sodium bicarbonate. The present invention assays the activity of RubisCO enzyme in a bacterial solution of Pseudomonas GD-9, and the RuBisCO enzyme activity is approximately 20.44 U / mg prot, indicating good carbon fixation capacity. In addition, the carbon fixation capacity of Pseudomonas GD-9 varies with different inorganic carbon sources. When an inorganic carbon source is added, the carbon fixation capacity increases to approximately 7.2 times and 2.4 times under the addition of NaHCO3 and Na2CO3, respectively.

[0028] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0030] Example 1 Isolation and Identification of Carbon-Fixing Pseudomonas sp. GD-9

[0031] The target soil is the rocky desertification photovoltaic field soil in Shilin County, Yunnan Province. The specific screening plan is as follows:

[0032] 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 shake it on a shaker (180 rpm, shake for 30 min) to make a soil suspension. Then dilute the soil suspension in a 10-fold gradient to 10 -4The soil dilution solution was diluted 200 mL and applied to a solid medium containing no organic carbon source. The culture medium was incubated at 28°C for 7 days. The solid medium composition was as follows: 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; trace element solution, 10 mL / L; agar, 20 g / L, pH 7.0. The trace element solution formula was as follows: 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.

[0033] 2. Pick different colonies with better morphology, growth and faster growth from a solid medium without an organic carbon source, and purify them multiple times on a new solid medium without an organic carbon source using the inoculation loop plate streak method until pure culture. In this example, 25 strains of carbon-fixing bacteria were initially isolated and purified by the dilution plate method. 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. Scanning electron microscopy was used to determine that the bacterial morphology of the strain was rod-shaped ( Figure 1 ), the colonies produced on solid culture medium are small, round, yellow, opaque, with a smooth and sticky surface and irregular edges ( Figure 2 ).

[0034] 3. The strain was further subjected to 16S rRNA sequencing: the genomic DNA of the strain was amplified by PCR using universal primers 27F: 5'-AGTTTGATCMTGGCTCAG-3' (SEQ ID NO. 2) and 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 3), and the amplified product was sequenced. The sequencing result is shown in SEQ ID NO. 1. Then, BLAST analysis was performed based on the 16S rRNA gene sequence of the strain using the NCBI database. The results showed that the strain had 100% homology with the 16S rRNA gene sequences of Pseudomonasasprosekii 2-4 (accession number MK277454.1), Pseudomonas fluorescens P147 (accession number MH518306.1), Pseudomonas uvaldensis 22119 (accession number OR426855.1), Pseudomonaspiscicola AC-H9 (accession number OQ421710.1), and Pseudomonas thivervalensis GHM35 (accession number OL944325.1), 99.93% homology with the 16S rRNA gene sequence of Pseudomonas canavaninivorans22121 (accession number OR426857.1), and 100% homology with the 16S rRNA gene sequence of Pseudomonasskilonensis. The 16S rRNA gene sequence of BMNS02 (accession number OR801638.1) and Pseudomonas liniFRN-1 (accession number OP520939.1) has a homology of 99.79%, and the 16S rRNA gene sequence of Pseudomonas koreensis D426 (accession number OR122192.1) and Pseudomonas bijieensis SP1 (accession number CP097108.1) has a homology of 99.64%. Figure 3The strain was identified as Pseudomonas sp. by 16S rRNA gene phylogenetic analysis, combined with its bacterial morphology and colony characteristics. However, 16S rRNA gene sequencing alone cannot confirm the strain's accurate classification. Furthermore, whole-genome sequencing was performed: the extracted whole genome was sent to Shanghai Paisonno Biotechnology Co., Ltd. for next-generation sequencing. The resulting sequence was compared with the whole-genome sequences of the aforementioned closely related reference strains for average nucleotide identity (ANI) analysis (the NCBI database failed to retrieve the full-genome sequences of Pseudomonas piscicola and Pseudomonas canavaninivorans). When the ANI value is greater than 95%, the two strains are considered to be the same species. The ANI results showed 81.58%, 80.52%, 88.62%, 86.77%, 87.27%, 82.28%, 82.05%, and 87.15%, respectively, indicating that the strain is distinct from the aforementioned closely related species and is a potential new species.

[0035] Example 2 Carbon-fixing enzyme activity of carbon-fixing bacteria Pseudomonas sp. GD-9

[0036] 1. Materials and Methods

[0037] 1.1 Test strain: Pseudomonas sp. GD-9, isolated and identified in Example 1.

[0038] 1.2 Detection of RubisCO carbon fixation enzyme activity in Pseudomonas sp. GD-9

[0039] In order to study the carbon fixation ability of Pseudomonas sp. GD-9, the colonies purified from the solid culture medium without an organic carbon source in Example 1 were picked and inoculated into 10 mL of liquid culture medium without an organic carbon source, and cultured at 28°C, 160 r / min, for 5 days. The bacterial solution at the end of the culture was taken for RubisCO enzyme activity determination, which was performed using the ribulose diphosphate carboxylase / oxygenase (RuBisCO) kit provided by Beijing Bo Ao Sen Biotechnology Co., Ltd. At the same time, the total protein content of the bacterial solution was measured using the BCA protein concentration determination kit, and the RuBisCO enzyme activity determination results were standardized. The specific measurement method refers to the kit instructions, and the determination results are shown in the table. Figure 4 In this example, in order to highlight the strong carbon fixation ability of Pseudomonas sp. GD-9, four other carbon-fixing bacteria isolated and purified in Example 1 were used for comparative study.

[0040] 1.3 Data Processing

[0041] The raw data of this experiment were preliminarily organized using Microsoft Excel Office 2016. The experimental results were plotted using GraphpadPism 9.5.

[0042] 2. Results and Analysis

[0043] The carbon fixation capacity of Pseudomonas sp. GD-9 is as follows Figure 4 As shown in the figure, its RuBisCO enzyme activity was about 20.44 U / mg prot, which was much higher than that of the other four carbon-fixing bacteria, indicating that it had good carbon fixation ability.

[0044] Example 3 Study on the carbon fixation ability of Pseudomonas sp. under different conditions

[0045] 1. Materials and Methods

[0046] 1.1 Test strain: Pseudomonas sp., isolated and identified as described in Example 1.

[0047] Carbon-free liquid culture 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 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.

[0048] Na2CO3 liquid culture medium: Add Na2CO3, 0.63g / L, to the carbon-free liquid culture medium.

[0049] NaHCO3 liquid culture medium: Add NaHCO3, 0.5g / L, to the carbon-free liquid culture medium.

[0050] NaNO2 liquid culture 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 7.0.

[0051] Na2S2O3 liquid culture medium: Replace NaNO2, 4.6 g / L in NaNO2 liquid culture medium with Na2S2O3, 5 g / L.

[0052] 1.2 Study on carbon fixation ability of Pseudomonas sp. under different inorganic carbon sources and different electron donors

[0053] The colonies purified from the solid medium without an organic carbon source in Example 1 were inoculated into 5 mL of carbon-free liquid medium. After culturing at 28°C and 160 rpm for 5 days, 400 μL of the culture medium was inoculated into 5 mL of carbon-free liquid medium, Na2CO3 liquid medium, NaHCO3 liquid medium, NaNO2 liquid medium, and Na2S2O3 liquid medium, with triplicate inoculations. After culturing at 28°C for 5 days, the abundance of the strains in the culture medium was determined. The specific method for measuring microbial abundance was as follows: SYBR-Green (10000×, Solebol) was diluted 100-fold and mixed with propidium iodide (PI, Solebol) at a ratio of 50:1 (SYBR-Green:PI, V:V) to produce a fluorescent dye. After passing 1 mL of the culture medium through a 300-mesh nylon sieve to remove large particles, 10 μL of the fluorescent dye was added and stained in the dark for 15 minutes. After vortexing, the viable bacterial count was determined.

[0054] 1.3 Data Processing

[0055] The experimental data were analyzed using CytExpert SRT software and then preliminarily organized using Microsoft Excel Office 2016. The experimental results were plotted using Graphpad Pism 9.5.

[0056] 2. Results and Analysis

[0057] Since the only carbon sources available to the strain during cultivation are atmospheric carbon dioxide and inorganic carbon sources in the culture medium, the increase in strain biomass is due to carbon fixation. Figure 5. The results showed that after 5 days of cultivation in the presence of only atmospheric carbon dioxide, the strain concentration of Pseudomonas sp. was 11470.0 Events / μL, indicating that it has a certain ability to fix atmospheric carbon dioxide. When an inorganic carbon source was added, the strain concentration increased, indicating that its carbon fixation ability was improved, which may be the result of the strain fixing carbon through other pathways. However, its utilization of the two different forms of inorganic carbon sources was quite different. Its strain concentration increased to about 7.2 times and 2.4 times with the addition of NaHCO3 and Na2CO3, respectively. When an electron donor was added, the carbon fixation ability of the strain was not improved, but was weakened to varying degrees, especially when NaNO2 was added. The results show that the strain can grow in extremely low-nutrient salt culture media, does not have to rely on electron donors, and has a higher carbon fixation potential in soil environments with high carbonate content, which is helpful for the development of strains for special environments (such as karst soils).

[0058] Example 4 Analysis of Carbon Fixation Pathways of Carbon-Fixing Bacteria Pseudomonas sp. GD-9

[0059] 1. Materials and Methods

[0060] The Pseudomonas sp. GD-9 isolated and identified in Example 1 was sent to Shanghai Paisonno Biotechnology Co., Ltd. for second-generation sequencing using the Illumina Novasek platform. The whole genome framework map was assembled and spliced. The bacteria were annotated and functionally identified using bioinformatics software and databases. The carbon fixation pathway of strain GD-9 was inferred from the KEGG database to provide a basis for studying the carbon fixation ability of the strain.

[0061] 2. Results and Analysis

[0062] Based on the KEGG database, the CO2 fixation pathway of strain GD-9 was clarified. First, CO2 is fixed by phosphoenolpyruvate to produce oxaloacetate, which is then converted to L-aspartate by aspartate aminotransferase, and then converted to threonine. Threonine is cleaved into glycine by threonine aldolase, and glycine is then converted to serine by glycine hydroxymethyltransferase, and finally converted into 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, after conversion to a variety of amino acids, ultimately achieves the renewal of phosphoenolpyruvate, and the synthesis and degradation of threonine is the core process in this cycle. When oxaloacetate is produced by fixing CO2, oxaloacetate generates malic acid under the catalysis of malate dehydrogenase, and malic acid is directly converted into pyruvate under the catalysis of malic enzyme to complete the carbon fixation cycle. In addition, biological carbon fixation is usually completed under the catalysis of a series of enzymes. Strain GD-9 can also realize the reaction of acetyl-CoA with HCO3 under the catalysis of acetyl-CoA carboxylase (ACC), an enzyme with higher 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 the phenomenon that the addition of inorganic carbon source NaHCO3 significantly improves the carbon fixation capacity of the strain.

[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An application of Pseudomonas GD-9 in fixing inorganic carbon, characterized in that: The Pseudomonas GD-9 is named as Pseudomonas Pseudomonas sp . , the deposit number is CGMCC No .31725; The inorganic carbon is sodium bicarbonate.

2. Use of a soil conditioner in improving the carbon sequestration capacity of karst soil, characterized in that: The soil conditioner comprises the Pseudomonas GD-9 of claim 1; The carbon is sodium bicarbonate.

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