Pseudomonas GD-9 with carbon sequestration capability and application thereof

Abstract

The invention provides pseudomonas GD-9 with carbon sequestration capacity and application of the pseudomonas GD-9, and relates to the technical field of microorganisms. The pseudomonas disclosed by the invention is classified and named as pseudomonas Pseudomonasp, and the preservation number of the pseudomonas Pseudomonasp is CGMCC (China General Microbiological Culture Collection Center) No.31725. The pseudomonas disclosed by the invention is separated from soil in a stony desertification photovoltaic field area in Stone Lin County in Yunnan province. Experiments show that the pseudomonas can keep good growth activity and carbon sequestration capacity in a salt culture medium with extremely low nutrition, and the carbon sequestration capacity of the pseudomonas is remarkably improved under the condition that only an inorganic carbon source NaHCO3 is added. The pseudomonas obtained through separation has important scientific significance for improving the carbon sequestration and sink increasing potential of soil and promoting high-quality development of the photovoltaic industry in the stony desertification fragile area.

Description

Technical Field

[0001] The 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] The soil in the rocky desertification-prone areas is shallow, calcium-rich and alkaline, and the ecosystem is very fragile, which seriously restricts the regional economic and social development. It is urgent to explore new models for the organic integration of energy, ecological restoration and characteristic industrial development.

[0003] At present, the research on key technologies of ecological photovoltaic in rocky desertification-vulnerable areas mainly focuses on the monitoring and evaluation of meteorological effects in photovoltaic areas and the monitoring and evaluation of soil heterogeneity, but lacks relevant research on the precise identification and screening of functional microorganisms in the soil of photovoltaic areas. It is urgent to analyze the carbon transformation pathways and strategies of key microorganisms and isolate and screen efficient carbon fixation indigenous functional microorganisms in photovoltaic areas, which has important potential application value in stimulating and enhancing the synergistic benefits of indigenous microorganisms in improving soil and fixing carbon. 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 classification name of the Pseudomonas GD-9 is Pseudomonas sp., and the preservation number is CGMCC No.31725.

[0007] Preferably, the nucleotide sequence of Pseudomonas 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 an application of the Pseudomonas GD-9 or the soil conditioner in improving the carbon fixation capacity of soil.

[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 strain 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 fields, 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 Microbiological Culture Collection Administration; the depository unit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No.31725; the deposit date is August 23, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

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

[0023] The present invention provides a Pseudomonas GD-9, wherein the classification name of the Pseudomonas GD-9 is Pseudomonas sp., and the preservation number is CGMCC No. 31725. The Pseudomonas GD-9 of the present invention is isolated and obtained from soil in a rocky desertification photovoltaic field area in Shilin County, Yunnan Province. The present invention uses a scanning electron microscope to determine that the bacterial morphology of the strain is rod-shaped, and the colonies produced by the strain on a solid culture medium are small, round, yellow, opaque, smooth and sticky on the surface, and irregular on the edge.

[0024]

[0025] The present invention also provides a soil conditioner, which includes the Pseudomonas GD-9. As an operative method, the active ingredient of the soil conditioner of the present invention includes Pseudomonas GD-9; the soil conditioner may also include an acceptable auxiliary material. As another operative method, the sole ingredient of the soil conditioner is Pseudomonas GD-9.

[0026] The present invention also provides an application of the Pseudomonas GD-9 or the soil conditioner in improving soil carbon fixation capacity. In the present invention, the soil is preferably karst soil. Experiments have shown that the Pseudomonas GD-9 described in the present invention can grow in a very low-nutrient salt culture medium without relying on electron donors, and has a higher carbon fixation potential in a soil environment with a high carbonate content, which helps to develop the application of strains in special environments (such as karst soils).

[0027] The present invention also provides an application 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 the bacterial solution of Pseudomonas GD-9, and the RuBisCO enzyme activity is about 20.44U / mg prot, which has good carbon fixation ability. In addition, the carbon fixation ability of Pseudomonas GD-9 in different inorganic carbon sources also varies. When an inorganic carbon source is added, the addition of NaHCO3 and Na2CO3 increases to about 7.2 times and 2.4 times, respectively.

[0028] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope 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 Bacteria 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 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 components of the solid culture medium are: 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; trace element solution, 10mL / L; agar, 20g / L, pH 7.0. The formula of the trace element solution is: FeCl2, 0.3g / L; FeSO4·7H2O, 0.3g / L, MnSO4·H2O, 0.15g / L; ZnSO4·7H2O, 0.25g / L; CoCl2, 0.2g / L.

[0033] 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. In this example, 25 strains of carbon-fixing bacteria were initially separated and purified 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. 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, smooth and sticky, with 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 sequence of Pseudomonasprosekii 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 Pseudomonas thivervalensis GHM35 (accession number OL944325.1). The 16S rRNA gene sequence homology of BMNS02 (accession number OR801638.1) and Pseudomonas liniFRN-1 (accession number OP520939.1) is 99.79%, and the 16S rRNA gene sequence homology of Pseudomonas koreensis D426 (accession number OR122192.1) and Pseudomonas bijieensis SP1 (accession number CP097108.1) is 99.64%. Figure 3The 16S rRNA gene phylogenetic analysis shown in the figure, combined with its bacterial morphology and colony characteristics, identified the strain as Pseudomonas 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 reference strains of the closely related species mentioned above for average nucleotide identity (ANI) analysis (where the NCBI database failed to query the whole genome sequences of Pseudomonas piscicola and Pseudomonas canavaninivorans). When the ANI value is greater than 95%, the two can be considered to be the same species. The ANI results were 81.58%, 80.52%, 88.62%, 86.77%, 87.27%, 82.28%, 82.05% and 87.15%, respectively, indicating that the strain is different from the closest species mentioned above 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 carbon fixation enzyme activity of Pseudomonas sp. GD-9 RubisCO

[0039] In order to study the carbon fixation ability of Pseudomonas sp. GD-9, the colonies purified from the solid culture medium without organic carbon source in Example 1 were picked and inoculated into 10 mL of liquid culture medium without organic carbon source, and cultured for 5 days at 28°C and 160 r / min. The bacterial liquid at the end of the culture was taken for RubisCO enzyme activity determination, which was determined using the ribulose diphosphate carboxylase / oxygenase (RuBisCO) kit provided by Beijing Bioson Biotechnology Co., Ltd. At the same time, the total protein content of the bacterial liquid 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 Figure 4 In order to highlight the strong carbon fixation ability of Pseudomonas sp. GD-9, this example uses the other 4 carbon fixation bacteria separated and purified in Example 1 for comparative study.

[0040] 1.3 Data processing

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

[0042] 2. Results and Analysis

[0043] Carbon fixation capacity of Pseudomonas sp. GD-9 Figure 4 As shown, its RuBisCO enzyme activity was about 20.44U / mg prot, which was much greater 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 carbon-fixing bacteria Pseudomonas sp. under different conditions

[0045] 1. Materials and methods

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

[0047] Carbon-free liquid culture medium: 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; trace element solution, 10mL / L; pH 7.0. The formula of the trace element solution is: FeCl2, 0.3g / L; FeSO4·7H2O, 0.3g / L, MnSO4·H2O, 0.15g / L; ZnSO4·7H2O, 0.25g / L; CoCl2, 0.2g / 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, 4.6g / 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 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 culture medium without organic carbon source in Example 1 were picked and inoculated into 5 mL carbon-free liquid culture medium. After culturing for 5 days at 28°C and 160 r / min, 400 μL was inoculated into 5 mL carbon-free liquid culture medium, Na2CO3 liquid culture medium, NaHCO3 liquid culture medium, NaNO2 liquid culture medium and Na2S2O3 liquid culture medium respectively, and three parallels were made. After culturing for 5 days at 28°C, the abundance of strains in the bacterial solution was detected. The specific method for measuring microbial abundance is as follows: SYBR-Green (10000×, Solebol) was diluted 100 times, and then mixed with propidium iodide (PI, Solebol) at 50:1 (SYBR-Green:PI, V:V) to obtain a 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 avoid light and dye for 15 minutes, and the number of viable bacteria was determined after vortexing.

[0054] 1.3 Data processing

[0055] The experimental data were analyzed in CytExpert SRT software and then preliminarily sorted using Microsoft ExcelOffice 2016. The experimental results were plotted using GraphpadPism 9.5.

[0056] 2. Results and Analysis

[0057] 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 carbon fixation ability of Pseudomonas sp. under different conditions are shown in 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 11470.0Events / 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 two different forms of inorganic carbon sources was quite different, and its strain concentration increased to about 7.2 times and 2.4 times under 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 under the condition of adding NaNO2. The results show that the strain can grow in extremely low-nutrient salt culture media without relying 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 in special environments (such as karst soils).

[0058] Example 4 Analysis of Carbon Fixation Pathway 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, 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-9 was inferred through 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 was fixed by phosphoenolpyruvate to generate oxaloacetate, which was then converted into L-aspartate by aspartate aminotransferase, and then converted into threonine. Threonine was cleaved into glycine by threonine aldolase, and glycine was then converted into 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 of multiple 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 when CO2 is fixed, oxaloacetate is converted into 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 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.

[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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Pseudomonas sp. GD-9, characterized in that The Pseudomonas GD-9 is classified and named as Pseudomonas sp., and its deposit number is CGMCC No.31725.

2. The Pseudomonas GD-9 according to claim 1, characterized in that The nucleotide sequence of Pseudomonas GD-9 is shown in SEQ ID NO.

1.

3. A soil conditioner, characterized in that: The soil conditioner comprises the Pseudomonas GD-9 according to claim 1 or 2.

4. Use of the Pseudomonas GD-9 according to any one of claims 1 to 2 or the soil conditioner according to claim 3 in improving the carbon fixation capacity of soil.

5. The use according to claim 4, characterized in that: The soil is karst soil.

6. Use of Pseudomonas GD-9 according to any one of claims 1 to 2 in fixing inorganic carbon.

7. The use according to claim 6, characterized in that: The inorganic carbon includes one or more of carbon dioxide, sodium carbonate and sodium bicarbonate.

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