Method for screening soil under cunninghamia lanceolata forest by carbon sequestration bacteria and application of method

Abstract

The invention discloses a method for screening soil under a cunninghamia lanceolata forest by carbon sequestration bacteria and application of the method. The carbon sequestration bacteria provided by the invention are Gemmobacter lutimaris bacteria, the carbon sequestration bacteria are separated and screened by adopting a mode of combining three culture mediums, namely a carbon-free inorganic liquid culture medium, a carbon-free inorganic solid culture medium and an LB solid culture medium, and compared with other bacteria screening technologies singly using the carbon-free inorganic culture medium, the method has the advantages of low cost, high efficiency, convenience in operation and wide application prospect. The strain is screened from the soil under the cunninghamia lanceolata forest in the karst region of Guizhou province, the strain has high carbon sequestration capacity, carbon can be effectively stored in the soil, and soil carbon adsorption and storage and accumulated carbon sequestration are facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial carbon sequestration, and relates to a method for screening soil under Chinese fir forests for carbon-fixing bacteria and its application. Background Art

[0002] With long-term intensive farming and overexploitation, the land is facing serious degradation, the soil organic carbon storage has decreased significantly, and the changes in the soil carbon pool have a significant impact on the atmospheric CO 2 concentration, and further affect global climate change. To mitigate global climate change, it is necessary to further enhance the ability of terrestrial ecosystems to fix atmospheric CO 2 in order to neutralize the emissions of anthropogenic CO 2 Therefore, there is an urgent need to find and develop efficient carbon sequestration technologies.

[0003] Compared with other carbon sequestration technologies, microbial carbon sequestration technology has the advantages of being green, economical, efficient and low-cost. Microorganisms drive the transformation of soil substances and can achieve carbon sequestration functions through anabolic metabolism. Changes in their community composition will directly affect the fixation of soil organic carbon; there are many types of soil microorganisms and they are widely distributed. Especially in relatively carbon-limited terrestrial ecosystems, microbial carbon sequestration is of great significance for maintaining its own functional diversity and the carbon cycle process of the ecosystem. And compared with plant-derived carbon, the carbon fixed by microorganisms can be directly assimilated by microorganisms and stored in the soil as more stable carbon such as microbial residue carbon after the death of microorganisms, which is conducive to soil carbon sequestration and cumulative carbon fixation. Carbon fixation can not only be achieved through plant photosynthetic carbon fixation, but also through the photosynthesis and chemosynthesis of autotrophic microorganisms to convert atmospheric CO 2 into organic matter to achieve microbial carbon sequestration.

[0004] The present invention screens carbon-fixing bacteria from the soil under Chinese fir forests. This carbon-fixing bacteria has strong carbon sequestration ability and can effectively store carbon in the soil, which is conducive to soil carbon sequestration and cumulative carbon fixation. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for screening soil under Chinese fir forests for carbon-fixing bacteria and its application. The screening method provided by the present invention is efficient, fast, convenient to operate, and is screened from the soil under Chinese fir forests in the karst area of Guizhou Province. Its bacterial strains have strong carbon sequestration ability and can effectively store carbon in the soil, which is conducive to soil carbon sequestration and cumulative carbon fixation.

[0006] The technical solution of the present invention:

[0007] A method for screening soil under Chinese fir forests for carbon-fixing bacteria includes the following steps:

[0008] (1) Sampling: The soil of Chinese fir forests in the karst area of Guizhou Province was selected as the sample for screening bacteria. Three plots of 20m * 20m were preset, and the five-point sampling method was adopted; before sampling, the soil drill, gloves, and sampling shovel were disinfected with 75% alcohol to prevent external microbial contamination; during sampling, a trial sampling was carried out around the sampling point to eliminate the influence of alcohol. Using a soil drill with a 30cm long and 5cm diameter drill bit, after removing the surface litter and humus layer, the soil sample at 0 - 20cm was collected and reserved for use;

[0009] (2) Decarbonization pretreatment: The soil sample collected in step (1) was mixed with deionized water at a ratio of 1:8 - 15, shaken at 50rpm for 20 - 30 hours, and filtered through a 0.45μm filter 1 - 3 times to remove the soluble organic carbon in the soil, obtaining the pretreated soil for reserve;

[0010] (3) Take 5g of the pretreated soil in step (2) and add it to 50mL of carbon-free inorganic liquid medium, and place it in a gas bath oscillator at 30℃ and 150rpm for culturing, and perform resuspension to obtain bacterial suspension 1; then take 1mL of bacterial suspension 1 and add it to 50mL of carbon-free inorganic liquid medium, and place it in a gas bath oscillator at 30℃ and 150rpm for culturing to obtain bacterial suspension 2;

[0011] (4) Take bacterial suspension 2 and streak it on a carbon-free inorganic solid medium, and place it in a constant temperature incubator at 30℃ for culturing. After the bacteria grow, select the strains with better growth and streak them on the plate again. Repeat this step 4 - 5 times to obtain single strains;

[0012] (5) Identify the single strain in step 4 as Gemmobacter lutimaris by 16sRNA, and at the same time use LB solid medium for enrichment culture, and conduct enzyme activity detection of the carbon fixation ability to determine that Gemmobacter lutimaris has carbon fixation activity.

[0013] The canopy density of the aforementioned Chinese fir forest is above 70%, and the understory structure is simple with few vegetation, which can avoid or reduce the interference to the original vegetation.

[0014] The aforementioned decarbonization pretreatment: The soil sample collected in step (1) was mixed with deionized water at a ratio of 1:10, shaken at 50rpm for 24 hours, and filtered through a 0.45μm filter 2 times to remove the soluble organic carbon in the soil, obtaining the pretreated soil for reserve.

[0015] The preparation method of the aforementioned carbon-free inorganic liquid medium is to add Na 2 HPO 4 0.4 - 0.6g, KH 2 PO 4 0.4 - 0.6g, MgSO 40.2 - 0.4 g, CaCl 2 0.15 - 0.25 g, NaHCO 3 0.4 - 0.6 g, NH 4 Cl 0.4 - 0.6 g, KNO 3 0.2 - 0.4 g, NaCl 0.3 - 0.5 g, trace element solution 1.5 - 2.5 mL and distilled water 1000 mL are mixed evenly, adjusted to pH 6.5 - 7.5 and sterilized at 115 - 125 °C for 25 - 35 min to obtain a carbon - free inorganic liquid medium; the preparation method of each 1 L of trace element solution is to mix FeCl 3 0.25 - 0.35 g, FeSO 4 ·7H 2 O 0.25 - 0.35 g, MnSO 4 ·H 2 O 0.12 - 0.18 g, ZnSO 4 0.12 - 0.16 g and CoCl 2 0.18 - 0.22 g evenly with 1000 mL of distilled water.

[0016] Specifically, the preparation method of the aforementioned carbon - free inorganic liquid medium is to mix Na 2 HPO 4 0.5 g, KH 2 PO 4 0.5 g, MgSO 4 0.3 g, CaCl 2 0.2 g, NaHCO 3 0.5 g, NH 4 Cl 0.5 g, KNO 3 0.3 g, NaCl 0.4 g, trace element solution 2 mL and distilled water 1000 mL are mixed evenly, adjusted to pH 7 and sterilized at 121 °C for 30 min to obtain a carbon - free inorganic liquid medium; the preparation method of the trace element solution is to mix FeCl 3 0.3 g, FeSO 4 0.3 g·7H 2 O, MnSO 4 ·H 2 O 0.15 g, ZnSO 4 0.14 g and CoCl 2 0.2 g evenly with 1000 mL of distilled water.

[0017] The preparation method of the carbon-free inorganic solid medium is to mix 900 - 1100 mL of the carbon-free inorganic liquid medium with 14 - 16 g of agar powder evenly, and sterilize it at 115 - 125 °C for 25 - 35 min to obtain the carbon-free inorganic solid medium.

[0018] Specifically, the preparation method of the carbon-free inorganic solid medium is to mix 1000 mL of the carbon-free inorganic liquid medium with 15 g of agar powder evenly, and sterilize it at 121 °C for 30 min to obtain the carbon-free inorganic solid medium.

[0019] The preparation method of the aforementioned LB solid medium is to mix 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl and 1000 mL of distilled water evenly, adjust the pH to 7, and then sterilize it at 121 °C for 30 min to obtain 1 L of LB medium.

[0020] An application of a carbon-fixing bacterium in promoting the carbon-fixing ability of soil, wherein the carbon-fixing bacterium is the aforementioned Gemmobacter lutimaris bacterium.

[0021] A carbon-fixing microbial inoculant, wherein the active ingredient of the carbon-fixing microbial inoculant includes the aforementioned Gemmobacter lutimaris bacterium.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention uses a combination of three media, namely, the carbon-free inorganic liquid medium, the carbon-free inorganic solid medium, and the LB solid medium, for the isolation and screening of carbon-fixing bacteria. Compared with other techniques that solely use carbon-free inorganic media for bacteria screening, this method has low cost, high efficiency, and convenient operation.

[0024] (2) The present invention first mentions that Gemmobacter lutimaris bacterium has a carbon-fixing effect and has ribulose-1,5-bisphosphate carboxylase / oxygenase activity, with the highest activity reaching 15.75 μmol / d / g, and the average of six groups of data reaching 13.26 μmol / d / g. The carbonic anhydrase activity reaches a maximum of 14.95 μmol / d / g, and the average of six groups of data reaches 14.04 μmol / d / g.

[0025] (3) Through the isolation and screening of autotrophic carbon-fixing microorganisms, the present invention can more clearly understand the microbial groups in the soil that play a carbon-fixing role, and can provide strain resources for using technologies such as synthetic biology to transform microbial strains and enhance the carbon-fixing potential of microorganisms, thereby enhancing the carbon-fixing ability of the soil. Description of the Drawings

[0026] Figure 1It is the phylogenetic tree of Gemmobacter lutimaris bacteria;

[0027] Figure 2 It is Gemmobacter lutimaris bacteria enriched by cultivation;

[0028] Figure 3 It is the ribulose-1,5-bisphosphate carboxylase / oxygenase activity and carbonic anhydrase activity of Gemmobacter lutimaris bacteria;

[0029] Figure 4 It is the morphology of Gemmobacter lutimaris bacteria. Specific implementation mode

[0030] The present invention will be further described in detail below in conjunction with embodiments and drawings, but the implementation modes of the present invention are not limited thereto. Reagents used in the embodiments can be conventionally purchased from the market without special instructions.

[0031] Example 1: Preparation of culture medium

[0032] (1) Preparation of carbon-free inorganic liquid medium: Mix Na 2 HPO 4 (0.5 g), KH 2 PO 4 (0.5 g), MgSO 4 (0.3 g), CaCl 2 (0.2 g), NaHCO 3 (0.5 g), NH 4 Cl(0.5 g), KNO 3 (0.3 g), NaCl(0.4 g), 2 mL of trace element solution and 1000 mL of distilled water evenly, adjust the pH to 7 and sterilize at 121 °C for 30 min to obtain the carbon-free inorganic liquid medium.

[0033] Preparation of trace element solution: Mix FeCl 3 (0.3 g), FeSO 4 ·7H 2 O(0.3 g), MnSO 4 ·H 2 O(0.15 g), ZnSO 4 (0.14 g) and CoCl 2 (0.2 g) with 1000 mL of distilled water evenly to obtain the trace element solution;

[0034] (2) Preparation of carbon-free inorganic solid medium: Mix 1000 mL of carbon-free inorganic liquid medium with 15 g of agar powder evenly, and sterilize it at 121 °C for 30 min to obtain the carbon-free inorganic solid medium.

[0035] (3) LB medium: Mix 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl and 1000 mL of distilled water evenly, adjust the pH to 7, and then sterilize it at 121 °C for 30 min to obtain 1 L of LB medium.

[0036] Example 2: Screening method for carbon-fixing bacteria

[0037] (1) Sampling: In the karst area, select a representative Chinese fir forest with a canopy density of over 70%, simple understory structure and few vegetation to avoid or reduce interference with the original vegetation. Preset 3 plots of 20 m * 20 m, and adopt the five-point sampling method. Before sampling, disinfect the soil drill, gloves and sampling shovel with 75% alcohol to prevent external microbial contamination. During sampling, conduct a trial sampling around the sampling point to eliminate the influence of alcohol. Use a soil drill with a drill bit of 30 cm in length and 5 cm in diameter to remove the surface litter and humus layer, and then collect soil samples at 0 - 20 cm, number them and indicate the sample name, time, location and sampler.

[0038] (2) Soil decarbonization pretreatment: Mix 5 g of soil samples under Chinese fir forest with deionized water at a ratio of 1:10, shake it at 50 rpm for 24 hours, and filter twice (filter 0.45 μm) to remove soluble organic carbon in the soil;

[0039] (3) Take 5 g of the pretreated soil and add it to 50 mL of carbon-free inorganic liquid medium, place it in a gas bath oscillator at 30 °C and 150 rpm for culturing. After observing the growth of bacteria, obtain bacterial suspension 1; take 1 mL of bacterial suspension 1 and add it to 50 mL of carbon-free inorganic liquid medium, place it in a gas bath oscillator at 30 °C and 150 rpm for culturing to obtain bacterial suspension 2; streak plate the bacterial suspension 2 onto the carbon-free inorganic solid medium and place it in an incubator at 30 °C for culturing. After the bacteria grow, select the strains with better growth and streak plate them again. Repeat this step four to five times to obtain a single strain AT1.

[0040] Example 3: Bacterial species identification method:

[0041] Samples of microbial genomes were extracted using a bacterial DNA extraction kit (product number D3350). The sample DNA was amplified using the primer pair 27F 5'-AGAGTTTGATCCTGGCTCAG-3' / 1492R 5'-GGTTACCTTGTTACGACTT-3'. In this experiment, the high-fidelity PCR polymerase product KOD OneTM PCR Master Mix (product number KMM-101) from TOYOBO Company was used for the PCR experiment. The PCR program consisted of 35 cycles (denaturation at 98°C for 10 s, annealing at 55°C for 8 s, and extension at 68°C for 2 s). 1% agarose gel electrophoresis was used to verify the PCR products, and the PCR products were sequenced using the Sanger method (ABI3730XL gene sequencer). The theoretical size of the PCR product band was approximately 1500 bp. Bidirectional primer sequencing was performed, and the sequencing results were spliced using DNAMAN software. Finally, the sequence information in the seq file obtained from the sequencing results was compared with Blast in the NCBI database.

[0042] Results of strain identification: The strain "AT1" had the highest similarity with Gemmobacter lutimaris YJ-T1-11, and the homology was 98.96% after removing the redundant sequences at both ends.

[0043] Table 1: Evaluation table of related species

[0044]

[0045] The one with the highest similarity in the comparison results was selected as the identification result and a phylogenetic tree was constructed, as Figure 1 shown.

[0046] Experimental Example 4: Determination of ribulose-1,5-bisphosphate carboxylase / oxygenase activity and carbonic anhydrase activity

[0047] (1) Gemmobacter lutimaris was enriched and cultured using LB liquid medium

[0048] Single colonies from the carbon-free inorganic solid medium were selected and placed in LB liquid medium for large-scale culture, and then centrifuged to obtain a precipitate, as shown in Figure 2 ;

[0049] (2) Determination of ribulose-1,5-bisphosphate carboxylase / oxygenase activity

[0050] The ribulose-1,5-bisphosphate carboxylase (Rubisco) kit from Jiangsu Enzyme Immunoassay Industry Co., Ltd. was used for the determination. The operation method was in accordance with the kit instructions, as follows:

[0051] Preparation for sample determination: Weigh approximately 0.1 g of the sample (Gemmobacter lutimaris), add 1 mL of Enzyme Labeling Reagent I, homogenize in an ice bath and then ultrasonically disrupt (ice bath, 200 W, disrupt for 3 s, intermittent for 7 s, total time 1 min), and then centrifuge at 8000 g for 10 min at 4 °C to obtain the supernatant for standby;

[0052] Determination steps: Preheat the spectrophotometer for more than 30 min, adjust the wavelength to 340 nm, and zero with distilled water; Mix Enzyme Labeling Reagent II and Enzyme Labeling Reagent III in a 1:1 ratio immediately before use; In a 1 mL quartz cuvette, add 50 μL of the supernatant, 50 μL of Enzyme Labeling Reagent IV, and 900 μL of the working solution, mix immediately, record the absorbance value A1 at 340 nm at 20 s and the absorbance value A2 at 5 min 20 s, calculate △A = A1 - A2, and repeat the determination of the sample six times.

[0053] (3) Determination of carbonic anhydrase activity

[0054] Use a carbonic anhydrase (CA) kit (Jiangsu Enzyme Immunoassay Industry Co., Ltd.) for determination. The operation method is in accordance with the kit instructions, specifically as follows:

[0055] Preparation for sample determination: Treatment of bacteria: Collect the bacteria (Gemmobacter lutimaris) into a centrifuge tube, discard the supernatant after centrifugation; According to the ratio of the number of bacteria or cells (10 4 individuals): extraction solution volume (mL) of 500 - 1000:1, ultrasonically disrupt the bacteria (ice bath, power 20% or 200 W, ultrasonic for 3 S, interval 10 S, repeat 30 times); Centrifuge at 8000 g for 10 min at 4 °C, take the supernatant, and place it on ice for further measurement.

[0056] Determination steps: Preheat the microplate reader for 30 min, adjust the wavelength to 405 nm; Preheat Reagent I and Reagent II at 25 °C for 10 min in advance; Take a 96-well plate, add 20 μL of the sample supernatant, 140 μL of Reagent I in sequence, and finally add 40 μL of Reagent II and mix immediately. Measure the absorbance value A1 at 3 min and the absorbance value A2 at 6 min at 405 nm, △A = A2 - A1.

[0057] (4) Results:

[0058] For ribulose-1,5-bisphosphate carboxylase / oxygenase activity and carbonic anhydrase activity, the results are shown in Table 2.

[0059] Table 2 Results of detection data

[0060]

Claims

1. A method for screening soil under a Chinese fir forest for carbon-fixing bacteria, characterized in that: The following steps are involved: (1) Sampling: Soil from a Chinese fir forest in the karst region of Guizhou Province was selected as a screening sample. Three 20 m*20 m sample plots were set up and a five-point sampling method was adopted. Before sampling, the soil drill, gloves, and sampling shovel were disinfected with 75% alcohol to prevent external microbial contamination. During sampling, trial sampling was conducted around the sampling point to eliminate the influence of alcohol. A soil drill with a length of 30 cm and a diameter of 5 cm was used to remove the surface litter and humus layer, and then soil samples at 0-20 cm were collected for later use. (2) Decarbonization pretreatment: the soil sample collected in step (1) was mixed with deionized water at a ratio of 1:8-15, shaken at 50 rpm for 20-30 hours, and filtered through a 0.45 μm filter 1-3 times to remove soluble organic carbon in the soil to obtain pretreated soil for later use; (3) taking 5 g of the pretreated soil in step (2) and adding it to 50 mL of carbon-free inorganic liquid culture medium, placing it in an air bath shaker at 30° C. and 150 rpm for cultivation, and resuspending it to obtain bacterial suspension 1; taking 1 mL of bacterial suspension 1 and adding it to 50 mL of carbon-free inorganic liquid culture medium, placing it in an air bath shaker at 30° C. and 150 rpm for cultivation, and obtaining bacterial suspension 2; (4) Take bacterial suspension 2 and streak it onto a carbon-free inorganic solid culture medium and place it in a 30°C constant temperature incubator for culture. After the bacteria grow out, select the strain with better growth and streak it again. Repeat this step 4-5 times to obtain a single strain; (5) The single strain in step 4 was identified as Gemmobacter lutimaris by 16sRNA, and enriched and cultured using LB solid medium, and the enzyme activity of carbon fixation ability was detected to determine that Gemmobacter lutimaris had carbon fixation activity.

2. The method for screening soil under a Chinese fir forest for carbon-fixing bacteria according to claim 1, characterized in that: In step (1), the canopy density of the Chinese fir forest is above 70%, and the understory structure is simple with little vegetation, thereby avoiding or reducing interference with native vegetation.

3. The method for screening soil under a Chinese fir forest for carbon-fixing bacteria according to claim 1, characterized in that: In step (2), the decarbonization pretreatment is as follows: the soil sample collected in step (1) is mixed with deionized water in a ratio of 1:10, shaken at 50 rpm for 24 hours, filtered twice with a 0.45 μm filter to remove soluble organic carbon in the soil, and a pretreated soil is obtained for later use.

4. The method for screening soil under a Chinese fir forest for carbon-fixing bacteria according to claim 1, characterized in that: In step (3), the carbon-free inorganic liquid culture medium is prepared by uniformly mixing Na2HPO4 0.4-0.6g, KH2PO4 0.4-0.6g, MgSO40.2-0.4g, CaCl2 0.15-0.25g, NaHCO3 0.4-0.6g, NH4Cl0.4-0.6g, KNO3 0.2-0.4g, NaCl0.3-0.5g, 1.5-2.5mL of trace element solution and 1000mL of distilled water, adjusting the pH to 6.5-7.5 and sterilizing at 115-125°C for 25-35min to obtain a carbon-free inorganic liquid culture medium; the preparation method of each 1L of trace element solution is to mix FeCl3 Mix 0.25-0.35g of FeSO4·7H2O 0.25-0.35g, 0.12-0.18g of MnSO4·H2O, 0.12-0.16g of ZnSO4 and 0.18-0.22g of CoCl2 with 1000mL of distilled water to obtain.

5. The method for screening soil under a Chinese fir forest for carbon-fixing bacteria according to claim 4, characterized in that: In step (3), the carbon-free inorganic liquid culture medium is prepared by uniformly mixing 0.5 g Na2HPO4, 0.5 g KH2PO4, 0.3 g MgSO4, 0.2 g CaCl2, 0.5 g NaHCO3, 0.5 g NH4Cl, 0.3 g KNO3, 0.4 g NaCl, 2 mL of trace element solution and 1000 mL of distilled water, adjusting the pH to 7 and sterilizing at 121° C. for 30 min to obtain a carbon-free inorganic liquid culture medium; the trace element solution is prepared by uniformly mixing 0.3 g FeCl3, 0.3 g FeSO4·7H2O, 0.15 g MnSO4·H2O, 0.14 g ZnSO4 and 0.2 g CoCl2 with 1000 mL of distilled water to obtain the obtained product.

6. The method for screening soil under a Chinese fir forest for carbon-fixing bacteria according to claim 1, characterized in that: In step (4), the carbon-free inorganic solid culture medium is prepared by uniformly mixing 900-1100 mL of carbon-free inorganic liquid culture medium with 14-16 g of agar powder, and sterilizing at 115-125° C. for 25-35 min to obtain a carbon-free inorganic solid culture medium.

7. The method for screening soil under a Chinese fir forest for carbon-fixing bacteria according to claim 6, characterized in that: In step (4), the carbon-free inorganic solid culture medium is prepared by uniformly mixing 1000 mL of carbon-free inorganic liquid culture medium with 15 g of agar powder, and sterilizing at 121° C. for 30 min to obtain a carbon-free inorganic solid culture medium.

8. The method for screening soil under a Chinese fir forest for carbon-fixing bacteria according to claim 1, characterized in that: In step (5), the LB solid culture medium is prepared by uniformly mixing 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl and 1000 mL of distilled water, adjusting the pH to 7, and sterilizing at 121° C. for 30 min to obtain 1 L of LB culture medium.

9. An application of carbon-fixing bacteria in promoting soil carbon fixation capacity, characterized in that: The carbon-fixing bacteria is the Gemmobacter lutimaris bacteria described in claim 1.

10. A carbon-fixing microbial agent, characterized in that: The active ingredient of the carbon-fixing microbial agent includes the Gemmobacter lutimaris bacteria according to claim 1.

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