Pseudomonas aeruginosa BT with soil carbon sequestration function of Chinese fir forest and application thereof

Abstract

The application discloses a Pseudomonas aeruginosa BT with a soil carbon sequestration function of a Chinese fir forest and an application thereof. The Pseudomonas aeruginosa BT is classified as Pseudomonas aeruginosa, and has been preserved in the Guangdong Microbial Culture Collection Center on January 15, 2025, with a preservation number of GDMCC No. 65797. The strain is derived from soil under a Chinese fir forest in a karst area, and is obtained through carbon-free inorganic medium enrichment, separation and purification, molecular identification and carbon sequestration related enzyme activity screening. The strain BT has 1,5-diphosphoribulose carboxylase / oxygenase activity and carbonic anhydrase activity, and can be used for promoting organic carbon accumulation of soil under a Chinese fir forest, improving soil organic carbon storage, and improving soil nutrient availability and soil physical environment. The carbon sequestration microbial inoculant prepared from the strain BT is used for spraying on a surface layer of soil under a Chinese fir forest, can improve soil SOC and SOCS, and has an application potential for soil carbon sequestration and sink increase of a karst Chinese fir forest.

Description

Technical Field

[0001] This invention belongs to the field of microbial carbon sequestration and forest soil carbon sequestration and enhancement technology, and relates to a Pseudomonas aeruginosa BT with carbon sequestration function in Chinese fir forest soil and its application. Background Technology

[0002] Soil organic carbon is a crucial component of the carbon pool in forest ecosystems. In karst regions, shallow soil layers, high rock exposure, and relatively weak soil water and fertilizer retention capacity make the accumulation of organic carbon in forest soils susceptible to limitations imposed by water, nutrients, and microbial activity. Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.) is a tall tree belonging to the genus Cunninghamia in the family Cupressaceae. Chinese fir plantations are one of the important types of plantations in southern my country, widely distributed in karst regions such as Guizhou. Improving the soil organic carbon content and storage in Chinese fir forests is of great significance for enhancing the carbon sink function of forest ecosystems, improving soil quality, and promoting the sustainable management of plantations.

[0003] Existing measures for carbon sequestration and enhancement in karst forest soils mainly focus on litter return to the field, biochar application, stand structure regulation, and nutrient regulation. While these measures can increase soil organic carbon accumulation to some extent, they suffer from long-term effects, significant dependence on site conditions, and potential antagonistic effects when combined. Microorganisms play a crucial role in soil carbon cycling; therefore, screening functional microorganisms with carbon sequestration potential and applying them to forest soil carbon sequestration and enhancement is an important supplementary technology direction for improving forest soil carbon sequestration.

[0004] Pseudomonas aeruginosa is a group of Gram-negative bacteria widely distributed in natural environments such as soil and water, exhibiting strong environmental adaptability and diverse metabolic functions. Previous studies have shown that some environmentally derived microorganisms have the potential to participate in soil carbon cycling and inorganic carbon transformation; however, no Pseudomonas aeruginosa strain with a clearly defined taxonomic position and validated carbon-fixing function derived from the soil of Chinese fir forests in karst regions has been reported.

[0005] Currently, there are still few carbon-fixing strains from karst Chinese fir forest understory soils that have clear storage information and have been validated through potted and field applications. Therefore, there is an urgent need to provide a Chinese fir forest soil carbon-fixing strain with a clearly defined source, defined function, and practical application potential, along with its application method. Summary of the Invention

[0006] The purpose of this invention is to provide a *Pseudomonas aeruginosa* strain BT with carbon sequestration function in Chinese fir forest soil and its applications. This strain has been preserved as biological material, possesses a clear strain identity and preservation number, and can be used for carbon sequestration and enhancement in Chinese fir forest soil.

[0007] The technical solution of the present invention: A strain of Pseudomonas aeruginosa with carbon sequestration function in Chinese fir forest soil, classified as Pseudomonas aeruginosa, was deposited at Guangdong Provincial Microbial Culture Collection Center on January 15, 2025, with accession number GDMCC No. 65797.

[0008] The aforementioned strain BT was obtained from the soil under Chinese fir forests in karst areas, and was obtained through enrichment in carbon-free inorganic culture medium, plate isolation, purification, and 16S rDNA molecular identification.

[0009] The aforementioned *Pseudomonas aeruginosa* BT strain can grow in carbon-free inorganic media and exhibits ribulose-1,5-bisphosphate carboxylase / oxygenase activity and carbonic anhydrase activity. During the screening of *Pseudomonas aeruginosa* BT strain, RubisCO activity and carbonic anhydrase activity were used as indicators of carbon fixation function. After comparison with candidate strains from different sources, the BT strain was determined to have superior carbon fixation function.

[0010] The aforementioned application of Pseudomonas aeruginosa BT in the preparation of carbon-fixing microbial agents can be used to increase carbon sequestration in karst Chinese fir forest soil, thereby improving the organic carbon content and organic carbon storage in the Chinese fir forest soil.

[0011] The aforementioned carbon-fixing microbial agents are bacterial liquids, bacterial suspensions, live bacterial preparations, freeze-dried bacterial powders, or combinations thereof.

[0012] The aforementioned carbon-fixing microbial agent was obtained by culturing *Pseudomonas aeruginosa* BT on LB medium or carbon-free inorganic medium. The specific culturing method for the carbon-fixing microbial agent was as follows: *Pseudomonas aeruginosa* BT was inoculated into LB solid medium plates and cultured at 28–32°C for 18–36 h; single colonies with consistent morphology and good growth were selected and inoculated into LB liquid medium, and cultured at 28–32°C and 150–200 rpm. -1 Under shaking conditions, culture for 12–24 h to obtain BT seed culture; inoculate the BT seed culture into fresh LB liquid medium at an inoculation rate of 1–10% by volume, and incubate at 28–32 °C and 150–200 rpm. -1 Continue culturing under the specified conditions for 12–24 hours to allow the BT strain to reach the logarithmic growth phase or the early stage of the stationary growth phase. After mixing with 50 mL of sterile distilled water, the carbon-fixing microbial culture BT is obtained.

[0013] The aforementioned method for carbon sequestration and enhancement of karst Chinese fir forest soil involves applying carbon-fixing microbial agents to the top 0-30cm layer of the Chinese fir forest soil, allowing Pseudomonas aeruginosa BT to enter the soil environment, thereby increasing the soil organic carbon content and soil organic carbon storage.

[0014] The aforementioned carbon-fixing microbial agent can be applied by surface spraying, watering, dripping, or trenching, and the application rate of the carbon-fixing microbial agent is 500-5000 mL·m -2 .

[0015] The aforementioned carbon-fixing microbial agent was applied at a rate of 2000 mL·m -2 .

[0016] The aforementioned karst fir forest is an artificial fir forest in the karst region, with a stand density of over 70% and soil consisting of yellow loam or clay loam.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) The strain BT provided by this invention is derived from the soil under the karst fir forest, has a clear ecological origin, and is suitable for the soil environment of fir forest.

[0018] (2) The strain BT provided by the present invention has been deposited as biological material with the accession number GDMCC No.65797. The source and identity of the strain are clear, which is conducive to repeated implementation and subsequent application.

[0019] (3) The strain BT provided by this invention has high carbon fixation-related enzyme activity, and the RubisCO activity of strain BT is 33.0 ± 2.3 μmol·d -1 ·g -1 The CA activity was 21.5 ± 1.4 μmol·d⁻¹. -1 ·g -1 The strain BT has high potential for carbon sequestration and can increase the soil organic carbon content and soil organic carbon storage in Chinese fir forests.

[0020] (4) The application method of the present invention is simple to operate and can be applied to the soil of Chinese fir forest by spraying on the soil surface, which is suitable for carbon sequestration and carbon sink enhancement in the management of karst Chinese fir forest.

[0021] (5) Both pot test and field test results showed that strain BT had an enhancing effect on soil SOC and SOCS. In the pot test, after 100 days of treatment with strain BT, the SOC reached 17.63±0.78 g·kg⁻¹ and the SOCS reached 3.18±0.24 kg·m⁻². In the field test, after 117 days of treatment with strain BT, the SOC reached 22.39 g·kg⁻¹. -1 SOCS can reach 6.57 kg·m -2 The values ​​were all higher than the control treatment, indicating that strain BT can promote soil carbon accumulation under controlled conditions in pots and under field Chinese fir forest soil conditions, and has good potential for practical application. Attached Figure Description

[0022] Figure 1A graph comparing the activities of carbon fixation-related enzymes among different candidate strain groups (data are expressed as mean ± SD, n=6; different letters indicate significant differences between groups, p<0.05). Figure 2 Representative colony morphology and culture diagram of strain BT; Figure 3 Phylogenetic tree of strain BT; Figure 4 Layout diagram for the BT pot validation experiment of strain; Figure 5 This is a schematic diagram showing the location and plot layout of the field validation test area for strain BT. Detailed Implementation

[0023] The present invention will be further described below with reference to the embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Unless otherwise specified in the embodiments, the conditions are performed according to conventional conditions in the art or the conditions recommended in the instructions for reagents and instruments.

[0024] Example 1: Culture medium preparation Preparation of carbon-free inorganic liquid culture medium: Mix 0.5g Na2HPO4, 0.5g KH2PO4, 0.3g MgSO4, 0.2g CaCl2, 0.5g NaHCO3, 0.5g NH4Cl, 0.3g KNO3, 0.4g NaCl, 2mL trace element solution and 1000mL distilled water evenly, adjust the pH to 7.0-7.5, and sterilize at 121℃ for 30min to obtain carbon-free inorganic liquid culture medium.

[0025] Preparation of trace element solution: FeCl3, FeSO4·7H2O, MnSO4·H2O, ZnSO4, and CoCl2 are mixed evenly with distilled water. The trace element solution is prepared from the following components: 0.03g FeCl3, 0.03g FeSO4·7H2O, 0.015g MnSO4·H2O, 0.014g ZnSO4, and 0.02g CoCl2. These are dissolved in distilled water and the volume is adjusted to 100mL to obtain the trace element solution.

[0026] Preparation of carbon-free inorganic solid culture medium: Add agar to carbon-free inorganic liquid culture medium to make the final concentration of agar 1.5%-2.0% (w / v), that is, add 1.5-2.0g of agar per 100mL of culture medium, or add 15-20g of agar per 1000mL of culture medium. After mixing evenly, sterilize at 121℃ for 30min. After cooling, pour into sterile petri dishes to obtain carbon-free inorganic solid culture medium.

[0027] Preparation of LB medium: Mix 10g tryptone, 5g yeast extract, 10g NaCl and 1000mL distilled water evenly, adjust the pH to 7.0-7.2, and sterilize at 121℃ for 30min to obtain LB medium; add 1.5%-2.0% agar when using solid culture.

[0028] Example 2: Isolation and Obtaining of Strains In the karst region of Guizhou, two planted Chinese fir forests—Shicang State-owned Forest Farm in Jinsha County and Boren Community in Huachu Town, Puding County—were selected as sampling areas for the screening and isolation of carbon-fixing microorganisms. Three 20m × 20m standard plots were set up in each sampling area, with appropriate spatial spacing between plots. A five-point sampling method was used for each plot, with sampling points set at the four corners and the center of the plot, collecting 0-20cm topsoil samples and forest litter samples.

[0029] The Shicang sampling area is located in Shicang State-owned Forest Farm, Jinsha County, Bijie City, Guizhou Province, with geographical coordinates of 106°08′10″E, 27°32′04″N. This area is a typical karst plantation of Chinese fir, with a canopy closure of over 70%, preferably over 80%, and a simple understory structure with sparse vegetation. The Boren sampling area is located in the understory area of ​​the plantation of Chinese fir in Boren Community, Huachu Town, Puding County, Guizhou Province, with geographical coordinates of 105°36′11″E, 26°12′26″N. This area belongs to a typical karst landform distribution area, with soil types mainly consisting of calcareous soil and yellow soil. The Chinese fir forest has a stable stand structure, a canopy closure of over 70%, low understory vegetation coverage, and minimal ecological disturbance.

[0030] Before sampling, the soil drill, sampling shovel, tweezers, gloves, and other sampling tools were disinfected with 75% ethanol. Before formal sampling, a trial sampling was conducted around the sampling point to eliminate the potential impact of residual ethanol on the sample microorganisms. For soil sampling, a 30cm long, 5cm diameter soil drill was used to remove surface litter and humus layer before collecting samples from the 0-20cm top layer. For litter sampling, surface litter from the Chinese fir forest was selected, and gravel, weeds, and other non-target impurities were removed before collection. The collected soil and litter samples were placed in sterile sampling bags and sealed for preservation, with the sample name, sampling time, sampling location, and sampling personnel information recorded.

[0031] After sampling, soil samples collected from five points within the same site were thoroughly mixed and used as starting materials for screening soil-derived microorganisms; litter samples collected from five points within the same site were also thoroughly mixed and used as starting materials for screening litter-derived microorganisms. If experiments could not be conducted immediately, the samples were stored at 4°C for a short period.

[0032] When pretreating soil samples, first remove impurities such as roots, gravel, dead branches and leaves, and mix thoroughly. To reduce the interference of in-situ soluble organic carbon on subsequent screening, mix soil samples with deionized water at a soil-to-water ratio of 1:8-15, preferably 1:9 or 1:10, and heat at 50 rpm. -1 Shake for 20-30 hours under the specified conditions, preferably 24 hours. After shaking, centrifuge and filter through a 0.45 μm filter membrane to obtain the pretreated soil sample.

[0033] When pretreating litter samples, first remove obvious soil, gravel, weeds, and other non-target impurities, and then chop or cut the litter into small pieces before mixing. Mix the sample with deionized water at a mass-to-volume ratio of 1:8-15, preferably 1:9 or 1:10, and heat at 50 rpm. -1 Shake for 20-30 hours under the specified conditions, preferably 24 hours. After shaking, centrifuge and filter through a 0.45 μm filter membrane to obtain the litter pretreated sample.

[0034] The pretreated soil and litter samples were inoculated into carbon-free inorganic liquid culture medium for enrichment culture to limit the growth of common heterotrophic microorganisms and promote the proliferation of functional microorganisms with carbon fixation potential. After enrichment culture, the culture solutions were serially diluted 10-fold and plated onto carbon-free inorganic solid culture medium and LB agar plates. During the culture period, colony morphology, color, size, transparency, edge characteristics, and growth rate were observed. Single colonies with typical morphology and good growth were selected for streak plating purification.

[0035] Candidate single colonies were purified by streak purification 2-5 times until a single strain with consistent colony morphology and stable growth was obtained. Among them, the candidate strain isolated from the soil sample of the planted Chinese fir forest in Boren Community, Huachu Town, Puding County was named AT; the candidate functional microbial material isolated from the surface litter sample of the planted Chinese fir forest in Boren Community, Huachu Town, Puding County was named AY; the candidate strain isolated from the soil sample of the Chinese fir forest in Shicang State-owned Forest Farm, Jinsha County was named BT; and the candidate functional microbial material isolated from the surface litter sample of the Chinese fir forest in Shicang State-owned Forest Farm, Jinsha County was named BY.

[0036] Example 3: Determination and Screening of Carbon Fixation-Related Enzyme Activities in Strains To evaluate the carbon fixation potential of candidate microbial materials from different sources, the activities of ribulose-1,5-bisphosphate carboxylase / oxygenase (RubisCO) and carbonic anhydrase (CA) were used as functional evaluation indicators. The candidate materials AT, BT, BY, and AY obtained in Example 2 were cultured in shake flasks, and the cultured cells were collected and centrifuged to obtain wet cell samples for the determination of carbon fixation-related enzyme activities.

[0037] For the RubisCO activity assay, approximately 0.1 g of bacterial sample was weighed, added to the extraction solution, homogenized in an ice bath and ultrasonically disrupted, and then centrifuged at 4℃ and 8000×g for 10 min. The supernatant was collected, and the absorbance change was measured at a wavelength of 340 nm. The RubisCO activity was calculated based on the absorbance change rate.

[0038] For CA activity determination, bacterial samples were weighed, and protein extraction and centrifugation were performed using the same method. The supernatant was collected, and the absorbance change was measured at a wavelength of 405 nm. The CA activity was then calculated.

[0039] The test results showed significant differences in the activities of carbon fixation-related enzymes among candidate materials from different sources. Regarding RubisCO activity, the BT strain exhibited the highest activity, at 32.99 ± 2.26 μmol·d⁻¹. -1 ·g -1 The candidate material sourced by BY is 30.15 ± 1.51 μmol·d⁻¹. -1 ·g -1 The functional microbial material derived from AY was 27.80 ± 0.82 μmol·d⁻¹. -1 ·g -1 The lowest level was observed in strain AT, at 13.26 ± 1.54 μmol·d⁻¹. -1 ·g -1 One-way ANOVA results showed that there were significant differences in RubisCO activity among the candidate materials (F=178.18, p<0.001).

[0040] The CA activity assay results showed that the BT strain had the highest activity, at 21.66 ± 1.39 μmol·d⁻¹. -1 ·g -1 The functional microbial material derived from AY was 21.12 ± 1.23 μmol·d⁻¹. -1 ·g -1 The candidate material sourced by BY was 17.50 ± 0.42 μmol·d⁻¹. -1 ·g -1 The lowest value was observed in strain AT, at 14.03 ± 0.61 μmol·d⁻¹. -1 ·g -1 One-way ANOVA results showed that there were significant differences in CA activity among different candidate materials (F=75.46, p<0.001).

[0041] Analysis of both RubisCO and CA activity indicators revealed that strain BT exhibited the highest levels in both key carbon fixation-related enzyme activities, indicating its strong inorganic carbon fixation and transformation capabilities. It is the superior carbon fixation strain screened in this study. While the RubisCO activity of AY-derived functional microbial materials was lower than that of strain BT, their CA activity was higher, demonstrating good potential for auxiliary applications in carbon fixation function evaluation.

[0042] In comparison, the overall carbon fixation function of AT strains and BY-derived candidate materials was relatively weak. Specifically, the AT strain exhibited the lowest RubisCO and CA activities and was not selected for further application evaluation. While the BY-derived candidate materials showed high RubisCO activity, their CA activity was relatively low, and subsequent molecular identification results indicated insufficient purification. Therefore, subsequent application validation will focus on the BT strain, which demonstrates superior carbon fixation function and good stability, as well as AY-derived functional microbial materials with potential for auxiliary carbon fixation, to conduct preparation and application evaluation of single-strain agents and mixed-strain agents (MIX).

[0043] Furthermore, a composite microbial agent MIX was prepared by mixing BT strains with functional microbial materials derived from AY in a certain proportion, which was used to evaluate the effects of different combinations of functional microorganisms on the soil carbon sequestration and enhancement of Chinese fir forests.

[0044] Example 4: Morphological observation and amplification sequencing identification of the strain The candidate strain BT, which performed well in the carbon fixation function evaluation, was identified by morphological observation and 16S rDNA amplification and sequencing. The colony morphology of strain BT is as follows: Figure 2 As shown. Genomic DNA of strain BT was extracted and purified, and PCR amplification was performed using universal 16S rDNA primers. Sequencing alignment and phylogenetic analysis were then conducted. Figure 3 The strain was identified and named *Pseudomonas aeruginosa* according to BT classification. This strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 15, 2025, with accession number GDMCC No. 65797.

[0045] During the functional evaluation of candidate strains, 16S rDNA amplification and sequencing analysis were performed on candidate samples AY and BY. Sequencing results showed significant overlap between the AY and BY sequences, indicating the possible presence of multiple amplification templates in the samples. This suggests insufficient purification, making them unsuitable for preservation and patent protection as single-function strains with a clear taxonomic status.

[0046] However, the functional microbial materials derived from AY exhibited high RubisCO and carbonic anhydrase activity in the carbon fixation function evaluation of Example 3, demonstrating good carbon fixation performance. Therefore, this study uses AY as a candidate functional microbial community with carbon fixation potential for subsequent evaluation of the combined application effects of carbon fixation microorganisms, rather than as the core preservation strain of this invention.

[0047] In contrast, the BT strain not only possesses high activity of carbon fixation-related enzymes, but also completed purification culture, 16S rDNA molecular identification, and strain preservation, demonstrating a clear taxonomic position and a basis for reproducible application. Therefore, this invention selects the BT strain as the core carbon fixation functional strain for subsequent agent preparation and application verification.

[0048] Example 5: Preparation of carbon-fixing microbial inoculum Based on the evaluation results of carbon fixation-related enzyme activities in Example 3, BT strains and AY-derived functional microbial materials with superior carbon fixation function and application value were selected to prepare carbon-fixing microbial agents. BT strains served as the core functional strains of this invention, while AY-derived functional microbial materials served as auxiliary functional materials, used to evaluate the impact of the composite microbial combination on soil carbon fixation.

[0049] Carbon-fixing microbial culture solutions BT, AY, and MIX (BT:AY = 1:1) were prepared separately. The specific methods are as follows: (1) Preparation of carbon-fixing microbial culture BT: The *Pseudomonas aeruginosa* BT strain obtained in Example 2 and identified in Example 4 was inoculated into LB solid medium plates and cultured at 28–32°C for 18–36 h. Single colonies with consistent morphology and good growth were picked and inoculated into LB liquid medium and cultured at 28–32°C and 150–200 r·min. -1 Under shaking conditions, BT seed culture was obtained for 12–24 h. The obtained BT seed culture was then inoculated into fresh LB liquid medium at an inoculation rate of 1%–10% by volume and incubated at 28–32℃ and 150–200 r·min. -1 Continue culturing under the specified conditions for 12–24 h to allow the BT strain to reach the logarithmic growth phase or the early stage of the stationary growth phase. After mixing with 50 mL of sterile distilled water, the carbon-fixing microbial culture BT is obtained.

[0050] (2) Preparation of carbon-fixing microbial culture solution AY: The carbon-fixing functional bacterial community obtained in Example 2 was activated and cultured in carbon-free medium and LB medium to obtain the functional bacterial culture solution of AY. The specific culture method is as follows: the culture material of AY was inoculated into LB solid medium and cultured at 28-32℃ for 18-36h. Colonies with good growth were picked and inoculated into LB liquid medium and cultured at 28-32℃ and 150-200r·min. -1 Under shaking conditions, culture for 12–24 h to obtain AY seed culture; inoculate the AY seed culture into fresh LB liquid medium at an inoculation rate of 1–10% by volume, and incubate at 28–32 °C and 150–200 rpm. -1 Continue culturing under the specified conditions for 12–24 hours to allow the AY strain to reach the logarithmic growth phase or the early stage of the stationary growth phase. After mixing with 50 mL of sterile distilled water, the carbon-fixing microbial culture AY is obtained.

[0051] (3) Preparation of carbon-fixing microbial compound bacterial solution MIX: BT strain and AY strain were brought to the logarithmic growth phase or the early stage of the stationary growth phase, respectively. They were then mixed at a volume ratio of BT:AY = 1:1 and thoroughly shaken to ensure uniform distribution of the two functional microorganisms. The mixed bacterial solution was then diluted with 50 mL of sterile distilled water to obtain the carbon-fixing microbial compound bacterial solution MIX.

[0052] Example 6: Validation of the application of carbon-fixing microbial inoculant in a container A truncated cone-shaped transparent plastic flowerpot was used as the test container, with an inner diameter of 15.5 cm at the top, an inner diameter of 11.0 cm at the bottom, and a vertical height of 24.5 cm. Drainage holes were provided at the bottom of the pot, and a double layer of low-mesh mesh was laid on the inside of the bottom of the pot to ensure smooth drainage and prevent soil particle loss.

[0053] After the soil was naturally air-dried, it was passed through a 10-mesh sieve to remove plant residues, gravel, and other impurities. The soil was then thoroughly mixed and placed into potted containers, with 2.00 kg of soil per pot. The pot walls were gently tapped to level the soil surface. A total of 12 potted samples were randomly divided into four treatment groups: a blank control group (CK), a carbon-fixing microbial agent BT treatment group (BT), an AY-derived functional microbial agent treatment group (AY), and a compound carbon-fixing microbial agent MIX treatment group (MIX). Each treatment had three replicates. The CK group was uniformly sprayed with an equal volume of sterile distilled water. The BT, AY, and MIX groups were each uniformly sprayed with 50 mL of their respective carbon-fixing microbial agent solution onto the soil surface, ensuring full contact between the solution and the soil environment. After each treatment, the soil was incubated under identical environmental conditions, and soil samples were collected according to the experimental design time to determine soil organic carbon content (SOC), soil organic carbon storage (SOCS), carbon-fixing enzyme activity, and other soil physicochemical indicators.

[0054] Water management was carried out using the soil moisture observation method during the experiment. The condition of the surface soil in the pots was observed every morning. When the surface soil color became significantly lighter, it felt dry to the touch, and it was loose, 50 mL of deionized water was uniformly poured into each pot. The watering frequency and the amount of water per pour were kept consistent for all treatments.

[0055] The pot experiment began on January 14, 2025, with samples taken on the 35th and 100th days to measure soil organic carbon, soil organic carbon storage, mineral-bound organic carbon, RubisCO activity, and CA activity.

[0056] The results showed that on day 100 of pot cultivation, all treatments with different carbon-fixing microbial agents exhibited a trend of promoting soil carbon accumulation. The soil SOC content in the blank control group (CK) was 15.71 ± 0.43 g·kg⁻¹. -1 SOCS was 2.74 ± 0.22 kg·m -2 The SOC content in the AY treatment group was 16.43 ± 0.90 g·kg. -1 SOCS was 2.83 ± 0.23 kg·m -2 The SOC content of the BT-treated group was 17.63 ± 0.78 g·kg. -1 SOCS was 3.18 ± 0.24 kg·m -2 The MIX compound microbial agent treatment group had the highest SOC content, at 18.25 ± 0.84 g·kg⁻¹. -1 SOCS reached 3.30±0.23 kg·m -2 .

[0057] Compared with the control group, the AY, BT, and MIX treatments all increased soil SOC and SOCS content. Specifically, the AY treatment increased SOC and SOCS by approximately 4.58% and 3.28%, respectively; the BT treatment by approximately 12.22% and 16.06%, respectively; and the MIX treatment by approximately 16.17% and 20.44%, respectively. The MIX treatment showed the highest increases in SOC and SOCS, indicating a possible synergistic effect between the BT and AY-derived functional microbial materials under pot cultivation conditions. However, considering the limited difference in the increase compared to the BT single-strain treatment, and the significant influence of inter-microbial interactions and environmental conditions on the compound microbial agent, the BT single-strain agent still demonstrated good application stability.

[0058] Comprehensive analysis shows that both BT and AY-derived functional microbial agents promote soil carbon accumulation, with BT exhibiting a stronger and more stable carbon sequestration promoting capacity. The MIX microbial agent, a combination of BT and AY-derived functional microbial materials, showed a certain synergistic promoting trend under pot conditions, but its improvement was limited compared to BT alone. Other physicochemical indicators are shown in Table 1.

[0059] Table 1. Results of Physicochemical Indicators for Potted Validation

[0060] Pot culture validation results showed that BT inoculant, AY-derived functional microbial inoculant, and MIX compound inoculant all promoted the increase of soil SOC and SOCS. Among them, the MIX treatment showed the highest increase in SOC and SOCS under pot culture conditions, indicating that there may be a certain synergistic promoting effect when BT strains are combined with AY-derived functional microbial materials. However, compared with BT inoculant alone, the difference in the increase of MIX treatment was relatively limited. Considering carbon fixation function, inoculant stability, and controllability in practical application, BT single inoculant still shows good application potential. The results of Example 3 showed that the BT strain had the highest level of RubisCO and CA activity, and had strong inorganic carbon fixation and transformation capabilities. At the same time, the BT strain has completed purification culture, 16S rDNA identification, and strain preservation, and has a clear taxonomic position and stable genetic background, which is conducive to the standardized production and quality control of inoculants. In contrast, although the MIX compound inoculant showed a certain enhancing trend under pot culture conditions, its effect may be affected by the strain ratio, environmental conditions, and inter-strain interactions. Therefore, for subsequent application validation and promotion of this invention, BT strains with clear sources and stable functions will be selected as the core carbon fixation functional strains.

[0061] Example 7: Application of strain BT in field soil of karst fir forest Field experimental plots were established in Chinese fir plantations in the karst region of Guizhou. The experimental forests were divided into middle-aged Chinese fir plantations with a canopy closure greater than 0.8, sparse understory vegetation, yellow soil, and clay loam soil.

[0062] The experiment was set up with four treatments: blank control group (CK), carbon-fixing microbial agent BT treatment group (BT), AY-derived functional microbial agent treatment group (AY), and BT+AY compound carbon-fixing microbial agent treatment group (MIX).

[0063] BT carbon-fixing microbial culture, AY-derived functional microbial culture, and BT+AY compound culture (MIX) were respectively prepared at 2000 mL·m -2 The appropriate amount of the treatment solution was diluted with natural water and applied evenly to the soil surface. The control group (CK) was sprayed with an equal amount of natural water. Each treatment had three replicate plots, each plot measuring 3 m × 3 m. Buffer zones were set up between plots and separated by partitions to reduce interference from the diffusion of moisture and bacterial solution between treatments.

[0064] 117 days after application, soil samples were collected from the 0-30 cm soil layer of each plot, and the soil organic carbon content (SOC), soil organic carbon storage (SOCS), total nitrogen, total phosphorus, total potassium, pH, alkaline available nitrogen, available phosphorus, available potassium, RubisCO activity, CA activity, bulk density, and soil moisture content were measured.

[0065] The results showed that after 117 days of application of carbon-fixing microbial agents to soil in wild Chinese fir forests, all different treatments had a certain impact on soil organic carbon accumulation. The SOC content of the blank control group (CK) soil was 15.74 g·kg⁻¹. -1 SOCS is 5.30 kg·m -2 The SOC content of the AY-derived functional microbial agent treatment group (AY) was 17.58 g·kg⁻¹. -1 SOCS is 6.96 kg·m -2 The BT-treated group had the highest SOC content, reaching 22.39 g·kg⁻¹. -1 SOCS is 6.57 kg·m -2 The SOC content of the MIX compound microbial agent treatment group was 17.95 g·kg. -1 SOCS is 5.02 kg·m -2 .

[0066] Compared with the control (CK) treatment, the AY treatment increased soil SOC content by approximately 11.68% and SOCS by approximately 31.32%; the BT treatment increased SOC content by approximately 42.25% and SOCS by approximately 23.96%, demonstrating a strong promoting effect on soil carbon accumulation; the MIX treatment increased SOC content by approximately 14.00%, but SOCS did not show an increasing trend. Overall, the BT single-agent treatment showed a more significant and stable effect on increasing soil SOC under natural field conditions.

[0067] Since SOC content can directly reflect the level of soil organic carbon accumulation per unit mass, and SOCS is also affected by factors such as soil bulk density and soil layer thickness, this study combines the SOC enhancement effect, carbon fixation-related enzyme activity, and strain application stability for a comprehensive evaluation.

[0068] Further analysis revealed that while the MIX compound microbial agent exhibited a certain synergistic promoting effect under pot conditions, its effectiveness in complex field environments was likely influenced by factors such as inter-strain competition, differences in environmental adaptability, and interactions within the in-situ soil microbial community, failing to demonstrate a sustained enhancing effect. In contrast, the BT strain, with its clearly defined origin, obtained through isolation and purification, possesses a distinct taxonomic position and stable carbon sequestration function; its single-strain application effectively promoted SOC accumulation in Cunninghamia lanceolata forest soil.

[0069] In addition, BT treatment also showed a good promoting effect on the improvement of some soil nutrients and physicochemical properties, including changes in indicators such as available nitrogen, available phosphorus, available potassium, soil moisture content and bulk density, further demonstrating that strain BT can adapt to the soil environment of karst fir forest and play a role in promoting soil carbon sequestration. (The results of physicochemical indicators are shown in Table 2).

[0070] Table 2. Results of physicochemical indicators in the field verification of karst fir forest

[0071] Comprehensive comparison revealed that BT single-strain treatment exhibited a more stable soil carbon accumulation promoting effect under natural field conditions, with a significantly higher SOC increase than AY-derived functional microbial agents and MIX compound agents. Although the MIX agent, formed by combining BT and AY-derived functional microbial materials, showed a certain synergistic promoting trend under pot conditions, in the complex field ecological environment, the effect of the compound agent may be affected by factors such as inter-strain competition, differences in environmental adaptability, and in-situ soil microbial community interactions, and did not show a sustained enhancing effect.

[0072] Although AY treatment improved SOCS values, no single strain of functional microorganisms derived from AY has been definitively identified, and their composition and taxonomic status remain unclear. Therefore, there are limitations in standardized production, quality control, and repeated application of the microbial agent. Considering the SOC enhancement effect, carbon fixation functional characteristics, clarity of functional strains, and application stability, the BT strain is more suitable as the core carbon fixation functional strain of this invention.

[0073] Therefore, this invention selects the single strain BT as the core carbon-fixing functional strain, rather than using the compound microbial agent MIX as the main application agent. The main reasons include: (1) The BT strain performed best in the evaluation of carbon fixation-related enzyme activities. The RubisCO activity and CA activity reached the highest level among the candidate strains. It has a strong inorganic carbon fixation and transformation ability and is an important functional bacterium that promotes soil carbon accumulation. (2) The BT strain has a clear origin. After isolation, purification, 16S rDNA molecular identification and preservation, it has a unique and stable strain identity, which can realize standardized production and quality control of the bacterial agent; (3) BT single bacterial agent showed a stable SOC enhancement effect under both pot and field conditions, while MIX compound bacterial agent showed a certain synergistic promotion trend under pot conditions, but did not show a continuous enhancement effect in complex field environments. Its application effect stability may be affected by strain interaction and environmental conditions.

[0074] In summary, the BT strain possesses a clear taxonomic status, strong carbon sequestration function, and stable application effect, making it a suitable functional microbial strain for carbon sequestration and carbon sink enhancement in karst fir forest soils.

[0075] Example 8: Quality control methods in the application of strain BT In the application of this invention, the strain can be confirmed to be in a normal growth state by observing the color, turbidity and growth status of the bacterial solution; the presence of obvious contamination by other bacteria can be detected by streak plating or coating; and the identity of the strain can be confirmed by 16S rDNA amplification and sequencing.

[0076] When necessary, OD600 determination, plate counting, or qPCR methods can be used to detect bacterial concentration or bacterial colonization. These detection methods are preferred quality control methods and are not essential limitations for the implementation of this invention. As long as the applied carbon-fixing microbial agent contains the preserved strain BT and can promote soil organic carbon accumulation in the Chinese fir forest soil environment, the purpose of this invention can be achieved.

[0077] The DNA sequence of Pseudomonas aeruginosa BT is as follows:

Claims

1. A type of Pseudomonas aeruginosa BT with soil carbon sequestration function in Chinese fir forests, characterized in that: The strain was classified as Pseudomonas aeruginosa in BT classification and was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 15, 2025, with accession number GDMCC No. 65797.

2. The *Pseudomonas aeruginosa* BT strain according to claim 1, characterized in that: The strain BT was obtained from the soil under Chinese fir forests in karst areas, and was obtained through enrichment in carbon-free inorganic culture medium, plate isolation, purification and 16S rDNA molecular identification.

3. The *Pseudomonas aeruginosa* BT strain according to claim 1, characterized in that: The *Pseudomonas aeruginosa* BT strain can grow in carbon-free inorganic culture medium and has ribulose-1,5-bisphosphate carboxylase / oxygenase activity and carbonic anhydrase activity.

4. The application of *Pseudomonas aeruginosa* BT according to claim 1 in the preparation of carbon-fixing microbial agents, characterized in that: Carbon-fixing microbial agents can be used to increase carbon sequestration in karst fir forest soils, thereby improving the organic carbon content and storage in the fir forest soils.

5. The application according to claim 4, characterized in that: The carbon-fixing microbial agent is a bacterial liquid, bacterial suspension, live bacterial preparation, freeze-dried bacterial powder, or a combination thereof.

6. The application according to claim 4, characterized in that: The carbon-fixing microbial agent is obtained by culturing *Pseudomonas aeruginosa* BT on LB medium or carbon-free inorganic medium. The specific culturing method for the carbon-fixing microbial agent is as follows: *Pseudomonas aeruginosa* BT is inoculated into LB solid medium plates and cultured at 28–32℃ for 18–36 h; single colonies with consistent morphology and good growth are picked and inoculated into LB liquid medium, incubated at 28–32℃ and 150–200 rpm. -1 Under shaking conditions, culture for 12–24 h to obtain BT seed culture; inoculate the BT seed culture into fresh LB liquid medium at an inoculation rate of 1–10% by volume, and incubate at 28–32 °C and 150–200 rpm. -1 Continue culturing under the specified conditions for 12–24 hours to allow the BT strain to reach the logarithmic growth phase or the early stage of the stationary growth phase. After mixing with 50 mL of sterile distilled water, the carbon-fixing microbial culture BT is obtained.

7. The application according to claim 6, characterized in that: The method for carbon sequestration and enhancement of karst Chinese fir forest soil involves applying carbon-fixing microbial agents to the top 0-30cm layer of the Chinese fir forest soil, allowing Pseudomonas aeruginosa BT to enter the soil environment, thereby increasing the soil organic carbon content and soil organic carbon storage.

8. The application according to claim 7, characterized in that: The carbon-fixing microbial agent can be applied by surface spraying, watering, dripping, or trenching, and the application rate is 500-5000 mL / m³. -2 .

9. The application according to claim 7, characterized in that: The application rate of the carbon-fixing microbial agent is 2000 mL·m -2 .

10. The application according to any one of claims 4-9, characterized in that: The karst fir forest is an artificial fir forest in the karst region, with a stand density of over 70% and soil consisting of yellow loam or clay loam.

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