Compound microbial agent as well as preparation method and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a compound microbial agent as well as a preparation method and application thereof. The invention provides a compound microbial agent. The effective components of the compound microbial agent are fermentation liquor of rhodobacter sphaeroides GTW-A1 and fermentation liquor of bacillus licheniformis GTW-B2, the preservation number of the rhodobacter sphaeroides GTW-A1 is CGMCC (China General Microbiological Culture Collection Center) No.33609, and the preservation number of the bacillus licheniformis GTW-B2 is CGMCC No.33610. The invention further discloses a preparation method of the The compound microbial agent can effectively increase the carbon reserve of soil, passivates the activity of heavy metals through the synergistic effect of bacterial strain metabolites, improves the soil structure and promotes ecological restoration of microorganisms.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a compound microbial agent and a preparation method and application thereof. BACKGROUND

[0002] Soil organic matter is one of the most important properties of arable land and the core of soil quality and function. In agricultural production, soil organic matter is a crucial determinant. For some major grain-producing areas, the average annual grain yield level is closely related to the average organic matter level of the arable land soil, but in terms of carbon density, the soil is generally lower than the world average. Therefore, increasing the carbon density of agricultural soil is of great significance to improving soil fertility and ensuring food production.

[0003] Long-term mineral resource exploitation activities, such as stripping, excavation, and stockpiling, often result in the original structure of the soil in the mining area being severely damaged, manifested as deterioration of soil physical properties, deterioration of soil chemical properties, and reduction of biological activity. These factors together significantly weaken the ability of the soil in the mining area to fix and store organic carbon. Many mining soils are also contaminated with heavy metals. High concentrations of heavy metal ions not only directly inhibit plant growth, limiting the contribution of vegetation restoration to carbon input, but also produce strong toxic stress on soil microorganisms, interfering with key ecological processes mediated by microorganisms, including organic matter decomposition, nutrient cycling, and the crucial carbon fixation process, thereby further hindering the establishment and stability of the soil carbon pool.

[0004] Rhodobacter sphaeroides is the earliest appearing prokaryotic microorganism with a primitive photosynthetic system on earth, and belongs to photosynthetic bacteria. The bacterial cells are non-toxic and rich in nutrients. Bacillus licheniformis is a type of facultative anaerobic saprophytic bacteria widely existing in nature. For a long time, this bacterium has been used as an industrial production bacterium for proteolytic enzymes, amylases, bioactive agents, antibiotics, and other special chemical substances, and has very low toxicity to human health and the environment. SUMMARY

[0005] The present application aims to provide a compound microbial agent with synergistic effect of photosynthetic carbon fixation and biological metabolism, which can effectively increase the soil carbon storage, and passivate the activity of heavy metals through the synergistic effect of the metabolic products of the strains, improve the soil structure and promote the ecological restoration of microorganisms.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions: On the one hand, the present application provides a compound microbial agent, which comprises Rhodobacter sphaeroides GTW-A1, deposited in the China General Microbiological Culture Collection Center, with the accession number CGMCC No. 33609.

[0007] Preferably, the compound microbial agent also includes Bacillus licheniformis GTW-B2, which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 33610.

[0008] Preferably, the effective components of the compound microbial agent are the fermentation broth of Rhodotorula glutinis GTW-A1 and the fermentation broth of Bacillus licheniformis GTW-B2.

[0009] Preferably, the ratio of the number of Rhodopseudomonas spp. GTW-A1 and Bacillus licheniformis GTW-B2 is 2:3 to 1:1.

[0010] Preferably, the ratio of the number of Rhodopseudomonas spp. GTW-A1 and Bacillus licheniformis GTW-B2 is 36:37.

[0011] More preferably, the viable cell count concentration of both the fermentation broth of Rhodopseudomonas spp. GTW-A1 and Bacillus licheniformis GTW-B2 is 0.5 × 10⁻⁶. 9 CFU / mL.

[0012] On the other hand, a method for preparing a compound microbial agent is provided, the method comprising the following steps: Rhodopseudomonas granulosus GTW-A1 and Bacillus licheniformis GTW-B2 were inoculated into the culture medium and cultured at 28-35℃ for 12-16h. Single colonies were picked and diluted with sterile ddH2O to prepare bacterial suspensions. The suspensions of Rhodotorula glutinis GTW-A1 and Bacillus licheniformis GTW-B2 were mixed and stirred evenly to form a composite seed solution. The compound seed culture was inoculated into the fermentation medium at a volume ratio of 1%, the initial pH was adjusted to 6-10, and the culture was shaken and cultured at 30-35℃ and 130-220rpm for 24h. The resulting culture solution is the compound microbial agent.

[0013] On the other hand, the present invention provides the application of the composite microbial agent in soil carbon sequestration.

[0014] On the other hand, the present invention provides the application of the composite microbial agent in the passivation of heavy metals in soil pollution.

[0015] The Rhodocytosporum GTW-A1 described in this invention was collected from alpine meadow soil at an altitude of 4800 meters in the Julong Copper Mine. Preservation information is as follows: Category Naming: Rhodobacter sphaeroides ; Preservation period: February 21, 2025; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections; Address of the depositary: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China; Accession number: CGMCC No.33609.

[0016] The Bacillus licheniformis GTW-B2 strain described in this invention was collected from alpine meadow soil at an altitude of 4400 meters in the Julong Copper Mine. Preservation information is as follows: Category Naming: Bacillus licheniformis ; Preservation period: February 21, 2025; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections; Address of the depositary: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China; Accession number: CGMCC No.33610.

[0017] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: (1) This invention innovatively constructs a microbial system that synergistically enhances photosynthetic carbon fixation and biological metabolism by combining Rhodopseudomonas spp. GTW-A1 with Bacillus licheniformis GTW-B2. Rhodopseudomonas spp. directly fixes atmospheric carbon dioxide through its photosynthetic mechanism, converting it into organic carbon storage substances such as polyhydroxyalkanoates, while Bacillus licheniformis promotes humus formation by decomposing organic matter, effectively improving the carbon fixation effect of the soil.

[0018] (2) In the preparation method of the present invention, the high activity and stability of the two strains are ensured by precisely controlling the culture conditions, so that the compound bacterial agent has the ability to be preserved for a long time. When this technology is applied to the remediation of mining areas, it can not only effectively increase the soil carbon storage, but also passivate the activity of heavy metals through the synergistic effect of the strain metabolites, improve the soil structure and promote the restoration of microbial ecology.

[0019] (3) Compared with traditional methods, this invention achieves a deep integration of carbon sequestration process and soil remediation function. Under the premise of no secondary pollution, it takes into account carbon cycle regulation, heavy metal risk control and soil physicochemical property optimization, providing an innovative solution for the ecological restoration of mining areas and the achievement of carbon neutrality goals.

[0020] (4) This invention utilizes the ability of photosynthetic bacteria to absorb and fix carbon dioxide, and the important role of Bacillus licheniformis in regulating soil carbon cycling. The two work synergistically to effectively enhance the carbon sequestration potential of mining soil. Photosynthetic bacteria, through their unique photosynthetic mechanism, can directly convert carbon dioxide in the air into organic matter and store it in the soil, thereby increasing the carbon content of the soil. Bacillus licheniformis, on the other hand, promotes the cycling and accumulation of carbon elements in the soil ecosystem by decomposing and transforming organic matter in the soil, further enhancing the soil's carbon sequestration capacity. The application of this complex microbial community can not only improve the absorption and fixation efficiency of carbon dioxide in mining soil, but also improve the physical and chemical properties and ecological environment of the soil, creating more favorable conditions for the survival and activities of soil microorganisms, thus forming a virtuous cycle and continuously enhancing the carbon sequestration potential of mining soil. This has important practical significance for addressing global climate change, achieving carbon neutrality goals, and ecological restoration of mining areas. Detailed Implementation

[0021] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially.

[0022] Example 1: Isolation, purification process and identification results of Rhodopseudomonas GTW-A1 The soil sample in this embodiment was a high-altitude meadow soil collected at an altitude of 4800 meters from the Julong Copper Mine. It was placed in a sterile sampling bag and stored at 4°C.

[0023] The aseptic enrichment medium used in this embodiment consists of the following components: sodium acetate 1.0 g / L, yeast extract 0.5 g / L, dipotassium hydrogen phosphate 0.5 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 0.2 g / L, sodium chloride 0.1 g / L, calcium chloride 0.05 g / L, and pH 7.0.

[0024] 1. Isolation and purification of bacterial strains Weigh 10g of the above soil sample and add it to a 250mL screw-top bottle containing 90mL of sterile enrichment medium. Mix thoroughly, then cover the surface of the liquid with sterile liquid paraffin to create an anaerobic environment. Place the screw-top bottle in a light incubator and incubate continuously for 6 days at 30°C and 2000-3000 lux light intensity. Observe the color change of the culture medium during this period; a pink or red color indicates the growth of the bacterial strain.

[0025] Perform serial dilutions of the culture medium, specifically diluting each solution to 10⁻⁶. -4 10 -5 10 -6Take 100 μL of culture medium at different dilutions and spread it onto a solid plate of enrichment medium supplemented with 1.5% agar. Place the plate in an anaerobic jar and incubate for 5 days at 30°C and 2000-3000 lux light.

[0026] Select healthy, typical pink to red single colonies from the agar plates and perform streak purification at least three times until a pure culture with consistent morphology is obtained. Inoculate the purified strain into liquid enrichment medium and culture under the same conditions, observing its growth and color. Select one vigorous, typical-colored strain and name it GTW-A1.

[0027] The purified strain GTW-A1 was inoculated into liquid culture medium (photosynthetic bacteria medium) and cultured at 30°C until the logarithmic growth phase. The bacterial suspension was mixed with an equal volume of 40% (v / v) sterile glycerol, aliquoted into cryovials, and stored long-term at -80°C. Simultaneously, slant cultures were prepared and stored at 4°C for later use.

[0028] 2. Morphological identification of strain GTW-A1 Colony morphology: After anaerobic light culture for 5 days on enrichment medium solid plates, strain GTW-A1 forms round, pink colonies with a diameter of 1-2 mm, neat edges, smooth and moist surface.

[0029] Cell morphology: A logarithmic growth phase bacterial suspension was Gram-stained and observed under a microscope (1000× oil immersion). The results showed that strain GTW-A1 was a Gram-negative bacterium with oval or short rod-shaped cells, approximately (0.8-1.2) μm × (1.5-2.5) μm in size. Cell motility was observed using the hanging drop method.

[0030] 3. Identification of the physiological and biochemical characteristics of strain GTW-A1 Temperature: The optimal growth temperature is 28-32°C, and it can grow in the range of 15-40°C.

[0031] pH: The optimal growth pH is 6.8-7.5, and it can grow in the pH range of 5.5-9.0.

[0032] Oxygen requirements: Facultative anaerobic bacteria; can perform photosynthetic growth under anaerobic light conditions; can also perform respiratory growth under aerobic dark conditions.

[0033] Osmotic pressure: It can grow in a medium containing 0-3% NaCl, with the optimal NaCl concentration being 0.5-1%.

[0034] Biochemical reactions: Refer to Bergey's Manual of Bacterial Identification or relevant microbial identification systems, such as API 20NE, for testing.

[0035] Oxidase: Positive (+); Catalase: Positive (+); Gelatin liquefaction: negative (-); Indole production: negative (-); Nitrate reduction: positive (+); Urease: Negative (-).

[0036] Sole carbon source utilization: It can utilize acetate, malate, succinate, lactate, and pyruvate as the sole carbon source for growth; it cannot or only weakly utilizes glucose, fructose, and mannitol.

[0037] Antibiotic sensitivity: Sensitive to streptomycin (10 μg / mL) and kanamycin (25 μg / mL); shows some tolerance to ampicillin (100 μg / mL).

[0038] Salt and alkali tolerance: It can grow at pH 8.5, but its growth is inhibited in 1% Na2CO3 medium.

[0039] 4. Molecular biological identification of the strain 16S rRNA gene amplification: Using extracted genomic DNA as a template, PCR amplification was performed using the universal primer pairs 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') for the bacterial 16S rRNA gene.

[0040] PCR reaction system (50 μL): template DNA 1 μL, 27F primer (10 μM) 1 μL, 1492R primer (10 μM) 1 μL, 2x Taq Master Mix 25 μL, ddH2O to make up to 50 μL.

[0041] PCR program: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 90 s, for a total of 30 cycles; 72°C final extension for 10 min. PCR products were detected by 1% agarose gel electrophoresis, showing a single bright band at approximately 1500 bp.

[0042] Sequencing and sequence analysis: The purified PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional sequencing. The obtained 16S rRNA gene sequence was then subjected to BLASTn alignment analysis in the NCBI database.

[0043] Results: The 16S rRNA gene sequence (approximately 1450 bp) of strain GTW-A1 showed a 99.8% sequence similarity to that of Rhodobacter sphaeroides. Based on morphological and physiological / biochemical characteristics, strain GTW-A1 was identified as a Rhodobacter sphaeroides. Rhodobacter sphaeroides ).

[0044] Example 2: Isolation, purification process and identification results of Bacillus licheniformis GTW-B2 The soil sample in this embodiment was a high-altitude meadow soil collected at an altitude of 4400 meters from the Julong Copper Mine. It was placed in a sterile sampling bag and stored at 4°C.

[0045] 1. Isolation and purification of bacterial strains Weigh 10g of the above soil sample, add it to 90mL of sterile physiological saline, mix thoroughly, and heat treat (80°C, 15 minutes) to enrich Bacillus.

[0046] The heat-treated suspension was serially diluted (10) -3 10 -4 10 -5 100 μL of bacterial suspension at different dilutions was plated onto selective separation medium plates. The medium was nutrient agar (NA) or LB agar, with a predetermined concentration of heavy metal ions (100 mg / L Pb) added. 2+ (from Pb(NO3)2) + 50 mg / L As(V) (from Na2HAsO4·7H2O) + 20 mg / L Cd 2+ (From CdCl2·2.5H2O).

[0047] From the above selective plates, select single colonies with good growth and different morphologies, purify them, and then spot-inoculate or streak-inoculate them onto the following two types of selection plates: (1) Nitrogen fixation potential screening plate: Ashby nitrogen-free medium agar plate; culture at 30-37°C for 6 days, observe whether there is colony growth; record the strains that can grow on nitrogen-free medium.

[0048] (2) Screening plate for organic matter decomposition ability: Starch hydrolysis plate (NA + 1% soluble starch); Protein hydrolysis plate (NA + 10% skim milk powder); Cellulose hydrolysis plates (basal salt medium + 0.5% CMC-Na); Incubate at 37°C for 48-72 hours, and detect the size of the hydrolysis zone using appropriate indicators (iodine solution, or direct observation of the clear zone, Congo red staining). Record the strains that produce obvious hydrolysis zones.

[0049] The Ashby nitrogen-free agar plate contains the following components: mannitol 10 g / L, K2HPO4 0.2 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.2 g / L, CaSO4·2H2O 0.1 g / L, CaCO3 5 g / L, agar 15 g / L, and pH 7.0-7.5.

[0050] The growth of each purified strain under heavy metal stress, its growth ability on nitrogen-free medium, and its performance on organic matter decomposition plates were comprehensively evaluated. A strain that simultaneously met the following criteria was selected: (a) vigorous growth on a primary screening plate containing multiple heavy metals; (b) ability to grow on nitrogen-free medium; and (c) production of a clear zone on starch and / or protein (and / or cellulose) decomposition plates. The strain with the best overall performance was named GTW-B2.

[0051] The purified strain GTW-B2 was inoculated into liquid culture medium (Bacillus broth) and cultured at 37°C until the logarithmic growth phase. It was then mixed with an equal volume of 40% (v / v) sterile glycerol and stored at -80°C. Slant agar or spore suspensions were prepared for later use.

[0052] 2. Morphological identification of strain GTW-B2 Morphological observation: Colony morphology (NA / LB plate, 37°C): 2-4 mm in diameter, grayish-white, opaque, with a dry, rough, wrinkled surface and irregular edges. Cell morphology (Gram staining): Gram-positive bacilli, (0.6-0.9) μm × (1.5-3.0) μm, capable of producing mesophyllary or sub-mesophyllary elliptical spores; sporangia show no obvious swelling. Motility was observed using the hanging drop method.

[0053] 3. Identification of the physiological and biochemical characteristics of strain GTW-B2 Optimal growth temperature: 37-50°C, pH 6.5-8.0; facultative anaerobic (aerobic is preferred); tolerates 0-7% NaCl. Biochemical reactions (detected using API or VITEK system): catalase (+), VP (+), nitrate reduction (+), citrate utilization (+), gelatin liquefaction (+), starch hydrolysis (+), casein hydrolysis (+), aescin hydrolysis (+). It can utilize various sugars and exhibits resistance to certain antibiotics.

[0054] 4. Molecular biological identification of strain GTW-B2 Genomic DNA was extracted from GTW-B2, and the 16S rRNA gene was amplified by PCR (primers 27F / 1492R) and sequenced. The sequence was then aligned with BLASTn using NCBI. The results showed that... Bacillus licheniformis Type strain ATCC 14580 TThe sequence similarity (NR_118996.1) was 99.9%. Based on morphological and physiological-biochemical characteristics, strain GTW-B2 was identified as Bacillus licheniformis. Bacillus licheniformis Bacillus licheniformis ).

[0055] Example 3: Study on the photosynthetic carbon fixation and soil organic carbon increase performance of strain GTW-A1 Photosynthetic carbon fixation capacity determination (laboratory pure culture): The strain GTW-A1 was inoculated into an inorganic salt medium (referring to Sistrom's basal medium, with organic carbon source removed and trace element solution added) using sodium bicarbonate (NaHCO3, 1.0 g / L) as the sole carbon source.

[0056] The inoculum size was set at 5% (v / v), and the cultures were placed under anaerobic light conditions (30°C, 3000 lux).

[0057] Control groups were set up: (a) no culture medium was inoculated; (b) the culture was inoculated with the bacterial strain but cultured in the dark; after 7 days of culture, the OD of the culture medium was measured. 660 The value reflects biomass.

[0058] Results: OD in the anaerobic light group 660 The OD values ​​were significantly higher than those of the dark control group and the uninoculated group (anaerobic light group). 660 =0.85±0.05, OD of the dark control group 660 =0.10±0.02), indicating that strain GTW-A1 can utilize light energy and inorganic carbon sources (HCO3-). - It grows using CO2 and has the ability to photosynthesize and fix carbon.

[0059] Increase the effect of soil organic carbon (pot simulation experiment): Test soil: collected from the topsoil field of Julong Copper Mine, air-dried and sieved (2 mm), and the initial organic carbon content was determined.

[0060] Preparation of inoculum: The strain GTW-A1 was cultured in enrichment medium until the late logarithmic growth phase (OD2). 660 ≈1.0), centrifuge to collect bacterial cells, wash twice with sterile physiological saline, and resuspend to a bacterial concentration of approximately 10. 8 CFU / mL.

[0061] Experimental treatment: Control group (CK): 100 mL of sterile saline was applied to each pot of soil (1 kg).

[0062] Treatment group (GTW): 100 mL of strain GTW-A1 bacterial suspension was applied to each pot of soil (1 kg). 3-5 replicates were set up for each treatment.

[0063] Cultivation conditions: Place the potted plants in a greenhouse, maintain the soil moisture content at 60-70% of field capacity, simulate the natural light / dark cycle (or set a light / dark cycle, 12h / 12h), and control the temperature at 25-30°C.

[0064] Index determination: After 60 days of cultivation, soil samples were taken from each pot, air-dried and ground, and the soil organic carbon content was determined by potassium dichromate titration method.

[0065] Results: The soil organic carbon content in the treatment group (GTW) was significantly higher than that in the control group (CK). The initial soil organic carbon content was 1.25%. After 60 days of cultivation, the organic carbon content in the CK group was 1.28±0.03%, and that in the GTW group was 1.45±0.04%, indicating that the application of strain GTW-A1 could effectively increase the soil organic carbon content, with an increase of approximately (1.45-1.28) / 1.28 ≈ 13.3%.

[0066] Example 4: Study on the organic matter decomposition and organic carbon accumulation performance of strain GTW-B2 Quantitative determination of extracellular enzyme activity: GTW-B2 was inoculated into liquid medium containing the corresponding inducing substrates (starch, casein, CMC-Na) and cultured in a shaker at 37°C.

[0067] Take samples periodically, centrifuge and collect the supernatant (crude enzyme solution).

[0068] The activities of amylase, protease, and cellulase were determined using standard methods (DNS method for reducing sugars and Folin-Ciocalteu method for tyrosine) (unit: U / mL).

[0069] Results: After 48 hours of induction culture, strain GTW-B2 showed amylase activity of 50±5 U / mL, protease activity of 80±8 U / mL, and cellulase activity of 15±2 U / mL, indicating that it has the ability to efficiently produce a variety of hydrolases.

[0070] Effects of soil organic carbon accumulation (soil incubation experiment): Test soil: Soil collected from locations similar to the screening samples or target soil requiring improvement, air-dried and sieved, and the initial organic carbon (TOC) and humic substances (humic acid HA, fulvic acid FA) content were determined.

[0071] Organic material addition: Add 2% (w / w) organic material (crushed corn stalks) to the soil.

[0072] Preparation and inoculation of bacterial agent: Prepare GTW-B2 bacterial suspension (approximately 10... 8 -10 9The experimental group was inoculated with 1 mL of bacterial suspension per 100 g of soil, while the control group was inoculated with an equal volume of sterile water.

[0073] Cultivation conditions: Adjust the soil moisture content to 60% of field capacity, place in a 28°C constant temperature incubator, keep aeration, and cultivate for 90 days.

[0074] Index determination: After the cultivation period, the contents of soil TOC, HA and FA were determined.

[0075] Results: After 90 days of cultivation, the soil TOC content in the GTW-B2 inoculated group was 1.85±0.08%, significantly higher than that in the control group (1.60±0.06%). P <0.05). More importantly, the humic acid (HA) content in the GTW-B2 treatment group increased by 30%, and the HA / FA ratio also increased significantly, indicating that GTW-B2 not only accelerated the decomposition of organic matter, but also promoted the transformation and accumulation of organic carbon into stable humic forms.

[0076] Example 5: Verification of the nitrogen fixation potential of strain GTW-B2 Growth validation in nitrogen-free medium: Strain GTW-B2 was inoculated into liquid Ashby nitrogen-free medium. An inoculation of the same initial amount of E. coli (which cannot fix nitrogen) was used as a negative control, and (if available) nitrogen-fixing bacterium Azotobacter vinelandii was used as a positive control.

[0077] Incubate at 30-37°C for 5-7 days, and monitor OD. 600 Value change.

[0078] Results: After 7 days of culture, the OD of GTW-B2 was... 600 The value reached 0.45±0.04, significantly higher than that of the almost non-growing E. coli control group (OD). 600 <0.05), indicating its potential to grow using atmospheric nitrogen as the sole nitrogen source. Positive control: A. vinelandii OD 600 It reached 0.60±0.05.

[0079] Assay for nitrogenase activity using the acetylene reduction method (ARA): GTW-B2 was inoculated into semi-solid nitrogen-free culture medium and placed in a sealed serum bottle.

[0080] After the strain has grown to a certain extent, some air is extracted and 10% (v / v) acetylene gas is injected.

[0081] Incubate at 30°C in the dark for 24 hours.

[0082] The amount of ethylene generated in the headspace sample was detected using gas chromatography (GC).

[0083] Results: The nitrogenase activity of strain GTW-B2 was determined to be 5.2 ± 0.8 nmol C2H4 / h / mg protein, clearly confirming its nitrogenase activity. No ethylene production was detected in the control group (uninoculated or heat-inactivated strains).

[0084] Example 6: Confirmation of the high metal content tolerance of strain GTW-B2 Methods: The minimum inhibitory concentration (MIC) of strain GTW-B2 against multiple heavy metals / metalloid ions was determined by liquid culture. LB broth was used as the basal medium.

[0085] Preparation of heavy metal / metal-like salt solutions: Prepare solutions of Pb(NO3)2, Na2HAsO4·7H2O (As(V)), CdCl2·2.5H2O, ZnSO4·7H2O, CuSO4·5H2O, and K2Cr2O7 (Cr 6+ ), sterile stock solution of NiCl2·6H2O.

[0086] MIC determination: LB medium containing gradient concentrations of metal / metal-like ions was prepared in 96-well plates, and GTW-B2 logarithmic-phase bacterial culture (final concentration 10) was inoculated. 5 -10 6 (CFU / mL), incubate at 37°C in a shaker for 48-72 hours, and measure OD. 600 Alternatively, visually assess the growth to determine the MIC.

[0087] Results: Strain GTW-B2 not only possesses nitrogen fixation potential and the ability to promote organic carbon accumulation, but also exhibits resistance to various heavy metal / metal-like ions, especially Pb. 2+ As(V) and Cd 2+ Its high tolerance. This multifunctional characteristic makes GTW-B2 extremely valuable for the bioremediation of heavy metal contaminated soil, the resource utilization of organic waste, and the development of novel multifunctional biofertilizers.

[0088] Example 7: Preparation and Application of Compound Microbial Agents 1. Preparation of compound microbial agents Rhodopseudomonas spp. GTW-A1 and Bacillus licheniformis GTW-B2 were inoculated into liquid culture medium and cultured at 28-35℃ for 12-16 h. Single colonies were picked and diluted with sterile ddH2O to prepare bacterial suspensions. The Rhodopseudomonas spp. GTW-A1 and Bacillus licheniformis GTW-B2 bacterial suspensions were adjusted to 1×10⁻⁶. 8CFU / mL, mixed at a bacterial strain ratio of 36:37, and stirred thoroughly to form a composite seed culture; the composite seed culture was inoculated into the fermentation medium at a volume ratio of 1%, the initial pH was adjusted to 6-10, and the culture was shaken at 30-35℃ and 130-220 rpm for 24 h. The resulting culture medium is the composite microbial agent. Before mixing, the viable cell concentration of the single-cell fermentation broth of Rhodophyton floccosum GTW-A1 and Bacillus licheniformis GTW-B2 was 0.5 × 10⁻⁶. 9 CFU / mL.

[0089] The liquid culture medium (photosynthetic bacteria culture medium) formula for Rhodopseudomonas glutathione GTW-A1 (per L of distilled water) is as follows: yeast extract 3.0 g, peptone 5.0 g, sodium acetate 2.0 g, magnesium sulfate 0.2 g, potassium dihydrogen phosphate 0.5 g, dipotassium hydrogen phosphate 0.3 g, sodium chloride 1.0 g, trace element solution 1 mL (containing FeSO4·7H2O 0.1 g, MnSO4·H2O 0.01 g, ZnSO4·7H2O 0.01 g, dissolved in 100 mL of distilled water), pH adjusted to 7.0-7.2, and autoclaved at 121℃ for 20 min.

[0090] The liquid culture medium (Bacillus medium) formula for Bacillus licheniformis GTW-B2 (per L of distilled water): 10.0 g tryptone, 5.0 g yeast extract, 5.0 g sodium chloride, 2.0 g glucose, 3.0 g calcium carbonate (added separately after sterilization), pH adjusted to 7.2-7.4, autoclaved at 121℃ for 20 min.

[0091] 2. Study on the synergistic effect of the compound microbial community GTW-A1+GTW-B2 in enhancing soil carbon sequestration in mining areas Test soil: Soil samples were collected from the topsoil field of the Julong Copper Mine. The soil samples were tested and found to have a low initial total organic carbon (TOC) content and may contain a certain concentration of heavy metals (within the tolerance range of the strain). Soil samples were air-dried, stones and plant debris were removed, and the samples were sieved through a 2 mm sieve for later use. The initial total organic carbon (TOC) content was determined.

[0092] Preparation of bacterial suspension: GTW-A1 and GTW-B2 cells were cultured in their respective suitable liquid media to the late logarithmic growth phase. The cells were collected by centrifugation, washed 2-3 times with sterile physiological saline (or phosphate buffer), resuspended, and adjusted to a concentration of 1.0 × 10⁻⁶. 8 CFU / mL, prepare compound bacterial suspension (A1+B2): Mix equal volumes of GTW-A1 and GTW-B2 bacterial suspensions evenly.

[0093] Potted plant experiment design: Take 1 kg of sieved mining soil and place it in a pot. Set up 5 replicates per treatment group: CK (control group): Add 100 mL of sterile saline (or buffer). A1 (single bacterial group): Add 100 mL of GTW-A1 bacterial suspension; B2 (single bacterial group): Add 100 mL of GTW-B2 bacterial suspension; A1+B2 (compound microbial group): Add 100 mL of an equal volume of compound microbial suspension (GTW-A1+GTW-B2); spray the microbial suspension evenly or mix it into the soil.

[0094] Cultivation conditions: Place the potted plants in a constant temperature incubation room or greenhouse at 25-28°C, according to the previously determined optimal conditions, and keep the soil properly aerated, replenish water regularly, maintain the soil moisture content at 25% of field capacity, and the cultivation period is 60 days.

[0095] Indicator Measurement: Soil samples were collected from each treatment group at day 0 of incubation and day 60 of incubation. The samples were air-dried, ground, and sieved through a 0.15 mm sieve. Total organic carbon (TOC) content was determined using the potassium dichromate titration method (or elemental analyzer). Soil humic components (such as humic acid HA and fulvic acid FA) were measured to assess organic carbon stability.

[0096] Results: See Table 1.

[0097] Table 1. Changes in total organic carbon (TOC) content in soil

[0098] As shown in Table 1, compared with the CK group, the A1 treatment resulted in a net increase of TOC of 0.17% - 0.05% = 0.12%; compared with the CK group, the B2 treatment resulted in a net increase of TOC of 0.25% - 0.05% = 0.20%; the expected summation of the single bacterial effect was 0.12% + 0.20% = 0.32%; while the A1 + B2 treatment resulted in a net increase of TOC of 0.50% - 0.05% = 0.45%, indicating that the compound microbial community GTW-A1 + GTW-B2 has a synergistic effect in improving the carbon sequestration effect of the mining area soil.

[0099] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A compound microbial agent, characterized in that, The compound microbial agent includes Rhodotorula glutinis GTW-A1, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33609.

2. The compound microbial agent according to claim 1, characterized in that, The compound microbial agent also includes Bacillus licheniformis GTW-B2, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33610.

3. The compound microbial agent according to claim 2, characterized in that, The effective components of the compound microbial agent are the fermentation broth of Rhodotorula glutinis GTW-A1 and Bacillus licheniformis GTW-B2.

4. The compound microbial agent according to claim 2 or 3, characterized in that, The ratio of the number of Rhodopseudomonas spp. GTW-A1 and Bacillus licheniformis GTW-B2 is 2:3 to 1:

1.

5. The compound microbial agent according to claim 4, characterized in that, The ratio of the number of Rhodopseudomonas spp. GTW-A1 and Bacillus licheniformis GTW-B2 was 36:

37.

6. The compound microbial agent according to claim 3, characterized in that, The viable cell count concentrations of both the fermentation broths of Rhodophyton floccosum GTW-A1 and Bacillus licheniformis GTW-B2 were 0.5 × 10⁻⁶. 9 CFU / mL.

7. A method for preparing a compound microbial inoculant, characterized in that, The preparation method includes the following steps: Rhodopseudomonas granulosus GTW-A1 and Bacillus licheniformis GTW-B2 were inoculated into the culture medium and cultured at 28-35℃ for 12-16h. Single colonies were picked and diluted with sterile ddH2O to prepare bacterial suspensions. The suspensions of Rhodotorula glutinis GTW-A1 and Bacillus licheniformis GTW-B2 were mixed and stirred evenly to form a composite seed solution. The compound seed culture was inoculated into the fermentation medium at a volume ratio of 1%, the initial pH was adjusted to 6-10, and the culture was shaken and cultured at 30-35℃ and 130-220rpm for 24h. The resulting culture solution is the compound microbial agent.

8. The application of the composite microbial agent according to any one of claims 1-3 in soil carbon sequestration.

9. The application of the composite microbial agent according to any one of claims 1-3 in the passivation of heavy metals in soil pollution.

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