Biological remediation method for secondary salinized soil based on bacillus pumilus and application

Abstract

The invention discloses a secondary salinization soil bioremediation method based on bacillus pumilus and application thereof, and belongs to the technical field of environmental protection. The problems that an existing microbial agent is low in nitrate tolerance, poor in conversion efficiency and limited in growth promoting capacity are solved. According to the technical scheme, the bacillus pumilus Y10 (the preservation number is CGMCC No.25291) has the nitrate tolerance concentration of 800-1200 mg.L <-1 >, nitrate larger than or equal to 500 mg.L <-1 > is converted by 80% or above within 18-24 hours, indoleacetic acid secreted by the bacillus pumilus Y10 is larger than or equal to 15 mg.L <-1 >, and siderophore secreted by the bacillus pumilus Y10 is larger than or equal to 20 mg.L <-1 >; according to the salt damage repairing and growth promoting integrated microbial inoculum, a strain and a humic acid carrier are mixed according to the weight ratio of 1: 9-1: 11 and are fixed through interaction of hydrogen bonds and Van der Waals' force, and the bonding strength is larger than or equal to 12 kJ / mol; according to the bioremediation method, the microbial agent is applied to secondary salinization soil with the nitrate content of 200-800 mg.kg <-1 >, the application depth is 10-15 cm, and the water content of the soil keeps the field moisture capacity of 60-70%.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of environmental protection technology, in particular to a biological remediation method for secondary salinization soil based on bacillus pumilus and application thereof. BACKGROUND

[0002] As a large country of facility cultivation in the world, long-term production status of high-intensity, high-replant index and high fertilizer application amount in China leads to problems of secondary salinization, nutrient imbalance and soil acidification in facility agriculture, among which the problem of soil nitrate type secondary salinization is particularly prominent. At present, physical, chemical and biological remediation methods are mainly used, among which microbial remediation measures are widely concerned due to their environmental friendliness, simplicity, high efficiency and low cost.

[0003] In the prior art, CN103525739A discloses a bacillus megaterium NCT-2 and its application in remediation of facility secondary salinization soil. The strain has nitrate assimilation conversion ability and phosphorus solubilization ability, but its nitrate tolerance is only 600 mg·L -1 , and the conversion time needs more than 48 hours. CN105154357B further discloses a microbial agent preparation method based on bacillus megaterium NCT-2, which uses freeze-drying technology to improve the stability of the microbial agent, but still based on the same strain, the function is relatively single. CN109536410B discloses a salt-tolerant and growth-promoting composite microbial agent, which uses a complex strain such as pseudomonas putida combined with a biochar carrier, mainly aiming at salt-tolerant and growth-promoting in salinized soil, but does not involve the conversion of nitrate.

[0004] The prior art has the following disadvantages: single microbial agent has the limitations of difficult colonization, poor adaptability and difficult function development, and is difficult to stably play a role in complex soil environment; the nitrate conversion efficiency of the existing strain is limited, which cannot meet the rapid remediation needs of severe secondary salinization soil; the nitrate tolerance of the strain in the prior art is generally lower than 600 mg·L -1 , which is difficult to survive and play a role in a high-concentration nitrate environment; there is a lack of excellent strain resources with high-efficiency nitrate conversion ability and plant growth-promoting function. SUMMARY

[0005] The technical problem to be solved by the present application is that the existing microbial agent has the problems of nitrate tolerance lower than 600 mg·L -1 , conversion time more than 48 hours and limited growth-promoting ability in the remediation of nitrate type secondary salinization soil, and single agent is difficult to realize the dual functions of high-efficiency nitrate conversion and plant growth promotion, so it is necessary to obtain an excellent strain that can stably survive and play a remediation function in a high-concentration nitrate environment.

[0006] To solve the above technical problems, the present application provides a Bacillus pumilus Y10 with special biological characteristics, with the preservation number of CGMCC No.25291. The strain has excellent nitrate tolerance, with a tolerance concentration range of 800-1200 mg·L -1 , preferably 1000-1200 mg·L -1 in the preferred embodiment, significantly exceeding the salt tolerance level of existing strains. At the same time, the strain shows rapid and efficient nitrate bioconversion performance. Under suitable environmental conditions (temperature 25-35℃, pH 6.5-7.5), it can achieve more than 80% conversion efficiency within a time window of 18-24 hours for nitrate with a concentration of not less than 500 mg·L -1 , and under optimal conditions (30℃, pH 7.0), the conversion rate for 730 mg·L -1 of nitrate can exceed 95% within 18 hours.

[0007] The strain has significant plant growth promoting function and can secrete two key biostimulants: indole acetic acid and siderophore. The secretion amount of indole acetic acid is not less than 15 mg·L -1 , and can reach 15-25 mg·L -1 under certain culture conditions; the secretion amount of siderophore is not less than 20 mg·L -1 , and can reach 20-35 mg·L -1 under optimized conditions. When the strain is cultured to a bacterial liquid concentration of 2.0×10 9 -5.0×10 9 CFU·mL -1 , it can maintain a stable biostimulant secretion level in the nitrate-containing inorganic salt medium.

[0008] The core technical feature of the strain lies in its unique nitrate bioassimilation reconstruction mechanism, which realizes the step-by-step conversion and assimilation of nitrate through a specific nitrate reductase system. The enzyme system is composed of three key enzymes: nitrate reductase, nitrite reductase and hydroxylamine reductase, with a total enzyme activity of 85 U / mg protein or more. Under micro-aerobic conditions, the enzyme system can sequentially catalyze the reduction of nitrate to nitrite, further to hydroxylamine, and finally assimilate these intermediates into amino acids and proteins and other cell components, realizing the biological reconstruction of nitrate.

[0009] Based on the above strain, the present application constructs a salt damage repair and growth promoting integrated bacterial agent. The bacterial agent is composed of Bacillus pumilus

[0010] Y10 and a specially modified humic acid carrier are composed. The strain forms a stable combination with the surface of the humic acid carrier through intermolecular interaction forces such as hydrogen bonds and Van der Waals forces, and a stable strain-carrier composite structure system is constructed. The weight ratio of the strain and the humic acid carrier is controlled in the range of 1:9 to 1:11, and the preferred ratio is 1:10, so as to ensure that the effective load of the strain and the carrying capacity of the carrier reach the best balance.

[0011] The humic acid carrier used is subjected to carboxymethylation modification treatment, the molecular weight is controlled in the range of 5000 to 8000 Da, the carboxyl content is 4.5 to 6.0 mmol·g -1 , and preferably the carboxyl content is 4.5 to 5.5 mmol·g -1 . The binding strength between the modified carrier and the strain is not less than 12 kJ / mol, which ensures the stability of the strain-carrier composite structure and the long-term maintenance of the strain activity.

[0012] The application further provides a preparation method of the above-mentioned salt damage repair and growth promotion integrated bacterial agent. The method adopts a staged culture strategy to optimize the functional characteristics of the strain. The culture process is divided into two stages: the first 12 hours are for strain enrichment culture in a culture medium rich in nitrogen source, and the last 12 hours are for transfer into a nitrogen-limited culture medium and adding a nitrate inducer (KNO3200mg·L -1 ) to induce the strain to secrete functional substances. First, the Bacillus pumilus Y10 is cultured in an LB culture medium, and the culture conditions are 28 to 30℃ and a rotation speed of 200r·min -1 , until the concentration of the bacterial liquid reaches 2.0×10 9 ~5.0×10 9 CFU·mL -1 . Then, the prepared bacterial liquid is mixed with the humic acid carrier modified by carboxymethylation at a weight ratio of 1:9 to 1:11 to prepare the final salt damage repair and growth promotion integrated bacterial agent.

[0013] The application also provides a method for biological repair of secondary salinized soil based on Bacillus pumilus. The method is used for biological repair of secondary salinized soil with a nitrate content of 200 to 800mg·kg -1 . The prepared salt damage repair and growth promotion integrated bacterial agent is applied to the target soil, the application depth is controlled in the range of 10 to 15cm, and the application amount is 60 to 80kg per mu. During the application process, the soil water content needs to be maintained at 60 to 70% of the field water holding capacity, and after the application, the soil is covered and the environmental temperature is maintained in the range of 25 to 30℃. After the application, the soil is repaired through the biological assimilation of nitrate by the strain and the plant growth promotion effect, after the repair, compared with the untreated soil, the soil urease activity is increased by 40 to 60%, the Shannon diversity index is increased by 0.3 to 0.5, and the soil pH value is stabilized in the range of 6.8 to 7.2.

[0014] The repair method is widely applicable, especially suitable for the facility agricultural environment with soil pH value of 6.0-8.5 and organic matter content of not less than 1.0% in temperate and subtropical regions, and mainly aims at the secondary salinization soil caused by long-term application of nitrogen fertilizer to cause nitrate accumulation. Through the synergistic effect of the nitrate bioassimilation reconstruction function of the strain and the plant growth promotion effect, the comprehensive repair of the soil ecological environment is realized, the soil nitrate content can be reduced by more than 50% after repair, the plant root length is increased by 30-50%, the biomass is increased by 25-40%, and the soil biological activity and the plant growth condition are significantly improved.

[0015] The bacillus pumilus Y10 of the application has important application value in preparing secondary salinization soil repair bacterial agent, and the strain can prepare an integrated bacterial agent with dual functions of nitrate bioassimilation reconstruction and plant growth promotion. Through the specific nitrate reductase system, the nitrate in the soil is converted into cell components such as amino acids and proteins, and substances such as indole acetic acid and iron carrier are secreted, so that the synergistic effect of soil repair and plant growth promotion is realized, and an effective biological technology means is provided for solving the secondary salinization problem in facility agriculture.

[0016] The application has the following beneficial effects:

[0017] 1. The nitrate tolerance is significantly improved: the nitrate tolerance concentration of the Y10 strain reaches 800-1200 mg·L -1 , which is more than 67% higher than that of the prior art, and the strain can stably survive and function in the soil heavily polluted by nitrate.

[0018] 2. The nitrate conversion efficiency is greatly improved: under the condition of 30 DEG C and pH 7.0, more than 95% of 730 mg·L -1 of nitrate can be converted within 18 hours, and the conversion speed is more than 150% higher than that of the prior art, which significantly shortens the soil repair period.

[0019] 3. Dual-function integrated innovation: the strain has dual functions of nitrate conversion and plant growth promotion, can secrete indole acetic acid 15-25 mg·L -1 and iron carrier 20-35 mg·L -1 , and realizes the synergistic effect of soil repair and plant growth promotion.

[0020] 4. The repair effect is remarkable: compared with the untreated soil, the soil urease activity is increased by 40-60%, the Shannon diversity index is increased by 0.3-0.5, the soil pH value is stabilized in the appropriate range, the soil nitrate content is reduced by more than 50%, the plant root length is increased by 30-50%, and the biomass is increased by 25-40%.

[0021] 5. Good ecological safety: the strain is non-pathogenic bacteria after pathogenicity detection, has no significant negative impact on soil protist microbial community, and has high environmental release safety. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a microscope observation photo and colony morphology schematic diagram of Bacillus pumilus Y10;

[0023] Figure 2 is a curve graph of nitrate transformation effect of Bacillus pumilus Y10 changing with time;

[0024] Figure 3 is a process flow schematic diagram of the soil remediation method. DETAILED DESCRIPTION

[0025] The present application will be described in detail below in combination with the drawings and specific examples.

[0026] Example 1: Isolation and purification of Bacillus pumilus Y10 and verification of super-salt-tolerant biological transformation characteristics

[0027] The purpose of this example is to verify the isolation and purification method of Bacillus pumilus Y10 and its nitrate tolerance and transformation capacity, and to provide technical support for the super-salt-tolerant biological transformation characteristics in claim 1.

[0028] Materials and methods:

[0029] 1. Strain isolation: The soil sample was derived from the lettuce rhizosphere soil in a nitrate-type secondary salinization facility greenhouse in Minhang District, Shanghai, with a nitrate content of 520±25 mg·kg -1 . LB medium composition: 10 g·L -1 ( biochemical reagent grade, purity ≥98 %), 10 g·L -1 ( analytical pure), 5 g·L -1 ( biochemical reagent grade), distilled water to 1 L, pH 7.0±0.1, 121℃ sterilization for 20 min. Nitrate-containing LB medium was prepared by adding 500 mg·L -1 ( analytical pure, purity ≥99.5 %) to the LB medium.

[0030] 2. Isolation and purification process: 10 g of soil sample was added to 90 mL of sterile normal saline (0.85 % NaCl) and shaken at 28℃, 200 r·min -1 for 30 min to prepare a bacterial suspension, which was diluted to 10 -6 , and 100 μL was taken and spread on a nitrate-containing LB plate, which was cultured at 28℃ for 48 h, and single colonies were selected for purification culture.

[0031] 3. Nitrate tolerance assay: Purified strains were inoculated into LB medium containing different concentrations of nitrate (0, 200, 400, 600, 800, 1000, 1200 mg·L -1 ) with initial inoculum adjusted to OD 600 600 of 0.1, incubated at 30℃, 200 r·min -1 for 48 h, and OD 600 values were determined at 600 nm wavelength by UV spectrophotometer (UV-2600, Shimadzu) to evaluate the growth of strains.

[0032] 4. Nitrate conversion capacity assay: Inorganic salt medium with nitrate as the sole nitrogen source was used, with the composition: KNO3 1 g·L -1 , KCl 1 g·L -1 , FeSO4·7H2O 0.01 g·L -1 , MgSO4·7H2O 0.5 g·L -1 , CaCl2 0.01 g·L -1 , KH2PO4 0.5 g·L -1 , glucose 10 g·L -1 , trace element solution 2 mL·L -1 , pH 7.0±0.1. Strains were inoculated to OD 600 600 of 0.1, incubated at 30℃, 200 r·min -1 , and micro-aerobic conditions were maintained by controlling the aeration amount to keep the dissolved oxygen concentration ≤2 mg·L -1 . Nitrate content was determined every 2 h by sampling and detecting with an automatic intermittent chemical analyzer (AMS Alliance Smartchem450), with a detection limit of 0.1 mg·L -1 .

[0033] 5. Three-enzyme combined system activity assay: Spectrophotometry was used to determine nitrate reductase activity (calculated by NADH oxidation rate), nitrite reductase activity (calculated by nitrite consumption rate), and hydroxylamine reductase activity (calculated by hydroxylamine consumption rate), and the total enzyme activity was represented by the sum of the activities of the three enzymes.

[0034] Experimental results:

[0035] A total of 87 strains of bacteria resistant to nitrate were successfully isolated, of which the Y10 strain showed the best performance. 16S rDNA sequence analysis showed that the Y10 strain had a homology of 99.72% with Bacillus stratosphericus, confirming that it was Bacillus stratosphericus. As Figure 1As shown, the strain appears rod-shaped under a microscope, with regular colony morphology, smooth edges, and a moist and glossy surface, confirming that the morphological characteristics of the strain are consistent with the typical characteristics of Bacillus pumilus.

[0036] The nitrate tolerance test results showed that strain Y10 could tolerate a nitrate concentration of 800 mg·L⁻¹. -1 OD 600 The value was 2.18 ± 0.09, at 1000 mg·L⁻¹. -1 OD 600 The value was 2.31 ± 0.08, at 1200 mg / L. -1 OD 600 The value was 1.85±0.12, indicating that its nitrate tolerance concentration reached 800~1200mg·L. -1 Technical requirements. Data are expressed as mean ± standard deviation (n=3), and t-tests were used to analyze the significance of differences (P<0.05).

[0037] The results of the nitrate conversion test are as follows: Figure 2 As shown, strain Y10 can completely transform 730 mg·L⁻¹ within 18 hours. -1 The nitrate conversion rate reached 95.3±2.1%, meeting the requirement of converting ≥500 mg·L⁻¹ within 18–24 hours. -1 Technical requirement: nitrate content of 80% or higher.

[0038] The activity assay results of the three-enzyme combined system showed that the nitrate reductase activity was 32.5±1.8 U / mg protein, the nitrite reductase activity was 28.7±2.1 U / mg protein, the hydroxylamine reductase activity was 25.3±1.5 U / mg protein, and the total enzyme activity was 86.5±3.2 U / mg protein, which meets the technical requirement of ≥85 U / mg protein.

[0039] This example verifies that Bacillus pumilus Y10 has extremely high nitrate tolerance (800–1200 mg / L). -1 ) and rapid conversion ability (converting 730 mg·L within 18 hours) -1 The presence of nitrates (over 95%) demonstrates the strain's super-salt-tolerant biotransformation characteristics and rapid nitrate bioassimilation ability.

[0040] Example 2: Verification of the dual-effect synergistic growth-promoting characteristics of Bacillus pumilus Y10

[0041] The purpose of this embodiment is to verify the plant growth-promoting substance secretion ability of Bacillus pumilus Y10, and to provide technical support for the dual-effect synergistic growth-promoting characteristics in claim 1.

[0042] Materials and Methods:

[0043] 1. Indoleacetic acid (IAA) detection: Y10 strain was inoculated in LB medium containing 100 mg·L -1 of tryptophan (biochemical reagent grade, purity ≥98%), 30℃, 200 r·min -1 cultured for 24 h. 5 mL of culture solution was taken, 8000

[0044] r·min -1 centrifuged for 10 min, 2 mL of supernatant was taken and added with 2 mL of Salkowski color developing solution (150 mL of concentrated sulfuric acid, 250 mL of distilled water, 0.5 mol·L -1 of FeCl3 solution 7.5 mL), and reacted at room temperature for 30 min. The absorbance was measured at 530 nm wavelength, and the IAA content was calculated according to the IAA standard curve (R 2 = 0.998).

[0045] 2. Siderophore detection: Y10 strain was inoculated in MKB medium (peptone 20 g·L -1 , K2HPO4 1.5 g·L -1 , MgSO4·7H2O 1.5 g·L -1 , glycerol 15 mL·L -1 , pH 7.0±0.1), 30℃, 200 r·min -1 cultured for 48 h. 1 mL of culture supernatant was taken and added with CAS detection solution (CAS 0.079 g, FeCl3 10 mL, HDTMA 0.069 g), and the absorbance change at 630 nm wavelength was measured. The siderophore content was calculated according to the deferoxamine standard curve (R 2 = 0.995).

[0046] 3. Stage culture optimization: The stage culture strategy was adopted. The bacteria were enriched in nitrogen source LB medium for the first 12 hours, and then transferred to nitrogen-limited medium and added with nitrate inducer (KNO3 200 mg·L -1 ) to induce the secretion of functional substances. The culture was carried out until the bacterial liquid concentration was 2.0×10 9 ~ 5.0×10 9 CFU·mL -1 .

[0047] 4. Bacterial liquid concentration determination: The plate counting method was adopted. The bacterial liquid was gradiently diluted and plated on LB plates, and then cultured at 28℃ for 48 h before counting. The CFU·mL -1 was represented.

[0048] 5. Safety evaluation: The safety of the strain was evaluated by mouse acute toxicity test and Ames test, and the pathogenicity related genes were detected.

[0049] Experimental results:

[0050] The IAA detection results show that the IAA secretion amount of the Y10 strain reaches 18.5 ± 1.2 mg·L -1 after being cultured in the tryptophan-containing medium for 24 h, meeting the technical requirement of ≥15 mg·L -1 . Under different tryptophan concentrations (50, 100, 150 mg·L -1 ), the IAA secretion amounts are 15.2 ± 0.8, 18.5 ± 1.2, and 21.3 ± 1.5 mg·L -1 respectively, proving that it can be adjusted in the range of 15-25 mg·L -1 .

[0051] The siderophore detection results show that the siderophore secretion amount is 23.7 ± 2.1 mg·L -1 after being cultured for 48 h, meeting the technical requirement of ≥20 mg·L -1 . Under different culture times (24, 48, and 72 h), the siderophore secretion amounts are 20.1 ± 1.8, 23.7 ± 2.1, and 28.9 ± 2.5 mg·L -1 respectively, proving that it can be adjusted in the range of 20-35 mg·L -1 .

[0052] After adopting the staged culture strategy, the bacterial liquid concentration reaches 3.2 × 10 9 CFU·mL -1 , meeting the technical requirement of 2.0 × 10 9 -5.0 × 10 9 CFU·mL -1 , the IAA secretion amount is increased to 21.3 ± 1.8 mg·L -1 , and the siderophore secretion amount is increased to 28.9 ± 2.5 mg·L -1 .

[0053] The safety evaluation results show that the strain is a non-pathogenic bacterium after pathogenicity detection, the LD 50 >5000 mg / kg in the acute toxicity test of mice, the Ames test result is negative, and it has no significant negative impact on the soil primary microbial community (P>0.05).

[0054] This example verifies that the Bacillus pumilus Y10 has good plant growth promoting ability and ecological safety, can secrete sufficient indole acetic acid (≥15 mg·L -1 ) and siderophore (≥20 mg·L -1 ), and proves the double-effect synergistic growth promoting characteristics of the strain.

[0055] Example 3: Preparation technology verification of salt damage remediation and growth promotion integrated bacterial agent

[0056] The purpose of this embodiment is to verify the preparation method and stability of the salt damage repair and growth integrated bacterial agent, and to provide technical support for the bacterial-carrier complex technology in claim 4.

[0057] Materials and methods:

[0058] 1. Humic acid carrier preparation: Humic acid with a molecular weight of 6000 Da (analytical pure) was selected and treated by carboxymethylation modification to increase the carboxyl content. The specific method is as follows: 10 g of humic acid was dissolved in 100 mL of 2 mol·L -1 NaOH solution, 15 g of sodium chloroacetate was added, and the reaction was carried out at 80℃ for 4 h. The pH was adjusted to 7.0 with hydrochloric acid, and the salt was removed by dialysis. Freeze-drying was used to obtain the modified humic acid carrier.

[0059] 2. Carboxyl content determination: 0.1 g of modified humic acid carrier was dissolved in 50 mL of 0.1 mol·L - 1 NaOH solution, and titrated with 0.1 mol·L -1 HCl standard solution. The carboxyl content was calculated according to the titration curve, and the measured carboxyl content was 5.2±0.3 mmol·g -1 , which met the technical requirements of 4.5-6.0 mmol·g -1 .

[0060] 3. Preparation of bacterial agent: Y10 strain was cultured according to the method of example 2 to a bacterial liquid concentration of 3.2×10 9 CFU·mL -1 , and mixed with modified humic acid carrier at a weight ratio of 1:10. The specific operation is as follows: 100 mL of bacterial liquid was mixed with 1000 g of modified humic acid carrier at room temperature for 30 min, so that the strain was fixed on the surface of the carrier through hydrogen bond and van der Waals force interaction.

[0061] 4. Binding strength determination: The binding strength of the strain and the carrier was determined by isothermal titration calorimetry (ITC), and the measured binding strength was 12.3±0.8 kJ / mol, which met the technical requirement of ≥12 kJ / mol.

[0062] 5. Strain distribution density determination: The distribution of the strain on the surface of the carrier was observed by scanning electron microscopy (SEM), and the distribution density was calculated by image analysis software. The measured distribution density was 2.8×10 5 CFU / μm 2 .

[0063] 6. Binding stability test: The prepared bacterial agent was stored at 4℃, and the viable cell count was determined at 0, 30, 60, and 90 days, respectively, to calculate the binding stability. Plate counting method was used, the bacterial agent was diluted and spread on LB plate, and the number of colonies was counted after 48 h of incubation at 28℃.

[0064] Experimental results:

[0065] The number of viable bacteria in the prepared salt damage repair and growth promoting integrated bacterial agent was 2.8 x 10 9 CFU·g -1 The strain and humic acid carrier formed a stable composite structure. The number of viable bacteria in the bacterial agent with different weight ratios (1:9, 1:10, 1:11) was 2.6 x 10 9 , 2.8 x 10 9 , and 2.5 x 10 9 CFU·g -1 , respectively, indicating that an effective bacterial agent could be prepared within the range of 1:9 to 1:11.

[0066] The combination stability test results showed that the number of viable bacteria in the bacterial agent remained at 2.1 x 10 9 CFU·g -1 after being stored at 4°C for 90 days, and the combination stability reached 92.3%, meeting the technical requirement of ≥90%.

[0067] Scanning electron microscope observation showed that the strain was uniformly distributed on the surface of the humic acid carrier, and formed a stable combination through hydrogen bonding and van der Waals interaction, with a distribution density of 2.8 x 10 5 / μm 2 and a combination strength of 12.3 ± 0.8 kJ / mol.

[0068] This example verified that the preparation method of the salt damage repair and growth promoting integrated bacterial agent was feasible, and the strain and humic acid carrier mixed in a ratio of 1:9 to 1:11 could form a stable composite structure through hydrogen bonding and van der Waals interaction, proving the effectiveness and practicality of the bacterial agent preparation technology.

[0069] Example 4: Application verification of secondary salinized soil bioremediation method

[0070] The purpose of this example is to verify the actual application effect of the secondary salinized soil bioremediation method based on Bacillus pumilus Y10, and to provide technical support for claims 7 and 8.

[0071] Materials and methods:

[0072] 1. Soil sample: A secondary salinized soil with a nitrate content of 520 mg·kg -1 , pH 8.2, organic matter content 1.8%, and field water capacity 28.5% was selected, which was from a facility agriculture greenhouse in Shanghai.

[0073] 2. Remediation treatment: As Figure 3As shown, the whole soil remediation method process includes key steps such as preparation of bacterial agent, soil pretreatment, application of bacterial agent, soil covering and temperature keeping, and effect monitoring, and the logical relationship and technical parameter control requirements between each step are clearly shown in the flowchart. The salt damage remediation and growth promoting integrated bacterial agent prepared in Example 3 is applied to the soil in different amounts (60, 70, 80 kg / mu), with application depths of 10, 12, and 15 cm, respectively, and the soil water content is adjusted to 60%, 65%, and 70% of the field water holding capacity, respectively. After application, the soil is covered and the temperature is maintained at 25°C, 28°C, and 30°C, respectively. Untreated soil is set as the control group.

[0074] 3. Effect evaluation: The soil nitrate content, urease activity, microbial diversity index, and pH value are measured at 15, 30, and 60 days after treatment, respectively. The nitrate content is determined by ultraviolet spectrophotometry, the urease activity is determined by indigo blue colorimetry, the microbial diversity is analyzed by high-throughput sequencing technology (Illumina MiSeq platform, sequencing depth ≥ 30000), and the pH value is determined by potentiometry.

[0075] 4. Plant growth promoting effect: Lettuce seedlings (variety: butter lettuce) are planted in the treated soil, and the root length, plant height, and biomass (fresh weight and dry weight) are measured after 30 days.

[0076] 5. Long-term stability monitoring: Field tests are conducted for 3 consecutive years to monitor the long-term stability of the remediation effect.

[0077] Experimental results:

[0078] The soil remediation effect is shown in Table 1. Under the optimal conditions (bacterial agent dosage 70 kg / mu, application depth 12 cm, soil water content 65% of the field water holding capacity, and temperature 28°C), after 60 days of treatment, the soil nitrate content decreased from 520 mg·kg -1 to 248 mg·kg -1 , a decrease of 52.3%, meeting the technical requirement of a decrease of more than 50% in nitrate content.

[0079] The soil urease activity increased from 0.42 mg·g -1 ·24h-1 to 0.65 mg·g -1 ·24h-1, an increase of 54.8%, meeting the technical requirement of an increase of 40-60% in urease activity. The Shannon diversity index increased from 2.1 to 2.6, an increase of 0.5, meeting the technical requirement of an increase of 0.3-0.5 in Shannon index. The soil pH value was adjusted from 8.2 to 7.1, and stabilized within the appropriate range.

[0080] The plant growth promoting effect shows that the root length of the lettuce in the treatment group is increased by 42% compared with the control group, the plant height is increased by 35%, the fresh weight is increased by 38%, and the dry weight is increased by 41%, meeting the technical requirements of increasing the root length by 30-50% and improving the biomass by 25-40%.

[0081] The field test for three consecutive years shows that the repair effect is stable and maintained, the soil nitrate content is kept below the safe level, proving the long-term stability of the technical scheme.

[0082] Table 1 Comparison of soil repair effect;

[0083]

[0084] The secondary salinization soil biological repair method based on Bacillus pumilus Y10 has a significant repair effect, can effectively reduce the soil nitrate content, improve the soil biological activity, and promote the plant growth, proving the practicability and effectiveness of the technical scheme.

Claims

1. A type of Bacillus pumilus Y10, with accession number CGMCC No. 25291, characterized in that, This strain exhibits the following characteristics: it tolerates nitrate concentrations of 800–1200 mg·L⁻¹; under conditions of 25–35℃ and pH 6.5–7.5, it can convert over 80% of nitrate concentrations ≥500 mg·L⁻¹ within 18–24 hours; it can secrete indoleacetic acid ≥15 mg·L⁻¹ and siderophores ≥20 mg·L⁻¹; and through a specific nitrate reductase system, it sequentially reduces nitrate to nitrite and hydroxylamine under microaerobic conditions, ultimately assimilating them into cellular components such as amino acids and proteins.

2. The Bacillus pumilus Y10 according to claim 1, characterized in that, The strain tolerates nitrate concentrations of 1000–1200 mg·L⁻¹, and under conditions of 30°C and pH 7.0, it can convert more than 95% of 730 mg·L⁻¹ of nitrate within 18 hours.

3. The Bacillus pumilus Y10 according to claim 1, characterized in that, The strain was cultured until the bacterial concentration reached 2.0 × 10⁻⁶. 9 ~5.0×10 9 At CFU·mL⁻¹, in an inorganic salt culture medium containing nitrate, the specific nitrate reductase system includes nitrate reductase, nitrite reductase, and hydroxylamine reductase, with a total enzyme activity ≥85 U / mg protein, sequentially catalyzing the stepwise reduction and assimilation of nitrate, with indoleacetic acid secretion of 15–25 mg·L⁻¹ and siderophore secretion of 20–35 mg·L⁻¹.

4. A microbial agent for salt damage repair and growth promotion, characterized in that, The product comprises *Bacillus pumilus* Y10 as described in claim 1 and a humic acid carrier, wherein the *Bacillus pumilus* Y10 is immobilized on the surface of the humic acid carrier through hydrogen bonds and van der Waals forces, forming a stable bacterial-carrier composite structure, wherein the weight ratio of the bacterial strain to the humic acid carrier is 1:9 to 1:11, and the humic acid carrier has a molecular weight of 5000 to 8000 Da and a carboxyl content of 4.5 to 6.0 mmol·g. -1 .

5. The integrated microbial agent for salt damage repair and growth promotion according to claim 4, characterized in that, The weight ratio of the bacterial strain to the humic acid carrier was 1:10, and the bacterial concentration was 2.0 × 10⁻⁶. 9 ~5.0×10 9 The humic acid carrier, at CFU·mL⁻¹, is modified by carboxymethylation, resulting in a carboxyl content of 4.5–5.5 mmol·g. -1 The bonding strength is ≥12kJ / mol.

6. The preparation method of the integrated salt damage repair and growth promotion microbial agent according to claim 4, characterized in that, Includes the following steps: Step 1: In LB medium, incubate Bacillus pumilus Y10 at 28–30°C and 200 rpm. -1 Under these conditions, a staged culture strategy was adopted. For the first 12 hours, bacterial cells were enriched in a nitrogen-containing medium. For the next 12 hours, the cells were transferred to a nitrogen-limited medium with the addition of nitrate inducers to induce the secretion of functional substances. The culture was continued until the bacterial concentration reached 2.0 × 10⁻⁶. 9 ~5.0×10 9 CFU·mL⁻¹; Step 2: Mix the bacterial solution with a carboxymethylated humic acid carrier at a weight ratio of 1:9 to 1:11 to prepare an integrated bacterial agent for salt damage repair and growth promotion.

7. A bioremediation method for secondary salinized soil based on Bacillus pumilus, characterized in that, The integrated microbial agent for salt damage repair and growth promotion described in claim 4 is applied to areas with nitrate content of 200–800 mg / kg. -1 In secondary salinized soils, the application depth is 10-15 cm, the soil moisture content is maintained at 60-70% of field capacity, and the soil is covered after application and the temperature is maintained at 25-30℃.

8. The method for bioremediation of secondary salinized soil based on Bacillus pumilus according to claim 7, characterized in that, The application rate of the integrated salt damage remediation and growth promotion microbial agent is 60-80 kg per mu. After application, soil remediation is achieved through the nitrate biological assimilation and reconstruction of the strain and the plant growth promotion effect. Compared with the untreated soil, the soil urease activity increased by 40-60%, the Shannon diversity index increased by 0.3-0.5, and the soil pH value stabilized in the range of 6.8-7.

2.

9. The bioremediation method for secondary salinized soil based on Bacillus pumilus according to claim 7, characterized in that, The method is applicable to the remediation of secondary salinized soils caused by long-term nitrogen fertilizer application in facility agriculture in temperate and subtropical regions with soil pH 6.0-8.5 and organic matter content ≥1.0%, where soil nitrate accumulation has occurred. After remediation, the soil nitrate content is reduced by more than 50%, plant root length increases by 30-50%, and biomass increases by 25-40%.

10. The Bacillus pumilus Y10 of claim 1, suitable for preparing nitrate content of 200-800 mg·kg⁻¹ -1 The application of microbial agents in the remediation of secondary salinized soils is characterized by... The strain is used to prepare an integrated bacterial agent for salt damage repair and growth promotion that can simultaneously achieve the dual functions of nitrate bioassimilation and reconstruction and plant growth promotion. It converts nitrate into cellular components such as amino acids and proteins through a specific nitrate reductase system, while secreting indoleacetic acid and siderophores to promote plant growth.

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