Citrobacter amalonaticus and application thereof in heavy metal pollution treatment

Abstract

The application discloses a citrobacter amalonaticus strain without malonic acid and application of the citrobacter amalonaticus strain in heavy metal pollution treatment. Citrobacter amalonaticus The strain has a preservation number of CGMCC NO.37781. The strain can effectively reduce the content of available lead and chromium in soil through the double mechanisms of adsorption on the surface of the strain and metabolism to produce organic acids such as citric acid, propionic acid and valeric acid to chelate heavy metals. The repair effect of the strain after fermentation culture for 5-7 days is optimal. The repair bacterial agent prepared from the strain is applied to the heavy metal contaminated soil at 2.5 kg per mu, the content of available lead can be reduced by more than 42.7%, and the content of available chromium can be reduced by more than 29.9%. The application realizes efficient and low-cost microbial repair of the heavy metal contaminated soil, has good economic and environmental benefits, and is suitable for large-area popularization and application.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a malondiic acid-free Citrobacterium and its application in the treatment of heavy metal pollution. Background Technology

[0002] With the development of industry and mining, heavy metal pollution has become a prominent global environmental problem. Heavy metals such as lead (Pb) and chromium (Cr) are highly toxic, difficult to degrade, and easily accumulate in the food chain, seriously threatening soil ecological security and human health. Remediation technologies for heavy metal-contaminated soil include physical remediation, chemical remediation, and bioremediation. Among these, microbial remediation has received widespread attention due to its advantages such as low cost, environmental friendliness, and ease of operation.

[0003] The main mechanisms for microbial remediation of heavy metal pollution include: adsorption through microbial cell walls, complexation with extracellular polymers, chelation and dissolution of metabolites (such as organic acids), and redox transformation. Citrobacter bacteria are common Enterobacteriaceae in the environment, and some strains possess the ability to produce organic acids and tolerate heavy metals. However, there is currently a lack of specialized strains and standardized application protocols that can efficiently adapt to heavy metal-polluted environments, stably produce acids, and effectively reduce the content of available lead and chromium in soil. Summary of the Invention

[0004] The purpose of this invention is to provide a malondioxycis-free Citrobacterium and its application in the treatment of heavy metal pollution.

[0005] A type of malonic acid-free citrate bacterium ( Citrobacter amalonaticus (), its accession number is CGMCCNO.37781, and it is named KM-11.

[0006] The malonic acid-free citrate bacillus ( Citrobacter amalonaticus Application of ) in the treatment of heavy metal pollution.

[0007] The heavy metal pollution remediation refers to the remediation of lead and / or chromium contaminated soil.

[0008] A soil remediation agent for heavy metal contaminated soil, comprising the aforementioned malondioxycitric acid-free citrobacter (Citrobacter malonic acid). Citrobacter amalonaticus ).

[0009] The remedial microbial agent is in powder form, with rice husk powder as the carrier, and a live bacteria content of 2-8 x 10⁻⁶. 9 CFU / g.

[0010] A method for preparing a microbial agent for remediating heavy metal contaminated soil includes the following steps: (1) The malonic acid-free citrate bacillus ( Citrobacter amalonaticus Inoculate into fermentation medium for liquid fermentation culture; (2) Ferment and culture for 5-7 days, then collect the fermentation culture; (3) Mix the fermentation culture with the carrier, dry it, and obtain the inoculum.

[0011] The fermentation medium mentioned in step (1) is a nutrient broth medium with a fermentation temperature of 28-37℃ and a pH of 6.0-7.5.

[0012] A method for remediating heavy metal contaminated soil, comprising applying the aforementioned microbial agent to the heavy metal contaminated soil.

[0013] The application rate of the microbial agent is 1.5-3.5 kg per mu.

[0014] The heavy metals are lead and / or chromium.

[0015] The present invention provides citrobacter free of malondialdehyde (Citrobacter malonic acid) Citrobacter amalonaticus Strain KM-11 was isolated and purified from soil samples that had been contaminated with heavy metals for a long period of time in a lead-zinc mining area. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.37781, and the deposit date is February 10, 2026.

[0016] The beneficial effects of this invention: The malondiic acid-free citrate bacillus screened by this invention ( Citrobacter amalonaticus This strain effectively reduces the content of available heavy metals in soil and decreases the risk of heavy metal migration and crop accumulation through a dual mechanism of adsorption on the bacterial surface and chelation with metabolic organic acids (citric acid, propionic acid, valeric acid, etc.). Shake-flask, pot, and field trials have verified that the strain exhibits the best remediation effect after 5-7 days of fermentation. Combined with standardized inoculants, it can economically and efficiently achieve large-scale remediation of lead- and chromium-contaminated farmland, demonstrating promising application prospects. Detailed Implementation

[0017] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0018] Example 1: Isolation and Identification of Citrobacter malonic acid-free KM-11 1. Strains Isolation The malonic acid-free citrate bacillus described in this invention ( Citrobacter amalonaticus Strain KM-11 was isolated from soil samples from a lead-zinc mining area that had been contaminated with heavy metals for a long period. 10 g of soil sample was weighed and added to 90 mL of sterile water. The mixture was shaken at 30°C and 150 rpm for 30 min. After standing, the supernatant was serially diluted and plated onto MacConkey agar plates, and incubated at 30°C for 24 h. Pink, smooth, and moist single colonies were picked and streaked onto nutrient agar plates for purification, yielding a pure culture, designated KM-11.

[0019] 2. Strain identification (1) Colony morphology When incubated at 30°C for 24 h on NA plates, the colonies are approximately 3 mm in size, round, slightly convex, with neat edges, milky white, translucent, with a smooth, moist, glossy, and wrinkle-free surface; on MacConkey agar, they appear pink (from lactose fermentation) and do not hemolyze.

[0020] (2) Cell morphology Gram-negative, rod-shaped, 1.0 × (2.0-6.0) μm in size, arranged singly or in pairs, with peritrichous flagella, highly motile, without spores or capsules.

[0021] (3) Physiological and biochemical characteristics Oxidase negative, catalase positive, nitrate reduction positive, indole negative, H2S negative, urease negative, gelatin liquefaction negative, citrate utilization positive; ferments glucose to produce acid and gas, ferments lactose, sucrose, and mannitol; methyl red positive, VP negative; optimal growth temperature 30℃, optimal pH 7.0, tolerant to NaCl 0-5%.

[0022] (4) 16S rRNA gene sequence Genomic DNA was extracted from strain KM-11, amplified by PCR using the universal bacterial primers 27F / 1492R, and sequenced to obtain a partial 16S rDNA sequence (SEQ ID NO: 1). This sequence was then subjected to BLAST alignment analysis in the GenBank database. Citrobacter amalonaticus The sequence similarity of the standard strain was 99.8%. Based on morphological and physiological / biochemical characteristics, strain KM-11 was identified as *Citrobacter amalgam*. Citrobacter amalonaticus ).

[0023] Example 2: Growth curve and acid production characteristics of strain KM-11 1. Growth curve determination The KM-11 strain was inoculated into nutrient broth liquid medium and cultured at 30℃ with shaking at 150 rpm. OD was measured every 4 h. 600The results showed that the 0-4 h period was the lag phase, the 4-16 h period was the logarithmic growth phase, the 16-36 h period was the stationary phase, and the bacterial culture entered the decline phase after 36 h. The OD value of the bacterial culture at the end of the logarithmic growth phase (16 h) was... 600 It can reach 1.8±0.1, with the maximum OD during the stable period. 600 It is 2.2 ± 0.1.

[0024] 2. Acid-producing characteristics Under the same culture conditions, fermentation broth was collected at different time points, and the supernatant was obtained by centrifugation. The types and contents of organic acids were determined by high-performance liquid chromatography (HPLC). Table 1 shows that the total organic acid level reached a relatively high level after 72 h (3 days) of culture. After 5-7 days of culture, the total organic acid level stabilized at 20-25 g / L, and the pH remained around 4.0. At this point, the metabolic activity of the bacteria was suitable for preparing the remediation agent. After 7 days of culture, the organic acid production no longer increased, and the bacteria began to age.

[0025] Table 1. Changes in total organic acid content and pH in KM-11 fermentation broth at different culture times.

[0026] Example 3: Effect of different culture cycles on heavy metal removal efficiency (shake flask experiment) 1. Test Methods Add lead (Pb) to a 250 mL Erlenmeyer flask 2+ ) and chromium (Cr 6+ 100 mL of simulated contamination solution (100 mg / L each) was inoculated into KM-11 fermentation cultures cultured for different time periods (1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 days), with an inoculation amount of 5% (v / v). After incubation at 30℃ and 150 rpm for 72 h with shaking, the supernatant was collected by centrifugation, and residual Pb was determined by atomic absorption spectrometry. 2+ and Cr 6+ Concentration, calculate removal rate. Use a blank control without inoculation for correction.

[0027] 2. Experimental Results Table 2. Effects of KM-11 fermentation products from different culture cycles on Pb 2+ and Cr 6+ Removal rate (%)

[0028] Note: The data in the table are the average of three replicates. Pb in the blank control is not included. 2+ and Cr 6+ The natural removal rate was <5%.

[0029] Table 2 shows that the KM-11 culture fermented for 5-7 days has a positive effect on Pb. 2+ and Cr6+ The best removal effect is achieved with Pb. 2+ The removal rate reached 46%-47%, Cr 6+ The removal rate reached 31%-32%. When the culture time was less than 5 days, the bacterial biomass and organic acid secretion were insufficient, resulting in poor removal efficiency; after 7 days, the bacterial cells aged and their metabolic activity decreased, leading to a gradual decrease in removal efficiency. This result is consistent with the acid production characteristics in Example 2.

[0030] Example 4 Indoor Potted Plant Experiment 1. Experimental Design Uncontaminated garden soil was collected, air-dried, sieved, and then lead nitrate and potassium dichromate were artificially added to bring the total lead content in the soil to 1000 mg / kg and the total chromium content to 1000 mg / kg (calculated as Cr). The soil was then allowed to age and reach equilibrium for 30 days. Each pot contained 2 kg of soil.

[0031] The following treatment was set up: CK (blank control): no bacterial agent was added, only an equal volume of sterile water was added; KM-11 treatment: Apply 50 mL of KM-11 fermentation liquid (equivalent to about 2.5 kg / mu of inoculant in the field, calculated based on soil dry weight) to each pot, which has been fermented and cultured for 6 days according to the method in Example 2, and add sterile water until the soil moisture content is 60% of the field capacity.

[0032] Each treatment was replicated three times and placed in a greenhouse (25-30℃, natural light). Regular watering was used to maintain humidity. Soil samples were collected after 60 days, and the contents of available lead and chromium in the soil were determined using the diethylenetriaminepentaacetic acid (DTPA) extraction method. The reduction rate was calculated.

[0033] 2. Experimental Results Table 3. Effects of KM-11 treatment on soil available heavy metal content in pot experiments.

[0034] Table 3 shows that after 60 days of application of the fermentation culture of strain KM-11, the content of available lead in the soil decreased by 42.7%, and the content of available chromium decreased by 29.9%. This indicates that KM-11 significantly reduced the bioavailability of lead and chromium in the soil through the dual effects of cell adsorption and organic acid chelation, thereby reducing their migration and crop uptake risks.

[0035] Example 5: Field Trial and Determination of Optimal Application Rate 1. Preparation of microbial agents The KM-11 strain was fermented and cultured for 6 days according to the method in Example 2. The fermentation broth was centrifuged to collect the bacterial cells, which were then mixed with sterilized rice husk powder (moisture content <10%) at a ratio of 1:4 and dried at low temperature to achieve a final viable cell content of 5×10⁻⁶. 9CFU / g yields the rice husk powder compound microbial agent.

[0036] 2. Overview of the test site The test site was located in contaminated farmland surrounding a lead-zinc smelter. The soil type was alluvial soil, with a pH of 6.8, an organic matter content of 1.5%, a total lead content of 856 mg / kg, and a total chromium content of 523 mg / kg.

[0037] 3. Experimental Design Seven treatments were set up with different inoculant application rates (including a blank control): 0 kg (CK), 1 kg, 2 kg, 2.5 kg, 3 kg, 4 kg, and 5 kg per acre. Each treatment plot was 30 m². 2 The study was conducted in a randomized block design with three replicates. The inoculum agent was mixed evenly with an appropriate amount of fine, dry soil and then manually applied, followed by rotary tillage to a depth of 15 cm to ensure thorough mixing. Soil samples from the 0–20 cm topsoil layer were collected before application and 90 days after application to determine the content of available lead and chromium. Routine field management was followed, without the application of other microbial agents.

[0038] 4. Test Results Table 4. Reduction rate of soil available Pb and Cr by KM-11 inoculant at different application rates (90 days after application)

[0039] Note: Data are mean ± standard deviation.

[0040] Table 4 shows that the application rate of the microbial agent is positively correlated with the remediation effect, but the rate of reduction slows down after the application rate exceeds 2.5 kg / mu. Considering the remediation effect, the cost of the microbial agent, and the labor input for transportation and application, 2.5 kg / mu is determined to be the optimal economic application rate. At this rate, the reduction rate of available lead is approximately 42%, and the reduction rate of available chromium is approximately 29%, which is in good agreement with the results of the pot experiment (Pb 42.7%, Cr 29.9%).

[0041] Example 6: Effects of KM-11 inoculant application on soil pH and enzyme activity 1. Test Methods In the field trial (Example 5), soil samples were collected from each treatment (CK, 2.5 kg / mu) at 30, 60 and 90 days after the application of the inoculant. Soil pH (water-soil ratio 2.5:1), urease activity (indophenol blue colorimetric method, expressed as NH3-N mg / g·d) and catalase activity (potassium permanganate titration method, expressed as 0.1M KMnO4 mL / g·h) were measured.

[0042] 2. Experimental Results Table 5 Effects of KM-11 inoculant (2.5 kg / mu) on soil pH and enzyme activity

[0043] Table 5 shows that after applying KM-11 inoculant, the soil pH slowly decreased from 6.82 to 6.22, which was due to the organic acid produced by the strain's metabolism. Although the acid production by the strain slightly decreased the soil pH, the secreted organic acids formed stable chelates with heavy metals, converting highly active exchangeable states into low-bioavailability organic bound states, thus reducing the content of available forms. Simultaneously, the activities of soil urease and catalase significantly increased, indicating that the application of the inoculant promoted the overall activity of soil microorganisms without producing toxic side effects, demonstrating good ecological safety.

[0044] Example 7: Effects of microbial agents on crop (Chinese cabbage) growth and heavy metal absorption. 1. Experimental Methods After the pot experiment (Example 4) was completed, Chinese cabbage seeds were sown in each pot, with 3 seedlings per pot. After 45 days of normal management, the plants were harvested, the fresh weight of the above-ground parts was measured, and the lead and chromium content in the plants was determined by atomic absorption spectrometry after nitric acid-perchloric acid digestion.

[0045] 2. Experimental Results Table 6. Effects of KM-11 treatment on growth and heavy metal absorption in Chinese cabbage.

[0046] Note: DW stands for dry weight. Data are mean ± standard deviation.

[0047] As shown in Table 6, after applying KM-11 microbial agent, the fresh weight of the aboveground parts of pak choi increased by 36.6%, the lead content in the plants decreased by 59.8%, and the chromium content decreased by 53.7%. This indicates that the microbial agent not only remediated the soil contaminated with heavy metals but also promoted crop growth and significantly reduced the absorption of heavy metals by crops, thus lowering health risks.

[0048] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A citrate-free bacterium (malonic acid) Citrobacter amalonaticus (), its accession number is CGMCCNO.37781, and it is named KM-11.

2. The citrobacterium malonicum of claim 1 ( Citrobacter amalonaticus Application of ) in the treatment of heavy metal pollution.

3. The application according to claim 2, characterized in that, The heavy metal pollution remediation refers to the remediation of lead and / or chromium contaminated soil.

4. A microbial agent for remediating heavy metal contaminated soil, characterized in that, Contains the malonic acid-free citrate bacillus as described in claim 1 ( Citrobacter amalonaticus ).

5. The heavy metal contaminated soil remediation microbial agent according to claim 4, characterized in that, The remedial microbial agent is in powder form, with rice husk powder as the carrier, and a live bacteria content of 2-8 x 10⁻⁶. 9 CFU / g.

6. A method for preparing a microbial agent for remediating heavy metal contaminated soil, characterized in that, Includes the following steps: (1) The malonic acid-free citrate bacillus described in claim 1 ( Citrobacter amalonaticus Inoculate into fermentation medium for liquid fermentation culture; (2) Ferment and culture for 5-7 days, then collect the fermentation culture; (3) Mix the fermentation culture with the carrier, dry it, and obtain the inoculum.

7. The preparation method of the heavy metal contaminated soil remediation microbial agent according to claim 6, characterized in that, The fermentation medium mentioned in step (1) is a nutrient broth medium with a fermentation temperature of 28-37℃ and a pH of 6.0-7.

5.

8. A method for remediating heavy metal-contaminated soil, characterized in that, The microbial agent of claim 4 is applied to heavy metal contaminated soil.

9. The method for remediating heavy metal contaminated soil according to claim 8, characterized in that, The application rate of the microbial agent is 1.5-3.5 kg per mu.

10. The method for remediating heavy metal contaminated soil according to claim 8, characterized in that, The heavy metals are lead and / or chromium.

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