Citrobacter braakii and its application in agricultural resource utilization of manganese mine slag

Abstract

The application discloses a citrobacter amalonaticus strain and application of the citrobacter amalonaticus strain in agricultural resource utilization of manganese mine slag. The strain can metabolically produce various organic acids such as citric acid, propionic acid and valeric acid, can efficiently dissolve the insoluble minerals in the manganese mine slag, and can convert the invalid state manganese, iron, silicon, calcium and magnesium in the slag into available nutrients absorbable by plants. Meanwhile, the organic acids can be chelated with heavy metal ions to reduce the bioavailability of the heavy metal ions. The application further provides a biological preparation containing the strain and a preparation method of the biological preparation. It is verified through experiments that the activation rate of the strain to the available manganese in the slag reaches 58.7%, the exchangeable manganese is reduced by 55.5%, the yield of pakchoi is increased by more than 94% after the biological preparation is applied, and the manganese content of the plant is reduced by about 40%. The application provides a green and efficient microbial solution for the agricultural resource utilization of the manganese mine slag.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Citrobacter malonic acid-free and its application in the agricultural resource utilization of manganese ore slag. Background Technology

[0002] Manganese ore slag is a solid waste generated during the mining and smelting of manganese ore. Large-scale stockpiling of manganese ore not only occupies land, but also allows heavy metals (such as Mn, Pb, and Cr) to leach into surrounding soil and water bodies with rainwater, causing serious environmental pollution. On the other hand, manganese ore slag contains abundant silicon, calcium, magnesium, iron, manganese, and various trace elements. If these mineral nutrients can be converted into effective forms that plants can absorb, it can be transformed from waste into a valuable resource, used as a soil conditioner or trace element fertilizer.

[0003] However, the mineral elements in manganese ore slag exist mostly in the form of insoluble silicates, oxides, or carbonates, which are difficult for plants to directly absorb and utilize. Traditional chemical activation methods (such as strong acid leaching) are costly and pose a significant risk of secondary pollution. Microbial activation technology has become a research hotspot due to its advantages of being green, mild, and sustainable. Organic acids produced by the metabolism of certain functional microorganisms can dissolve insoluble minerals in the slag, releasing available nutrients. Simultaneously, these organic acids can chelate or complex with heavy metal ions, reducing their bioavailability and environmental migration capacity.

[0004] Currently, there are few reported strains that can adapt to the extreme environments of manganese ore slag, such as high salinity, high metal content, and low organic matter, and efficiently produce acid to activate nutrients. Therefore, screening and developing specialized strains suitable for the agricultural resource utilization of manganese ore slag, and clarifying their optimal application conditions, is of significant practical importance. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Citrobacter malonic acid-free and its application in the agricultural resource utilization of manganese ore slag.

[0006] A strain of Citrobacter amalgam ( Citrobacter amalonaticus (), its accession number is CGMCCNO.37781, and it is named KM-11.

[0007] The malonic acid-free citrate bacillus ( Citrobacter amalonaticus Application of manganese ore slag in agricultural resource utilization.

[0008] The malonic acid-free citrate bacillus ( Citrobacter amalonaticus The organic acids produced by metabolism function in the following ways: dissolving insoluble minerals in manganese ore slag, converting unavailable manganese, iron, silicon, calcium, and magnesium in the slag into readily available nutrients that plants can absorb, and chelating with heavy metal ions in the slag to reduce the bioavailability of heavy metals.

[0009] A biological agent for agricultural applications of manganese ore slag, comprising the aforementioned malondiic acid-free Citrobacter ( Citrobacter amalonaticus ).

[0010] The biological agent is in powder form, with rice husk powder as the carrier, and a live bacteria content of 5-10 × 10⁻⁶. 9 CFU / g.

[0011] The preparation method of the biological agent for agricultural application of manganese ore slag is carried out according to the following steps: (1) The citrobacter malonic acid-free bacteria described in claim 1 ( Citrobacter amalonaticus Inoculate the seed culture into liquid culture medium and incubate at 28-37℃ with shaking for 16-24 h to obtain the seed culture. (2) Inoculate the seed culture into the fermentation medium and culture at 28-37℃ with shaking for 5-7 days to obtain the fermentation culture; (3) Mix the fermentation culture with the sterilized carrier and dry at low temperature to obtain the biological agent.

[0012] The liquid culture medium in step (1) is nutrient broth medium or LB medium.

[0013] The fermentation medium in step (2) is a nutrient broth medium or a slag-specific medium, with an inoculation amount of 5-10% for the seed liquid and a shaking speed of 150-200 rpm.

[0014] In step (3), the mixing mass ratio of the fermentation culture to the carrier is 1:(3-5), and the carrier is rice husk powder.

[0015] A method for agricultural improvement of manganese ore slag involves mixing nutrient supplements with manganese ore slag, adjusting the moisture content to 20-30%, and inoculating with malondiic acid-free Citrobacter (Citrobacter malonic acid). Citrobacter amalonaticus Seed liquid, piled up for fermentation for 10-30 days.

[0016] The malonic acid-free citrate bacillus ( Citrobacter amalonaticus OD of seed liquid 600 It is 1.4-1.6.

[0017] The manganese ore slag and nutritional supplements are mixed at a mass ratio of (70-85):(15-30).

[0018] The nutritional supplement consists of starch, soybean flour, and rice husk powder, wherein the mass ratio of starch to soybean flour is (6-8):(4-2), and rice husk powder accounts for 30-50% of the total mass of the nutritional supplement.

[0019] 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.

[0020] The beneficial effects of this invention are as follows: The malondialdehyde-free Citrobacter KM-11 and its biologically modified preparation provided by this invention can efficiently activate mineral nutrients such as silicon, calcium, magnesium, iron, and manganese in manganese ore slag, while passivating heavy metals and reducing their bioavailability. This strain is highly adaptable and can grow well and stably produce acid in the high-salt, high-metal, and low-organic-matter environment of ore slag. The optimal substrate formula (80% ore slag, 7% starch, 3% soybean flour, 10% rice husk powder, and 25% moisture) is low in cost and easy to operate. This invention provides a green, economical, and efficient microbial technology approach for the agricultural resource utilization of manganese ore slag, and has good prospects for industrial application. Detailed Implementation

[0021] 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.

[0022] 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.

[0023] 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.

[0024] (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.

[0025] (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%.

[0026] (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 ).

[0027] 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. 600 The 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.

[0028] 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 cells was suitable for preparing the inoculum. After 7 days of culture, the organic acid production no longer increased, and the cells began to age.

[0029] Table 1. Changes in total organic acid content and pH in KM-11 fermentation broth at different culture times.

[0030] Example 3: Optimization of the best fermentation substrate ratio for manganese ore slag 1. Experimental materials The manganese ore slag was extracted from a manganese mine in Inner Mongolia and crushed through a 60-mesh sieve. Its chemical composition is: MnO2 18.3%, SiO2 32.5%, CaO 8.2%, MgO 3.5%, Fe2O3 12.1%, total manganese (as Mn) 11.6%, and available manganese (DTPA extracted) only 0.23%. Nutritional supplements include: starch (corn starch, commercially available), soybean flour (soybeans crushed through an 80-mesh sieve), and rice husk powder (crushed through a 40-mesh sieve).

[0031] 2. Experimental Design Using slag as a base, different proportions of starch, soybean flour, and rice husk powder were added, and the moisture content was adjusted to 25%. Seed culture of strain KM-11 (OD) was then inoculated. 600 =1.5), inoculum size 5% (v / w), mixed well and incubated at 30℃ for 14 days. The available manganese content (DTPA extraction method) and viable cell count in the fermented system were used as evaluation indicators. Nine treatments were set up, with three replicates for each treatment. The optimal ratio results are shown in Table 2.

[0032] Table 2. Effects of different matrix ratios on available manganese and viable bacteria count in KM-11 activated slag.

[0033] Note: Increase rate of available manganese (%) = (available manganese content after treatment - initial available manganese content) / initial available manganese content × 100%. The initial available manganese content is 0.23 g / kg.

[0034] As shown in Table 2, treatment 2 (80% slag, 7% starch, 3% soybean flour, 10% rice husk powder, 25% moisture) showed the highest increase in available manganese (58.6 ± 2.5%), and the viable bacterial count also reached 3.4 × 10⁻⁶. 8 CFU / g. This ratio provides the strain with a suitable carbon-to-nitrogen ratio (C / N approximately 25:1) and good aeration, which is beneficial for strain growth and acid production. Therefore, the optimal substrate ratio was determined to be: 80% manganese ore slag, 7% starch, 3% soybean flour, 10% rice husk powder, and a system moisture content of 25%.

[0035] Example 4: Activation effect of strain KM-11 on multiple nutrients in manganese ore slag Using the optimal ratio (treatment 2) and fermentation conditions determined in Example 3, a blank control (CK, with an equal volume of sterile water added) without inoculation was set up, and each treatment was replicated three times. Samples were taken after 14 days of fermentation, and the contents of available manganese, available iron, available silicon, available calcium, and available magnesium in the slag were determined, and the activation rate was calculated. The results are shown in Table 3.

[0036] Table 3. Activation effect of strain KM-11 on multiple elements in manganese ore slag

[0037] Note: Activation rate (%) = (active content after KM-11 treatment - active content after CK treatment) / active content after CK treatment × 100%.

[0038] As shown in Table 3, after treatment with KM-11, the effective content of manganese, iron, silicon, calcium and magnesium in the slag was significantly increased, with activation rates of 58.7%, 73.3%, 71.2%, 57.0% and 65.1%, respectively. This indicates that the organic acids produced by the bacterial strain can effectively dissolve the insoluble mineral components in the slag and greatly improve its agricultural nutrient availability.

[0039] Example 5: Passivation effect of strain KM-11 on heavy metals in manganese ore slag The experimental materials and treatment were the same as in Example 4. After 14 days of fermentation, the speciation of manganese, lead, and chromium in the slag was analyzed using the Tessier continuous extraction method, with a focus on determining the changes in the content of exchangeable (bioavailable) forms. The results are shown in Table 4.

[0040] Table 4. Effects of KM-11 treatment on the exchangeable heavy metal content in slag.

[0041] Note: Exchangeable state reduction rate (%) = (content after CK treatment - content after KM-11 treatment) / content after CK treatment × 100%.

[0042] As shown in Table 4, after treatment with strain KM-11, the exchangeable contents of Mn, Pb, and Cr in the slag decreased by 55.5%, 55.9%, and 53.3%, respectively. This indicates that the organic acids produced by the strain's metabolism form stable chelates with heavy metal ions, converting them into organically bound or residual states, thereby significantly reducing the bioavailability and environmental migration risk of heavy metals.

[0043] Example 6: Preparation of biological modification agent and pot plant validation test 1. Preparation of biologically modified formulations Following the optimal ratio of Example 3 (80% slag, 7% starch, 3% soybean flour, 10% rice husk powder), KM-11 seed culture (5% inoculum) was added, and the moisture content was adjusted to 25%. Fermentation was carried out at 30℃ for 14 days. After fermentation, the material was dried at a low temperature below 40℃, pulverized, and passed through a 60-mesh sieve to obtain the biological improvement preparation (powder). Plate counting showed a viable cell count of (3.2±0.3)×10⁻⁶. 8 CFU / g.

[0044] 2. Pot experiment The tested soil was infertile sandy loam (0.6% organic matter). The following treatments were established: CK1, blank soil (no materials added); CK2, soil + unfermented slag substrate (same ratio as the optimum, but without inoculation); T1, soil + fermented material from treatment 2 of Example 3 (i.e., biological amendment), added at 10% of soil mass; T2, soil + 80% slag + 7% starch + 3% soybean flour + 10% rice husk powder + KM-11 seed liquid (same as T1, but directly mixed without drying after fermentation), added at 10% of soil mass (dry matter equivalent).

[0045] Each pot contained 2 kg of soil, with 5 replicates per treatment. After thorough mixing, the soil was potted and allowed to equilibrate for 7 days before sowing Chinese cabbage (variety: Shanghai bok choy), leaving 3 seedlings per pot. Standard management was implemented, and the plants were harvested after 45 days of growth. The aboveground fresh weight, plant height, and manganese content of the aboveground parts were measured. The results are shown in Table 5.

[0046] Table 5. Effects of different treatments on the growth and manganese absorption of Chinese cabbage

[0047] Note: The yield increase rate is calculated based on CK1; DW is dry weight.

[0048] Table 5 shows that the addition of KM-11 biological amendments (T1 and T2) significantly promoted the growth of Chinese cabbage, with plant height and fresh weight increasing by more than 90% compared to the control. At the same time, the manganese content in the plants decreased by 39-41% compared to CK1. This indicates that the formulation can effectively activate the nutrients in the slag for plant absorption and reduce the translocation of excess manganese to the aboveground parts through passivation, thus possessing excellent dual functions of "activation-passivation".

[0049] 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 strain of Citrobacter amalgam ( 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 manganese ore slag in agricultural resource utilization.

3. The application according to claim 2, characterized in that, The malonic acid-free citrate bacillus ( Citrobacter amalonaticus The organic acids produced by metabolism function in the following ways: dissolving insoluble minerals in manganese ore slag, converting unavailable manganese, iron, silicon, calcium, and magnesium in the slag into readily available nutrients that plants can absorb, and chelating with heavy metal ions in the slag to reduce the bioavailability of heavy metals.

4. A biological agent for agricultural application of manganese ore slag, characterized in that, Contains the malonic acid-free citrate bacillus as described in claim 1 ( Citrobacter amalonaticus ).

5. The biological agent for agricultural application of manganese ore slag according to claim 4, characterized in that, The biological agent is in powder form, with rice husk powder as the carrier, and a live bacteria content of 5-10 × 10⁻⁶. 9 CFU / g.

6. The method for preparing the biological agent for agricultural application of manganese ore slag as described in claim 4, characterized in that, Follow these steps: (1) The malonic acid-free citrate bacillus described in claim 1 ( Citrobacter amalonaticus Inoculate the seed culture into liquid culture medium and incubate at 28-37℃ with shaking for 16-24 h to obtain the seed culture; (2) Inoculate the seed culture into the fermentation medium and culture at 28-37℃ with shaking for 5-7 days to obtain the fermentation culture; (3) Mix the fermentation culture with the sterilized carrier and dry at low temperature to obtain the biological agent.

7. The method for preparing the biological agent for agricultural application of manganese ore slag according to claim 6, characterized in that, The liquid culture medium in step (1) is nutrient broth culture medium or LB culture medium; the fermentation culture medium in step (2) is nutrient broth culture medium or slag-specific culture medium, the inoculation amount of seed liquid is 5-10%, and the shaking speed is 150-200 rpm; the mixing mass ratio of fermentation culture to carrier in step (3) is 1:(3-5), and the carrier is rice husk powder.

8. A method for agricultural improvement of manganese ore slag, characterized in that, The nutritional supplements were mixed with manganese ore slag, and the moisture content was adjusted to 20-30%. The mixture was then inoculated with the malonic acid-free citrate bacterium described in claim 1. Citrobacter amalonaticus Seed liquid, piled up for fermentation for 10-30 days.

9. The method for agricultural improvement of manganese ore slag according to claim 8, characterized in that, The malonic acid-free citrate bacillus ( Citrobacter amalonaticus OD of seed liquid 600 It is 1.4-1.

6.

10. The method for agricultural improvement of manganese ore slag according to claim 8, characterized in that, The manganese ore slag and nutritional supplements are mixed at a mass ratio of (70-85):(15-30). The nutritional supplements consist of starch, soybean flour, and rice husk powder, wherein the mass ratio of starch to soybean flour is (6-8):(4-2), and rice husk powder accounts for 30-50% of the total mass of the nutritional supplements.

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