Strain with bauxite dealkalization function and application thereof

Abstract

The application discloses a strain with a red mud dealkalization function and application thereof, and the classification name of the BRY1 strain is Acinetobacter baumannii Acinetobacter baumannii , the preservation number of which is GDMCC No 66371, the preservation date is May 22, 2025, and the preservation unit is Guangdong Microbial Culture Collection Center. The BRY1 strain provided by the application has strong alkali resistance and high acid production capacity, and can quickly and effectively neutralize alkaline substances in red mud. The BRY1 strain is obtained by being separated and screened from a specific environment, i.e., a red mud storage yard, and has good adaptability and dealkalization specificity to the red mud environment.

Description

Technical Field

[0001] This invention belongs to the field of industrial solid waste biological treatment technology and environmental microbiology, specifically involving a strain with red mud dealkalization function and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Red mud is a highly alkaline solid waste generated during alumina production, with a pH value as high as 10-13. It contains various alkaline substances (such as NaOH, Na₂CO₃, NaAl(OH)₄, etc.) and trace amounts of heavy metals. Large-scale stockpiling of red mud not only occupies land but also poses environmental safety risks and ecological hazards. The strong alkalinity of red mud is a major bottleneck restricting its large-scale resource utilization (such as in building materials, roadbed materials, and soil treatment).

[0004] Currently, the main methods for red mud dealkali removal include: water washing, which consumes a large amount of water and easily generates alkaline wastewater; acid neutralization, which uses inorganic acids (such as sulfuric acid and hydrochloric acid), which is costly and poses equipment corrosion and safety risks; CO2 carbonation, where the reaction rate and dealkali removal efficiency are sometimes limited; and biological dealkali removal, which utilizes acids or CO2 produced by microbial metabolism to neutralize alkaline substances, and has the potential for low cost, environmental friendliness, and sustainability. However, existing biological dealkali removal technologies suffer from problems such as poor strain adaptability, low dealkali removal efficiency, complex processes, or high costs.

[0005] Therefore, developing efficient, specific, and adaptable functional strains for red mud dealkalization and establishing their application processes are of great significance for promoting the biological harmless treatment and resource utilization of red mud. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a strain with red mud dealkalization function and its application.

[0007] The technical solution adopted in this invention is as follows: In a first aspect of the invention, a BRY1 strain with red mud dealkalization function is provided, and the BRY1 strain is classified as Acinetobacter baumannii. Acinetobacter baumannii Its accession number is GDMCC No. 66371, the accession date is May 22, 2025, and the depositary institution is Guangdong Microbial Culture Collection Center (Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou).

[0008] In a second aspect of the invention, a microbial mixture is provided, characterized in that it contains at least the BRY1 strain of the present invention, said microbial mixture may contain other strains with red mud dealkalization function and microorganisms symbiotic with the BRY1 strain of the present invention.

[0009] In one or more embodiments of the present invention, the microorganism comprising other strains with red mud dealkalization function and coexisting with the BRY1 strain of the present invention is a BRY2 strain, a BRY5 strain, or a BRY9 strain, wherein: The BRY2 strain is *Acinetobacter argyi*. Acinetobacter soli Its accession number is GDMCC No. 66372, the accession date is May 22, 2025, and the depositary institution is Guangdong Microbial Culture Collection Center; The BRY5 strain is Acinetobacter pitera. Acinetobacter pittii Its accession number is GDMCC No. 66373, the accession date is May 22, 2025, and the depositary institution is Guangdong Microbial Culture Collection Center; The BRY9 strain is *Codonopsis sacchari*. Kosakonia cowanii Its accession number is GDMCC No.66374, the accession date is May 22, 2025, and the depositary institution is Guangdong Microbial Culture Collection Center.

[0010] In a third aspect of the invention, a product comprising the BRY1 strain or the microbial mixture of the present invention is provided, the product being a microbial agent.

[0011] The active ingredient of the product is the bacterial cell, fermentation broth, or fermentation supernatant of the BRY1 strain.

[0012] In a fourth aspect of the invention, a fermentation broth comprising the BRY1 strain is provided.

[0013] In a fifth aspect of the invention, a fermentation method for the BRY1 strain is provided, wherein the BRY1 strain is inoculated into a fermentation medium for fermentation culture.

[0014] In a sixth aspect of the invention, the use of the BRY1 strain, the microbial mixture, and / or the product in red mud dealkalization is provided.

[0015] In a seventh aspect of the invention, a method for dealkalizing red mud is provided, the method comprising the step of contacting the red mud to be dealkalized with the BRY1 strain or the microbial mixture or the product.

[0016] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) The BRY1 strain provided by this invention has strong alkali resistance and high acid production capacity, and can quickly and effectively neutralize alkaline substances in red mud. The BRY1 strain was isolated and screened from a specific environment - a red mud dump, and has good adaptability to the red mud environment and dealkali specificity.

[0017] (2) The red mud dealkalization process mainly relies on microbial metabolism, without the need for large amounts of expensive chemical agents, thus avoiding secondary pollution; industrial and agricultural waste (such as straw and molasses) can be used as a nutrient source, further reducing costs.

[0018] (3) The red mud dealkali process has mild conditions, relatively simple equipment requirements, and is easy to scale up and industrialize.

[0019] (4) The red mud after dealkalization broadens the ways of resource utilization (such as building material additives), and valuable elements that may be enriched in the dealkalization liquid can be recovered. Attached Figure Description

[0020] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 pH changes in culture media inoculated with different acid-producing bacteria under different alkaline conditions.

[0022] Figure 2 pH changes in culture media inoculated with different acid-producing bacteria under different salinity conditions.

[0023] Figure 3 Changes in cell dry weight of acid-producing bacteria under different alkaline and saline conditions.

[0024] Figure 4 The production of organic acids by acid-producing bacteria under optimal growth conditions.

[0025] Figure 5 The effects of different bacterial strains inoculated into red mud on the regulation of red mud alkalinity were investigated. Mixed samples consisted of four strains. Detailed Implementation

[0026] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0027] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0028] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0029] Example 1: Screening and performance determination of four rhizosphere functional strains of red mud pioneer plants. 1. Screening of rhizosphere strains from red mud and pioneer plants Red mud and pioneer plant roots were selected from typical red mud dumps in China. Red mud samples and rhizosphere red mud samples were collected and transported to the laboratory in sterile bags. Red mud samples from different red mud microdomains and plant rhizospheres were collected and homogenized. 5 g of red mud sample was weighed and added to 50 ml of sterile physiological saline (0.90% NaCl). The mixture was incubated at 28 ℃ and 120 rpm with shaking for 30 min, then allowed to stand. The supernatant was used as the primary bacterial suspension. 1 mL of the primary bacterial suspension was inoculated into LB liquid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0) and incubated at 28 ℃ and 120 rpm with shaking for 18 h. The enriched bacterial suspension was then serially diluted (10... -3 ~10 -5 The cultures were inoculated onto LB agar plates (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar powder, pH 7.0) using the streak plating method and incubated at 28 °C for 48 h. Single colonies with significantly different morphologies were selected for isolation and purification. The purified culture was then mixed with an equal volume of 50% sterile glycerol and incubated at -80 °C.

[0030] 2. Screening of acid-producing strains The rhizosphere strains obtained from the initial screening were inoculated into bromocresol green differential medium (20 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, 50 g / L NaCl, 20 g / L agar, 0.022 g / L bromocresol green, pH 7.0) and cultured at 28 ℃ for 48 h. If the green color of the medium turned lighter or yellow, it indicated that the strain had acid-producing function.

[0031] 3. Morphological identification BRY1 strain forms round, pale yellow, opaque colonies with a flat interior, moist and smooth surface, and regular edges. Under a microscope, they are often arranged in pairs, mostly coccobacilli, without obvious flagella or spores. BRY2 strain forms round, pale yellow, opaque colonies with a raised interior, moist and smooth surface, and regular edges. Under a microscope, they appear rod-shaped, without obvious flagella or spores. BRY5 strain forms round, white, opaque colonies with a relatively flat interior, dry and rough surface, and slightly irregular edges. Under a microscope, they appear short rod-shaped, without obvious flagella or spores. BRY9 strain forms round, yellow, opaque colonies with a flat interior, moist and smooth surface, and regular edges. Under a microscope, they appear rod-shaped, without obvious flagella or spores.

[0032] 4. Physiological and biochemical identification BRY1 strain is a Gram-negative, obligate aerobic bacterium that can grow normally at temperatures ranging from 37°C to 42°C. It is negative for oxidases, positive for catalase, and negative for nitrate reduction. It can utilize glucose as a carbon source. BRY2 strain is a Gram-negative, aerobic bacterium that is negative for oxidases, positive for catalase, and cannot reduce nitrate. It can utilize glucose, maltose, and mannitol as carbon sources and produce acid. BRY5 strain is a Gram-negative, aerobic bacterium that can grow at temperatures ranging from 37°C to 44°C. It is negative for oxidases, positive for catalase, and can reduce nitrate to nitrite. It can utilize glucose, lactose, and citrate. BRY9 strain is a Gram-negative, facultative anaerobic bacterium with an optimal growth temperature of 37°C. It is positive for methyl red and can ferment glucose to produce acid and gas.

[0033] 5. Molecular biological identification Bacterial DNA was extracted using a bacterial genomic DNA extraction kit and amplified using universal primers 27F (5´-AGAGTTTGATCCTGGCTCAG-3´) and 1492R (5´-GGTTACCTTGTTACGACTT-3´). The total reaction mixture (25 μL) consisted of 5 μL dNTP mixture, 2 L MgCl2, 2.5 μL buffer, 1 μL Taq DNA polymerase, 0.25 μL 27F primer, 0.25 μL 1492R primer, 0.5 μL BSA, 12.5 μL dH2O, and 1 μL template DNA. The amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ for 30 s, 52℃ for 30 s, 72℃ for 1 min, for 32 cycles; and a final extension at 72℃ for 10 min. The PCR products were detected and purified by agarose gel electrophoresis. The purified qualified samples were sequenced by Beijing Ruiboxingke Biotechnology Co., Ltd., and the successfully sequenced nucleotide sequences were uploaded to the NCBI Blast database for comparison and analysis. Finally, a phylogenetic tree was constructed using MEGA 11 to determine the phylogenetic relationship of the strains.

[0034] Homology comparison and phylogenetic analysis using NCBI GenBank revealed that BRY1 is related to other genera belonging to the same family. Acinetobacter baumannii AB-S3 (accession number: OQ881018.1) is located on the same branch of the phylogenetic tree, and the two share 68% 16S rRNA sequence similarity. Therefore, strain BRY1 is identified as Acinetobacter baumannii. Acinetobacter baumannii BRY2 and others belong to the same category Acinetobacter soli NR 044454.1 (accession number: OP216732.1) is located on the same branch of the phylogenetic tree, and the two share 76% 16S rRNA sequence similarity. Therefore, strain BRY2 is identified as Acinetobacter tumefaciens. Acinetobacter soli BRY5 and others belong to the same category Acinetobacter pittii CIP 70.29 and GL (accession numbers: NR 116774.1 and OQ947067.1, respectively) are located on the same branch of the phylogenetic tree, and the 16S rRNA sequence of strain BRY5 has 100% similarity to CIP 70.29 and GL. Therefore, strain BRY5 is identified as Acinetobacter petrietes. Acinetobacter pittii BRY9 and others belong to the same category Kosakonia cowanii DYH22 (accession number: PP528640.1) is located on the same branch of the phylogenetic tree, and the two have 100% 16S rRNA sequence similarity. Therefore, strain BRY9 is identified as Coccidia sacchariformis. Kosakonia cowanii .

[0035] Example 2 Performance determination of acid-producing bacteria (1) Determination of salt and alkali tolerance of acid-producing bacteria Four acid-producing bacteria, BRY1, BRY2, BRY5, and BRY9, were inoculated into acid-producing medium (20 g / L glucose, 5 g / L peptone, and 5 g / L yeast extract) using glucose as the carbon source. Alkali tolerance was determined by setting pH gradients of 8, 9, 10, and 11. Salt tolerance was determined by adding NaCl concentrations of 10, 25, 40, and 55 g / L to the acid-producing medium. To control for single variables, salt-alkali stress experiments were conducted separately with three replicates. The acid-producing bacteria were inoculated into media with different salt-alkali gradients and cultured at 150 rpm and 28 °C until the pH of the medium stabilized. The pH of the medium was measured every two days using a pH meter. After culture, the medium was centrifuged to remove the supernatant, and the bacterial precipitate was dried at 80 °C to constant weight before determining the dry weight of the acid-producing strains.

[0036] like Figure 1 and Figure 2 The results showed that under different initial pH and NaCl concentrations, the pH of the culture medium decreased with culture time. In the alkali tolerance test, the pH decreased rapidly within the first three days of culture, finally stabilizing in the range of 4.15-5.97 on day 17. Strains BRY1, BRY2, and BRY5 showed slight fluctuations from day 3 to day 13 before gradually stabilizing. The final stable pH values ​​of the four strains, arranged in descending order, were basically consistent with the initial pH order of each system. In the salt tolerance test, the pH also decreased rapidly in the first three days, stabilizing between 4.10 and 5.46 on day 15. At an initial NaCl concentration of 55 g / L, all four strains exhibited the highest stable pH value, and no significant differences were observed among the other salt concentration gradients. Figure 3 In different initial pH media, the dry weight of the four acid-producing bacteria reached its maximum value at pH 9 or 10. However, in media with different NaCl concentrations, the dry weight of the bacteria generally decreased with increasing salt concentration.

[0037] (2) Determination of the organic acid secretion performance of acid-producing bacteria High-performance liquid chromatography (HPLC) was used to determine the organic acids secreted by acid-producing bacteria in an acid-producing medium with glucose as the substrate under optimal saline-alkaline conditions. After centrifugation to remove bacterial cells, the test bacterial solution was filtered through a 0.22-micron filter and then mixed with a standard mixed organic acid solution (containing oxalic acid, tartaric acid, lactic acid, malic acid, acetic acid, citric acid, fumaric acid, and succinic acid) for detection. The chromatographic conditions were as follows: Ultimate AQ-C18 column, 5 μm (250 mm × 4.6 mm, 5 μm); mobile phase: 50 mM K₂HPO₄ solution (pH = 2.5): methanol = 97.5: 2.5; injection volume: 10 μL; column temperature: 30 ℃; detection wavelength: 210 nm; flow rate: 0.7 mL / min.

[0038] like Figure 4 When four acid-producing bacteria were cultured using glucose as the carbon source, strain BRY2 produced the highest organic acid yield, reaching 1071.13 mg / L, followed by strain BRY1 at 1032.82 mg / L. The organic acid compositions of these two strains were similar, with malic acid as the main product, at concentrations of 823.88 mg / L and 792.09 mg / L, respectively. They also secreted small amounts of citric acid, tartaric acid, acetic acid, and trace amounts of oxalic acid. Strain BRY5 produced a total organic acid yield of 758.96 mg / L, with acetic acid (558.26 mg / L) and citric acid (185.92 mg / L) being the main components. Strain BRY9 produced the lowest total organic acid yield at 484.75 mg / L, with citric acid being the dominant component (448.77 mg / L). The control group (CK, acid-producing medium sterilized by high temperature and autoclave) had an organic acid content of only 190.96 mg / L, less than half the yield of strain BRY9.

[0039] Example 3: Effect of the strain on the alkalinity of red mud The experiment was conducted in plastic containers, with 200 g of red mud placed in each container. The experiment included six treatments, each repeated three times: (1) blank control red mud; (2) inoculated with acid-producing bacteria BRY1; (3) inoculated with acid-producing bacteria BRY2; (4) inoculated with acid-producing bacteria BRY5; (5) inoculated with acid-producing bacteria BRY9; and (6) mixed inoculated with BRY1, BRY2, BRY5, and BRY9. The specific experimental steps were as follows: the four acid-producing bacteria BRY1, BRY2, BRY5, and BRY9 were cultured in the logarithmic growth phase (OD). 600=1.0), inoculated into red mud at a rate of 100 ml per inoculation, once a month, with mixed inoculation: 25 mL for each strain. After inoculation, stir regularly to ensure uniform and thorough reaction, and cure for 4 months, during which time deionized water is added by weighing to maintain a constant moisture content. A blank control red mud sample was prepared by adding the same amount of deionized water throughout the process. After curing, red mud samples were collected and air-dried, and the pH of the red mud in deionized water (solid-liquid ratio 1:5) was measured using a pH meter.

[0040] like Figure 5 Significant differences in pH values ​​were observed in red mud after treatment with different acid-producing bacteria. The BRY1 and mixed strain treatments showed the best alkalinity-reducing effect, lowering the red mud pH to 7.57, a significant decrease of 1.64 units compared to the blank control group (pH=9.21). Although the pH of the blank control group decreased slightly due to CO2 neutralization, the alkalinity remained at a relatively high level (pH=9.21), confirming the crucial role of acid-producing bacteria. Among single strains, BRY2 showed the second best alkalinity-reducing effect (pH=7.77); BRY5 and BRY9 had weaker alkalinity-reducing abilities, resulting in red mud pH values ​​of 8.86 and 8.52 respectively after treatment, with little difference from the blank control group, indicating limited effects on red mud alkalinity regulation.

[0041] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A BRY1 strain with red mud dealkalization function, characterized in that, The taxonomic name of the BRY1 strain is Acinetobacter baumannii (ATCC 19606®) Acinetobacter baumannii , and its preservation number is GDMCC No: 66371, the preservation date is May 22, 2025, and the preservation unit is Guangdong Microbial Culture Collection Center.

2. A microbial mixture, characterized in that, It contains at least the BRY1 strain as described in claim 1.

3. The microbial cocktail of claim 2, wherein, The microbial mixture also contains other strains with red mud dealkalization function and microorganisms symbiotic with the BRY1 strain, namely BRY2, BRY5 and BRY9 strains, wherein the BRY2 strain is Acinetobacter spp. (…). Acinetobacter soli The accession number is GDMCC No: 66372, the accession date is May 22, 2025, and the depositary institution is Guangdong Microbial Culture Collection Center; The BRY5 strain is Acinetobacter pitera (… Acinetobacter pittii The accession number is GDMCC No: 66373, the accession date is May 22, 2025, and the depositary institution is Guangdong Microbial Culture Collection Center; The BRY9 strain is *Codonopsis sacchari* (Sacchariformis). Kosakonia cowanii The accession number is GDMCC No: 66374, the accession date is May 22, 2025, and the depositary institution is Guangdong Microbial Culture Collection Center.

4. A product comprising the BRY1 strain of claim 1 or a mixture of microorganisms of any one of claims 2 to 3.

5. The product of claim 4, wherein This product is a microbial inoculant, and the active ingredients of the product are the bacterial cells and fermentation broth of the BRY1 strain described in claim 1.

6. A fermentation broth comprising the BRY1 strain of claim 1.

7. A fermentation method of the BRYl strain according to claim 1, characterized by, The BRY1 strain was inoculated into a fermentation medium for fermentation culture.

8. The application of the BRY1 strain of claim 1, the microbial mixture of any one of claims 2-3, and / or the product of claim 4 or 5 in red mud dealkalization.

9. A method of dealkalization of red mud, characterized in that, The method includes the step of contacting the red mud requiring dealkalization with the BRY1 strain of claim 1, or the microbial mixture of any one of claims 2-3, or the product of claim 4 or 5.

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