A strain of talaromyces verruculosus with red mud dealkalization function, a compound microbial agent and application thereof

Abstract

The present application provides a strain of Tuberolysidiopsis sp. with bauxite dealkalization function, a compound microbial agent and application thereof, belonging to the field of industrial solid waste biological treatment technology and environmental microorganisms; specifically, a strain of Tuberolysidiopsis sp. DYZC, which has been preserved in the China General Microbiological Culture Collection Center on June 5, 2026, with a preservation number of CGMCC No. 42726; a compound microbial agent containing the Tuberolysidiopsis sp. DYZC; and a preparation method of the compound microbial agent is provided; and the application of the Tuberolysidiopsis sp. DYZC or the compound microbial agent in bauxite dealkalization.

Description

Technical Field

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

[0002] With the increasing global demand for aluminum and the development of the alumina industry, the accumulated amount of red mud has been increasing year by year. The main type of red mud stockpiled in my country is Bayer process red mud, which produces approximately 0.3-2 tons of red mud per ton of alumina produced, currently exceeding 4 billion tons, with an annual increase of about 180 million tons. However, the comprehensive utilization rate of red mud has long been low, with most of it undergoing natural sedimentation treatment in on-site storage ponds. The main mineral components of red mud include orthosilicate, hydrated calcium silicate, hydrated garnet, calcite, nepheline, hydrated sodium silicate, and perovskite. The leachate produced during stockpiling typically has a pH value exceeding 12 and contains high concentrations of aluminum, chlorine, fluorine, sodium, nitrates, and sulfates, which can easily lead to soil salinization and water pollution. Due to its high alkalinity and salinity, red mud poses a serious threat to the environment and ecosystems. Furthermore, red mud also contains heavy metals such as arsenic, lead, zinc, copper, nickel, and chromium, which pose potential hazards to the environment and human health.

[0003] Currently, the main technologies for the harmless treatment and resource utilization of red mud include physical, chemical, and biological methods. Physical methods, primarily water washing for dealkalization, suffer from problems such as high water consumption, generation of highly alkaline wastewater, high treatment costs, and difficulty in large-scale application. Chemical methods, often employing acid neutralization and carbonation, can rapidly reduce alkalinity, but they consume large amounts of reagents, cause severe equipment corrosion, and are prone to secondary pollution. Furthermore, the treated red mud often fails to meet long-term safe utilization requirements, presenting a challenge in balancing dealkalization efficiency, economic cost, and environmental friendliness. Biological dealkalization relies on microbial metabolism to produce acid to neutralize alkaline substances. It offers advantages such as mild conditions, environmental friendliness, low cost, and no secondary pollution, making it a research hotspot in the field of red mud treatment. However, existing biological dealkalization technologies still have shortcomings such as long microbial cultivation cycles and poor remediation timeliness, making it difficult to meet the needs of engineering applications.

[0004] While there are reports on microorganisms that contribute to the dealkalization of red mud, the number of publicly disclosed microbial strains is limited, and their effects are also limited. Therefore, developing efficient dealkalization strains with strong alkali tolerance and adaptability, and constructing efficient biological dealkalization technologies, is of great significance for achieving large-scale harmless disposal and resource utilization of red mud. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a strain of *Basilus valerianus* with red mud dealkalization function, a compound microbial agent, and their applications.

[0006] The technical solution of this invention is as follows: A strain of *Varicospora var.* Talaromyces verruculosus DYZC was deposited on June 5, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 42726.

[0007] A compound microbial agent containing the aforementioned *Dysporum vulgare* DYZC.

[0008] Preferably, the compound microbial agent further includes *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae DYZA, Aspergillus tabineus ( Aspergillus tubingensis At least one of FCZB; Saccharomyces cerevisiae ( Saccharomyces cerevisiae The accession number for DYZA is CGMCC 2.9348; Aspergillus tabineus ( Aspergillus tubingensis The accession number of FCZB is CGMCC 3.40180.

[0009] Preferably, the compound microbial agent contains the above-mentioned *Gnaphalium affine* DYZC, *Saccharomyces cerevisiae* DYZA, and *Aspergillus tabineus* FCZB.

[0010] Preferably, the total number of bacteria in the compound microbial agent is 1×10⁻⁶. 7 More than 1 / mL.

[0011] Preferably, in the compound microbial agent, the ratio of the number of Saccharomyces cerevisiae DYZA, the number of Pterocaryonium vulgare DYZC, and the number of Aspergillus tabineum FCZB spores is 1:(0.5-2):(0.5-2).

[0012] The preparation method of the above-mentioned compound microbial agent includes the following steps: (1) Saccharomyces cerevisiae DYZA, Basilaria verruciformis DYZC and Aspergillus tabinei FCZB were inoculated onto their respective liquid culture media. Saccharomyces cerevisiae DYZA was cultured to the logarithmic growth phase, and Basilaria verruciformis DYZC and Aspergillus tabinei FCZB were cultured for 24-48 h to obtain seed cultures of Saccharomyces cerevisiae DYZA, Basilaria verruciformis DYZC and Aspergillus tabinei FCZB, respectively. (2) The seed cultures of Saccharomyces cerevisiae DYZA, Basilaria verruciformis DYZC and Aspergillus tabineus FCZB obtained in step (1) were inoculated into the corresponding expansion culture medium at an inoculation rate of 5-10% by volume, and the culture was expanded to obtain Saccharomyces cerevisiae DYZA, Basilaria verruciformis DYZC and Aspergillus tabineus FCZB culture. (3) Detect the number of Saccharomyces cerevisiae DYZA, Saccharomyces vulgaris DYZC, and Aspergillus tabineus FCZB obtained in step (2), and mix them according to the ratio of Saccharomyces cerevisiae DYZA, Saccharomyces vulgaris DYZC, and Aspergillus tabineus FCZB spores 1:(0.5-2):(0.5-2) to obtain a compound microbial agent.

[0013] Preferably, in step (1) or step (2), the culture medium formulation and culture conditions are as follows: The formulas for the liquid culture medium and expansion culture medium of Saccharomyces cerevisiae DYZA are as follows: 8-10g yeast extract, 18-20g peptone, 18-20g glucose, and water to a final volume of 1000mL. The formulas for liquid culture medium and expansion medium of *Gnaphalium affine* DYZC and *Aspergillus tabineus* FCZB are: 6-8 g potato extract powder, 18-20 g glucose, and water to a final volume of 1000 mL. The cultivation conditions described in step (1) are as follows: Saccharomyces cerevisiae DYZA was cultured at 25-30℃ and 150-180 rpm until the logarithmic growth phase. DYZC of *Basilaria verruciformis* was cultured at 25-28℃ and 150-180 rpm for 24-48 hours. Aspergillus tabineus FCZB was cultured at 28-30℃ and 150-180 rpm for 24-48 hours. The conditions for scaling up the culture described in step (1) are as follows: Saccharomyces cerevisiae DYZA was cultured at 25-30℃ and 150-180 rpm for 16-24 hours. DYZC of *Basilaria verruciformis* was cultured at 25-28℃ and 150-180 rpm for 36-48 hours. Aspergillus tabineus FCZB was cultured at 28-30℃ and 150-180 rpm for 36-48 hours.

[0014] Application of the above-mentioned *Dysporum salina* DYZC, the above-mentioned compound microbial agent, or the compound microbial agent prepared by the above method in red mud dealkalization.

[0015] Preferably, in the application, at least one of straw and urea is added during the red mud dealkalization process.

[0016] The beneficial effects of the present invention include at least the following: 1. This invention provides a fungus called DYZC, which maintains a strong acid-producing capacity in a system with a high amount of red mud added, and the acid-producing performance of DYZC is superior to that of Saccharomyces cerevisiae DYZA and Aspergillus tabineus FCZB.

[0017] 2. The present invention also provides a compound microbial agent. The present invention found that by mixing three strains of Saccharomyces cerevisiae DYZA, Bassilago farfara DYZC and Aspergillus tabineus FCZB, the three strains not only do not produce antagonistic reactions, but also have a significant effect on reducing the alkalinity of red mud. Compared with single strains, the compound microbial agent provided by the present invention has stronger system pH stability when used for red mud dealkalization. Attached Figure Description

[0018] Figure 1 This is a microscopic image of the Gram staining results of strain DYZA.

[0019] Figure 2 This is a microscopic image of the Gram staining results of strain DYZC.

[0020] Figure 3 This is a microscopic image of the Gram staining results of strain FCZB.

[0021] Figure 4 This is a graph showing the pH changes in the culture medium for strain DYZA under optimal growth conditions.

[0022] Figure 5 This is a graph showing the pH changes in the culture medium for strain DYZC under optimal growth conditions.

[0023] Figure 6 This is a graph showing the pH changes in the culture medium for strain FCZB under optimal growth conditions.

[0024] Figure 7 The pH changes in the culture medium inoculated with strain DYZA under different red mud addition conditions are shown in the figure.

[0025] Figure 8 The pH changes in the culture medium inoculated with strain DYZC under different red mud addition conditions are shown in the figure.

[0026] Figure 9 The pH changes in the culture medium inoculated with strain FCZB under different red mud addition conditions are shown in the figure.

[0027] Figure 10 The pH changes of the culture medium inoculated with the mixed bacterial agent under different red mud addition conditions are shown in the figure.

[0028] Figure 11 The pH change of the culture medium under long-term remediation with mixed bacterial agents under the condition of 40% red mud addition is shown in the figure.

[0029] Figure 12 The pH changes in the culture medium inoculated with strain DYZC and its related species under conditions of 40% red mud addition. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0031] Unless otherwise specified, all contents in the following embodiments are based on the prior art; the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0032] 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.

[0033] Example 1 Screening and biochemical identification of acid-producing bacteria 1. Culture medium formulation 1.1 Potato glucose aqueous medium: 6g potato extract powder, 20g glucose, distilled water to a final volume of 1000mL, dispensed, and autoclaved at 121℃ for 15 minutes. Add 20g agar to this medium, keeping everything else unchanged, to obtain potato glucose agar medium (PDA).

[0034] 1.2 YPD medium: 10g yeast extract, 20g glucose, 20g peptone, 1000mL distilled water, dispensed, sterilized at 115℃ for 30min, ready for use. Add 18.0g agar to this medium, keeping everything else unchanged, to obtain YPD solid medium.

[0035] 1.3 Bromothymol Blue Identification Medium: 6g potato extract powder, 20g glucose, 20g agar, 5g bromothymol blue, distilled water to a final volume of 1000mL, dispensed, and autoclaved at 121℃ for 15 minutes.

[0036] 1.4 Red mud addition 40% culture medium: 6g potato extract powder, 20g glucose, 400g red mud, distilled water to a final volume of 1000mL, dispensed, and autoclaved at 121℃ for 15 minutes. Changing the amount of red mud added to this culture medium to 100g, 200g, and 300g, while keeping other parameters unchanged, results in red mud addition 10%, 20%, and 30% culture media, respectively.

[0037] 2. Isolation of native bacteria in red mud Red mud samples were collected from typical red mud dumps in China, sealed in sterile bags, and transported to the laboratory under light-proof and low-temperature conditions. Red mud samples from different sampling points were collected and homogenized. 5g of each sample was added to 50mL of sterile physiological saline (0.90% NaCl), and incubated at 28℃ and 120rpm with shaking for 30min. The mixture was then allowed to stand, and the supernatant was used as the primary bacterial suspension. 1mL of the primary bacterial suspension was inoculated onto potato dextrose agar and incubated at 30℃ and 150rpm for 18–24h. The enriched bacterial suspension was then serially diluted (10-10). -4 ~10 -7 The diluted solution was spread onto PDA solid medium plates using the "plate spread method," and allowed to stand for 20 minutes to allow the plates to absorb the bacterial solution. The plates were then incubated at 30°C for 48–72 hours. Single colonies with significantly different morphologies were selected and inoculated onto PDA solid medium plates using the "streak plating method" for isolation and purification three times. The purified bacterial solution was then mixed with an equal volume of 50% sterile glycerol and stored at -80°C.

[0038] 3. Screening of acid-producing strains The native red mud bacteria obtained from the initial screening were inoculated into bromothymol blue identification medium and incubated at 30°C inverted mode for 48-72 hours. If the green of the medium lightens or changes from green to yellow, it indicates that the strain has acid-producing function.

[0039] Physiological and biochemical identification indicators of 4 strains After inoculating the obtained strains onto culture media and culturing for 48–72 h, the colony growth and morphology were observed and recorded. Cell morphology was observed and recorded under a microscope, and physiological and biochemical tests were performed on each strain. The main tests conducted included methyl red test, VP test, indole reaction, urease test, and glucose and lactose fermentation tests. Specific experimental results are shown in Table 1. Gram staining results for each strain are shown in [Table 1]. Figure 1 , Figure 2 , Figure 3 .

[0040] On YPD solid medium, strain DYZA is milky white, opaque, round or nearly round, with raised, moist and viscous colonies that are creamy, with complete and neat edges, without wrinkles or fluffy aerial hyphae.

[0041] On PDA solid medium, strain DYZC appears as a fluffy to powdery substance, relatively dense, with a slightly raised center. As the culture time increases, the sporulation area gradually turns grayish-green to olive-green, with a darker center and lighter-colored hyphae on the periphery. The reverse side of the colony is yellowish-brown to brownish-yellow, without diffuse pigments.

[0042] On PDA solid medium, strain FCZB appears as velvety to flocculent, with well-developed and clearly visible aerial hyphae. As the culture time increases, the sporulation area gradually turns dark brown to blackish brown, with a slightly raised center, neat edges, and a white hyphal ring; the reverse side of the colony is pale yellow to yellowish brown, without obvious diffuse pigmentation.

[0043] Table 1 Physiological and biochemical characteristics of strain DYZA, strain DYZC, and strain FCZB

[0044] Positive: +; Negative: -.

[0045] 5. Molecular biological identification Strains DYZA, DYZC, and FCZB were inoculated into potato dextrose agar and cultured overnight at 30°C and 150 rpm on a shaker. The cells were then collected by centrifugation at 12,000 rpm. Genomic DNA was extracted from each strain using the Chrysalis Fungal Genomic DNA Extraction Kit. PCR amplification was performed using primers ITS1 and ITS4.

[0046] The primer sequences are as follows: ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' SEQ ID NO.1; ITS4: 5'-TCCTCCGCTTATTGATATGC-3' SEQ ID NO. 2.

[0047] After purification, the PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with sequences in the NCBI database for homology analysis. The results showed that the sequence of the isolated and purified strain DYZA was similar to that of *Saccharomyces cerevisiae* with accession number NC_001144. Saccharomyces cerevisiae The strain DYZC shares 99% homology with *T. verrucous*, which is the closest in evolutionary distance and also shows high homology with other species of the same species. The sequence of strain DYZC is similar to that of *T. verrucous* (accession number MN077562.1). alaromyces verruculosus The homology reached 99%, the closest evolutionary distance, and it also showed high homology with other species to which this strain belongs; the sequence of strain FCZB is similar to that of Aspergillus tabineum with accession number MG745309.1. Aspergillus tubingensis It has 99% homology, is the closest in evolutionary distance, and also has high homology with other species to which this species belongs.

[0048] Ultimately, based on the physiological and biochemical characteristics of these three acid-producing bacteria combined with molecular verification, it was determined that the three bacteria involved in this invention, DYZA, DYZC, and FCZB, are *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae ), Wartsporium ( Talaromyces verruculosus ), Tabing Aspergillus ( Aspergillus tubingensis).

[0049] The preservation information for the three strains is as follows: The DYZC strain is *Vallisneria* (…). Talaromyces verruculosus It was deposited on June 5, 2026, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 42726.

[0050] The DYZA strain is a brewer's yeast ( Saccharomyces cerevisiae The accession number of the China General Microbiological Culture Collection Center is CGMCC 2.9348, and it can be obtained by the public from the collection center.

[0051] FCZB strain is Aspergillus tabineus ( Aspergillus tubingensis The accession number of the China General Microbiological Culture Collection Center is CGMCC 3.40180, and it can be obtained by the public from the collection center.

[0052] Example 2 Performance testing of acid-producing bacteria Saccharomyces cerevisiae DYZA was inoculated into YPD liquid medium and cultured at 28℃ and 150 rpm until the logarithmic growth phase to obtain Saccharomyces cerevisiae DYZA seed culture. Bassilago farfara DYZC was inoculated into PDA liquid medium and cultured at 28℃ and 150 rpm for 36 h to obtain Bassilago farfara DYZC seed culture. Aspergillus tabineum FCZB was inoculated into PDA liquid medium and cultured at 28℃ and 150 rpm for 36 h to obtain Aspergillus tabineum FCZB seed culture. The Saccharomyces cerevisiae DYZA seed culture, Bassilago farfara DYZC seed culture, and Aspergillus tabineum FCZB seed culture were each transferred at a 1% (v / v) inoculation rate to Erlenmeyer flasks containing 100 ml of their respective liquid medium, establishing aerobic and anoxic culture groups. The aerobic culture group bottles were sealed with sterile, breathable sealing film and incubated at 28°C and 150 rpm for 48 hours on a shaker; the anoxic culture group bottles were sealed with sterile rubber stoppers and incubated at 28°C and 150 rpm for 48 hours on a shaker. Samples were taken every 12 hours to test the pH of the culture medium and record the acid production of individual bacteria. The results are shown below. Figure 4 , Figure 5 , Figure 6 .

[0053] Depend on Figure 4 , Figure 5 , Figure 6As can be seen, the pH of the culture medium decreases with the growth and metabolism of the bacteria. Saccharomyces cerevisiae DYZA, when cultured under anaerobic conditions for 24 hours, can lower the pH of the culture medium to a minimum of 3.8; Bassilago farfara DYZC, when cultured under anaerobic conditions for 48 hours, can lower the pH of the culture medium to a minimum of 4.1; and Aspergillus tabineus FCZB, when cultured under anaerobic conditions for 48 hours, can lower the pH of the culture medium to a minimum of 4.6.

[0054] Example 3 Salt and alkali tolerance test of acid-producing bacteria 1. Preparation of compound microbial inoculum ZH 1.1 Activation of microbial strains Saccharomyces cerevisiae DYZA was inoculated into YPD liquid medium and cultured at 28℃ and 150 rpm until the logarithmic growth phase to obtain Saccharomyces cerevisiae DYZA seed culture; Bassilago farfara DYZC was inoculated into PDA liquid medium and cultured at 28℃ and 150 rpm for 36 h to obtain Bassilago farfara DYZC seed culture; Aspergillus tabineum FCZB was inoculated into PDA liquid medium and cultured at 28℃ and 150 rpm for 36 h to obtain Aspergillus tabineum FCZB seed culture.

[0055] 1.2 Large-scale culture of bacterial strains The *Saccharomyces cerevisiae* DYZA seed culture obtained in step 1.1 was inoculated into YPD liquid medium at a volume ratio of 10%, and cultured at 28°C and 150 rpm for 24 h to obtain *Saccharomyces cerevisiae* DYZA culture. The *Gnaphalium affine* DYZC seed culture obtained in step 1.1 was inoculated into PDA liquid medium at a volume ratio of 10%, and cultured at 28°C and 150 rpm for 48 h to obtain *Gnaphalium affine* DYZC culture. The *Aspergillus tabineus* FCZB seed culture obtained in step 1.1 was inoculated into PDA liquid medium at a volume ratio of 10%, and cultured at 28°C and 150 rpm for 48 h to obtain *Aspergillus tabineus* FCZB culture.

[0056] 1.3 Microbial strain compounding Take 1.5 ml of the DYZA strain bacterial suspension from step 1.2 and measure the OD value of the bacterial suspension using a UV-Vis spectrophotometer at a wavelength of 600 nm, then adjust the OD value to approximately 0.8. Take the DYCZ and FCZB strain bacterial suspensions from step 1.2 and measure the spore concentration in the bacterial suspensions using a hemocytometer. Adjust the bacterial counts of the *Saccharomyces cerevisiae* DYZA, *Gnaphalium affine* DYZC, and *Aspergillus tabine* FCZB suspensions to be equal using fresh culture medium. Mix them according to a 1:1:1 ratio to obtain the composite microbial suspension (i.e., mixed inoculum, ZH). The total bacterial count of the composite microbial inoculum is 1 × 10⁻⁶. 7per mL.

[0057] Salt and alkali tolerance test of 2 bacterial strains and mixed bacterial solutions 2.1 Take the bacterial suspensions of the three acid-producing strains (Saccharomyces cerevisiae DYZA, Basilaria verruciformis DYZC, Aspergillus tabineus FCZB) and the mixed bacterial suspension ZH, respectively, and inoculate them into the culture medium with a mass fraction of 10% red mud at a volume fraction of 5%. Conduct salt and alkali tolerance tests at 28℃ and 150 rpm for 48 h to obtain the bacterial suspensions of each system. During the period, the pH value of the system is measured every 12 h, and the pH change data is recorded.

[0058] 2.2 Take samples of the bacterial solutions from each system in step 2.1 and inoculate them into a culture medium containing 20% ​​red mud at a volume fraction of 5%. Continue to conduct salt and alkali tolerance tests at 28℃ and 150 rpm for 48 hours to obtain the bacterial solutions of each system. During this period, take samples every 12 hours to detect the pH value of the system and record the pH change data.

[0059] 2.3 Take samples of the bacterial solutions from each system in step 2.2 and inoculate them into a culture medium containing 30% red mud at a volume fraction of 5%. Conduct a continuous salt and alkali tolerance test at 28℃ and 150 rpm for 48 hours to obtain the bacterial solutions of each system. During this period, take samples every 12 hours to detect the pH value of the system and record the pH change data.

[0060] 2.4 Samples of the bacterial solutions from each system in step 2.3 were taken and inoculated into a culture medium containing 40% red mud at a volume fraction of 5% to initiate a high-alkaline environment experiment. After initial incubation at 28℃ and 150rpm for 48h, 10% red mud and 5% fresh culture medium were added to the system every 48h to maintain a high-alkaline environment. The process lasted for 3 cycles, and the pH value of each system was continuously monitored throughout to determine the pH regulation ability of the bacterial strains and mixed bacterial solutions.

[0061] See results Figure 7 , Figure 8 , Figure 9 Tables 2, 3, and 4 show that the three acid-producing strains (Saccharomyces cerevisiae DYZA, Aspergillus verrucosum DYZC, and Aspergillus tabineus FCZB) exhibited strong salt and alkali tolerance during gradient salt and alkali tolerance culture. The culture groups with 40% red mud addition for each strain could maintain the pH of the system at 6.4-7.1 for 48 hours. The acid-producing performance of Aspergillus verrucosum DYZC was better than that of Saccharomyces cerevisiae DYZA and Aspergillus tabineus FCZB. In all groups, it showed a rapid response to reduce the pH of the system within 12 hours. Moreover, the lowest pH value of the Aspergillus verrucosum DYZC system was lower than that of the Saccharomyces cerevisiae DYZA and Aspergillus tabineus FCZB systems.

[0062] The results of salt and alkali tolerance culture of the compound bacterial solution on culture media with different amounts of red mud are shown in the figure. Figure 10 As shown in Table 5, the results indicated that the composite bacterial solution exhibited strong salt and alkali tolerance in all culture groups, rapidly responding to acid production and lowering the system pH within 12 hours, maintaining it between 6.4 and 6.9. Combining the salt and alkali tolerance test results of single strains and composite bacterial solutions, within 48 hours of the salt and alkali tolerance test, compared with Aspergillus tabinei FCZB, the composite bacterial solution lowered the system pH to between 5.6 and 6.5 within 12 hours. The acid production efficiency of each group of Aspergillus tabinei FCZB was lower than that of the composite bacterial solution, and it could lower the system pH to a minimum of 6.1-7.2 within 24 hours. Compared with Saccharomyces cerevisiae DYZA and Bassilago farfara DYZC, it also had the ability to rapidly respond to acid production and lower the system pH within 12 hours. However, comparing the data detected after 48 hours, the compound bacterial solution showed strong system pH stability. After the system pH reached its lowest value, each group of single bacteria showed a slow upward trend in system pH. When the system pH of each single bacteria group with 40% red mud was detected after 10 days of fermentation, the pH of the Saccharomyces cerevisiae DYZA system rose to 8.36, the pH of the Basidiomyces var. verrucosum DYZC system rose to 7.642, and the pH of the Aspergillus tabineus FCZB system was 7.885. Meanwhile, the pH of the compound bacterial solution system was 6.64, indicating a dynamic equilibrium period.

[0063] Table 2. pH changes in culture medium inoculated with Saccharomyces cerevisiae DYZA under different red mud addition conditions.

[0064] Table 3. pH changes in culture medium inoculated with *Dysporum verrucosum* DYZC under different red mud addition levels.

[0065] Table 4. pH changes in culture medium inoculated with Aspergillus tabineus FCZB under different red mud addition conditions.

[0066] Table 5. pH changes in culture medium inoculated with compound microbial agent (ZH) under different red mud addition conditions.

[0067] Example 4 Long-term monitoring of the effect of mixed bacterial agents on regulating the alkalinity of red mud The mixed bacterial culture ZH prepared in step 1.3 of Example 3 was inoculated at a volume fraction of 5% into a 1000ml Erlenmeyer flask containing 800ml of PDA medium with 40% red mud. The pH of the system was continuously monitored, and the system was incubated under anaerobic conditions until the pH reached dynamic equilibrium. The results are shown in […]. Figure 11In liquid culture medium with 40% red mud addition, the pH decreased from 10.0-10.5 to 6.4-6.6 in 2-4 days, and the pH of the system maintained a dynamic equilibrium between 6.8-7.2 in 5-11 days.

[0068] Example 5 The effect of mixed microbial agents on the remediation of alkalinity in red mud The mixed bacterial solution ZH prepared in step 1.3 of Example 3 was mixed with red mud at a weight ratio of 1:20 to form a pile. 12% sterilized crushed wheat straw and 0.42% urea were added to the total system to maintain a moisture content of 20-50 wt%. After 30 days of remediation, the pH of the pile system decreased from 10.4 to a stable level of 7.0-8.0.

[0069] Example 6 Salt and alkali resistance test of DYZC-related bacteria of *Varicospora virosa* 1. Preparation of bacterial culture 1.1 Activation of microbial strains *Varicospora virosa* DYZC and its related strains *Varicospora virosa* DYZC-2 and DYZC-3 (laboratory-preserved strains obtained from red mud) were inoculated into PDA liquid medium and cultured at 28°C and 150 rpm for 36 h to obtain *Varicospora virosa* DYZC seed culture, *Varicospora virosa* DYZC-2 seed culture, and *Varicospora virosa* DYZC-3 seed culture.

[0070] 1.2 Large-scale culture of bacterial strains The seed cultures of *Spirulina verrucosum* DYZC, DYZC-2, and DYZC-3 obtained in step 1.1 were inoculated into PDA liquid medium at a volume ratio of 10%, and cultured at 28°C and 150 rpm for 48 hours to obtain *Spirulina verrucosum* DYZC, DYZC-2, and DYZC-3 bacterial cultures.

[0071] 2. Salt and alkali resistance test Three strains of *Spirosporium verruciformis* (DYZC, DYZC-2, and DYZC-3) were inoculated into PDA liquid culture medium containing 40% red mud at a volume fraction of 5%. A salt and alkali tolerance test was conducted at 28℃ and 150 rpm for 48 hours. The pH value of the system was measured every 12 hours, and pH changes were recorded. The results are shown below. Figure 12 .

[0072] like Figure 12The results showed that, in the salt and alkali tolerance test, *Spirometra virosa* DYZC maintained a strong acid-producing ability in the system with high red mud addition, reducing the system pH to 6.3 within 12 hours and stabilizing it between 6.3 and 6.9; *Spirometra virosa* DYZC-3's acid-producing ability was inhibited in the system with high red mud addition, and the system pH decreased slightly, fluctuating dynamically between 9.6 and 9.7; *Spirometra virosa* DYZC-2 almost lost its acid-producing ability in the system with high red mud addition, and the system pH showed an upward trend with the continuous release of alkaline substances from the red mud.

[0073] This invention provides a *Basilaria verruciformis* DYZC strain that maintains strong acid-producing ability even in systems with high red mud addition. This invention also provides a composite microbial agent. Through mixing *Saccharomyces cerevisiae* DYZA, *Basilaria verruciformis* DYZC, and *Aspergillus tabineus* FCZB strains, this invention found that the three strains not only do not exhibit antagonistic reactions but also significantly reduce the alkalinity of red mud. Compared to single strains, the composite microbial agent provided by this invention exhibits stronger pH stability when used for red mud dealkalization.

Claims

1. A strain of *Spirosporium verruciformis* ( Talaromyces verruculosus DYZC was deposited on June 5, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 42726.

2. A compound microbial agent, characterized in that, Contains *DYZC*, the form of *Basilaria verruciformis* as described in claim 1.

3. The compound microbial agent as described in claim 2, characterized in that, Also includes Saccharomyces cerevisiae ( Saccharomyces cerevisiae DYZA, Aspergillus tabineus ( Aspergillus tubingensis At least one of FCZB; Saccharomyces cerevisiae ( Saccharomyces cerevisiae The accession number for DYZA is CGMCC 2.9348; Aspergillus tabineus ( Aspergillus tubingensis The accession number of FCZB is CGMCC 3.40180.

4. The compound microbial agent as described in claim 3, characterized in that, It contains *Gnaphalium affine* DYZC, *Saccharomyces cerevisiae* DYZA, and *Aspergillus tabineus* FCZB.

5. The compound microbial agent as described in claim 3, characterized in that, The total number of bacteria in the compound microbial agent is 1×10⁻⁶. 7 More than 1 / mL.

6. The compound microbial agent as described in claim 4, characterized in that, The ratio of the number of Saccharomyces cerevisiae DYZA spores, the number of Sporobacter var. verrucosa DYZC spores, and the number of Aspergillus tabineus FCZB spores was 1:(0.5-2):(0.5-2).

7. The method for preparing the composite microbial agent according to any one of claims 2-6, characterized in that, Includes the following steps: (1) Saccharomyces cerevisiae DYZA, Basilaria verruciformis DYZC and Aspergillus tabinei FCZB were inoculated onto their respective liquid culture media. Saccharomyces cerevisiae DYZA was cultured to the logarithmic growth phase, and Basilaria verruciformis DYZC and Aspergillus tabinei FCZB were cultured for 24-48 h to obtain seed cultures of Saccharomyces cerevisiae DYZA, Basilaria verruciformis DYZC and Aspergillus tabinei FCZB, respectively. (2) The seed cultures of Saccharomyces cerevisiae DYZA, Basilaria verruciformis DYZC and Aspergillus tabineus FCZB obtained in step (1) were inoculated into the corresponding expansion culture medium at an inoculation rate of 5-10% by volume, and the culture was expanded to obtain Saccharomyces cerevisiae DYZA, Basilaria verruciformis DYZC and Aspergillus tabineus FCZB culture. (3) Detect the number of Saccharomyces cerevisiae DYZA, Saccharomyces vulgaris DYZC, and Aspergillus tabineus FCZB obtained in step (2), and mix them according to the ratio of Saccharomyces cerevisiae DYZA, Saccharomyces vulgaris DYZC, and Aspergillus tabineus FCZB spores 1:(0.5-2):(0.5-2) to obtain a compound microbial agent.

8. The preparation method according to claim 7, characterized in that, In step (1) or step (2), the culture medium formulation and culture conditions are as follows: The formulas for the liquid culture medium and expansion culture medium of Saccharomyces cerevisiae DYZA are as follows: 8-10g yeast extract, 18-20g peptone, 18-20g glucose, and water to a final volume of 1000mL. The formulas for liquid culture medium and expansion medium of *Gnaphalium affine* DYZC and *Aspergillus tabineus* FCZB are: 6-8 g potato extract powder, 18-20 g glucose, and water to a final volume of 1000 mL. The cultivation conditions described in step (1) are as follows: Saccharomyces cerevisiae DYZA was cultured at 25-30℃ and 150-180 rpm until the logarithmic growth phase. DYZC of *Basilaria verruciformis* was cultured at 25-28℃ and 150-180 rpm for 24-48 hours. Aspergillus tabineus FCZB was cultured at 28-30℃ and 150-180 rpm for 24-48 hours. The conditions for scaling up the culture as described in step (2) are as follows: Saccharomyces cerevisiae DYZA was cultured at 25-30℃ and 150-180 rpm for 16-24 hours. DYZC of *Basilaria verruciformis* was cultured at 25-28℃ and 150-180 rpm for 36-48 hours. Aspergillus tabineus FCZB was cultured at 28-30℃ and 150-180 rpm for 36-48 hours.

9. The application of the *Dysporum salina* DYZC of claim 1, the composite microbial agent of any one of claims 2-6, or the composite microbial agent prepared by the method of any one of claims 7-8 in red mud dealkalization.

10. The application as described in claim 9, characterized in that, At least one of straw and urea is added during the red mud dealkalization process.

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