Enterobacter chengduensis and application thereof

Abstract

The present application relates to the technical field of microorganism and environmental pollution, and discloses an enterobacter chenduensis (Enterobacter chenduensis) Enterobacter chengduensis ) and application thereof. The present application provides an enterobacter chenduensis SR-12, which can take the sulfate in soil as an electron acceptor and reduce it to hydrogen sulfide (H2S), and S 2‑ generated in the reduction process can combine with cadmium (Cd 2+ ) in soil to generate extremely insoluble cadmium sulfide (CdS) precipitate, thereby effectively reducing the cadmium content in soil and further reducing the accumulation of cadmium in crops, which has important significance for realizing safe utilization and sustainable repair of cadmium-polluted farmland, especially cadmium-polluted paddy fields.

Description

Technical Field

[0001] This invention relates to the field of microorganisms and environmental pollution, specifically to a strain of Enterobacter chengduensis and its applications. Background Technology

[0002] Cadmium (Cd) is a highly toxic heavy metal that can continuously enter farmland soil through mining, industrial waste discharge, and improper application of agricultural inputs. Cadmium has strong migration capabilities and high bioavailability in soil. Currently, cadmium-contaminated soil remediation methods are mainly divided into two categories: physicochemical remediation and microbial bioremediation. Physical remediation technologies such as soil replacement and electrokinetic remediation involve large engineering projects and high costs, and severely damage the original soil aggregate structure and topsoil. Chemical passivation is simple to operate and widely used, but conventional passivation materials have weak binding force to cadmium ions, and passivation products are easily affected by fluctuations in the field environment such as soil pH, redox potential, and alternating wet and dry conditions, leading to easy reactivation of heavy metals and insufficient long-term remediation stability. In contrast, microbial in-situ remediation has advantages such as minimal soil disturbance, readily available raw materials, low remediation costs, and no secondary pollution, and has become a research hotspot in the field of heavy metal remediation in farmland in recent years.

[0003] The core pathways of microbial passivation and fixation of soil cadmium can be divided into two categories: First, relying on the adsorption of free Cd² by active functional groups such as hydroxyl, carboxyl, and amino groups on the cell wall and extracellular polymeric substances (EPS). + Secondly, by regulating the rhizosphere microenvironment through microbial metabolism, cadmium ions are induced to precipitate as insoluble phosphates, carbonates, and sulfides, thus reducing the bioavailability of cadmium at its source. Among these, sulfate-reducing bacteria (SRB) anaerobicly reduce sulfate to form S². - S² - with Cd² + It rapidly combines to generate cadmium sulfide (CdS) with extremely low solubility, exhibiting a significant passivation effect, making it a highly promising functional microorganism in the field of heavy metal remediation.

[0004] Existing sulfate-reducing bacterial systems are mainly divided into two categories: one is traditional strictly anaerobic SRBs such as desulfovibrio, and the other is facultative anaerobic sulfate-reducing strains of Enterobacteriaceae, such as those disclosed in prior art document CN115161229A. Enterobacter chengduensis SFRB19-1. Existing SRB remediation systems generally suffer from multiple technical shortcomings, which restrict their large-scale application in paddy fields: 1. The strain has an inherent defect in its ability to adapt to the environment. Traditional desulfurizing Vibrio bacteria are obligate strict anaerobes with extremely poor oxygen tolerance. Even trace amounts of oxygen introduced during field application and soil wetting / drying cycles cause significant bacterial inactivation, making them completely unsuitable for the microaerophilic environment of paddy fields with alternating flooding and drying. While existing reparative Enterobacteriaceae (such as Enterobacter Chengdu SFRB19-1) are facultative anaerobes and can grow in anaerobic systems for short periods, their genomes do not carry... sqr The sulfide oxidation and detoxification gene continuously produces H2S, which accumulates intracellularly, resulting in severe metabolic autotoxicity. With prolonged culture and remediation time, the bacterial activity rapidly declines, leading to poor long-term colonization in the field and a short remediation duration.

[0005] 2. There is a lack of technology for long-lasting preservation of microbial agents, and a gap in industrial-scale supporting field application technologies. Traditional desulfurizing Vibrio bacteria are highly sensitive to aerobic environments and temperature fluctuations, and have stringent requirements for storage and transportation conditions. Conventional freeze-drying and room-temperature storage processes cannot guarantee the number of viable bacteria. Existing SRB-related research is mostly limited to small-scale laboratory liquid shake-flask tests, and no cell protection coating process suitable for freeze-drying and long-term room-temperature storage has been developed. The bacterial agents have short shelf lives, and a large number of viable bacteria die off during storage and transportation. At the same time, there is a lack of standardized application processes for cadmium-contaminated red soil paddy fields in southern China, and a lack of yield-increasing verification data in small-scale fields. They can only achieve cadmium removal in the laboratory and cannot simultaneously restore the productive capacity of contaminated farmland, thus limiting their industrialization and promotion value.

[0006] 3. Lacking a self-detoxification mechanism at the molecular level, the sustainability of repair is difficult to improve. All publicly available data are currently available in the NCBI GenBank and RefSeq whole genome databases. Enterobacter No naturally occurring functional sqr homologous genes were detected in the genomes of the strains (including Enterobacter chengensis), which could not autonomously oxidize and decompose intracellular toxic hydrogen sulfide, and could not overcome the product inhibition bottleneck commonly found in SRB at the molecular level. The repair cycle and passivation stability were difficult to further improve.

[0007] In summary, existing SRB remediation strains cannot simultaneously meet the multiple requirements of long-term desulfurization and passivation, long-term storage and transportation stability, and increased yield and efficiency in the field. Summary of the Invention

[0008] This invention provides a strain of Enterobacter chengduensis and its application to solve the problems existing in the prior art, thereby achieving efficient and stable remediation of cadmium-contaminated water and soil.

[0009] Firstly, this invention provides a strain of Enterobacter chengensis (Chengdu Enterobacter). Enterobacter chengduensis ), which is either (a) or (b) below: (a) Enterobacter Chengdu Enterobacter chengduensisSR-12 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 20, 2025, with accession number GDMCC No. 66031; (b) A strain whose genome is at least 85%, 90%, 95%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to that of the strain described in (a).

[0010] In one alternative embodiment, the Chengdu Enterobacter genome carries the sqr sulfide oxidation detoxification gene.

[0011] Secondly, the present invention also provides a microbial agent, including the aforementioned Enterobacter chengiana, or its bacterial suspension, or its culture medium, or its fermentation broth, or its fermentation broth supernatant, or its inactivated bacteria, or its metabolites, or its dry powder preparation.

[0012] In one optional embodiment, the microbial agent includes freeze-dried bacterial powder, rice straw powder, cellulosic enzyme, hydrolyzed soybean meal powder, urea, and / or rice bran powder.

[0013] In one optional embodiment, the freeze-dried bacterial powder comprises the aforementioned Enterobacter chengensis, trehalose, skim milk powder, ferrous ammonium sulfate, and / or Tris-HCl buffer.

[0014] In one optional embodiment, the concentration of live bacteria in the microbial agent is 30 to 50 billion CFU / g.

[0015] In one optional embodiment, the concentration of viable bacteria in the microbial agent is 4.631 billion CFU / g.

[0016] Thirdly, the present invention also provides a method for preparing the aforementioned microbial agent, the method comprising: culturing the Chengdu Enterobacter in a culture medium.

[0017] In one alternative embodiment, the culture medium comprises a carbon source; the carbon source includes at least one of sodium lactate, glucose, ethanol, and sucrose.

[0018] In one alternative embodiment, the culture medium comprises a nitrogen source; the nitrogen source includes at least one of yeast extract, urea, potassium nitrate, and tryptone.

[0019] In one alternative embodiment, the culture medium further comprises at least one of K2HPO4, NaCl, and KCl.

[0020] In one optional embodiment, the Chengdu Enterobacter is cultured in a culture medium at a pH of 5.0 to 8.0.

[0021] Fourthly, the present invention also provides the application of the aforementioned Enterobacter Chengduis, the aforementioned microbial agent, or the microbial agent prepared by the preparation method of the aforementioned microbial agent in the remediation of cadmium-contaminated soil or the reduction of cadmium accumulation in crops.

[0022] Fifthly, the present invention also provides a method for remediating cadmium-contaminated soil, the method comprising: applying the microbial agent to the cadmium-contaminated soil to be remediated.

[0023] In one alternative implementation, every 100 m 2 Apply 30-60 kg of the microbial agent described above to the cadmium-contaminated soil to be remediated.

[0024] In one alternative implementation, every 100 m 2 54.236 kg of the microbial agent described herein was applied to the cadmium-contaminated soil to be remediated.

[0025] The technical solution of this invention has the following advantages: This invention provides a strain of Enterobacter chengiana (Chengdu Enterobacter). Enterobacter chengduensis The strain, named Enterobacter chengensis SR-12, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 20, 2025, with accession number GDMCC No. 66031.

[0026] The Chengdu Enterobacter SR-12 described in this invention has the following advantages: 1. Unique sqr sulfide detoxification gene, solving the metabolic autotoxicity defect of desulfurization Vibrio (core original highlight) Traditional desulfurizing Vibrio continuously accumulates H2S during sulfate reduction. Lacking an efficient sulfide detoxification mechanism, high concentrations of sulfides inhibit bacterial metabolism and damage cell membrane structure, leading to a rapid decline in activity and repair failure in the later stages of the strain's life cycle. Furthermore, conventional Enterobacteriaceae lack sulfur-metabolizing genes and cannot efficiently desulfurize. The SR-12 strain of this invention specifically carries the sqr functional gene in the Enterobacterial background, encoding a thioquinone oxidoreductase that continuously oxidizes and removes intracellular toxic sulfides, maintaining sulfur metabolic homeostasis. This unique molecular mechanism is absent in common Enterobacteriaceae and rare in desulfurizing Vibrio, enabling the strain to tolerate high-sulfur, highly toxic repair environments, exhibiting metabolic stability far superior to traditional desulfurizing Vibrio.

[0027] 2. Due to its unique "growth-function decoupling" metabolic characteristics, desulfurization vibrio does not possess long-term repair capabilities. Conventional desulfurizing Vibrio bacteria strictly follow the metabolic pattern of "synchronous increase and decrease in desulfurization function with cell growth." Once the strain enters its decline phase, its desulfurization function is rapidly lost, with only a short-term effect during the logarithmic phase, resulting in a short repair cycle and no sustained passivation ability in the later stages. The SR-12 strain of this invention possesses a unique decoupled kinetic characteristic of growth stagnation and functional activation: during the cell decline phase, biomass decreases, but sulfate reduction efficiency continuously increases, with maximum desulfurization efficiency occurring in the mid-to-late stages, achieving one-time addition, later-stage synergistic effect, and long-term passivation. This metabolic characteristic is a unique advantage not possessed by desulfurizing Vibrio bacteria, completely solving the technical bottleneck of traditional remediation bacteria being "effective in the early stages and ineffective in the later stages."

[0028] 3. Its environmental resistance and field colonization ability are significantly better than those of *Vibrio desulfurans*. Existing desulfurization vibrio is a typical obligate strict anaerobic microorganism, highly sensitive to environmental stresses such as oxygen, temperature fluctuations, soil salinity, and humidity levels. It can only survive in completely anaerobic environments; it is rapidly inactivated upon contact with trace amounts of oxygen, resulting in extremely low colonization success rates after field application. Its sulfate reduction remediation effect fluctuates greatly, limiting its practical application value in the field. The SR-12 strain of this invention belongs to the Enterobacteriaceae family and has fundamental physiological and metabolic differences from desulfurization vibrio. Its core advantage is stable growth and proliferation even under aerobic conditions. Even in microaerobic or intermittently aerobic alternating wet and dry environments in farmland, it maintains good growth and does not die off in large numbers due to the presence of oxygen. In summary, SR-12 exhibits outstanding comprehensive stress resistance, including tolerance to mild aerobic conditions, salinity, and environmental fluctuations. It can be stably colonized in complex farmland soils for a long period. Its overall field remediation stability and environmental adaptability are far superior to environmentally sensitive desulfurization vibrio, making it more suitable for in-situ remediation of contaminated farmland.

[0029] 4. It can prepare long-lasting preservative agents, overcoming the industry pain point that desulfurization Vibrio cannot be stored for a long time. Traditional desulfurizing Vibrio bacteria are fragile, intolerant of drying and salt, and easily inactivated, making it impossible to prepare solid bacterial agents. Their shelf life is typically less than 1-2 months, completely failing to meet the needs of industrial-scale storage, transportation, and commercialization. The SR-12 strain of this invention has a stable bacterial structure, and combined with a unique pre-coating process using a protective agent, it can stably prepare solid composite bacterial agents with a shelf life of up to 6 months at room temperature. It exhibits high viable cell retention and stable function, a commercial advantage that desulfurizing Vibrio systems cannot achieve.

[0030] 5. Compatible with agricultural and forestry solid waste slow-release systems, and even more suitable for in-situ long-term remediation of farmland. Desulfurization vibrio has stringent requirements for nutrient substrates, only adapting to small-molecule, fast-acting carbon sources (sodium lactate, sodium acetate). It suffers from rapid substrate consumption and poor remediation sustainability, making it incompatible with slow-release carbon source systems in agriculture and forestry. The SR-12 strain of this invention features a flexible metabolic pathway, efficiently utilizing slow-release short-chain organic acids produced by enzymatic hydrolysis of rice straw for continuous energy supply. Combined with a gradient nitrogen source system of soybean meal hydrolysate and urea, it can achieve long-term, steady-state, and continuous sulfate reduction passivation effects in soil, making it more suitable for in-situ long-term remediation in farmland.

[0031] 6. The fermentation process is simple, has greater capacity for mass production, and does not have strict anaerobic requirements. Desulfurization Vibrio fermentation requires extreme anaerobic conditions, has a long cultivation cycle, low cell yield, and extremely high production costs, making it difficult to scale up industrially. The SR-12 strain of this invention offers mild cultivation conditions, high fermentation yield, controllable process, and is suitable for large-scale production. It can produce highly active functional bacterial mud at low cost and in large quantities, with industrial value far exceeding that of traditional desulfurization Vibrio remediation systems.

[0032] Meanwhile, this invention innovates a long-term alternative carbon source in the field: a rice straw + cellulose complex enzyme hydrolysis system. After enzymatic hydrolysis, the straw can continuously generate short-chain organic acids such as lactic acid, acetic acid, and propionic acid, which are consistent with the substrates of sodium lactate metabolism and perfectly match the SRB sulfate reduction metabolic pathway.

[0033] In summary, compared with existing mainstream desulfurized Vibrio remediation technologies, this invention provides for the first time an Enterobacter remediation system carrying a unique sqr detoxification gene, possessing growth-function decoupling and long-lasting metabolic characteristics, high stress resistance, long-term preservation capability, and suitability for large-scale field production applications. It overcomes the common defects of desulfurized Vibrio, such as sensitivity and easy inactivation, poor preservation, short remediation cycle, and difficulty in industrialization. It combines molecular innovation, metabolic uniqueness, and industrial practicality, and has outstanding substantive features and significant progress.

[0034] Biological Preservation Information: Strain name: Enterobacter chengduensis SR-12; Strains classified as: Enterobacter chengensis Enterobacter chengduensis ; Preservation institution: Guangdong Provincial Center for Microbial Culture Collection; The depository is abbreviated as GDMCC. Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; Registration number at the Depository Center: GDMCC No. 66031; Preservation date: March 20, 2025. Attached Figure Description

[0035] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0036] Figure 1 This is a colony morphology diagram of strain SR-12 on solid purification medium; Figure 2 This is a morphological diagram of strain SR-12.

[0037] Figure 3 The phylogenetic tree of strain SR-12 was constructed based on the 16S rDNA gene using the maximum likelihood method.

[0038] Figure 4 These are the results of comparative gene analysis of the sulfate reduction metabolic pathway in strain SR-12.

[0039] Figure 5 This describes the effect of initial sulfate concentration on strain growth and sulfate removal rate.

[0040] Figure 6 This relates to the effect of carbon source on strain growth and sulfate removal rate.

[0041] Figure 7 This relates to the effect of nitrogen source on strain growth and sulfate removal rate.

[0042] Figure 8 The effect of inorganic salts on strain growth and sulfate removal rate Figure 9 This describes the effect of pH on bacterial growth and sulfate removal rate.

[0043] Figure 10 This study investigated the effects of the gaseous oxygen environment on bacterial growth and sulfate removal rate.

[0044] Figure 11 This is a time-dynamic graph showing the growth of the strain and the sulfate reduction efficiency.

[0045] Figure 12 This is a diagram showing the passivation effect of strain SR-12 on available cadmium in soil. Detailed Implementation

[0046] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0047] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0048] The culture media and reagents involved in this invention are as follows: (1) Liquid culture medium A: 0.5 g K2HPO4, 1.0 g NH4Cl, 1.0 g Na2SO4, 0.1 g CaCl2·2H2O, 2.0 g MgSO4·7H2O, 1.0 g yeast extract, 5.0 g 70% sodium lactate, add distilled water to a final volume of 1000 mL, and adjust the pH of the culture medium to 7.0~8.0. After fully dissolving each component, transfer to an Erlenmeyer flask, seal, and autoclave at 121 ℃ for 20 min. After sterilization, cool to 50 ℃ for later use. Separately prepare a 1% ferrous ammonium sulfate solution, sterilize it by microporous membrane filtration, and mix it with 5 mL of sterilized ferrous ammonium sulfate solution per 100 mL of culture medium before use.

[0049] (2) Solid purification medium: K2HPO4 0.5g, NH4Cl 1.0g, Na2SO4 1.0g, CaCl2·2H2O 0.1g, MgSO4·7H2O 2.0g, yeast extract 1.0g, 70% sodium lactate 5.0g, agar 20g / L, distilled water 1000 mL. After dissolving, pour into Erlenmeyer flasks, seal, and sterilize by steam at 121℃ for 20 min. After sterilization, cool to 50℃ for later use. Prepare a 1% ferrous ammonium sulfate solution separately, sterilize by microporous membrane filtration, and mix with 5 mL of sterilized ferrous ammonium sulfate solution per 100 mL of medium before use.

[0050] (3) Liquid culture medium B: 0.5 g K2HPO4, 1.0 g NH4Cl, 1.0 g yeast extract, 0.1 g CaCl2·6H2O, 2.0 g MgSO4·7H2O, 1.0 g Na2SO4, 0.002 g FeSO4·7H2O, 0.5 g 70% sodium lactate, and 1000 mL distilled water. After dissolving, pour into an Erlenmeyer flask, seal, and sterilize by steaming at 121℃ for 20 min. Separately prepare a 0.6% cysteine ​​hydrochloride solution, sterilize by filtration through a 0.22 μm filter membrane, and add it to the culture medium at a ratio of 10% before use.

[0051] (4) Solid culture medium C: K2HPO4 0.5 g, NH4Cl 1.0 g, yeast extract 1.0 g, CaCl2·6H2O 0.1 g, MgSO4·7H2O 2.0 g, Na2SO4 1.0 g, FeSO4·7H2O 0.002 g, 70% sodium lactate 0.5 g, agar 20 g / L, distilled water 1000 mL. After dissolving, pour into Erlenmeyer flasks, seal, and steam sterilize at 121℃ for 20 min. Separately prepare a 0.6% cysteine ​​hydrochloride solution, filter it through a 0.22 μm filter membrane for sterilization, and add it to the culture medium at a ratio of 10% before use.

[0052] (5) Basic growth medium: Dissolve 0.5 g K2HPO4, 1.0 g NH4Cl, 1.0 g yeast extract, 2.0 g Na2SO4, and 2.0 g 70% sodium lactate in 1000 mL of distilled water, pour into an Erlenmeyer flask, seal, and sterilize by steaming at 121℃ for 30 min. Adjust the pH to 7.0. Prepare a 0.6% cysteine ​​hydrochloride solution, sterilize by filtration through a 0.22 μm filter membrane, and add it to the culture medium at a ratio of 10% before use.

[0053] (6) Fermentation medium: Dissolve 0.5 g K2HPO4, 1.0 g NH4Cl, 1.0 g yeast extract, 2.0 g Na2SO4, and 2.0 g 70% sodium lactate in 1000 mL of distilled water, pour into an Erlenmeyer flask, seal, and sterilize by steaming at 121℃ for 30 min. Prepare a 0.6% cysteine ​​hydrochloride solution, sterilize by filtration through a 0.22 μm filter membrane, and add it to the medium at a ratio of 10% before use.

[0054] Example 1 Isolation of strains (1) Sample collection: A red soil paddy field with excessive heavy metals was collected in Guixi County, Yingtan City. The silt collected from 5cm underground was placed in a sterile sample bag, the air was removed and the sample was sealed and stored at 4℃ and transported to the laboratory.

[0055] (2) Enrichment culture of SRB microorganisms: Prepare 300 mL of liquid culture medium A, dispense it into 500 mL blue cap bottles, sterilize the culture medium by high pressure steam sterilization, after cooling, weigh 50 g of soil sample and add it to the liquid culture medium in the blue cap bottle, shake well, add 20 mL of sterilized liquid paraffin, cover the bottle and carry out enrichment culture.

[0056] (3) Strain strain isolation, including the following steps: ① Sample dilution and plating: Add 2 mL of the original bacterial solution to a test tube containing 8 mL of sterile water and shake thoroughly. Perform 10-fold serial dilutions (e.g., 10-fold) using sterile physiological saline or phosphate buffer (PBS). -1 Up to 10 -3① Take 200 μL of each dilution and spread it on a purified solid medium plate. Each dilution has 3 replicates. After the bacterial solution is absorbed, invert the plate into an anaerobic jar, remove the air and inject nitrogen, and anaerobic culture at 30°C. ② Pick black single colonies with obvious morphological differences and streak them onto a purified solid medium plate. Repeat 2-3 times until a pure culture is obtained. ③ Observe the morphology of the cells under a microscope and freeze them with glycerol.

[0057] Example 2 Identification and Preservation of Strains 1. Morphological identification The bacterial strain isolated in Example 1 was inoculated into liquid culture medium B, shaken at 150 rpm, and anaerobic cultured at 30°C for 48 h to obtain a bacterial suspension; the obtained bacterial suspension was diluted 10... 5 -10 6 Take 15 μL and spread it evenly on solid culture medium C using a spreader. Then, incubate it anaerobically at a constant temperature of 30℃ for 3 days and observe the morphological characteristics of individual colonies. Single colonies corresponding to strain SR-12 are milky white, opaque, and viscous with smooth edges (as shown in the image). Figure 1 (As shown). Simultaneously, strain SR-12 was stained with crystal violet and safranin counterstaining solutions, respectively. After washing and drying, the color changes were observed (e.g., ...). Figure 2 As shown in the figure, the results indicate that the bacterium is a Gram-negative bacterium.

[0058] In summary, based on the 10th and 12th editions of the "Handbook of Clinical Microbiology", it is determined that it conforms to the morphological characteristics of Enterobacter chengensis.

[0059] 2. Molecular biological identification gDNA was extracted from strain SR-12 and sequence alignment was performed for identification. The specific procedures are as follows: SR-12 strain was inoculated into liquid medium B and placed in an anaerobic incubator, where it was cultured at 30 °C and 120 r / min in the dark with shaking for 48 h. Genomic DNA (gDNA) was extracted from the bacterial precipitate according to the OMEGA bacterial DNA extraction kit instructions, and the purified DNA samples underwent quality control. Genomic DNA was quantified using a TBS-380 fluorometer (Turner BioSystems Inc., Sunnyvale, CA). High-quality DNA samples (OD) were selected. 260 / 280 =1.8~2.0, total DNA >6 μg) as template for subsequent experiments.

[0060] Using the extracted genomic DNA of strain SR-12 as a template, PCR amplification was performed using universal primers for bacterial 16S rDNA. The primer sequences are as follows: Upstream primer: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.2) Downstream primer: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO.3) PCR amplification was performed using a 25 μL standard system: 12.5 μL 2×Taq PCR Master Mix, 1.0 μL upstream primer (10 μmol / L), 1.0 μL downstream primer (10 μmol / L), 1.0 μL template gDNA, and 9.5 μL sterile ultrapure water. The reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 90 s, for a total of 35 cycles; final extension at 72℃ for 10 min, and storage at 4℃.

[0061] Five μL of PCR product was subjected to 1.0% agarose gel electrophoresis. After observing a clear and single target band using a UV gel imaging system, the amplified product was sent to Shanghai Lingen Biotechnology Co., Ltd. for bidirectional sequencing. After splicing and correction, the complete 16S rDNA sequence of strain SR-12 was obtained, and the sequence information is shown in SEQ ID NO.1.

[0062] The obtained 16S rDNA sequence of the strain was entered into the National Center for Biotechnology Information (NCBI) database, and homology analysis was performed using the BLAST online alignment program against publicly available standard strain sequences in the database. The results showed that the 16S rDNA sequence of strain SR-12 shared ≥99.8% homology with *Enterobacter chengduensis* WCHECl-C4 (GenBank accession number: KY979143.1). Based on the 16S rRNA sequence results, sequence alignment was performed using the 16S-based ID program on the EzBioCloud website. Sequences from closely related species and outgroups were selected and downloaded and saved in FASTA format. Subsequently, MEGA 7.0 software was used to align the target sequence with the reference sequence, and a phylogenetic tree was constructed using the maximum likelihood (ML) method. Figure 3 ).

[0063] Based on a comprehensive analysis of colony and cell morphology, physiological and biochemical reactions, and phylogenetic tree, the strain SR-12 Chengdu Enterobacter was identified as isolated in this invention.

[0064] 3. Whole-genome sequencing and comparative genomics analysis Whole-genome sequencing was performed on the SR-12 strain of this invention, and the complete sqr coding sequence (SEQ ID NO.4) was obtained through gene annotation. Using the complete sqr coding region as the query sequence, BLASTn / BLASTp was used to search the NCBi GenBank and RefSeq microbial whole genome databases, and the search scope included all uploaded data. Enterobacter , Citrobacter , Klebsiella Genomic searches of Enterobacteriaceae strains with E-value ≤ 1e-5 and sequence similarity ≥ 60% yielded no matches of Enterobacteriaceae-derived sqr homologous functional genes (e.g., ...). Figure 4 (As shown).

[0065] The sqr gene encodes thioquinone oxidoreductase (SQR, EC 1.8.5.4), an enzyme belonging to the glutathione reductase family of membrane-bound flavoproteins that can oxidize and decompose the metabolite hydrogen sulfide, relieve toxic inhibition, and allow the strain to maintain sulfate reduction activity for a long time.

[0066] sqr is a functional gene unique to the SR-12 strain. Common Enterobacteriaceae do not contain this homologous gene and cannot achieve efficient sulfide detoxification. This gene endows the strain with excellent sulfur metabolism tolerance and metabolic sustainability, which is the core molecular basis for the long-term remediation of heavy metal pollution in soil by microbial agents.

[0067] 4. Biological Preservation strain Enterobacter chengduensis SR-12 was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on March 20, 2025, and classified as Enterobacter Chengduense (Chengdu Enterobacter). Enterobacter chengduensis The collection number is GDMCC No.66031, the collection address is 5th Floor, Building 59, No.100 Xianlie Middle Road, Guangzhou, and it has been in the collection for thirty years.

[0068] Experiment 1: Fermentation optimization of growth and sulfate degradation conditions for strain SR-12 1. Effect of initial sulfate concentration on cell growth and sulfate reduction efficiency The sulfur metabolism activity of sulfate-reducing bacteria is highly dependent on the sulfate substrate supply level of the system. Too low a substrate concentration limits the strain's energy production, while too high a concentration can easily cause osmotic stress and inhibit cell proliferation. This experiment used gradient initial sulfate concentrations to investigate the dual effects of substrate content on the growth and sulfate-reducing capacity of SR-12 under uniform anaerobic culture conditions.

[0069] (1) Activation of strain: The SR-12 strain preserved at low temperature was inoculated into sterile liquid culture medium B, placed in an anaerobic culture tank to create an anaerobic environment, and activated by shaking at 30 ℃ and 120 r / min for 48 h in the dark to prepare SR-12 seed liquid.

[0070] (2) Gradient substrate culture experiment: Using liquid culture medium B as the basic culture system, only the amount of sodium sulfate added was adjusted to set five gradient substrate concentrations of 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L, and 3.0 g / L, respectively, while keeping the other culture medium components completely consistent. The seed culture obtained in step (1) was inoculated into each group of culture medium at an inoculation rate of 1% (v / v), and transferred to an anaerobic culture tank. The culture was carried out at 30 ℃ and 120 r / min in the dark with shaking for 72 h. After the culture period, the OD of each group of bacterial culture was measured. 600 The value represents the bacterial biomass. The residual sulfate concentration in the culture medium was detected simultaneously and the sulfate removal rate was calculated. This was used to screen the optimal sodium sulfate substrate concentration that is suitable for both the proliferation and sulfate reduction functions of the SR-12 strain.

[0071] Method for calculating sulfate removal rate: Using the initial sulfate concentration of each culture medium before inoculation as a baseline, the sulfate content of the system before and after inoculation was measured, and the sulfate removal rate was calculated using the following formula: η=( C0-C t ) / C0×100% In the formula: η is the sulfate removal rate (%); C0 is the initial sulfate concentration (mg / L) before inoculation of the culture medium; C t The concentration (mg / L) of residual sulfate in the system after 72 hours of cultivation.

[0072] The results are as follows Figure 5 As shown, with the gradual increase of sodium sulfate concentration in the culture medium, the biomass and sulfate removal rate of strain SR-12 both exhibited a pattern of first increasing and then decreasing. At a sodium sulfate concentration of 2.0 g / L, the strain's OD... 600 The concentration reached 0.72, and the sulfate removal rate was 41.21%, both of which were the highest among all groups. Excessively high substrate concentrations can inhibit bacterial growth and sulfur metabolism activity. Overall, 2.0 g / L is the optimal substrate concentration for the growth and sulfate reduction of SR-12 strain under the conditions of this experiment.

[0073] 2. Effects of different carbon sources on strain growth and sulfate reduction efficiency The core function of sulfate-reducing bacteria is to convert sulfate ions (SO4) into sulfate groups. 2- ) is reduced to sulfide ions (S 2-These carbon sources then react with heavy metal ions to form sulfide precipitates. To comprehensively evaluate the synergistic effects of different carbon sources on the "growth-function" of the strain, the OD of the bacterial culture was measured after culturing with different carbon sources during the strain's culture phase. 600 (Characterizing growth) and the concentration of residual sulfate in the culture medium (calculating removal rate).

[0074] (1) Activation: The Chengdu Enterobacter SR-12 strain preserved at low temperature was inoculated into sterile liquid culture medium B and placed in an anaerobic culture tank to construct an anaerobic culture environment. The strain was continuously cultured in the dark for 48 h under constant temperature shaking conditions of 30℃ and shaking speed of 120 r / min to obtain the seed liquid of the activated strain.

[0075] (2) Carbon source screening and culture: The basic growth medium was used as the control group, and 2 g / L sucrose, ethanol, glucose, and sodium lactate were added as single external carbon sources, respectively, while the other components of each group remained unchanged. The seed culture obtained in step (1) was inoculated into each group's culture medium at an inoculation rate of 1% (v / v), and transferred to an anaerobic culture tank. The culture was carried out at 30 ℃ and 120 r / min in the dark with shaking for 72 h. After the culture period, the OD of each group of bacterial culture was measured. 600 The value represents the bacterial biomass, and the residual sulfate concentration in the culture medium is detected simultaneously and the sulfate removal rate is calculated to screen the optimal carbon source type suitable for the growth and sulfate reduction function of strain SR-12.

[0076] Method for calculating sulfate removal rate: Using the initial sulfate concentration of each culture medium before inoculation as a baseline, the sulfate content of the system before and after inoculation was measured, and the sulfate removal rate was calculated using the following formula: η=( C0-C t ) / C0×100% In the formula: η is the sulfate removal rate (%); C0 is the initial sulfate concentration (mg / L) before inoculation of the culture medium; C t The concentration (mg / L) of residual sulfate in the system after 72 hours of cultivation.

[0077] The results are as follows Figure 6 As shown, under different carbon source conditions, the growth rate of the strain showed a certain positive correlation with the sulfate removal rate, but the correspondence was not completely linear. The strain grew best when sodium lactate was used as the carbon source (OD). 600 =1.21), with a sulfate removal rate of 52.5%; the highest sulfate removal rate (55.3%) was achieved when glucose was used as the carbon source, followed by cell growth (OD). 600 =0.70); ethanol, although it resulted in the worst bacterial growth (OD). 600Although the sulfate removal rate was 51.7% (=0.10), it still exhibited low growth and high activity metabolic characteristics; sucrose showed the worst effect. Therefore, sodium lactate is suitable as the main carbon source for fermentation production.

[0078] 3. Effects of different nitrogen sources on strain growth and sulfate reduction efficiency Nitrogen sources are essential raw materials for the synthesis of proteins and nucleic acids in microorganisms and play a crucial regulatory role in the growth and metabolism of microbial strains. To comprehensively evaluate the synergistic effects of different nitrogen sources on the "growth-function" of microorganisms, OD values ​​of the bacterial culture were measured after culturing with different nitrogen sources during the strain cultivation phase. 600 (Characterizing growth) and the concentration of residual sulfate in the culture medium (calculating removal rate).

[0079] (1) Activation: The Chengdu Enterobacter SR-12 strain preserved at low temperature was inoculated into sterile liquid culture medium and placed in an anaerobic culture tank to construct an anaerobic culture environment. The strain was continuously cultured in the dark for 48 h under constant temperature shaking conditions of 30℃ and shaking speed of 120 r / min to obtain the seed liquid of the activated strain.

[0080] (2) Nitrogen source screening and cultivation: Based on the optimal carbon source, a single-factor optimization experiment of nitrogen source types was further carried out. The experiment used the same basic growth culture medium system as described above. Tryptone, urea, potassium nitrate and yeast extract were selected as single exogenous nitrogen sources, and the nitrogen source addition amount of each experimental group was 1 g / L. The other culture medium components, sterilization methods and anaerobic system construction conditions of each group were completely consistent.

[0081] The SR-12 seed culture prepared in step (1) was inoculated into the culture media of different nitrogen sources at an inoculation rate of 1% (v / v), and placed in anaerobic incubators for continuous culture at 30 ℃ and 120 r / min for 72 h. After the culture was completed, the OD of each group of bacterial culture was measured. 600 The values ​​were used to characterize the biomass and growth and proliferation level of the strain, and the residual sulfate ion concentration in the culture medium of each group was detected to calculate the sulfate removal effect under different nitrogen source conditions, and to screen the optimal nitrogen source type suitable for the growth and sulfate reduction function of strain SR-12.

[0082] Method for calculating sulfate removal rate: Using the initial sulfate concentration of each culture medium before inoculation as a baseline, the sulfate content of the system before and after inoculation was measured, and the sulfate removal rate was calculated using the following formula: η=( C0-C t ) / C0×100% In the formula: η is the sulfate removal rate (%); C0 is the initial sulfate concentration (mg / L) before inoculation of the culture medium; C tThe concentration (mg / L) of residual sulfate in the system after 72 hours of cultivation.

[0083] The results are as follows Figure 7 As shown: the strain grows best when yeast extract is used as the nitrogen source (OD). 600 =1.2), with a sulfate removal rate of 52.5%; when urea was used as the nitrogen source, the sulfate removal rate reached as high as 71.2%, the highest among the four groups, and the bacterial growth was also better (OD). 600 =1.00); tryptone, although its bacterial growth is extremely poor (OD). 600 =0.11), but the sulfate removal rate was as high as 71.2%, the highest reduction efficiency among all treatment groups, exhibiting extreme "low growth, ultra-high activity" metabolic characteristics; potassium nitrate had the worst effect (removal rate 18.5%, OD 600 =0.31). Therefore, yeast extract is suitable as the main nitrogen source for fermentation production to obtain high biomass, while tryptone (or urea), although inhibiting cell proliferation, can significantly stimulate sulfate reduction function and can be used as a functional induction nitrogen source in remediation applications.

[0084] 4. Effects of different inorganic salts on bacterial growth and sulfate reduction efficiency To investigate the differential regulatory effects of different inorganic salts on the cell proliferation and sulfate reduction remediation function of SR-12 strain, and to provide experimental basis for optimizing the inorganic salt system for fermentation propagation agents and in-situ remediation of highly saline-polluted farmland, this study added equal amounts of K2HPO4, NaCl, and KCl to a uniform basic growth medium. After cultivation, the OD of the bacterial solution was measured. 600 The effects of three types of inorganic salts on the growth performance and desulfurization remediation activity of the strain were systematically compared, using the concentration of residual sulfate in the culture medium (to characterize growth rate) and the concentration of residual sulfate in the culture medium (to calculate removal rate).

[0085] (1) Activation: The Chengdu Enterobacter SR-12 strain preserved at low temperature was inoculated into sterile liquid culture medium B and placed in an anaerobic culture tank to construct an anaerobic culture environment. The strain was continuously cultured in the dark for 48 h under constant temperature shaking conditions of 30℃ and shaking speed of 120 r / min to obtain the seed liquid of the activated strain.

[0086] (2) Inorganic salt screening and cultivation: Based on the optimal carbon and nitrogen sources, further single-factor optimization experiments on inorganic salt types were conducted. The experiment used the same basic growth medium system as described above. Dipotassium hydrogen phosphate, sodium chloride, and potassium chloride were selected as single exogenous inorganic salts, with an inorganic salt addition amount of 0.5 g / L in each experimental group. The other medium components, sterilization methods, and anaerobic system construction conditions were completely consistent in each group.

[0087] The SR-12 seed culture prepared in step (1) was inoculated into experimental culture media containing different inorganic salts at an inoculation rate of 1% (v / v). The media were then placed in anaerobic incubators and cultured continuously for 72 h at a constant temperature and shaking rate of 30 ℃ and 120 r / min. After the culture was completed, the OD values ​​of the bacterial cultures in each group were measured. 600 The values ​​were used to characterize the biomass and growth and proliferation level of the strain, and the residual sulfate ion concentration in the culture medium of each group was detected to calculate the sulfate removal effect under different inorganic salt conditions, and to screen the optimal inorganic salt type suitable for the growth and sulfate reduction function of strain SR-12.

[0088] Method for calculating sulfate removal rate: Using the initial sulfate concentration of each culture medium before inoculation as a baseline, the sulfate content of the system before and after inoculation was measured, and the sulfate removal rate was calculated using the following formula: η=( C0-C t ) / C0×100% In the formula: η is the sulfate removal rate (%); C0 is the initial sulfate concentration (mg / L) before inoculation of the culture medium; C t The concentration (mg / L) of residual sulfate in the system after 72 hours of cultivation.

[0089] The results are as follows Figure 8 As shown, the sulfate removal rates under the three inorganic salt conditions remained at a high level (K₂HPO₄ 52.5%, NaCl 58.3%, KCl 55.3%), indicating that the sulfate reduction function of this strain has strong tolerance to different inorganic salts. However, the growth rate (OD) of the strain... 600 Significant differences exist between different inorganic salts: OD in the K2HPO4 group 600 The OD value was 0.69, indicating good bacterial growth; the OD values ​​of the NaCl and KCl groups were also high. 600 The values ​​were only 0.19 and 0.14, respectively, indicating severe growth inhibition. This further validates the phenomenon of "growth-function decoupling"—in the presence of high concentrations of chlorides (NaCl, KCl), although the proliferation of the strain was inhibited, its sulfate reductase system could still maintain normal or even highly efficient catalytic activity (removal rates reached 58.3% and 55.3%, respectively). Therefore, K2HPO4, as an inorganic salt and buffer salt, is most beneficial to the fermentation production of the strain; and in the application of high-salt soil remediation, even if the colonization of the strain is limited, its passivation function can still be effectively exerted.

[0090] 5. Effects of pH on bacterial growth and sulfate reduction efficiency pH is a key environmental factor affecting the metabolic activity of microorganisms, directly influencing enzyme activity, cell membrane permeability, and the dissociation state of substances. To investigate the regulatory mechanism of initial pH on the growth and sulfate reduction function of strain SR-12, and to clarify the optimal acid-base environment for the strain's growth and functional expression, this experiment conducted a single-factor optimization experiment on initial pH.

[0091] Based on the aforementioned optimal carbon source, nitrogen source, and inorganic salt-determined basal culture medium system, the content of each component, inoculum size, culture temperature, shaking speed, culture time, and anaerobic culture conditions were kept completely consistent, with only the initial pH value of the culture medium being adjusted. The initial pH of the culture medium was adjusted to four gradients: 5.0, 6.0, 7.0, and 8.0, respectively, using dilute hydrochloric acid and sodium hydroxide solutions. After inoculation with seed culture, the culture was anaerobic and cultured in the dark at 30 ℃ and 120 r / min for 72 h. After the culture was completed, the OD of each group of bacterial cultures was simultaneously measured. 600 The value represents the biomass of the strain. The residual sulfate concentration in the culture medium of each group was detected and the sulfate removal rate was calculated. The effect of the initial pH on the growth performance and sulfate reduction function of strain SR-12 was comprehensively evaluated, and the optimal initial culture pH conditions were screened.

[0092] Method for calculating sulfate removal rate: Using the initial sulfate concentration of each culture medium before inoculation as a baseline, the sulfate content of the system before and after inoculation was measured, and the sulfate removal rate was calculated using the following formula: η=( C0-C t ) / C0×100% In the formula: η is the sulfate removal rate (%); C0 is the initial sulfate concentration (mg / L) before inoculation of the culture medium; C t The concentration (mg / L) of residual sulfate in the system after 72 hours of cultivation.

[0093] The results are as follows Figure 9 As shown, the response patterns of bacterial cell proliferation and sulfate reduction performance to pH exhibit significant differentiation. SR-12 showed optimal growth at neutral pH 7.0, with a high OD... 600 The concentration reached 1.649, with a sulfate removal rate of 57.04%, and the bacterial biomass reached its peak value for the entire group; the strain exhibited good growth at pH 6.0, and the OD... 600 The concentration was 1.356, the sulfate removal rate was 54.45%, and the overall desulfurization performance was stable; bacterial cell proliferation was significantly inhibited at pH 5.0, and the OD... 600The concentration of sulfate was only 0.616, but the sulfate removal rate was as high as 64.82%, the highest among all treatment groups, exhibiting the special metabolic characteristics of "low biomass and ultra-high unit desulfurization activity." This indicates that although a weakly acidic environment hinders the cell proliferation of the strain, it can significantly enhance the catalytic efficiency of the intracellular sulfate reductase system. Under weakly alkaline conditions of pH 8.0, the growth of the strain was severely inhibited, and the OD... 600 With a concentration of only 0.304, the sulfate removal rate was 56.70%, and both bacterial proliferation and overall desulfurization efficiency showed a significant decline.

[0094] Based on the combined scenarios of fermentation production and in-situ remediation, the conclusion is that when preparing remediation agents by large-scale fermentation, the optimal initial pH is 7.0, which can achieve high-density cell expansion. When carrying out in-situ remediation of acidic paddy fields and acidified contaminated soils with a pH close to 5.0, even if the colonization and proliferation of the strains are limited, they can still perform their core functions of sulfate reduction and heavy metal passivation exceptionally well, and the strains have a wide range of acid-base environment adaptability.

[0095] 6. Effects of gaseous oxygen environment on bacterial growth and sulfate reduction efficiency Based on the aforementioned optimal carbon source, nitrogen source, and inorganic salt-determined basal culture medium system, the content of each component, pH value, inoculum size, culture temperature, shaking speed, and culture time were kept completely consistent, with only the gaseous oxygen concentration in the culture system being changed as a single variable. Anaerobic culture tanks with evacuation and displacement capabilities were used to control the gaseous oxygen content within the tanks, setting up three gaseous environments: strictly anaerobic (oxygen content <0.1%), microaerophilic (oxygen content 5%), and atmospheric pressure aerobic. After inoculation with SR-12 seed culture, all groups were uniformly placed at 30 ℃, 120 r / min, and cultured in the dark with shaking for 72 h. After the culture period, the OD of the bacterial culture was measured. 600 Characterize the biomass of the strain; simultaneously detect the residual sulfate concentration in the culture medium and calculate the sulfate removal rate. Comprehensively evaluate the growth level and sulfate reduction capacity of SR-12 under different gas phase oxygen environments, and screen the optimal gas phase culture conditions suitable for the strain's proliferation and sulfur metabolism functions.

[0096] Method for calculating sulfate removal rate: Using the initial sulfate concentration of each culture medium before inoculation as a baseline, the sulfate content of the system before and after inoculation was measured, and the sulfate removal rate was calculated using the following formula: η=( C0-C t ) / C0×100% In the formula: η is the sulfate removal rate (%); C0 is the initial sulfate concentration (mg / L) before inoculation of the culture medium; C t The concentration (mg / L) of residual sulfate in the system after 72 hours of cultivation.

[0097] The results are as follows Figure 10 As shown, the concentration of gaseous oxygen exhibits completely different regulatory mechanisms on the cell proliferation and sulfate reduction performance of strain SR-12, with significant differences in their responses. From the perspective of cell growth: under strictly anaerobic, microaerophilic, and aerobic conditions, the OD of the strain... 600 The biomass values ​​for the three groups were 0.550, 0.562, and 0.498, respectively. The overall biomass values ​​were similar across the three groups, with optimal cell proliferation observed under microaerophilic conditions. The fully aerobic environment only slightly inhibited bacterial growth. This result demonstrates that SR-12 possesses strong oxygen tolerance, surviving and proliferating normally even in aerobic, alternating wet and dry farmland soil environments, exhibiting outstanding environmental adaptability. Regarding sulfate reduction function: oxygen has a strong inhibitory effect on the strain's desulfurization metabolism. Only the strictly anaerobic group achieved efficient sulfate reduction, with a sulfate removal rate of 52%. Once trace amounts of oxygen were present, the reduction activity decreased sharply, with sulfate removal rates in the microaerophilic and aerobic groups reaching only 2% and 7%, respectively. Combining these two sets of indicators, it can be concluded that the SR-12 strain can only perform its core remediation functions of sulfate reduction and cadmium passivation under strictly anaerobic conditions. However, the strain exhibits high oxygen tolerance, maintaining good proliferation under both microaerophilic and aerobic conditions, demonstrating its ability to adapt to the complex habitat of alternating wet and dry conditions and dynamic fluctuations in oxygen content in farmland soil, indicating its potential for practical field application.

[0098] 7. Temporal dynamics of strain growth and sulfate reduction efficiency To clarify the growth and reproduction patterns of strain SR-12 of this invention, and to determine the optimal harvest time for inoculant fermentation and the field planting window, the activated strain was inoculated into optimized medium B (using sodium lactate as the carbon source) at an inoculum rate of 5% (v / v). Under optimal growth conditions, it was anaerobically cultured at 30℃, and OD was measured every 12 hours. 600 The value was continuously monitored for 84 hours, and a growth curve was plotted.

[0099] To clarify the growth and reproduction dynamics of strain SR-12, accurately determine the optimal fermentation and harvest time of the compound microbial agent, and identify the optimal window period for field colonization and functional expression, a dynamic experiment on the growth sequence of the strain was conducted. This experiment used the optimal culture medium system obtained from previous screening, fixing the initial pH of the medium at 7.0 (the optimal pH condition for strain growth and functional expression). Sodium lactate was used as the sole optimal carbon source. The optimized complete fermentation medium formula was: K₂HPO₄ 0.5 g, NH₄Cl 1.0 g, yeast extract 1.0 g, Na₂SO₄ 2.0 g, 70% sodium lactate 2.0 g, and distilled water to a final volume of 1000 mL. After autoclaving at 121 ℃ for 30 min, the initial pH of the medium was adjusted to 7.0. Before use, 0.6% cysteine ​​hydrochloride filter sterilization solution was added at a volume fraction of 10% to construct an anaerobic culture system.

[0100] The activated SR-12 seed culture was inoculated into the above-mentioned optimal culture medium at a volume fraction of 1% and incubated statically under a constant temperature anaerobic environment at 30 ℃. Samples were taken every 12 hours during the culture period to measure the OD of the bacterial culture. 600 The values ​​were used to characterize the dynamic changes in bacterial biomass in real time, and the residual sulfate ion concentration in the culture medium was simultaneously detected at each time point to calculate the sulfate removal rate. The continuous monitoring period was 84 h. Based on the monitoring data, the growth kinetic curve of strain SR-12 was plotted, and the temporal matching relationship between the growth and proliferation patterns of the strain and its sulfate reduction function was analyzed simultaneously. The logarithmic growth phase, stationary phase, and high-efficiency functional expression phase of the strain were identified, providing core experimental basis for the timing of industrial fermentation and harvesting of the microbial agent and the window period for field remediation application.

[0101] The results are as follows Figure 11 As shown: Strain growth (OD) 600 The removal rates of ions and sulfate exhibit different kinetic characteristics over time. During the 0-12 h lag phase, growth is slow and function is not yet initiated; during the 12-48 h logarithmic phase, both are highly coupled and rise synchronously, with OD reaching a peak at 48 h. 600 The concentration reached 1.15, with a sulfate removal rate of 25%; growth reached its peak (OD) after 48-60 hours. 600 =1.18), sulfate removal rate reached 49%. It enters the decay phase after 60-84 hours, OD... 600 The concentration dropped to 0.80, but the sulfate removal rate rose to a maximum of 69.5%, exhibiting a typical decoupling phenomenon of "growth stagnation and functional activation". Therefore, the optimal harvest time for microbial fermentation is 48-60 h.

[0102] Example 3: A method for preparing a microbial inoculant. This embodiment utilizes the SR-12 strain preserved in Example 2 to prepare a microbial inoculant, which can be subsequently applied to soil remediation. The specific method is as follows: (1) Preparation of strain seed liquid The optimized culture medium was used to prepare the fermentation base solution, with the following formula: 0.5 g K₂HPO₄, 1.0 g NH₄Cl, 1.0 g yeast extract, 2.0 g Na₂SO₄, and 2.0 g 70% sodium lactate, which were then diluted to 1000 mL with distilled water. After preparation, the medium was autoclaved at 121 °C for 30 min. After cooling, the initial pH of the medium was adjusted to 7.0. Before use, 0.6% cysteine ​​hydrochloride solution (filtered through a 0.22 μm filter) was added at a volume fraction of 10% to construct an anaerobic culture system. Chengdu Enterobacter SR-12, preserved at low temperature, was inoculated into the above culture medium and placed in an anaerobic incubator. The culture temperature was set at 30 °C, and the shaking speed at 120 r / min. The culture was carried out under constant temperature and anaerobic shaking for 48 h to obtain a stable, non-contaminated primary seed culture of SR-12.

[0103] (2) Large-scale fermentation and preparation of functional microbial mud The prepared SR-12 primary seed culture was inoculated into the sterilized fermentation medium at a volume fraction of 5%, and fermented at a constant temperature of 30 ℃ under anaerobic conditions with stirring at 120 r / min for 78 h (optimal functional harvest window). After fermentation, the entire fermentation broth was placed in a low-temperature high-speed centrifuge and centrifuged at 8000 r / min for 10 min to completely remove the fermentation supernatant. The accumulated bacterial precipitate at the bottom was collected, which is the highly active SR-12 functional bacterial sludge, for later use.

[0104] (3) Preparation of compound heavy metal pollution remediation microbial agent This invention employs a two-stage production process: "freeze-drying the microbial cells separately, followed by low-temperature compounding with nutrient substrates." The application standard is 1.0 g per kilogram of contaminated red soil, and the wet functional microbial mud feeding standard is as follows: Weigh out 1.00 g of wet bacterial sludge, 0.089 g of trehalose, and 0.111 g of skim milk powder according to the specified ratio. Dissolve in sterile water, add 0.2 wt% ferrous ammonium sulfate and 0.12 mol / L Tris-HCl buffer to adjust the pH of the system to 7.0, and stir at 32 ℃ for 40 min to complete the bacterial cell coating pretreatment. Spread the bacterial solution evenly on a tray and pre-freeze at -40 ℃ for 6 h. Dry using a gradient temperature and gradient pressure vacuum freeze-drying program until the moisture content is ≤3%, pulverize and pass through an 80-mesh sieve to obtain 0.20 g of SR-12 freeze-dried bacterial powder. The bacterial cell survival rate during the freeze-drying process was 70%, and the initial viable cell concentration of the freeze-dried pure bacterial powder was 48.3 billion CFU / g.

[0105] Weigh 1.20 g of 40-60 mesh rice straw powder, 0.012 g of cellulose complex enzyme, 0.05 g of soybean meal hydrolysate, 0.04 g of urea, and 0.30 g of 40-60 mesh rice bran powder, and premix them in a low-temperature mixer for 20 min. Then add the aforementioned 0.20 g of SR-12 freeze-dried bacterial powder, and gently stir for 8 min under light-protected conditions at ≤25 ℃. After mixing evenly, vacuum seal the mixture in an aluminum foil bag with a thickness of 15 mil or more to obtain the finished compound repair bacterial agent. The initial viable bacterial concentration of the finished product is 4.631 billion CFU / g.

[0106] For every 100 m² of Jiangxi red soil rice experimental plot (26,000 kg of dry topsoil), apply 54.236 kg of this compound microbial agent in one application. After tilling and mixing, keep it in 10 cm shallow water for 14 days for anaerobic activation. This can achieve long-term passivation of cadmium in the soil and increase rice yield.

[0107] Experimental Example 2: Storage Stability Test of SR-12 Strain Bacterial Compound (1) Test sample The test sample was the composite remedial bacterial agent of this invention; Comparative Example 1: Blank lyophilized bacterial agent without protectant, lyophilization survival rate 35.6%, initial viable cell concentration 1.552 billion CFU / g; Comparative Example 2: Commercially available sulfate-reducing bacterial agent, initial viable cell concentration 2.57 billion CFU / g; Control sample: Liquid bacterial solution prepared according to patent CN116640693A, equivalent to a solid viable cell concentration of approximately 100 million CFU / g. This method lacks data on lyophilization process and storage stability.

[0108] (2) Accelerated storage test Accelerated testing was conducted on samples from each group in a constant temperature and humidity chamber at 37 ℃ and 65% relative humidity. Samples were taken at 0, 10, 30, and 100 days, and the viable bacterial concentration was determined using the plate coating method to calculate the viable bacterial retention rate. Based on the Arrhenius model conversion, storage at 37 ℃ for 100 days is equivalent to storage at room temperature (25 ℃) for approximately 230 days.

[0109] The viable bacterial concentration and retention rate of each group of bacterial agents are detailed in Table 1.

[0110] The bacterial agent of this invention exhibits significant advantages in storage stability. After 100 days of accelerated storage, its viable cell retention rate reached 72.60%, with a viable cell concentration of 3.362 billion CFU / g; in contrast, the viable cell retention rates of the blank bacterial agent and the commercially available bacterial agent were only 4.10% and 18.10%, respectively, indicating severe degradation of bacterial activity.

[0111] This invention utilizes a composite protective system composed of trehalose, skim milk powder, and ferrous ammonium sulfate, achieving a freeze-drying survival rate of up to 70%. The porous carriers of straw and rice bran effectively isolate oxygen and moisture, further slowing down bacterial cell death. Compared to CN116640693A, which only allows for the preparation of liquid bacterial solutions and cannot be stored for long periods, this product is a solid dosage form, enabling storage and transportation at room temperature.

[0112] Calculations show that this microbial agent can be stored stably at room temperature for more than 7.5 months, with sufficient live bacteria content, which can meet the needs of large-scale application in the remediation of rice fields in high temperature and humidity in southern China.

[0113] Table 1. Stability test data of each group of bacterial agents under accelerated storage at 37℃

[0114] Experiment Example 3: Verification Test on the Passivation Effect of Composite Remediation Microbial Agent on Available Cadmium in Cadmium-Contaminated Soil In this experiment, artificial cadmium-contaminated simulated soil was prepared. The SR-12 composite remediation microbial agent prepared in Example 3 was used to conduct a soil passivation remediation test. The passivation and stabilization effect of the microbial agent of this invention on available cadmium in the soil was quantitatively investigated. The specific experimental procedures are as follows: (1) Preparation of cadmium-contaminated aged soil Weigh 140 g of air-dried and sieved test soil and place it in a 250 mL blue-capped reagent bottle. Add 116 mL of deionized water and mix well. Accurately weigh 2.04 g of cadmium chloride dihydrate (CdCl2). Dissolve 2,5H₂O in 500 mL of deionized water to prepare Cd²⁺. + A cadmium stock solution with a mass concentration of 2 mg / mL was used. The soil cadmium pollution level was set at 10 mg / kg. 700 μL of cadmium stock solution was added to the soil system in the bottle, and the mixture was thoroughly stirred to ensure even distribution of heavy metal ions in the soil matrix. The reagent bottle was gently shaken to expel internal air bubbles, and the cap was loosened. The bottle was then placed in a dark, ambient environment at room temperature and pressure for 60 days. During the aging process, air was not excluded to maintain an aerobic environment, allowing exogenous cadmium to fully adsorb and complex with soil colloids, achieving a stable equilibrium in the distribution of various heavy metal forms and completing the aging of the contaminated soil.

[0115] (2) Experimental treatment grouping After 60 days of soil aging, the bottle caps were tightened, and four parallel experimental treatments were set up. The materials in each group were thoroughly mixed with the soil: Treatment 1: Blank control group, no exogenous remediation materials were added; Treatment 2: Sodium sulfite treatment group, 20 mmol / kg sodium sulfate was added to the soil; Treatment 3: Microbial agent treatment group, 10 g / kg of the compound remediation microbial agent of this invention was added to the soil; Treatment 4: Microbial agent-sodium sulfate synergistic treatment group, 20 mmol / kg sodium sulfate and 10 g / kg of the compound remediation microbial agent of this invention were added simultaneously.

[0116] (3) Repair culture and detection methods After each group of materials was thoroughly mixed with the soil, a 2–3 cm layer of sterile liquid paraffin was added to isolate the air and create an anaerobic reducing environment. The bottle caps were then tightened and sealed, and the mixture was placed in a constant temperature and humidity biochemical incubator at 25 ℃ and 70% relative humidity for 60 days in the dark. After the remediation culture period, the available cadmium content in the soil of each group was determined using the DTPA extraction method. The results of the available cadmium detection in the soil of each group are shown in the figure below. Figure 8 .

[0117] (4) Experimental results and mechanism analysis To clarify the passivation and remediation capabilities and synergistic effects of the SR-12 sulfate-reducing bacteria and sodium sulfate substrate of this invention on cadmium-contaminated soil, quantitative analysis was performed on the detection data. Figure 12 The available cadmium content in the soil for each group was as follows: 3.96 mg / kg for the blank control group, 2.622 mg / kg for the sodium sulfate alone group, 2.347 mg / kg for the SR-12 inoculant alone group, and 1.835 mg / kg for the sodium sulfate + SR-12 inoculant synergistic group. Based on the available cadmium content of the blank group, the passivation rate was calculated as follows: 33.78% for the sodium sulfate alone group, 40.73% for the SR-12 inoculant alone group, and a 53.66% passivation rate for the sodium sulfate + SR-12 inoculant synergistic group.

[0118] The results show that the combined application of sodium sulfate and SR-12 microbial agent resulted in the best cadmium passivation effect in the soil, with a significantly higher passivation rate than either of the two single treatments, indicating a clear synergistic effect. Sodium sulfite can serve as a sulfur metabolism substrate, simultaneously activating both native soil sulfate-reducing bacteria and the exogenous SR-12 strain. The SR-12 strain carries the sqr sulfide detoxification gene, which can alleviate the autotoxic stress caused by hydrogen sulfide production and continuously and stably carry out sulfate reduction reactions to generate sulfides. The long-term sulfur-producing passivation effect of the exogenous SR-12 strain complements the remediation capacity of native microorganisms, converting active cadmium in the soil into stable and insoluble cadmium sulfide precipitates. This constructs a synergistic remediation system of "substrate activation of native microbial communities + long-term passivation by specific functional bacteria," fundamentally reducing the bioavailability of cadmium in the soil.

[0119] Experiment Example 4: Effects of SR-12 Compound Microbial Agent on the Growth and Cadmium Accumulation of Cadmium-Contaminated Rice The collected test soil was dried under natural conditions and then ground. Impurities such as stones and plant / animal remains were removed using a 2mm nylon sieve. After determining the total cadmium content of the soil, CdCl₂·2.5H₂O solution was added to adjust the soil to a cadmium-contaminated level of 5 mg / kg. The soil was then allowed to age naturally for 60 days to obtain the soil to be remediated. Pot experiments were conducted using round plastic containers (bottom diameter 27.5 cm, diameter 37.5 cm, height 33 cm), with each container containing 8 kg of the cadmium-contaminated soil to be remediated. Four parallel experimental treatments were set up, and the materials in each group were thoroughly mixed with the soil: Treatment 1: Blank control group, no exogenous remediation materials were added; Treatment 2: Sodium sulfite treatment group, 2.5 mmol / kg sodium sulfate was added to the soil; Treatment 3: Microbial agent treatment group, 10 g / kg of the compound remediation microbial agent of this invention was added to the soil; Treatment 4: Microbial agent-sodium sulfate synergistic treatment group, 2.5 mmol / kg sodium sulfate and 10 g / kg of the compound remediation microbial agent of this invention were added simultaneously.

[0120] As shown in Table 2, the biomass of brown rice, aboveground parts, and roots in the blank control group was only 46.98 g, 43.03 g, and 17.59 g, respectively. Adding sodium sulfate alone or applying SR-12 inoculant alone significantly increased the biomass of each part of the rice. Under the combined treatment of sodium sulfate and SR-12 inoculant, the biomass of brown rice, aboveground parts, and roots reached the highest values ​​among all groups, at 73.18 g, 59.23 g, and 29.05 g, respectively. Compared with the blank group, the combined treatment increased brown rice biomass by 55.77% and root biomass by 65.15%. This indicates that exogenous sodium sulfate can serve as a metabolic substrate for the SR-12 strain, improving the rhizosphere microenvironment of rice under cadmium stress. The combined use of the strain and sulfate can significantly alleviate the growth inhibition of rice by cadmium, simultaneously promoting the accumulation of root, straw, and grain biomass, thus achieving yield increase.

[0121] The cadmium content in different parts of rice is shown in Table 3. In the blank control group, the cadmium content in brown rice was as high as 0.298 mg / kg, in the aboveground parts 1.809 mg / kg, and in the roots 2.214 mg / kg. After applying sodium sulfate alone, the cadmium content in brown rice decreased to 0.126 mg / kg; after applying SR-12 inoculant alone, the cadmium content in brown rice further decreased to 0.103 mg / kg; the combined treatment group (sodium sulfate and inoculant) had the lowest cadmium content in brown rice, only 0.094 mg / kg, a decrease of 68.46% compared to the blank control group. The variation pattern of cadmium content in the aboveground parts was consistent with that in brown rice, with the combined treatment group having the lowest aboveground cadmium content (0.983 mg / kg). The cadmium accumulation in the roots of rice increased synchronously with the addition of exogenous sulfate and inoculant, with the combined treatment group having the highest cadmium content in the roots (2.357 mg / kg). The above patterns indicate that the SR-12 strain utilizes sulfate to undergo a reduction reaction, converting active cadmium in the soil into insoluble cadmium sulfide precipitate. At the same time, it promotes the adsorption and retention of cadmium by rice roots, with a large amount of cadmium fixed in the roots, significantly inhibiting the transport of cadmium from the roots to straw and edible brown rice. Sulfate and SR-12 bacterial agent have a significant synergistic passivation effect, and the cadmium reduction ability is better than that of single remediation materials.

[0122] Table 2 Rice biomass (g)

[0123] Table 3. Cadmium content in different parts of rice (mg / kg)

[0124] Experimental Example 5: In-situ remediation effect of SR-12 compound microbial agent on cadmium-contaminated rice. To verify the soil cadmium passivation effect, field planting stability, and farmland yield and quality improvement capabilities of the SR-12 sulfate-reducing bacteria in the complex environment of actual farmland, and to distinguish it from the one-sidedness of the single controllable conditions in the laboratory, a real field plot experiment was conducted to verify the actual remediation efficacy and application value of the strain under complex field stresses such as natural climate, competition from indigenous microorganisms, and fluctuations in soil physicochemical properties. This provides real and reliable field data support for the large-scale promotion and application of this technology in farmland.

[0125] (1) Overview of the test site The experimental site was selected from typical cadmium-contaminated paddy fields in Jiangxi Province. The regional farming pattern was a conventional double-cropping rice planting system, which is consistent with the mainstream planting characteristics of paddy fields in southern China. The soil in the experimental plots was generally mildly to moderately contaminated with cadmium. The soil physicochemical properties were uniform, the terrain was flat, and the water and fertilizer irrigation conditions were consistent, eliminating interference from plot differences. During the experiment, conventional field cultivation, pest and disease control, and irrigation and fertilization management were uniform throughout the entire process to ensure the uniqueness of the experimental variables.

[0126] (2) Experimental design and group setting The experiment adopted a randomized plot design, with a single plot area of ​​100 m². Isolation ridges were set between plots to prevent cross-flow of water and fertilizer. Two treatment groups were set up, with three biological replicates for each group, as follows: Blank control group (CK): conventional cultivation management, without the application of any remediation strains or remediation substrates, maintaining the natural growth state of the farmland; SR-12 remediation treatment group (T): uniform application of the SR-12 sulfate-reducing bacteria matching remediation system of this invention, evenly spread according to the predetermined farmland application dosage, relying on the exogenous functional bacteria combined with the indigenous microorganism synergistic remediation system to carry out in-situ passivation remediation.

[0127] (3) Field application method In the pre-transplanting land preparation stage for rice, anhydrous sodium sulfate was applied exogenously based on the available sulfur content of the farmland, bringing the sodium sulfate content in the soil to 390 mg / kg. The material was then evenly mixed into the topsoil at a tillage depth of 20 cm. After tillage, the field was flooded, with the water layer completely covering the soil surface and maintaining a stable depth of approximately 3 cm, followed by anaerobic activation for 7 days. Subsequently, the compound remediation microbial agent of this invention was evenly applied at a ratio of 10 g / kg of soil, and after a second tillage and mixing, the field was kept under the same shallow flooding conditions for another 7 days of activation. The 3 cm shallow water layer created a stable micro-anaerobic environment in the rice rhizosphere, meeting the sulfur metabolism physiological needs of the SR-12 strain and fully stimulating its sulfate reduction capacity. Utilizing the strain's long-term metabolic characteristics of growth-function decoupling, it continuously converted available cadmium in the soil into insoluble precipitates, achieving stable passivation of heavy metals. After the activation period, all experimental plots adopted the conventional rice planting and management model of Jiangxi Province, maintaining consistent water and fertilizer, pest and disease control, and irrigation measures to ensure that the experiment only involved a single variable: the remediation treatment.

[0128] (4) Core detection indicators and measurement methods Available cadmium content in soil: Soil samples from the topsoil of each plot were collected during the rice harvest period to test the residual amount of available cadmium in the soil and evaluate the actual passivation and remediation effect in the field. Actual rice yield: Rice was harvested, dried, and weighed separately in each plot, and the actual yield of each plot was calculated and converted into standard yield per mu (unit of land area).

[0129] The actual yield and cadmium accumulation detection results at harvest are shown in Table 4: In the 100 m² blank control plot, the rice yield was 177.5 kg, equivalent to 1183.33 kg / mu, with a cadmium content of 0.553 mg / kg; in the 100 m² SR-12 microbial agent remediation plot, the rice yield was 237.7 kg, equivalent to 1584.67 kg / mu, with a cadmium content of only 0.362 mg / kg. Compared to the blank control group, the SR-12 treatment group showed a significant increase in rice yield, increasing by 401.34 kg / mu, a yield increase rate of 33.92%, demonstrating a remarkable yield-increasing effect. Simultaneously, the cadmium content in the rice decreased significantly, with a reduction of 34.54%, indicating that the SR-12 strain can fix available cadmium in the soil through sulfate reduction, reducing the absorption and accumulation of heavy metal cadmium by rice grains. In summary, the SR-12 microbial agent of this invention has both yield-increasing and cadmium-reducing remediation effects, and has good promotional value in the practical application of cadmium-contaminated paddy fields.

[0130] After in-situ remediation using the SR-12 strain of this invention, the productivity of contaminated farmland significantly increased, far exceeding the production level of conventional paddy fields in the region. The core mechanism is as follows: On the one hand, relying on its unique sqr sulfide detoxification gene and growth-function decoupled metabolic characteristics, the SR-12 strain stably exerts its sulfate reduction function in complex field environments, continuously passivating active cadmium in the soil, reducing heavy metal biotoxicity, and relieving the inhibitory effect of cadmium stress on rice root growth and nutrient absorption. On the other hand, the strain's remediation system can optimize the rhizosphere soil microecological environment, activate indigenous functional microorganisms, improve soil tillage performance, and achieve multiple synergistic benefits of "heavy metal passivation and detoxification + soil microecological improvement + crop growth promotion and yield increase."

[0131] Table 4. Effects of microbial agents on yield increase and cadmium reduction

[0132] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A strain of Enterobacter Chengduis ( Enterobacter chengduensis ), characterized in that, It is either (a) or (b) below: (a) Enterobacter Chengdu Enterobacter chengduensis SR-12 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 20, 2025, with accession number GDMCC No. 66031; (b) A strain whose genome is at least 85%, 90%, 95%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to that of the strain described in (a).

2. The *Enterobacter chengensis* according to claim 1, characterized in that, The genome of the Chengdu Enterobacter carries the sqr sulfide oxidation detoxification gene.

3. A microbial inoculant, characterized in that, Includes the Chengdu Enterobacter as described in claim 1 or 2, or its bacterial suspension, or its culture medium, or its fermentation broth, or its fermentation broth supernatant, or its inactivated bacteria, or its metabolites, or its dry powder preparation; Optionally, the microbial agent includes freeze-dried bacterial powder, rice straw powder, cellulosic enzyme, hydrolyzed soybean meal powder, urea and / or rice bran powder; Optionally, the freeze-dried bacterial powder includes the *Enterobacterium chengensis*, trehalose, skim milk powder, ferrous ammonium sulfate, and / or Tris-HCl buffer as described in claim 1 or 2. Optionally, the concentration of live bacteria in the microbial agent is 30-50 billion CFU / g; Optionally, the concentration of live bacteria in the microbial agent is 4.631 billion CFU / g.

4. The method for preparing the microbial inoculant according to claim 3, characterized in that, The preparation method includes culturing the Enterobacter chengensis of claim 1 or 2 in a culture medium.

5. The method for preparing the microbial inoculant according to claim 4, characterized in that, The culture medium contains a carbon source; the carbon source includes at least one of sodium lactate, glucose, ethanol and sucrose.

6. The method for preparing the microbial inoculant according to claim 4 or 5, characterized in that, The culture medium contains a nitrogen source; the nitrogen source includes at least one of yeast extract, urea, potassium nitrate and tryptone.

7. The method for preparing the microbial inoculant according to any one of claims 4-6, characterized in that, The culture medium also includes at least one of K2HPO4, NaCl, and KCl.

8. The method for preparing the microbial inoculant according to any one of claims 4-7, characterized in that, The *Enterobacter chengshanense* was cultured in a medium at a pH of 5.0–8.

0.

9. The application of the microbial agent prepared by the method of preparing the Enterobacter cylindrica of Chengdu as described in claim 1 or 2, the microbial agent as described in claim 3, or the microbial agent as described in any one of claims 4-8 in the remediation of cadmium-contaminated soil or the reduction of cadmium accumulation in crops.

10. A method for remediating cadmium-contaminated soil, characterized in that, The method includes: applying the microbial agent of claim 3 to the cadmium-contaminated soil to be remediated; Optional, per 100 m 2 Apply 30-60 kg of the microbial agent described above to the cadmium-contaminated soil to be remediated; Optional, per 100 m 2 54.236 kg of the microbial agent described herein was applied to the cadmium-contaminated soil to be remediated.

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