Heavy metal-tolerant cupriavidus strain resistant to acid stress, application and bacterial agent thereof

Abstract

The present application relates to the field of agricultural microorganisms and heavy metal pollution prevention, and provides an acid-stress-tolerant metal-tolerant cupriavidus strain, application and microbial agent thereof.The strain is preserved in the China General Microbiological Culture Collection Center, has the acid-tolerant property of stable growth in a pH 3.0 to 7.0 environment, and has the ability to reduce the concentration of various heavy metal ions such as chromium, manganese, copper, arsenic, cadmium and lead.In the wheat and rice hydroponic germination test, the strain can significantly promote root growth and alleviate the inhibition of various heavy metal stresses on root development, and the heavy metal ion removal rate can reach a high level.The present application can be used for heavy metal pollution treatment and agricultural green and safe production under acidic conditions.

Description

Technical Field

[0001] This invention relates to the fields of agricultural microorganisms and heavy metal pollution control, and particularly to an acid-stress-resistant and heavy metal-tolerant copper-loving bacterium strain, its application, and its inoculant. Background Technology

[0002] Heavy metal pollution in soil has become a global environmental problem, posing a persistent threat to agricultural production, food security, and public health. Toxic heavy metals such as arsenic, cadmium, cobalt, chromium, copper, nickel, and lead are difficult to degrade in soil, easily accumulate in arable land environments over long periods, and can enter the food chain through the soil-crop system, increasing the risk of human exposure. Related studies indicate that a significant proportion of farmland soil globally is contaminated with these heavy metals. The contaminated arable land area is large, affecting a large population, and the risks of heavy metal pollution exhibit both widespread and insidious characteristics.

[0003] Heavy metal pollution in China's soils is a particularly prominent problem, exhibiting a complex pattern of acidification and heavy metal pollution in the southern regions. Acidic soils are widely distributed in the red soil regions of southern China, the mountainous areas of southwest China, and some facility agriculture areas. Low soil pH often triggers a chain reaction of nutrient imbalances, enhanced activation and migration of heavy metals, and decreased soil microbial activity, thereby exacerbating crop stress and reducing the safety of agricultural products. Simultaneously, soil acidification in my country is showing a trend of expanding scope and deepening severity. The overlapping of acidification and heavy metal pollution poses a more severe challenge to agricultural safety in southern regions.

[0004] Heavy metal toxicity has a significant inhibitory effect on crop growth, typically manifesting as inhibited seed germination, reduced germination rate, and slow seedling growth. At the physiological level, it can also cause adverse effects such as disrupted cell division, impaired photosynthetic systems, disordered nutrient absorption and transport, increased oxidative stress, and water imbalance. Especially in acidic environments, the bioavailability of heavy metals increases, leading to more pronounced crop damage and resulting in reduced yields, lower quality, and even an increased risk of agricultural products exceeding safety standards.

[0005] The remediation of heavy metal pollution in soil currently mainly includes physicochemical remediation, bioremediation, and integrated ecological restoration. Physicochemical methods typically offer rapid treatment and noticeable short-term effects, but they also suffer from high costs, potential damage to soil structure, and the introduction of secondary pollution, making them unsuitable for large-scale arable land. In contrast, bioremediation technologies utilize plants, microorganisms, or combinations thereof to remove, immobilize, or transform heavy metals. These technologies offer advantages such as relatively low cost, environmental friendliness, and applicability to large areas of mildly to moderately polluted sites, making them a crucial direction for current soil heavy metal remediation. However, the adaptability of existing bioremediation strains to acidic soil conditions, their stable remediation capacity under conditions of multiple heavy metal coexistence, and their synergistic effects on crop growth still require further improvement. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a copper-tolerant bacterium that is resistant to acid stress and multiple heavy metal stresses. Therefore, embodiments of this invention provide an acid-resistant copper-tolerant bacterium strain, its application, and a fungal agent.

[0007] In a first aspect, embodiments of the present invention provide an acid-stress-resistant, heavy metal-resistant copper-loving bacterium strain, the strain being named *Copper-loving Bacteria* (…). Cupriavidus metallidurans The heavy metal-tolerant copper-loving bacteria (xm1225) is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36356 and deposit date of October 27, 2025.

[0008] Preferably, the 16S rDNA sequence of the strain is shown in SEQ ID NO.1.

[0009] Preferably, the strain is acid-resistant and can grow in an acidic environment with a pH value of 3.0 to 7.0.

[0010] Preferably, the strain is a short rod-shaped, non-spore-forming Gram-negative bacterium.

[0011] Secondly, embodiments of the present invention also provide the application of the acid-stress-resistant and heavy metal-tolerant copper-loving bacteria described in the first aspect in promoting plant root growth, wherein the plants include wheat and rice.

[0012] Thirdly, embodiments of the present invention also provide the application of the acid-stress-resistant and heavy metal-resistant copper-loving bacteria described in the first aspect in the degradation of heavy metals, wherein the heavy metals include one or more of chromium, manganese, copper, arsenic, cadmium, and lead.

[0013] Fourthly, embodiments of the present invention also provide a microbial agent, including the acid-stress-resistant and heavy metal-resistant copper-loving bacteria described in the first aspect.

[0014] In summary, the beneficial effects of the present invention are as follows:

[0015] The heavy metal-tolerant copper-loving bacterium xm1225 provided by this invention has significant acid stress resistance and can maintain good growth in environments with a pH value of 3.0 to 7.0, making it suitable for low pH application scenarios such as acidic soils in the south.

[0016] The strain of this invention exhibits tolerance and significant removal capacity for multiple heavy metal ions. It can survive under high stress conditions where multiple heavy metals such as chromium, manganese, copper, arsenic, cadmium, and lead coexist, and reduce the concentration of the corresponding heavy metal ions in the solution, which is beneficial for the remediation of complex polluted environments. The strain of this invention also has plant growth-promoting effects, significantly promoting root growth in crops such as wheat and rice, and effectively alleviating the inhibition of plant root growth by heavy metal stress, thereby improving the germination and early growth capacity of crops in polluted environments.

[0017] The strains of this invention are derived from the natural environment, have low application costs, do not introduce chemical residues, are environmentally friendly, and can be used for large-scale green remediation and safe production in agricultural production scenarios. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments of the present invention will be briefly introduced below. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort, and these are all within the protection scope of the present invention.

[0019] Figure 1 This is a scanning electron microscope image of the acid-stress-resistant and heavy metal-resistant copper-loving bacteria strain described in the embodiments of the present invention.

[0020] Figure 2 This is a comparison diagram of the growth of the strains of the present invention under different pH conditions in plate culture.

[0021] Figure 3 This is a growth curve diagram of the strain of the present invention under different pH conditions.

[0022] Figure 4 This is a trend graph showing the removal effect of the strains in this invention on various heavy metal ions.

[0023] Figure 5 This is a comparison diagram of the taproot length of wheat in different treatment groups in this embodiment of the invention.

[0024] Figure 6 This is a comparison diagram of the taproot length of rice in different treatment groups in this embodiment of the invention.

[0025] Figure 7 This is a comparison chart of the removal rates of various heavy metal ions in the hydroponic solution of the fourth group of wheat experiments in this embodiment of the invention.

[0026] Figure 8 This is a comparison chart of the removal rates of various heavy metal ions in the hydroponic solution of the fourth group of rice experiments in this embodiment of the invention. Detailed Implementation

[0027] The features and exemplary embodiments of various aspects of the present invention will now be described in detail. To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only configured to explain the present invention and are not configured to limit the present invention. For those skilled in the art, the present invention can be practiced without some of these specific details. The following description of the embodiments is merely intended to provide a better understanding of the present invention by illustrating examples of the invention.

[0028] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising..." does not exclude the presence of additional identical elements in the process, method, article, or apparatus that includes said element.

[0029] Example 1

[0030] Please see Figure 1 This invention provides an acid-stress-resistant, heavy metal-resistant copper-pollinated bacterium strain, named *Copper-pollinated Bacteria spp.* Cupriavidus metallidurans The heavy metal-tolerant copper-loving bacteria (xm1225) is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36356 and deposit date of October 27, 2025.

[0031] Please see Figure 1 This embodiment provides an acid-stress-resistant, heavy metal-resistant copper-loving bacterium strain, named *Copper-loving Bacteria spp.* Cupriavidus metallidurans xm1225. This strain was isolated from a naturally sourced sample. During the isolation process, colonies were first formed by streak culturing on a medium, followed by multiple streak purifications to obtain morphologically consistent single colonies, ensuring the purity and genetic stability of the strain. Under standard culture conditions, the obtained strain can form regular, milky-white, round colonies with neat edges and opaque surfaces, facilitating identification on plates and continuous subculturing.

[0032] In terms of performance, this strain exhibits outstanding acid stress tolerance, maintaining growth activity under acidic conditions, making it suitable for applications where microbial activity is easily inhibited in low-pH environments. Simultaneously, this strain can survive and maintain its proliferative capacity under stress conditions with multiple heavy metals coexisting, making it suitable for reducing the content of heavy metal ions such as chromium, manganese, copper, arsenic, cadmium, and lead in culture systems, demonstrating its adaptability and application potential in environments polluted by multiple heavy metals. Furthermore, in comparative experiments during the plant germination stage, this strain showed a promoting effect on crop root growth under both heavy metal-free and heavy metal stress conditions, indicating that it can not only maintain biological activity under pollution stress but also positively influence early plant growth, thus possessing dual application value in environmental remediation and crop growth promotion.

[0033] To ensure the availability and reproducibility of the strain, the strain described in this embodiment has been deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 36356. Through this accession information, the strain possesses a clear identification and traceable origin. Those skilled in the art can obtain the strain based on the accession number and verify its morphological characteristics and functional properties, thereby supporting subsequent implementation methods for applications such as acid resistance, multimetal resistance, and plant growth promotion.

[0034] Preferably, the 16S rDNA sequence of the strain is shown in SEQ ID NO.1.

[0035] Specifically, the 16S rDNA sequence of the strain, SEQ ID NO.1, is as follows:

[0036]

[0037] To obtain the 16S rDNA sequence and perform molecular-level identification of the strain, this embodiment employs a 16S rDNA amplification and sequencing process using genomic DNA as a template. The process includes sample preparation, DNA extraction, PCR amplification, product electrophoresis verification, product purification and recovery, Sanger sequencing, and sequence alignment analysis.

[0038] In the sample preparation stage, bacterial cells in an actively growing state are preferably selected as starting material. After the strain is cultured on nutrient agar medium until single colonies form, a single colony is picked and inoculated into nutrient broth medium and cultured under suitable temperature conditions to achieve sufficient biomass to meet the sample volume requirements for subsequent DNA extraction. After obtaining the bacterial culture, the bacterial precipitate is preferably collected by centrifugation. The supernatant is discarded, and the bacterial cells are washed to reduce the impact of residual culture medium on subsequent enzymatic reactions.

[0039] During the DNA extraction stage, a DNA extraction kit manufactured by Qiagen Corporation is preferably used to extract genomic DNA from the strain. Specifically, the collected bacterial cells are mixed with lysis buffer to fully lyse the cells and release nucleic acids. Protein removal and nucleic acid purification are then performed according to the kit's procedure. Finally, the DNA is dissolved in sterile water or elution buffer to obtain genomic DNA. The obtained genomic DNA serves as a template for subsequent PCR amplification, effectively characterizing the genetic information of the strain.

[0040] During the PCR amplification stage, a universal 16S rDNA primer pair is preferably selected to amplify the 16S rDNA fragment of the strain. The upstream primer 27F has a sequence of 5'--AGAGTTTGATCMTGGCTCAG-3', and the downstream primer 1492R has a sequence of 5'-TACGGYTACCTTGTTACGACTT-3'. The preferred PCR reaction system is a 50 μL system, comprising 2 μL of genomic DNA, 2 μL each of the upstream and downstream primers (10 μM concentration), 25 μL of ExTaq DNA polymerase reagent, and the remainder made up to 50 μL with sterile water. This system configuration ensures amplification efficiency while reducing the probability of non-specific amplification.

[0041] In the PCR program setup, the pre-denaturation step is preferably set to 94℃ for 5 min to ensure complete denaturation of the template DNA. This is followed by cyclic amplification, with the preferred cycling parameters being 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, and 72℃ extension for 90 seconds, for a total of 35 cycles. The annealing temperature promotes specific binding of the primers to the template, and the extension time ensures complete synthesis of the target fragment. After cycling, a 72℃ extension for 10 min is preferred to ensure any incomplete extension is completed. Finally, the sample is stored at 4℃ for subsequent analysis. This procedure yields an amplified product close to the full-length 16S rDNA, facilitating subsequent classification and identification.

[0042] During the amplification product validation stage, 5 μL of PCR product was preferably subjected to 1% agarose gel electrophoresis to confirm the presence of the amplified product and whether the fragment length met expectations. Electrophoresis results showed the target fragment was approximately 1.5 kb, indicating that the amplified fragment matched the length of the 16S rDNA target region. To ensure the quality of subsequent sequencing signals, the PCR amplification product was preferably recovered and purified to remove primers, free dNTPs, enzymes, and impurities, thereby reducing unwanted peaks and background interference.

[0043] During the sequencing stage, the purified PCR product was preferably sent to a third-party sequencing institution for Sanger sequencing to obtain highly accurate nucleotide sequence information. The sequencing result yielded an effective sequence length of 1433 bp, which was compiled and defined as SEQ ID NO.1. To further determine the taxonomic position of this strain, the 16S rDNA sequence was preferably subjected to database alignment analysis, specifically using BLAST alignment to perform a similarity search between SEQ ID NO.1 and 16S rDNA reference sequences in publicly available databases. The alignment results showed that the sequence was similar to that of copper-tolerant heavy metal bacteria. Cupriavidus metallidurans The similarity was 99.7%, which supports the identification results of the strain at the molecular level and can be corroborated by its morphological observation results.

[0044] Preferably, the strain is acid-resistant and can grow in an acidic environment with a pH value of 3.0 to 7.0.

[0045] Specifically, the strain provided in this embodiment is acid-resistant and can grow in an acidic environment with a pH value of 3.0 to 7.0. This embodiment verifies the acid resistance of the strain through two methods: solid culture and liquid growth curve.

[0046] See Figure 2After streaking culture of the strain on culture media with different pH conditions, it can be observed that clear colony growth bands can be formed under pH 3, pH 5 and pH 7 conditions, indicating that the strain still has good survival and proliferation ability under strong acid conditions, and can not only grow under near neutral conditions.

[0047] See Figure 3 To further quantify the growth performance under different pH conditions, the strain was cultured in liquid culture systems at pH 3, pH 5 and pH 7, and the OD600 of the bacterial solution was measured at different culture times to characterize the changes in bacterial biomass.

[0048] Depend on Figure 3 As can be seen, all curves showed a gradual upward trend in the early stages of cultivation, entering a significant proliferation phase after approximately 21 hours. By 30 hours, OD600 reached a high level. The proliferation rate was relatively faster under pH 7 conditions, while under pH 3 conditions, although there was a slight lag in the early stages, continuous growth still occurred, reaching a stable and high biomass level. The results of solid culture and liquid growth curves corroborated each other, indicating that this strain has stable growth ability within the pH range of 3.0 to 7.0, possesses good acid stress tolerance, and is suitable for applications in acidic environments.

[0049] The present invention also provides a method for preparing the aforementioned copper-tolerant heavy metal bacterium strain, the method comprising:

[0050] S1. The natural mineral water sample is left to stand in a container for a first preset time to form a biofilm at the bottom of the container, and the biofilm is taken as a separation material using a sterile inoculation loop.

[0051] The natural mineral water source sample used in this step is preferably a soil sample from a natural mineral water source in Yongchun County, Quanzhou City, to ensure consistency between the strain source and the application scenario. The soil sample is mixed with sterile water or extracted and then placed in a container to stand for a first preset time, allowing microorganisms in the sample to gradually attach to and accumulate on the bottom or inner wall of the container, forming a biofilm. Compared to directly taking the supernatant, the biofilm more easily accumulates bacterial communities with strong attachment and growth capabilities and good environmental adaptability, which is beneficial for obtaining target strains with tolerant characteristics in subsequent isolation processes. Using a sterile inoculation loop to collect the biofilm as the separation material reduces the risk of introducing exogenous contaminants and improves sampling stability, allowing subsequent streak culturing to more effectively obtain dominant strains from the water source soil.

[0052] S2. The separated material is inoculated into a nutrient broth culture medium for enrichment, wherein a heavy metal standard solution is added to the nutrient broth in advance, and the heavy metal standard solution is filtered through a sterile filter membrane with a preset pore size before addition.

[0053] In this step, the separation material is the biofilm obtained in S1, which contains various environmental microorganisms. The separation material is inoculated into a nutrient broth medium for enrichment. The purpose is to rapidly expand the number of microorganisms in the liquid system, allowing strains with the target traits that were initially present in low concentrations in the sample to proliferate and form a bacterial solution suitable for subsequent serial dilution separation. The nutrient broth provides nutrients such as peptone and beef extract, which support bacteria to enter a growth state in a short time, thereby improving enrichment efficiency and shortening the screening cycle.

[0054] Unlike conventional enrichment, this step involves pre-adding heavy metal standard solutions to the nutrient broth. This applies selective pressure during the enrichment stage, making it difficult for microorganisms intolerant to heavy metals such as chromium, manganese, copper, arsenic, cadmium, and lead to proliferate or inhibiting their growth. Conversely, microbial communities with tolerance or the ability to adsorb and fix heavy metals are more likely to dominate the system. This setup reduces the number of candidates during subsequent plate separation, increases the probability of obtaining strains resistant to multiple heavy metals, and also makes the single colonies obtained in subsequent screening more closely resemble the target application environment.

[0055] Before being added, the heavy metal standard solution is filtered through a sterile filter membrane with a preset pore size. This process sterilizes the heavy metal solution and prevents the introduction of contaminants into the standard solution or preparation process, which could lead to contamination of the enrichment system with non-target bacteria and affect the screening judgment.

[0056] In one embodiment, the preset pore size is typically 0.22 μm, which is used to trap microorganisms such as bacteria while allowing metal ions to pass through. This achieves sterilization of the solution without changing the composition of heavy metal ions, ensuring the controllability and repeatability of enrichment culture.

[0057] S3. The nutrient broth medium inoculated with the separation material is shaken and cultured for a second preset time under preset shaking culture conditions. After the culture is completed, the cultured bacterial solution is serially diluted according to the preset serial dilution factor to obtain the diluted solution.

[0058] The shaking culture in this step is used to further enrich and expand the number of target strains in a liquid system under heavy metal stress, allowing heavy metal-tolerant bacteria to gain a competitive advantage. Shaking culture can continuously improve the homogenization and mass transfer efficiency of the solution, ensuring sufficient contact between the bacteria and heavy metal ions, avoiding localized differences in conditions caused by bacterial sedimentation, and simultaneously increasing dissolved oxygen levels, which is beneficial for the bacteria to maintain metabolic activity and complete proliferation under stress conditions. The second preset time setting is used to ensure that the enrichment culture reaches a bacterial count level suitable for plate separation, usually allowing the system to enter the logarithmic growth phase or near the stationary phase, thus providing a sufficient number of viable bacteria for subsequent dilution and separation.

[0059] Serial dilution after cultivation is used to reduce the bacterial concentration in the culture medium to a level where it can form dispersed single colonies on solid culture media. If no dilution is performed before plating or streaking, the excessively high bacterial concentration will cause colony fusion, making it difficult to obtain single colonies and affecting subsequent purification. By preset serial dilution factors, colonies of appropriate quantity and dispersed distribution can be obtained at different dilutions, making it easier to pick single colonies with different morphologies and reducing the risk of missed screening.

[0060] Preferably, the preset gradient dilution factor includes 10. -3 10 -4 and 10 -5 Specifically, sterile water or sterile saline can be used as the diluent, and serial dilutions can be performed in tenfold series to obtain 10... -3 Diluent, 10 -4 Diluent and 10 -5 The diluent is then used for subsequent inoculation onto nutrient agar medium to obtain initial colonies with better separation results. S4. The diluent is inoculated onto nutrient agar medium using the streak plate method and cultured under preset culture conditions to obtain initial colonies, which include single colonies with different morphologies.

[0061] In this step, the streak plating method involves using a sterile inoculating loop or needle to streak the diluted solution onto the surface of a solid culture medium in a stepwise manner, dispersing the bacteria from high-density areas to low-density areas, thus forming spatially separated single colonies after incubation. Inoculating the diluted solution onto nutrient agar medium and culturing under preset conditions allows the surviving bacteria in the diluted solution to colonize, grow, and form observable colonies on the solid surface. Nutrient agar medium provides a basic carbon and nitrogen source and growth factors, suitable for the recovery and growth of environmentally sourced bacteria and the manifestation of colony morphology, facilitating subsequent picking and purification. The preset incubation conditions are preferably 36°C for 24 to 48 hours to promote full colony development. The initial colonies formed after incubation typically vary in color, shape, edge regularity, and transparency, resulting in an initial colony set containing single colonies of different morphologies, providing candidate sources for subsequent purification and screening.

[0062] S5. Select single colonies with different morphologies and perform streaking purification again. Repeat the purification until single colonies with consistent morphology are obtained.

[0063] This step is used to obtain pure culture strains. Specifically, colonies with different morphologies obtained from S2 are picked and streaked again onto fresh nutrient agar medium to further dilute and isolate the bacteria, allowing them to reform into single colonies. This streaking purification process is repeated until the colony morphology is stable and consistent after continuous subculturing, reducing interference from mixed or associated bacteria. The purified strain forms regular, milky-white, round colonies with neat edges and opaque surfaces on nutrient agar, which serves as an important indicator of successful purification and strain stability.

[0064] S6. After multiple purifications, single colonies were screened again to obtain acid-stress-resistant and heavy metal-resistant copper-loving bacteria strains.

[0065] This step applies selective pressure to the purified strain to screen for target strains that simultaneously possess tolerance to acid stress and multiple heavy metal stresses. Preferably, the repeatedly purified strain is inoculated into a culture system containing multiple heavy metal ions, and cultured under conditions where ions such as chromium, manganese, copper, arsenic, cadmium, and lead coexist and at high concentrations. This makes it difficult for intolerant strains to survive or proliferate, thus preserving tolerant strains. After cultivation, the bacterial solution is streaked back into nutrient agar medium to obtain strains that can still form single colonies after the heavy metal stress. Further, the growth ability of the candidate strain under low pH conditions is verified. The candidate strain can maintain growth activity in an environment with a pH value of 3.0 to 7.0, thus confirming the acid-stress-resistant, heavy metal-tolerant copper-loving bacterium strain xm1225.

[0066] Preferably, the re-screening of single colonies after multiple purifications to obtain the acid-stress-resistant, heavy metal-tolerant copper-loving bacterium strain includes:

[0067] S61. The heavy metal standard solution is added to the container in advance, and the single colony after multiple purifications is inoculated into the container. The heavy metal standard solution includes chromium, manganese, copper, arsenic, cadmium and lead, and the ion concentration of each heavy metal in the heavy metal standard solution is 0.5 g / L.

[0068] In this step, the container can be an Erlenmeyer flask or other containers. By pre-setting a culture system with multiple heavy metal ions coexisting within the Erlenmeyer flask, a complex stress condition is applied to the strains, ensuring that the screening process aligns with the target application scenario. The single colonies purified multiple times serve as the inoculum source, guaranteeing that a single strain, rather than a mixed bacterial community, is introduced into the screening system, avoiding misinterpretation of tolerance from symbiotic or mixed bacteria as characteristics of the target strain. Chromium, manganese, copper, arsenic, cadmium, and lead are added simultaneously and each is set to an ion concentration of 0.5 g / L, giving the screening system strong selective pressure. This effectively distinguishes common environmental bacteria from candidate strains with high tolerance, thereby increasing the probability of obtaining strains resistant to multiple heavy metal stresses. This step also makes the changes in metal ion concentration after subsequent culture comparable, facilitating the evaluation of strain tolerance and removal capacity under the same conditions.

[0069] S62. Under the preset oscillation culture conditions, the container is oscillated and cultured for the second preset time to obtain the cultured bacterial solution;

[0070] This step involves shaking the culture to ensure sufficient contact between the bacteria and the heavy metal-containing culture system, preventing localized concentration differences caused by bacterial sedimentation and thus distributing the screening pressure more evenly throughout the system. Shaking also improves dissolved oxygen levels and mass transfer efficiency, which is beneficial for the bacteria's metabolism and proliferation under high stress conditions, thus more accurately reflecting the bacteria's survival ability in a complex heavy metal environment. The second preset time is set to ensure a sufficiently long culture period, allowing the bacteria in the heavy metal solution to reach an ideal growth state, supporting subsequent screening and purification while ensuring the stability of heavy metal removal during the culture process. Depending on actual needs, the second preset time is typically set between 24 and 48 hours, sufficient for adequate bacterial proliferation and a stable bacterial concentration, providing a sufficient number of bacteria for subsequent dilution and separation.

[0071] S63. The bacterial solution is streaked onto nutrient agar medium and cultured to obtain the acid-stress-resistant heavy metal copper-loving bacterium strain.

[0072] This step involves re-inoculation and colony establishment. Its purpose is to transfer the viable S42-selected bacteria from the liquid system to a solid culture medium to form single colonies, thereby obtaining a pure culture strain that can be stably passaged and preserved. Streak inoculation allows for stepwise dilution and dispersion of the bacterial solution, enabling surviving bacteria to form single, separable colonies on the plate, facilitating picking and further purification for confirmation. Nutrient agar, as a universal culture medium, supports the recovery of surviving strains without introducing additional selection pressure, allowing for more direct observation of the stability and consistency of colony morphology, and obtaining target strains for subsequent acid tolerance verification, 16S rDNA identification, and functional evaluation. The single colonies obtained through the above re-inoculation culture, combined with their ability to grow even under low pH conditions, confirm them as the acid-stress-resistant, heavy metal-tolerant copper-loving bacterium strain.

[0073] In a preferred embodiment, the preset pore size of the present invention is 0.22 μm, the first preset time is 7 days, the second preset time is 48 hours, and the preset culture conditions include culture at a temperature of 30-40 degrees Celsius for 24-48 hours; the preset shaking culture conditions include culture at a temperature of 37 degrees Celsius and a speed of 160 rpm; the components of the nutrient agar medium per liter include 10.0 g peptone, 3.0 g beef extract, 5.0 g sodium chloride, and 15.0 g agar; the components of the nutrient broth medium per liter include 10.0 g peptone, 3.0 g beef extract, and 5.0 g sodium chloride.

[0074] Specifically, in this embodiment, the first preset time refers to the time during which the natural mineral water sample is left to stand in the container in step S1. This time is set to 7 days, which is sufficient for the microorganisms in the water sample to form a biofilm at the bottom of the container through natural sedimentation and adhesion. A biofilm is a structure in which microorganisms attach and aggregate into a thin layer, making it suitable for the separation and enrichment of microorganisms.

[0075] The second preset time is 48 hours, which is applicable to the shaking culture time of the nutrient broth medium inoculated with the isolated material in step S2. The 48-hour culture time ensures that the bacteria can proliferate sufficiently in a nutrient-rich environment, especially for the growth of microorganisms under heavy metal stress conditions. Sufficient time can ensure the activity of microorganisms and support subsequent screening.

[0076] The preset culture conditions include incubation at 30-40 degrees Celsius for 24-48 hours. These conditions ensure that the strains can complete colony formation and biomass accumulation within a relatively stable temperature window during isolation, purification, and re-inoculation. 30-40 degrees Celsius is a commonly used growth temperature range for this type of environmentally derived bacteria. Within this range, both bacterial metabolic activity and growth rate are considered, allowing colonies to form within 24-48 hours with clear morphological characteristics, facilitating the identification of morphological differences, picking single colonies, and determining whether purification is complete. Setting the culture time to 24-48 hours covers the process of the strain transitioning from the adaptation phase to the logarithmic growth phase and forming observable colonies. Furthermore, it avoids excessive colony fusion, interference from other bacteria, or blurred morphological characteristics caused by excessively long culture times, thereby improving the reliability and reproducibility of purification and screening.

[0077] The preset shaking culture conditions include cultivation at 37 degrees Celsius and 160 rpm. 37 degrees Celsius is the standard bacterial culture temperature, which can simulate the optimal growth temperature in many environments, promoting rapid bacterial reproduction. 160 rpm refers to the shaking speed of the culture flask during cultivation, with stirring performed at a rate of 160 times per minute. This shaking condition effectively increases the dissolution and diffusion of oxygen and nutrients, improves the contact efficiency between the bacteria and the heavy metal solution, and promotes the growth and metabolism of microorganisms under heavy metal stress conditions.

[0078] The nutrient agar medium comprises, per liter, 10.0 g peptone, 3.0 g beef extract, 5.0 g sodium chloride, and 15.0 g agar. This medium is a commonly used nutrient-type solid culture medium formulation. Peptone and beef extract serve as the main organic nitrogen and growth factor sources, providing the strain with nutrients such as amino acids, peptides, and vitamins, supporting the rapid recovery of growth and the formation of stable colonies during the isolation and purification stages. Sodium chloride is used to maintain the ionic strength and osmotic pressure of the medium, reducing the impact of osmotic stress on strain growth and resulting in more stable and easily identifiable colony morphology. Agar acts as a solidifying agent, forming solid plates that support streak plating, allowing different bacterial cells to spatially separate into single colonies. This provides a foundation for subsequent multiple purification processes, re-inoculation, and the stable acquisition of the target strain.

[0079] Nutrient broth medium has a similar composition to agar medium, but does not contain agar, therefore it is a liquid medium. Its components include peptone (10.0 g / L) to provide a nitrogen source for the microorganisms, beef extract (3.0 g / L) to provide essential vitamins and other nutrients, and sodium chloride (5.0 g / L) to maintain the osmotic pressure of the liquid medium. This liquid medium supports bacterial growth in a liquid environment and is suitable for enrichment culture procedures.

[0080] The above conditions ensure the optimal growth environment for the strain in different culture steps, and support the rapid proliferation of bacteria and their adaptability to heavy metal stress through reasonable culture conditions and temperature management, while enabling them to form bacterial cells with high activity and degradation ability.

[0081] Example 2

[0082] The present invention also provides the application of the acid-stress-resistant heavy metal-resistant copper-loving bacteria of Example 1 in the degradation of heavy metals, wherein the heavy metals include one or more of chromium, manganese, copper, arsenic, cadmium and lead.

[0083] The strain's ability to reduce heavy metal ions was evaluated by conducting cultivation experiments under various heavy metal coexistence conditions and using ICP-MS to detect the concentration of each heavy metal ion in the culture system. Please refer to [link to relevant documentation]. Figure 4 The graph shows the trend of the concentration of each heavy metal ion as a function of culture time.

[0084] The experimental steps in this embodiment are as follows:

[0085] 1) Stock culture: Single colonies cultured on nutrient agar medium for 48 hours were picked and inoculated into nutrient broth tubes, and cultured at 36°C for 24 hours to obtain the stock culture of the strain. This step ensures that the bacteria are in a highly active state, serving as the inoculum source for subsequent heavy metal degradation experiments.

[0086] 2) Construction and inoculation of the multimetal system: 10 mL of the above stock solution was inoculated into a 250 mL Erlenmeyer flask. 90 mL of heavy metal solution was pre-added to the flask, with the initial concentrations of chromium, manganese, copper, arsenic, cadmium, and lead ions in the solution ranging from 0.3 g / L to 0.6 g / L. The strain was cultured at 36°C to ensure sufficient contact between the strain and heavy metal ions in a stressful environment with multiple heavy metals, thus demonstrating its degradation effect under combined pollution conditions.

[0087] 3) Detection and Trend Mapping of Heavy Metal Ion Concentration: After 24 hours of cultivation, the concentration of each heavy metal ion in the culture system was detected using ICP-MS. Detection was performed every 3 days during subsequent cultivation to obtain metal ion concentration data at different cultivation time points. Instrument conditions: RF power 1500W, plasma gas flow rate 15L / min, carrier gas flow rate 0.8L / min, auxiliary gas flow rate 0.4L / min, nebulizer temperature 2℃, sampling depth 8mm, acquisition mode: Spectrum. Based on the detection data, trend graphs of each ion concentration changing over time were plotted, as shown below. Figure 4 As shown.

[0088] Depend on Figure 4It is evident that, under conditions where multiple heavy metal ions coexist and their initial concentrations are all within the range of 0.3 g / L to 0.6 g / L, the concentrations of chromium, manganese, copper, arsenic, cadmium, and lead ions in the culture system show a continuous decreasing trend with prolonged culture time. Particularly in the early stages of culture, the concentrations of multiple metal ions decreased significantly, exhibiting a rapid reduction characteristic. At subsequent detection time points, the concentrations of each metal ion remained at a low level and further decreased, indicating that the strain can maintain a stable effect under high stress conditions of multiple heavy metals and simultaneously reduce the concentrations of multiple heavy metal ions. This demonstrates that the strain possesses significant heavy metal degradation capabilities in multi-metal coexistence systems, effectively removing or reducing multiple heavy metal ions.

[0089] Example 3

[0090] This invention also provides the application of the acid-stress-resistant and heavy metal-resistant copper-loving bacteria described in Example 1 in promoting plant root growth, wherein the plants include wheat and rice.

[0091] This embodiment provides the application of the acid-stress-tolerant and heavy metal-tolerant copper-loving bacteria described in Example 1 in promoting plant root growth. The plants include wheat and rice. A hydroponic germination experiment was conducted under fertilizer- and pesticide-free conditions by setting up control groups including sterile water, heavy metal stress, bacterial agent treatment, and heavy metal stress combined with bacterial agent treatment. After cultivation, the length of the seedling taproot and the residual heavy metal content in the hydroponic solution were measured to verify the effectiveness of the strain in promoting plant growth and alleviating heavy metal poisoning.

[0092] 1) Experimental materials and treatment groups

[0093] Preparation of inoculum:

[0094] Single colonies were picked from nutrient agar medium and cultured for 48 hours. They were then inoculated into 50 mL of nutrient broth and cultured at 37°C for 24 hours to obtain the bacterial culture for inoculation.

[0095] Plant materials and treatment:

[0096] Several wheat and rice seeds were selected, carefully chosen, and their surfaces were disinfected by wiping with 75% alcohol and rinsing with sterile water 3 to 4 times. The treated seeds were then placed in a sterile hydroponic system for germination. After germination, seeds with uniform size and consistent growth were selected for grouped experiments.

[0097] Experimental Groups:

[0098] Taking wheat as an example, 40 seeds were selected and divided into 4 groups of 10 seeds each. The same grouping method was used for rice, as follows:

[0099] Group 1 was the sterile water control group, in which seeds were placed in a hydroponic system and only sterile water was added.

[0100] Group 2 was the heavy metal stress group, in which the seeds were placed in a mixed solution of heavy metals containing chromium, manganese, copper, arsenic, cadmium and lead, with each heavy metal ion having a concentration of 50 μg / L.

[0101] Group 3 is the bacterial culture group, in which the seeds are placed in sterile water after inoculation with the bacterial solution, and the inoculation amount is 2%.

[0102] Group 4 was a group subjected to heavy metal stress and supplemented with microbial agents. The seeds were placed in a mixed solution containing the heavy metals, with each heavy metal ion concentration of 50 μg / L, and inoculated with the microbial solution at an inoculation amount of 2%.

[0103] Cultivation conditions:

[0104] Each group of experiments was placed in the dark at 25℃ for 10 days.

[0105] 2) Data Acquisition and Measurement Methods

[0106] Main root length measurement:

[0107] After 10 days of cultivation, the length of the taproot of each group of seedlings was measured and the average value was calculated to characterize the root development.

[0108] Determination and removal rate calculation of heavy metal residues in hydroponic solutions:

[0109] The concentration of heavy metal ions (Ct) in the supernatant after the fourth culture was completed was determined by ICP-MS, and the removal rate was calculated with an initial concentration (C0) of 50 μg / L. The removal rate satisfies the following formula:

[0110] Removal rate = (C0-Ct) / C0×100%.

[0111] Please see Figure 5 The average taproot lengths of wheat in each group were as follows: Group 1: 6.65 cm, Group 2: 3.72 cm, Group 3: 10.45 cm, and Group 4: 10.06 cm. It can be seen that heavy metal stress significantly inhibits wheat root growth, with Group 2 showing a significantly shorter root length than the control group. After inoculation with the microbial agent, wheat roots grew significantly, with both Group 3 and Group 4 showing significantly longer root lengths than the control group. Furthermore, Group 4 maintained a root length close to that of Group 3 under heavy metal stress, indicating that the strain can effectively alleviate root growth inhibition caused by heavy metal stress.

[0112] Please see Figure 6The average taproot lengths of the rice groups were as follows: Group 1: 12.32 cm, Group 2: 8.79 cm, Group 3: 19.91 cm, and Group 4: 20.44 cm. Consistent with the wheat results, the root lengths of the heavy metal stress groups decreased significantly, while the roots grew significantly after the addition of the microbial agent. Furthermore, the taproot length of Group 4, which received the microbial agent under heavy metal stress, was similar to that of Group 3, which only received the microbial agent. This indicates that the strain also significantly promotes root growth in rice and can reduce the adverse effects of heavy metal toxicity on root development.

[0113] Please see Figure 7 and Figure 8 The detection of residual heavy metal ions in the fourth group of hydroponic solutions showed that the strain had a significant removal effect on chromium, manganese, copper, arsenic, cadmium, and lead ions in the hydroponic system, with high removal rates for each heavy metal ion, and an overall removal rate of over 99%. This result indicates that the strain can not only promote root growth in plant growth systems but also reduce the concentration of heavy metal ions in hydroponic solutions, thereby reducing the heavy metal exposure level of plants during cultivation.

[0114] As can be seen from the above results, this embodiment has at least the following beneficial effects:

[0115] The strain can significantly promote the elongation of the taproot of wheat and rice and improve the root development level during the germination stage under heavy metal-free conditions, thus demonstrating stable plant growth-promoting potential.

[0116] Under stress conditions where multiple heavy metal ions coexist, the strain can still significantly increase the length of the main root of crops, thereby eliminating or significantly reducing the root growth inhibition caused by heavy metal stress and improving the early growth ability of crops in polluted environments.

[0117] The strain can significantly reduce the concentration of heavy metal ions such as chromium, manganese, copper, arsenic, cadmium, and lead in hydroponic solutions, achieving a removal rate of over 99%. This reduces the toxic effects of heavy metals on plants at the source, achieving a synergistic effect of plant growth promotion and heavy metal removal. It is suitable for green remediation and safe agricultural production scenarios in environments polluted by heavy metals.

[0118] Example 4

[0119] This invention also provides a microbial agent, including the acid-stress-resistant and heavy metal-resistant copper-loving bacterium strain described in Example 1.

[0120] This invention also provides a microbial agent, including the acid-stress-resistant and heavy metal-tolerant copper-loving bacterium strain described in Example 1. The microbial agent uses the strain as its active ingredient. By culturing the strain to obtain bacterial cells and forming a formulation that is easy to apply and store, it can stably release bacterial activity in practical application scenarios, thereby achieving the functional output of heavy metal removal and plant growth promotion.

[0121] In terms of preparation and use, the microbial agent is preferably composed of an active bacterial solution of the strain. Specifically, the bacterial solution can be obtained by culturing the strain in a nutrient broth medium, and then used as a source of inoculum for treating culture systems containing heavy metals or for inoculating plant germination and hydroponic systems. The inoculum can be added by volume fraction, for example, 2% inoculation in a hydroponic system in plant experiments, allowing the bacteria to quickly establish dominance in the hydroponic environment, continuously contacting heavy metal ions such as chromium, manganese, copper, arsenic, cadmium, and lead, and reducing their concentration in the solution phase, while promoting plant root development. Since the strain can maintain growth activity in an environment with a pH value of 3.0 to 7.0, the microbial agent can still maintain an effective bacterial count and functional stability under acidic conditions, adapting to the application requirements of the acidic environment in the south where heavy metals are more easily activated.

[0122] Compared to relying solely on chemical passivating agents or single growth-promoting bacterial agents, the aforementioned microbial agent possesses the advantage of multiple functionalities. On one hand, the bacterial strains in the agent can survive under high stress conditions where multiple heavy metals coexist and reduce the concentration of various heavy metal ions, thereby mitigating the toxic effects of heavy metals on plants. On the other hand, the strains exhibit the effect of promoting root growth in wheat and rice under both heavy metal-free and heavy metal stress conditions, allowing the agent to simultaneously address pollution and promote crop growth during the germination stage. Therefore, the aforementioned microbial agent can be used for heavy metal pollution control in acidic environments, early plant growth promotion, and heavy metal stress mitigation, exhibiting environmental friendliness, strong applicability, and relatively low application costs.

[0123] It should be clarified that the present invention is not limited to the specific configurations and processes described above and shown in the figures. For the sake of brevity, detailed descriptions of known methods are omitted here. In the above embodiments, several specific steps are described and shown as examples. However, the method process of the present invention is not limited to the specific steps described and shown. Those skilled in the art can make various changes, modifications, and additions, or change the order of steps, after understanding the spirit of the present invention.

[0124] It should also be noted that the exemplary embodiments mentioned in this invention describe methods or systems based on a series of steps or apparatus. However, this invention is not limited to the order of the steps described above; that is, the steps can be performed in the order mentioned in the embodiments, or in a different order, or several steps can be performed simultaneously.

[0125] The above description is merely a specific embodiment of the present invention. Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working processes of the systems, modules, and units described above can be referred to the corresponding processes in the foregoing method embodiments, and will not be repeated here. It should be understood that the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the protection scope of the present invention.

Claims

1. A copper-tolerant bacterium strain resistant to acid stress, characterized in that, The strain was named *Copper-tolerant heavy metal bacterium* (Bacteria spp.). Cupriavidus metallidurans The strain xm1225 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 36356.

2. The application of the acid-stress-resistant and heavy metal-resistant copper-pollinated bacterium strain according to claim 1, characterized in that, The strain is used to promote root growth in plants, including wheat and rice.

3. The application of the acid-stress-resistant and heavy metal-resistant copper-pollinated bacterium strain according to claim 1, characterized in that, The strain is used to degrade heavy metals in soil, including one or more of chromium, manganese, copper, arsenic, cadmium, and lead.

4. A microbial inoculant, characterized in that, Including the acid-stress-resistant, heavy metal-resistant copper-loving bacteria strain as described in claim 1.

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