Hexavalent chromium removing bacterium Brucella daejoensis XM2 and application thereof

Abstract

The invention relates to a hexavalent chromium removing bacterium Brucella daejoensis XM2 and an application thereof, and relates to an environmental microorganism, in particular to a hexavalent chromium removing bacterium Brucella daejoensis XM2 and an application thereof. The strain is named as Brucella daejoensis XM2, and the preservation number of the Brucella daejoensis XM2 is CCTCC (China Center For Type Culture Collection) NO: M 20242533. The strain is separated from an industrial polluted environment, is obtained through gradient domestication and screening, and has high tolerance and efficient reducing capacity to Cr (VI). The Cr (VI) removal agent can remove 50 mg / L of Cr (VI) and 100 mg / L of Cr (VI) within 24 hours under the conditions that the pH is 7.0 and the temperature is 37 DEG C, has high environmental adaptability, and can effectively remove Cr (VI) at the temperature of 30-40 DEG C and the pH is 6.5-9. 80% of Cr (VI) in the industrial wastewater is removed within 72 hours at the temperature of 45 DEG C and the pH value of 4.0. The strain can be applied to biological treatment of chromium-containing wastewater, is suitable for an industrial continuous system, and is low in cost and free of secondary pollution.

Description

Technical Field

[0001] The present invention relates to the field of environmental microbial technology, and in particular to a strain of hexavalent chromium removal bacteria. Brucella daejeonensis XM2 and its application in the treatment of chromium-containing wastewater are particularly suitable for industrial continuous treatment systems. Background Art

[0002] Chromium (Cr) in nature usually exists in the form of trivalent chromium (Cr(III)) and hexavalent chromium (Cr(VI)) (Thatoi H, Das S, Mishra J, RathBP, Das N. Bacterial chromate reductase, a potential enzyme forbioremediation of hexavalent chromium: A review [J]. J Environ Manage. 2014Dec; 146: 383-399.). Cr(VI) is an internationally recognized carcinogenic metal. The discharge of "three wastes" from industries such as leather making, electroplating, metallurgy, and chemical industry inevitably leads to chromium pollution (DAS BK, DAS PK, DAS BP, et al. Green technology to limit the effects of hexavalent chromium contaminated waterbodies on public health and vegetation at industrial sites [J]. Journal of Applied Biology&Biotechnology, 2021, 9(2): 28-35.).

[0003] Cr(VI) is difficult to degrade, posing a serious threat to the ecological environment and public health. Studies have shown that long-term digestive tract exposure to Cr(VI) can cause oral tumors, small intestinal cancer, and adenomas in animals; damage to the human digestive and reproductive systems; and even serious health problems such as digestive tract tumors and birth defects. With the accelerating pace of industrialization, the safe and effective removal of heavy metal pollutants like Cr(VI) has become an urgent environmental science issue that needs to be addressed in the national economic and social development.

[0004] Cr(VI) is highly biotoxic, while Cr(III) is an essential trace element for human metabolism. Converting harmful Cr(VI) into beneficial Cr(III) is a simple and feasible solution for Cr(VI) pollution remediation. Currently, Cr pollution remediation technologies primarily include physical, chemical, and biological remediation techniques. Physical and chemical remediation techniques, however, suffer from limitations such as high operating costs and energy consumption, and the tendency to generate secondary pollution. These limitations hinder their practical application in heavy metal pollution control, hindering their ability to meet the requirements of green, low-carbon, and sustainable pollution control. In recent years, bioremediation, particularly microbial remediation, has attracted widespread attention from researchers worldwide due to its ease of operation, low cost, and lack of secondary pollution. Over billions of years of evolution, microorganisms have gradually developed a system capable of withstanding external environmental pressures. Microbial remediation leverages their adsorption, degradation, and redox properties to transform pollutants in the environment into low- or even non-toxic forms, thereby achieving environmental remediation. Compared to physical and chemical remediation, this technology is more resource-efficient and environmentally friendly, and holds great promise for future application. Summary of the Invention

[0005] The first object of the present invention is to provide a Cr(VI) removal bacterium Brucella daejeonensis XM2.

[0006] The second object of the present invention is to provide a Cr(VI) removal bacterium Brucella daejeonensis Application of XM2 in the treatment of Cr(VI) pollution in water bodies. This strain can efficiently reduce Cr(VI) to low-toxic Cr(III), making it suitable for bioremediation of chromium-containing wastewater.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a Cr(VI) removal bacterium, which is classified and named Brucella datianensis Brucella daejeonensis XM2 was deposited in China Center for Type Culture Collection (CCTCC NO: M 20242533), isolated from sewage sludge in Xiamen Industrial Park, and its 16S rDNA sequence is shown in SEQ ID NO: 1 in the sequence listing.

[0009] This strain was isolated from a sewage outfall near the mudflats of Binhai West Avenue in Tong'an District, Xiamen, Fujian Province (24.69°N, 118.16°E). Sludge samples were collected using sterile sampling cups, placed in an ice box, and transferred to the microbiology laboratory within 24 hours for the next stage of research.

[0010] Sterilize 100 mL of LB liquid culture medium, cool it to room temperature, and add Cr(VI) mother liquor treated with a sterile filter membrane to make the Cr(VI) concentration in the liquid culture medium system 50 mg / L. Add 10 g of sludge sample to the LB culture medium containing Cr(VI) in a clean bench, mix it thoroughly, and place it in a constant temperature shaker at 37°C and 180 rpm for shaking culture for 7 days. Pipette 10% of the bacterial solution and transfer it to the new culture medium, and increase the Cr(VI) concentration to 100 mg / L, and continue to culture under the same culture conditions for 7 days. Then transfer the bacterial solution produced in the previous process to the LB liquid culture medium containing 200 mg / L Cr(VI) at a 10% inoculation volume and continue to culture for 7 days. The final bacterial solution is gradient diluted and spread on the LB solid culture medium. After multiple separation and purification, the pure Brucella daejeonensis XM2 single bacteria.

[0011] The 16s rDNA sequence of strain XM2 was identified to be Brucella daejeonensis The similarity of MJ11 is 97.99%, and they are on the same branch in the phylogenetic tree, so they are named Brucella datianensis. Brucella daejeonensis XM2. This strain was deposited in the China Center for Type Culture Collection (CCTCC) on November 12, 2024, at China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the collection number of CCTCC NO: M 20242533.

[0012] To accurately quantify the Cr(VI) removal efficiency of strain XM2, a single XM2 colony was streaked onto LB solid medium, the plate was inverted in a constant-temperature incubator, and cultured at 37°C for 48 hours. A single XM2 colony was picked from the LB solid plate and inoculated into LB liquid medium for overnight culture, creating the XM2 seed culture. This seed culture was inoculated at a 4% (v / v) inoculum into medium containing 50 mg / L, 100 mg / L, and 200 mg / L Cr(VI), respectively. LB liquid medium without Cr(VI) served as a control. Cultures were incubated at pH 7, 37°C, and 180 rpm. Samples of the culture medium were removed every 12 hours, and the Cr(VI) removal efficiency of strain XM2 was measured and calculated over a 72-hour period.

[0013] The above experiments showed that strain XM2, under the conditions of pH 7, temperature 37°C, and rotation speed 180 rpm, achieved a 97.90% removal rate for 50 mg / L Cr(VI) after 12 hours of cultivation, and 100% after 24 hours. Within 12 hours, the removal rate for 100 mg / L Cr(VI) reached 74.69%, and after 24 hours, the removal rate reached 100%. Within 48 hours, the removal rate for 200 mg / L Cr(VI) reached 27.68%, and after 72 hours, the removal rate reached 37.32%.

[0014] The LB solid medium composition is as follows: 10 g tryptone, 5 g yeast extract powder, 10 g sodium chloride, 15 g / L agar powder, 1 L deionized water, pH 6.9-7.1. The LB liquid medium composition is as follows: 10 g tryptone, 5 g yeast extract powder, 10 g sodium chloride, 1 L deionized water, pH 6.9-7.1.

[0015] To investigate the impact of environmental factors, the removal efficiency was evaluated at 30 different pH values ​​(6.5, 7.0, 7.5, 8.0, 8.5, and 9.0) and different incubation temperatures (30°C, 35°C, 40°C, 45°C, and 50°C). Cr(VI) content was measured every 24 hours using dibenzoylhydrazide spectrophotometry. The removal efficiency was calculated (see Example 4 for the specific formula), and contour maps were constructed to determine the optimal range of conditions for strain XM2.

[0016] The results of Cr(VI) removal by strain XM2 under different pH and temperature conditions showed that XM2 could effectively remove Cr(VI) at 30℃~40℃ and pH between 6.5~9, indicating that strain XM2 has strong environmental adaptability and a wide range of applications.

[0017] To further investigate the Cr(VI) removal mechanism of strain XM2, fresh seed liquid was inoculated at a 4% (v / v) inoculum into LB liquid medium containing Cr(VI). A Cr(VI-free) control was used. Cultures were maintained at pH 7, 37°C, and 180 rpm for 48 hours before collection. Following pretreatment, the strain's structure and surface elements were characterized using scanning electron microscopy (SEM), energy dispersive spectroscopy (EDX), and transmission electron microscopy (TEM).

[0018] Characterization analysis results revealed significant changes in bacterial morphology after Cr(VI) treatment, with strain XM2 exhibiting porous damage, severe rupture, and pronounced wrinkling. EDX analysis revealed Cr adsorption on the surface of XM2, suggesting that biosorption contributes to Cr(VI) removal by XM2. TEM results revealed the appearance of dark, electron-dense particles, including black plaques, following Cr(VI) treatment, likely due to the accumulation of intracellular reduction products.

[0019] To investigate the removal efficiency of strain XM2 in real-world wastewater, a 4% (v / v) inoculum of seed liquid was inoculated into industrial wastewater containing Cr(VI). Four groups of nutrient support systems were established, including glucose, lactose, galactose, and beef extract. Industrial wastewater without any carbon source was used as a control. Cultures were maintained at a pH of 4, a temperature of 37°C, and a rotation speed of 180 rpm. Samples of the bacterial culture were taken every 12 hours, and the Cr(VI) removal efficiency of strain XM2 in industrial wastewater was measured and calculated over a 48-hour period.

[0020] The results showed that strain XM2 had a better repair effect with the support of beef extract, and the removal rate fluctuated between 45% and 55%, which was significantly higher than that of the other three groups and the control group. The removal rate of other nutritional supports was only maintained between 20% and 25%.

[0021] To optimize the removal efficiency of strain XM2 in real-world wastewater, the effects of pH and temperature on its remediation of industrial wastewater were investigated, using beef extract, a carbon source with excellent removal performance. Six inoculums were inoculated with a 4% (v / v) seed solution into Cr(VI)-containing industrial wastewater. A pH gradient of 4 to 9 was established, with the inoculum without carbon source added serving as the control. Cultures were maintained at 37°C and 180 rpm. Under optimal pH conditions, a temperature gradient of 20°C to 45°C, with 5°C intervals, was established for six inoculum groups. A control group was established with the inoculum without carbon source added, and culture was maintained at 180 rpm.

[0022] The results showed that, with the support of beef extract as a carbon source, XM2 was most effective at removing Cr(VI) at pH levels of 4 to 5, with a maximum removal rate of 67.21% within 72 hours at pH 4. Under the optimal culture conditions of pH 4, at temperatures ranging from 25°C to 45°C, strain XM2 achieved a Cr(VI) removal rate exceeding 50% within 72 hours. The 45°C experimental group achieved the highest Cr(VI) removal rate, reaching nearly 80% within 72 hours.

[0023] Compared with the prior art, the present invention has the following outstanding advantages and technical effects:

[0024] The present invention has relatively completely completed the implementation case study of chromium removal functional strains from screening, laboratory effect research, mechanism research to industrial wastewater remediation application. Brucella daejeonensis Under laboratory conditions, XM2 can completely remove 50 mg / L and 100 mg / L of Cr(VI); it has a good removal effect in an environment with a pH of 6.5-9 and a temperature range of 30℃-40℃, with a wide range of applications, which is conducive to subsequent sewage remediation; the characterization results suggest that XM2's removal of Cr(VI) is mainly based on biological adsorption, and may also have biological reduction; the industrial wastewater remediation results show that the strain XM2 has a good removal effect under the support of the carbon source of beef extract, in an acidic environment with a pH of 4-5 and a temperature range of 25℃-45℃, among which the optimal removal rate is achieved at pH=4 and temperature of 45℃, and it can remove about 80% of Cr(VI) in industrial wastewater; through industrial wastewater treatment application and optimization research, the practicality of XM2 in complex environments has been verified. The strain can be used for the biological remediation of chromium-containing wastewater in extreme acidic and high-temperature environments. The optimal remediation conditions of pH=4 and temperature of 45℃ are easy to achieve, low-carbon and environmentally friendly, and are especially suitable for industrial continuous systems, with the advantages of low cost and no secondary pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the colony macroscopic morphology of the strain of the present invention.

[0026] Figure 2 Phylogenetic analysis tree of the strains of the present invention.

[0027] Figure 3 The removal rates of the strain of the present invention for different concentrations of Cr(VI) at different times.

[0028] Figure 4 It is a contour map of the removal of Cr(VI) by the strain of the present invention under different temperature and pH conditions.

[0029] Figure 5 These are SEM images of the bacterial cells of the strain of the present invention before and after Cr(VI) treatment.

[0030] Figure 6 TEM images of the bacterial strain of the present invention before and after Cr(VI) treatment.

[0031] Figure 7 The figure shows the removal of Cr(VI) from industrial wastewater by the strain of the present invention with the support of different carbon sources.

[0032] Figure 8 The figure shows the removal of Cr(VI) from industrial wastewater by the strain of the present invention under different pH conditions.

[0033] Figure 9The figure shows the removal of Cr(VI) from industrial wastewater by the strain of the present invention at different temperatures. DETAILED DESCRIPTION

[0034] The following is intended to further illustrate the present invention in conjunction with the accompanying drawings, rather than to limit the present invention.

[0035] Example 1: Morphological characteristics of strain XM2

[0036] A single colony was streaked onto LB solid medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 15 g / L, pH 6.9-7.1) and the plate was inverted and incubated in a 37°C incubator for 48 hours. The macroscopic morphology of the colonies was observed visually. The results showed that the colonies had a smooth and moist surface, regular edges, no halo, and a slightly raised center (see Figure 1 The SEM results of the characterization analysis in the subsequent Example 5 also showed that the colony morphology of strain XM2 was highly consistent with the typical spherical short rod-shaped characteristics of Brucella.

[0037] Example 2: Acclimation, enrichment, and screening of strain XM2

[0038] (1) Given that Cr(VI) pollution is generally accumulative in the environment, the sludge samples used in this study were collected from a sewage outlet near the beach on Binhai West Avenue in Tong'an District, Xiamen City, Fujian Province (24.69°N, 118.16°E). Tong'an Industrial Concentration Zone is a comprehensive industrial base for the clothing, footwear, leather, chemical fiber, and nonferrous metal industries that Xiamen has long focused on developing, and is therefore potentially contaminated by Cr(VI) (Baidu Encyclopedia. Tong'an Industrial Concentration Zone [EB / OL]. https: / / baike.baidu.com / item / %E5%90%8C%E5%AE%89%E5%B7%A5%E4%B8%9A%E9%9B%86%E4%B8%AD% E5%8C%BA / 7502536?fromtitle=%E5%8E%A6%E9%97%A8%E5%90%8C%E5%AE%89%E5%B7%A5%E4% B8%9A%E9%9B%86%E4%B8%AD%E5%8C%BA&fromid=3615881 ; Yu Mingxiao. Research on the Transformation and Development Strategy of Tongan Industrial Concentration Zone [D]. Huaqiao University, 2019). Therefore, microorganisms in sludge samples may have the potential to convert Cr(VI) to Cr(III) under natural selection under external environmental pressure, thereby achieving Cr(VI) removal. In this example, three enrichment cycles were set up to acclimate and screen for highly efficient Cr(VI)-removing bacteria. In the first enrichment cycle, 10 g of sludge sample was aseptically added to LB liquid culture medium containing 50 mg / L Cr(VI) and incubated in a constant temperature shaker at 37°C and 180 rpm for 7 days.

[0039] (2) After the first enrichment cycle, the culture was transferred to a new culture medium at a 10% (v / v) inoculation volume, and the Cr(VI) concentration was increased to 100 mg / L. The culture was continued under the same culture conditions for 7 days. This process was the second enrichment cycle.

[0040] (3) After the second enrichment cycle, after 7 days of cultivation, the culture was transferred to LB liquid medium containing 200 mg / L Cr(VI) at a 10% (v / v) inoculation volume and cultured for another 7 days. This process was the third enrichment cycle.

[0041] (4) The suspension produced in the third round of enrichment is the bacterial stock solution. -6 To dilute the final concentration, the strain was evenly spread on a solid LB plate, which was then inverted and incubated at 37°C for approximately 48 hours. After repeated isolation and purification, the single strain XM2 was obtained.

[0042] The components of LB solid medium are: 10 g tryptone, 5 g yeast extract powder, 10 g sodium chloride, 15 g / L agar powder, 1 L deionized water, pH 6.9-7.1. The components of LB liquid medium are: 10 g tryptone, 5 g yeast extract powder, 10 g sodium chloride, 1 L deionized water, pH 6.9-7.1.

[0043] Example 3: Phylogenetic Analysis

[0044] In this example, the 16S rDNA sequence of strain XM2 was analyzed to determine its taxonomic status and clarify its relationship to other known strains. DNA was extracted from a freshly cultured strain XM2 using a genomic DNA extraction kit according to the manufacturer's instructions. The extracted DNA was dissolved in sterile deionized water, its integrity was verified by 1% agarose gel electrophoresis, and its concentration and purity were determined using a UV spectrophotometer to ensure that the DNA quality met the requirements of subsequent experiments. Universal primers were used to amplify the 16S rDNA of strain XM2 by PCR. The PCR reaction system (total volume 50 μL) consisted of 2 μL template DNA, 25 μL 2× Taq PCR-Super Mix (+dye), 1 μL each of the upstream and downstream primers (10 μM), and the volume was made up to 50 μL with sterile deionized water. PCR reaction conditions included initial denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 60 seconds. The final extension was performed at 72°C for 10 minutes. PCR products were examined by 1% agarose gel electrophoresis to detect the appearance of specific bands, which were approximately 1300 bp in size.

[0045] After purifying the 16S rDNA amplified fragment using a PCR product purification kit, it was sent to a professional sequencing company for bidirectional sequencing to ensure the accuracy of the sequencing results. The 16S rDNA sequence measured using the EZbiocloud (https: / / www.ezbiocloud.net / ) database is as follows:

[0046]

[0047] Similarity comparison analysis was performed with existing nucleic acid sequences. Phylogenetic analysis was performed using MEGA-X software. ClustalW algorithm was used for alignment to ensure accurate sequence alignment. After alignment, the neighbor-joining method was used to construct a phylogenetic tree. During the construction process, an appropriate model (such as the Kimura 2-parameter model) was selected to calculate genetic distances, and 1000 bootstrap tests were performed to evaluate the reliability of the phylogenetic tree branches. The phylogenetic analysis tree of strain XM1 was finally obtained, see Figure 2 .

[0048] from Figure 2 It can be seen that the 16s rDNA sequence of strain XM2 is similar to Brucella daejeonensis s MJ11 has a similarity of 97.99% and forms a branch together, so it is named Brucella datianensis Brucella daejeonensis XM2.

[0049] Example 4: Cr(VI) removal efficiency test and condition optimization of strain XM2

[0050] To verify the removal effect of the strain on Cr(VI), a single colony of XM2 was streaked onto LB solid medium, and the plate was inverted in a constant temperature incubator. After culturing at 37°C for 48 h, a single colony was picked and inoculated into 100 mL of LB liquid medium. The culture was carried out overnight at pH = 7, temperature 37°C, and rotation speed 180 rpm until the logarithmic growth phase (OD 600 =0.6-0.8) to obtain XM2 seed liquid. Fresh seed liquid was inoculated at a 4% (v / v) inoculum into 100 mL of LB liquid medium containing 50 mg / L, 100 mg / L, and 200 mg / L Cr(VI), respectively. Cultures were maintained at pH 7, 37°C, and 180 rpm. LB liquid medium without Cr(VI) served as a control, with three replicates per group. Cultures were collected at 0, 12, 24, 36, 48, 60, and 72 h, and Cr(VI) concentrations were determined using the diphenylcarbazide spectrophotometric method (GB 7467-1987).

[0051] Calculation of Cr(VI) removal rate:

[0052] The OD of the Cr(VI) test solution measured at the initial time of the experiment 540 Recorded as A0, the OD measured at the time of testing 540 The OD of the solution without Cr(VI) is recorded as A1. 540 Denote it as C. Substitute it into formula (1) to calculate the removal rate of Cr(VI) by the strain at the time of testing, which is recorded as X.

[0053] X= (1)

[0054] The Cr(VI) removal efficiency of strain XM2 is shown in Figure 3 At 37°C and 180 rpm, strain XM2 achieved a 97.90% removal rate for 50 mg / L Cr(VI) after 12 h of culture; and 100% after 24 h. Within 12 h, the removal rate for 100 mg / L Cr(VI) reached 74.69%, and 100% after 24 h; within 48 h, the removal rate for 200 mg / L Cr(VI) reached 27.68%, and 37.32% after 72 h. The above results indicate that Brucella datianensis Brucella daejeonensis XM2 has a good removal effect on Cr(VI), and can completely remove 50 mg / L and 100 mg / L Cr(VI) within 24 h. Its chromium removal performance is better than that of other reported Brucella species (Urvashi Thacker, Rasesh Parikh, Yogesh Shouche, Datta Madamwar, Reduction of chromate by cell-free extract of Brucella sp. isolated from Cr(VI) contaminated sites [J]. Bioresource Technology, 2007, 98(8): 1541-1547.).

[0055] To investigate the impact of environmental factors, the removal efficiency was evaluated at 30 different pH values ​​(6.5, 7.0, 7.5, 8.0, 8.5, and 9.0) and different culture temperatures (30°C, 35°C, 40°C, 45°C, and 50°C). Fresh seed liquid was inoculated at a 4% (v / v) inoculum into 100 mL of LB liquid medium containing 100 mg / L Cr(VI). The culture was incubated at 180 rpm for 72 hours. The Cr(VI) content was measured every 24 hours using dibenzoylhydrazide spectrophotometry. The removal efficiency was calculated (see Example 4 for the specific formula) and contour plots were constructed to determine the optimal range of conditions for strain XM2.

[0056] Cr(VI) removal results of strain XM2 under different pH and temperature conditions (see Figure 4 ) showed that XM2 could effectively remove Cr(VI) at 30℃~40℃ and pH between 6.5 and 9, indicating that strain XM2 has strong environmental adaptability and a wide range of applications.

[0057] Experimental Example 5: Study on the Cr(VI) removal mechanism of strain XM2

[0058] To investigate the removal mechanism of strain XM2, fresh seed liquid was inoculated at a 4% (v / v) inoculum into 300 mL of LB liquid medium containing 50 mg / L and 100 mg / L Cr(VI), respectively. LB without Cr(VI) served as a control. The culture was incubated at pH 7, 37°C, and 180 rpm for 48 h. An appropriate amount of the culture was centrifuged at 5000 rpm at 4°C for 10 min to collect the appropriate amount of cells. The cells were resuspended in 1× PBS and pipetted until homogenous. The cells were centrifuged at 5000 rpm at 4°C for 10 min and washed three times to obtain the cell samples. 4°C pre-cooled glutaraldehyde fixative was slowly added along the tube wall, and the cells were then refrigerated at 4°C for overnight fixation. The cells were then sent to a specialist company for scanning electron microscopy (SEM), energy dispersive spectroscopy (EDX), and transmission electron microscopy (TEM).

[0059] SEM can be used to observe the general morphology of XM2 bacteria (see Figure 5 Compared with the control group, the morphological characteristics of the bacteria after Cr(VI) treatment changed significantly. In the control group without Cr(VI), the bacteria were short rod-shaped, with fewer wrinkles on the surface and a relatively plump and rounded appearance. At 50 mg / L Cr(VI), the surface of the bacteria showed varying degrees of depression and distortion, and the rod-shaped structure was less obvious. At the higher concentration of 100 mg / L Cr(VI), pore-like damage appeared on the cell surface, the bacteria were severely ruptured, wrinkles were extremely obvious, and the length of the bacteria increased significantly, indicating that higher concentrations of Cr(VI) have a greater impact on the morphology and growth of the bacteria.

[0060] In order to verify whether the element Cr is adsorbed on the bacterial surface, EDX spectrum analysis was further performed. Table 1 shows the percentage of each element content on the bacterial surface of the strain of the present invention before and after Cr(VI) treatment.

[0061] Table 1

[0062]

[0063] No Cr (0.00%) was detected in the untreated control. The normalized mass of Cr in the 50 mg / L and 100 mg / L Cr(VI)-treated groups was 0.05% and 0.11%, respectively. This indicates that a small amount of Cr is adsorbed on the surface of bacterial strain XM2, and the amount adsorbed is positively correlated with the external Cr(VI) concentration, suggesting that biosorption may be one of the mechanisms by which strain XM2 removes Cr(VI).

[0064] Heavy metals can interfere with the growth and metabolism of bacteria, causing changes in the internal structure of bacteria. Transmission electron microscopy (TEM) can be used to observe changes in the internal morphology of bacteria under Cr(VI) stress (see Figure 6 After Cr(VI) treatment, some dark electron-dense particles such as black spots appeared in strain XM2, which may be related to the accumulation of reduction products in the cells.

[0065] Example 6: Application and Optimization of Strain XM2 in Treatment of Chromium-Containing Wastewater

[0066] Industrial wastewater samples were collected from the pretreatment wastewater system of an electronics industry company located in Torch Park, Maxiang Subdistrict, Xiang'an District, Xiamen, Fujian Province (24.63°N, 118.24°E). The company uses Cr(VI) as a raw material, and irregular monitoring of the wastewater revealed a Cr(VI) content of approximately 50 mg / L. The wastewater was acidic, with a pH of approximately 4. Samples were collected in clean, dried PE bottles, placed in an ice box, and immediately transported to the laboratory for storage at 4°C.

[0067] First, the effects of different carbon sources on the Cr(VI) removal efficiency of the XM2 strain were evaluated, and the optimal carbon source was selected for subsequent optimization. Four carbon source groups (glucose, fructose, galactose, and beef extract) were set up, all at a carbon source concentration of 20 g / L. A control group without carbon source addition was also established, with three replicates per group. Fresh seed liquid was inoculated at a 4% (v / v) inoculum into 10 mL of industrial wastewater containing different carbon sources. The cultures were cultured at pH 4, 37°C, and 180 rpm. The culture suspensions were removed at 0, 12, 24, 36, and 48 hours. The Cr(VI) concentration was determined using the diphenylcarbazide spectrophotometric method (GB 7467-1987), and the removal efficiency was calculated. The specific calculation formula is shown in Example 4.

[0068] The results of Cr(VI) removal by strain XM2 under different carbon sources (see Figure 7) showed that the Cr(VI) removal rates of the glucose, fructose and galactose groups all reached the highest value of about 30% at 24 h; however, the removal efficiency began to decline after 36 h, and dropped to about 20% at 48 h, which was presumably due to carbon source exhaustion or metabolic poisoning; the beef extract group maintained a stable removal efficiency from 12 h to 48 h, with the removal rate fluctuating between 45% and 55%, which was significantly higher than that of the other three groups and the control group, which may be because beef extract provided strain XM1 with a variety of mixed carbon sources; the control group always maintained a low level of removal rate of 20% to 25%, verifying the positive promoting effect of beef extract as a carbon source on the Cr(VI) removal process.

[0069] In order to optimize the removal effect of strain XM2 in actual wastewater, the effects of pH and temperature on the remediation of industrial wastewater by XM2 were explored with the support of beef extract, a carbon source with good removal effect.

[0070] Six groups (4, 5, 6, 7, 8, and 9) with different pH culture conditions were set up, with an inoculum system without carbon source added serving as the control group. Three replicates were set up in each group. Fresh seed liquid was inoculated at a 4% (v / v) inoculum into 10 mL of industrial wastewater containing different carbon sources. Cultures were maintained at 37°C and 180 rpm. The culture suspensions were removed at 0, 12, 24, 36, 48, and 72 hours. Cr(VI) concentrations were measured using the diphenylcarbazide spectrophotometric method (GB 7467-1987), and the removal rate was calculated. The specific calculation formula is shown in Example 4.

[0071] The results of industrial wastewater removal by strain XM2 under different pH conditions (see Figure 8 ) showed that, with the support of beef extract, the Cr(VI) removal efficiency of XM2 gradually decreased with increasing pH. XM2 effectively removed Cr(VI) from wastewater in the acidic pH range of 4–5, achieving the highest removal efficiency at pH 4, with a 72-hour removal rate of 67.21%. However, after the pH exceeded 7, the removal efficiency remained below 10%, likely due to precipitation of Cr(VI) in the wastewater under alkaline conditions, which hindered the strain's Cr(VI) removal function. Therefore, strain XM2 exhibited excellent Cr(VI) removal capabilities in acidic environments of 4–5, with the highest removal efficiency at pH 4, suggesting that strain XM2 is particularly suitable for treating acidic industrial wastewater.

[0072] Under optimal culture conditions of pH 4, six temperature gradients (20°C, 25°C, 30°C, 35°C, 40°C, and 45°C) were established. A control group was established with an inoculum system without carbon source added. Three replicates were set up in each group. Fresh seed liquid was inoculated at a 4% (v / v) inoculum into 10 mL of industrial wastewater containing different carbon sources and incubated at 180 rpm. The culture suspension was removed at 0, 12, 24, 36, 48, and 72 hours. Cr(VI) concentrations were measured using the diphenylcarbazide spectrophotometric method (GB 7467-1987), and the removal rate was calculated. The specific calculation formula is shown in Example 4.

[0073] The removal results of industrial wastewater by strain XM2 at different temperatures (see Figure 9 ) showed that within 12 hours, Cr(VI) removal rates exceeded 15% in both the control and experimental groups at all temperatures, reaching approximately 20% in the 40°C and 45°C experimental groups. At 24 hours, XM2's removal rate significantly increased to approximately 40% at all temperatures, reaching nearly 45% at 45°C. Removal rates slowed from 24 to 36 hours, reaching 65% at 48 hours in the 45°C experimental group. After 72 hours, the 45°C experimental group achieved the highest removal rate, reaching nearly 80%. Removal rates also exceeded 50% at 72 hours in all other temperature groups. This indicates that this strain maintains high activity even at high temperatures (40°C–45°C), demonstrating excellent environmental adaptability and suitability for use in high-temperature industrial wastewater discharge environments in southern China.

[0074] The above embodiments are only preferred embodiments of the present invention and should not be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent of the present invention.

Claims

1. A hexavalent chromium removal bacterium Brucella daejeonensis XM2, featuring: The hexavalent chromium removal bacteria Brucella daejeonensis XM2 was named Brucella datianensis Brucella daejeonensis XM2, deposited in China Center for Type Culture Collection, with the deposit number CCTCC NO: M 20242533, the Brucella datianensis Brucella daejeonensis The 16S rDNA sequence of XM2 is shown in SEQ ID NO:

1.

2. As claimed in claim 1, a hexavalent chromium removal bacterium Brucella datianensis Brucella daejeonensis Application of XM2 in the treatment of chromium-containing wastewater.

3. The use according to claim 2, characterized in that The specific method of the application is: Brucella daejeonensis XM2 was inoculated into wastewater containing Cr(VI) at a controlled temperature of 45 °C, pH = 4, a rotation speed of 180 rpm, and an inoculation amount of 4% (v / v).

4. The use according to claim 2, characterized in that The concentration of Cr(VI) in the chromium-containing wastewater is 10 mg / L to 200 mg / L.

5. The use according to claim 2, characterized in that Brucella daejeonensis XM2 was applied to the bioreactor in an immobilized form to achieve continuous Cr(VI) removal.

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