Hexavalent chromium removing bacterium Brevibacterium optitidis XM1 and application thereof

Abstract

The invention relates to a hexavalent chromium removing bacterium Brevibacterium otidis XM1 and application thereof, and relates to the technical field of environmental microorganisms, in particular to a hexavalent chromium removing bacterium Brevibacterium otidis XM1 and application thereof. The strain is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M 20242532, and the 16S rDNA sequence of the strain is as shown in SEQ ID NO: 1. 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). Under the conditions that the pH is 7.0 and the temperature is 37 DEG C, the removal rate of 50 mg / L Cr (VI after 48 hours reaches 97.87%, and the removal rate of 72 hours reaches 100%. 90% of Cr (VI) in the industrial wastewater can be removed within 72 hours under the conditions that the pH is 4.0 and the temperature is 45 DEG C. The strain can be applied to biological treatment of chromium-containing wastewater, and is low in cost and free of secondary pollution.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental microorganisms, in particular to a hexavalent chromium removal bacterium Brevibacteriumotitidis XM1 and application thereof in the treatment of chromium-containing wastewater, and is particularly suitable for an industrial continuous treatment system. 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 remediating Cr(VI) pollution. 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 aligned with the emerging development concepts of resource conservation and environmental friendliness, and holds great promise for future applications. Summary of the Invention

[0005] The first object of the present invention is to provide a Cr(VI)-removing bacterium Brevibacterium otitidis XM1.

[0006] A second objective of the present invention is to provide a Cr(VI)-removing bacterium, Brevibacterium otitidis XM1, for use in the treatment of Cr(VI) contamination in water. This strain can efficiently reduce Cr(VI) to the less 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)-removing bacterium, which is classified and named Brevibacterium otitidis XM1, preserved in the China Center for Type Culture Collection (CCTCC NO: M 20242532), isolated from Xiamen industrial sludge, and has a 16S rDNA sequence as shown in SEQ ID No: 1 in the sequence table.

[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] 100 mL of LB liquid medium was sterilized, cooled to room temperature, and then a sterile-filtered Cr(VI) stock solution was added to the liquid medium to a Cr(VI) concentration of 50 mg / L. In a clean bench, 10 g of sludge sample was added to the LB medium containing Cr(VI), mixed thoroughly, and incubated in a constant-temperature shaker at 37°C and 180 rpm for 7 days. 10% of the bacterial suspension was transferred to fresh medium, and the Cr(VI) concentration was increased to 100 mg / L. Incubation continued under the same conditions for 7 days. A 10% inoculum of the bacterial suspension from the previous step was then transferred to LB liquid medium containing 200 mg / L Cr(VI) and incubated for another 7 days. The resulting bacterial suspension was serially diluted and plated onto LB solid medium. Multiple isolation and purification procedures were performed to obtain pure Brevibacterium otitidis XM1.

[0011] Identification revealed that the 16s rDNA sequence of strain XM1 shared 98.98% similarity with Brevibacterium otitidis NCFB 3053(T), and the strain was located on the same branch in the phylogenetic tree. Therefore, the strain was named Brevibacterium otitidis XM1. This strain was deposited with the China Center for Type Culture Collection (CCTCC) on November 12, 2024, at the China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China. The collection number is CCTCC NO: M 20242532.

[0012] To accurately quantify the Cr(VI) removal efficiency of strain XM1, a single XM1 colony was streaked onto LB solid medium, the plate was inverted in a constant-temperature incubator, and incubated at 37°C for 48 hours. A single XM1 colony was picked from the LB solid plate and inoculated into LB liquid medium overnight to create the XM1 seed solution. This seed solution 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. The culture was maintained at pH 7, 37°C, and 180 rpm. Samples of the culture solution were removed every 12 hours, and the Cr(VI) removal efficiency of strain XM1 was measured and calculated over a 72-hour period.

[0013] The above experiment measured that: after being cultured for 48 hours under the conditions of pH = 7, temperature of 37°C and rotation speed of 180 rpm, the strain XM1 of the present invention had a removal rate of 97.87% for 50 mg / L Cr(VI), and a removal rate of 100% after 72 hours; the removal rate of 100 mg / L Cr(VI) reached 39.55% within 48 hours, and the removal rate reached 52.85% within 72 hours; the removal rate of 200 mg / L Cr(VI) reached 30.93% within 48 hours, and the removal rate reached 33.29% within 72 hours.

[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 further investigate the Cr(VI) removal mechanism of strain XM1, 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).

[0016] The characterization analysis results showed that: SEM analysis found that the morphological characteristics of the bacteria changed significantly after treatment with Cr(VI), the surface depression of strain XM1 intensified, accompanied by shrinkage, and the volume of the bacteria decreased and accompanied by bacterial rupture; EDX analysis found that Cr element was adsorbed on the surface of XM1 bacteria, suggesting that biological adsorption was involved in the removal of Cr(VI) by XM1; TEM results showed that some dark electron-dense particles such as black spots appeared after treatment with Cr(VI), which may be related to the accumulation of reduction products in the cells.

[0017] To investigate the removal efficiency of strain XM1 in real-world wastewater, a 4% (v / v) inoculum of seed culture 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. Culture samples were taken every 12 hours, and the Cr(VI) removal efficiency of strain XM1 in industrial wastewater was measured and calculated over a 48-hour period.

[0018] The results showed that strain XM1 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 30%.

[0019] To optimize the removal efficiency of strain XM1 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 efficiency. 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.

[0020] The results showed that, with the support of beef extract as a carbon source, XM1 was highly effective at removing Cr(VI) at pH levels of 4 to 6, with a maximum removal rate of 64.30% within 72 hours at pH 4. Under the optimal culture conditions of pH 4, at temperatures ranging from 25°C to 45°C, strain XM1 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 90% within 72 hours.

[0021] Compared with the prior art, the present invention has the following outstanding advantages and technical effects:

[0022] The present invention relatively completely completes the implementation case study of chromium removal functional strains from screening, laboratory effect research, mechanism research to industrial wastewater remediation application. Experiments found that the Brevibacterium otitidis XM1 of the present invention had a removal rate of 97.87% for 50 mg / L Cr(VI) after 48 hours and a removal rate of 100% after 72 hours; characterization analysis results suggested that the removal of Cr(VI) by XM1 was mainly through biological adsorption, and may also be accompanied by biological reduction; industrial wastewater remediation results showed that strain XM1 had a good removal effect under the support of beef extract carbon source, in an acidic environment of pH 4-6 and a temperature range of 25℃-45℃, among which the optimal removal rate was achieved at pH=4 and temperature of 45℃, and it could remove about 90% of Cr(VI) in industrial wastewater; through industrial wastewater treatment application and optimization research, the practicality of XM1 in complex environments was verified. The strain can be used for the bioremediation 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 particularly suitable for industrial continuous systems, with the advantages of low cost and no secondary pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the colony macroscopic morphology of the strain of the present invention.

[0024] Figure 2 This is the phylogenetic analysis tree of the strains of the present invention.

[0025] Figure 3 The removal rates of the strain of the present invention for different concentrations of Cr(VI) at different times.

[0026] Figure 4 These are SEM images of the bacterial cells of the strain of the present invention before and after Cr(VI) treatment.

[0027] Figure 5 These are TEM images of the strain of the present invention before and after Cr(VI) treatment.

[0028] Figure 6 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.

[0029] Figure 7 The figure shows the removal of Cr(VI) from industrial wastewater by the strain of the present invention under different pH conditions.

[0030] Figure 8 The figure shows the removal of Cr(VI) from industrial wastewater by the strain of the present invention at different temperatures. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the following embodiments will be further described in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention. On the contrary, the present invention encompasses any substitutions, modifications, equivalent methods and solutions made within the spirit and scope of the present invention as defined by the claims. Furthermore, in order to provide the public with a better understanding of the present invention, certain specific details are described in detail in the following detailed description of the present invention. Those skilled in the art can fully understand the present invention without the description of these details.

[0032] Example 1: Morphological characteristics of strain XM1

[0033] A single colony was streaked onto LB solid medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 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 h. Visual observation of the macroscopic morphology of the colonies revealed a round, pale yellow-green color with a smooth, moist surface, regular edges, no halo, and a slightly raised center (see ). Figure 1The SEM results of the characterization analysis in the subsequent Example 5 also showed that the colony morphology of strain XM1 was highly consistent with the typical characteristics of Brevibacterium.

[0034] Example 2: Acclimation, enrichment, and screening of strain XM1

[0035] (1) Given that Cr(VI) pollution is generally accumulative in the environment, the sludge samples used in the present invention were collected from a sewage outlet near the Binhai West Avenue beach (industrial concentration zone) 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 long-term development of clothing, shoes, hats, leather, chemical fiber, non-ferrous metals and other industries in Xiamen City, and there is a possibility of Cr(VI) pollution (Yu Mingxiao. Research on the Transformation and Development Strategy of Tong'an Industrial Concentration Zone [D]. Huaqiao University, 2019). Therefore, the microorganisms in the sludge sample may have the potential to convert Cr(VI) into Cr(III) under the natural selection of external environmental pressure, thereby achieving the removal of Cr(VI). In this example, three rounds of enrichment cycles were set up to domesticate and screen high-efficiency Cr(VI) removal bacteria. In the first enrichment cycle, 10 g of sludge sample was aseptically added to LB liquid medium containing 50 mg / L Cr(VI) and cultured in a constant temperature shaker at 37°C and 180 rpm for 7 days.

[0036] (2) After the first enrichment cycle, the culture was transferred to a new culture medium at a 10% inoculum volume, and the concentration of Cr(VI) 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.

[0037] (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% inoculation volume and cultured for another 7 days. This process was the third enrichment cycle.

[0038] (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 XM1 was obtained.

[0039] (5) A single colony was inoculated into sterilized LB liquid medium and cultured in a constant temperature shaker at 37°C and 180 rpm until the strain reached the logarithmic growth phase. The seed solution of strain XM1 was obtained and inoculated into LB liquid medium containing 50 mg / L and 100 mg / L Cr(VI) at a 4% (v / v) inoculum. The medium without Cr(VI) was used as a control. The culture was cultured at pH = 7, temperature 37°C, and rotation speed 180 rpm. The bacterial solution samples were taken every 24 h, the Cr(VI) concentration in the LB medium was measured, and the Cr(VI) removal rate of strain XM1 was calculated.

[0040] 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.

[0041] Example 3: Phylogenetic Analysis

[0042] In this example, the 16S rDNA sequence of strain XM1 was analyzed to determine its taxonomic status and clarify its relationship to other known strains. DNA was extracted from a freshly cultured strain XM1 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 XM1 by PCR. The PCR reaction system (50 μL total volume) 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 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 60 s, followed by a final extension at 72°C for 10 min. PCR products were examined by 1% agarose gel electrophoresis for the appearance of specific bands, which were approximately 1400 bp in size.

[0043] 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 measured 16S rDNA sequence (such as SEQ ID NO: 1 in the sequence list) was compared with the existing nucleic acid sequence using the EZbiocloud (https: / / www.ezbiocloud.net / ) database for similarity comparison analysis. Phylogenetic analysis was performed using MEGA-X software. The ClustalW algorithm was used for alignment to ensure accurate alignment of the sequences. After the alignment was completed, 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 the genetic distance, and 1000 Bootstrap tests were performed to evaluate the reliability of the phylogenetic tree branches. Finally, the phylogenetic analysis tree of strain XM1 was obtained, see Figure 2 .

[0044] from Figure 2 It can be seen that the 16s rDNA sequence of strain XM1 is 98.98% similar to that of Brevibacterium otitidis NCFB3053(T), and they form a clade together, so it was named Brevibacterium otitidis XM1.

[0045] Example 4: Cr(VI) removal efficiency test of strain XM1

[0046] To verify the removal efficiency of the strain for Cr(VI), a single colony of XM1 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 XM1 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).

[0047] Calculation of Cr(VI) removal rate:

[0048] 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.

[0049] (1)

[0050] The Cr(VI) removal efficiency of strain XM1 is shown in Figure 3 At 37°C and 180 rpm, strain XM1 had a good removal effect on 50 mg / L Cr(VI). After 48 h of culture, the removal rate of strain XM1 on 50 mg / L Cr(VI) reached 97.87%; within 72 h, the removal rate reached 100%; as the concentration of Cr(VI) increased, the removal effect decreased, and within 48 h, the removal rate on 100 mg / L Cr(VI) reached 39.55%, and within 72 h, the removal rate reached 52.85%; within 48 h, the removal rate on 200 mg / L Cr(VI) reached 30.93%, and within 72 h, the removal rate reached 33.29%. The above results indicate that Brevibacterium otitidis Brevibacterium otitidis XM1 has a significant removal effect on Cr(VI), and can completely remove 50 mg / L Cr(VI) within 72 h. Algoriphagus sp. CM16, the removal rate of 50 mg / L Cr(VI) reached 85.3% within 48 h (WenZhao Li, Meng Wang, Dong Xiao, et al. Exploration on the mechanismof Cr(VI) bioreduction by a novel cold and salt tolerant bacteriaAlgoriphagus sp. CM16 isolated from marine sediment [J]. EnvironmentalTechnology & Innovation, 2025). Silva et al. found Arthrobacter viscosusThe removal rate of 50 mg / L Cr(VI) reached 96% within 7 days (SILVA B, FIGUEIREDO H, QUINTELAS C, et al. Improved biosorption for Cr(VI) reduction and removal by Arthrobacter viscosus using zeolite [J]. International Biodeterioration & Biodegradation, 2012, 74(1):116-23.). Although there are cases of chromium removal by Brevibacterium in these chromium-removing bacteria phylogenetic tree studies, no research on the chromium removal performance of Brevibacterium otitidis has been found. This shows that strain XM1 is the first Brevibacterium otitidis to be discovered to have chromium removal performance.

[0051] Experimental Example 5: Investigation of the Cr(VI) removal mechanism of strain XM1

[0052] To investigate the removal mechanism of strain XM1, 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 bacterial 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 specialized company for scanning electron microscopy (SEM), energy dispersive spectroscopy (EDX), and transmission electron microscopy (TEM) examination.

[0053] SEM can observe the general morphology of XM1 bacteria (see Figure 4). 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 slight wrinkles on the surface and relatively plump, which is consistent with the typical characteristics of Bacillus brevis, and once again verified the morphological analysis results of strain XM1 in Example 1. In 50 mg / L Cr(VI), the wrinkles on the surface of the bacteria were obvious, and the shape became more irregular, the length became shorter, and the edges of some bacteria became blurred and accompanied by bacterial rupture; in 100 mg / L Cr(VI), the degree of concavity of the cell surface was aggravated, accompanied by atrophy, and the volume of the bacteria became smaller, but the rupture situation was not much different from that of the 50 mg / L Cr(VI) group, which indicates that high concentrations of Cr(VI) have a certain toxicity to strain XM1.

[0054] In order to verify whether the element Cr is adsorbed on the surface of the bacteria, EDX spectrum analysis was further performed. The results of the proportion of the content of each element on the surface of the bacteria of the strain of the present invention before and after Cr(VI) treatment are shown in Table 1.

[0055] Table 1

[0056]

[0057] 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 0.02% and 0.07%, respectively. This indicates that a small amount of Cr is adsorbed on the surface of bacterial strain XM1, 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 XM1 removes Cr(VI).

[0058] 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 5 After Cr(VI) treatment, strain XM1 showed spherical white matter and black plaques, suggesting that the spherical white matter may appear under conditions of extreme nutritional imbalance, while some dark electron-dense particles such as black plaques may be related to the accumulation of intracellular reduction products.

[0059] Example 6: Application and optimization of strain XM1 in the treatment of chromium-containing wastewater

[0060] 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.

[0061] First, the effects of different carbon sources on the Cr(VI) removal efficiency of the XM1 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 collected 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.

[0062] The removal results of strain XM1 under different carbon source support (see Figure 6 ) showed that the Cr(VI) removal rates of the glucose, fructose and galactose groups all reached the highest value at 24 h, which was about 30%; 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.

[0063] In order to optimize the removal effect of strain XM1 in actual wastewater, the effects of pH and temperature on the remediation of industrial wastewater by XM1 were explored with the support of beef extract, a carbon source with good removal effect.

[0064] 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.

[0065] The results of industrial wastewater removal by strain XM1 under different pH conditions (see Figure 7 ) showed that, with the support of beef extract, the Cr(VI) removal efficiency of XM1 gradually decreased with increasing pH. XM1 effectively removed Cr(VI) from wastewater in the acidic pH range of 4 to 6, achieving the highest removal efficiency at pH 4, with a removal rate of 64.30% after 72 hours. However, after the pH exceeded 7, the removal efficiency remained below 10%, likely due to the precipitation of Cr(VI) in the wastewater under alkaline conditions, which affected the strain's Cr(VI) removal function. Therefore, strain XM1 exhibited excellent Cr(VI) removal capabilities in acidic environments of 4 to 6, with the highest removal efficiency at pH 4, suggesting that strain XM1 is particularly suitable for treating acidic industrial wastewater.

[0066] Under the optimal culture conditions of pH 4, six temperature gradients of 5°C were established (20°C, 25°C, 30°C, 35°C, 40°C, and 45°C). 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.

[0067] The removal results of industrial wastewater by strain XM1 at different temperatures (see Figure 8) showed that within 12 hours, the Cr(VI) removal rate 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, XM1's removal rate significantly increased to approximately 40% at all temperatures, reaching nearly 50% at 45°C. At 48 hours, the 45°C experimental group achieved a 65% removal rate. After 72 hours, the 45°C experimental group achieved the highest removal rate, approaching 90%. All groups at other temperatures also achieved removal rates exceeding 50% within 72 hours. This indicates that this strain can remove Cr(VI) from wastewater in temperatures ranging from 25°C to 45°C and maintains high activity even at high temperatures (40°C to 45°C). This strain possesses excellent environmental adaptability and is suitable for use in high-temperature industrial wastewater discharge environments in southern China.

[0068] 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, Brevibacterium otitidis XM1, characterized by: The hexavalent chromium-removing bacterium Brevibacterium otitidis XM1 is named Brevibacterium otitidis XM1 and is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20242532. The 16S rDNA sequence of the Brevibacterium otitidis XM1 is shown in the sequence listing as SEQ ID NO:

1.

2. Use of the hexavalent chromium removal bacterium Brevibacterium otitidis XM1 as claimed in claim 1 in the treatment of chromium-containing wastewater.

3. The use according to claim 2, characterized in that The specific application method is: inoculating Brevibacteriumotitidis XM1 into wastewater containing Cr(VI), controlling the temperature at 45°C, pH=4, the rotation speed at 180 rpm, and the inoculation amount at 4% (v / v).

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

5. The use according to claim 2, characterized in that Brevibacterium otitidis XM1 was immobilized in a bioreactor to achieve continuous Cr(VI) removal.

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