Burkholderia and application thereof

Abstract

The invention relates to Burkholderia orbicola and application thereof, the Burkholderia orbicola is preserved in Guangdong Microbiological Culture Collection Center on September 4, 2025, the preservation number is GDMCC No: 66914, and the Burkholderia orbicola is classified and named as Burkholderia orbicola. The composite material has high tolerance to copper, lead and cadmium, the adsorption rate to copper can reach 44.59% in 24 h, the adsorption rate to lead can reach 98.71%, and the adsorption rate to cadmium can reach 48.39%. Therefore, the adsorbent can be used for resisting stress of heavy metals and adsorbing and removing copper, lead and cadmium pollution.

Description

Technical Field

[0001] This application relates to the field of heavy metal remediation technology, and in particular to a strain of Burkholderia and its application. Background Technology

[0002] To control the spread of heavy metal pollution, wastewater containing heavy metals needs to be pretreated to reduce the pollution load. Early mainstream pretreatment technologies were mainly physicochemical methods, including chemical precipitation, ion exchange, membrane separation, and reverse osmosis. However, these technologies generally have drawbacks: chemical precipitation easily generates secondary solid waste, ion exchange resin regeneration is costly, and membrane separation and reverse osmosis equipment have high investment and energy consumption. In contrast, adsorption technology, due to its high cost-effectiveness and environmental friendliness, has become one of the preferred methods for removing heavy metal ions from aqueous solutions.

[0003] Against this backdrop, microbial adsorption technology has become a research hotspot. Microorganisms such as bacteria, fungi, and algae are rich in functional groups such as amino and carboxyl groups, which can bind to metal ions through mechanisms such as electrostatic adsorption and complexation, achieving efficient immobilization and separation. Compared with traditional technologies, this technology has significant advantages: high adsorption efficiency; low cost, as microorganisms can be cultivated using industrial fermentation waste and agricultural waste; environmentally friendly, with no secondary pollution, and the adsorbed microorganisms can also recover metals and recycle themselves through desorption. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a strain of Burkholderia and its applications.

[0005] The first objective of this invention is to provide a strain of Burkholderia (… Burkholderia orbicola )FZHZ-CPCA1.

[0006] A second objective of this invention is to provide the aforementioned Burkholderia ( Burkholderia orbicola Application of FZHZ-CPCA1 in the adsorption and / or removal of heavy metals.

[0007] A third objective of this invention is to provide the aforementioned Burkholderia ( Burkholderia orbicola Application of FZHZ-CPCA1 in mitigating heavy metal pollution.

[0008] The fourth object of the present invention is to provide the aforementioned Burkholderia ( Burkholderia orbicola Application of FZHZ-CPCA1 in the treatment of heavy metal polluted water bodies.

[0009] The fifth object of the present invention is to provide the aforementioned Burkholderia ( Burkholderia orbicola Application of FZHZ-CPCA1 in the remediation of heavy metal contaminated soil.

[0010] The sixth object of the present invention is to provide the aforementioned Burkholderia ( Burkholderia orbicola Application of FZHZ-CPCA1 in the preparation of heavy metal removal agents.

[0011] The seventh objective of this invention is to provide a heavy metal removal agent or bactericide.

[0012] An eighth object of the present invention is to provide a method for adsorbing and / or removing heavy metals.

[0013] The ninth objective of this invention is to provide a method for mitigating heavy metal pollution.

[0014] The tenth objective of this invention is to provide a method for treating water or soil polluted by heavy metals.

[0015] To achieve the above objectives, the present invention is implemented through the following technical solution: A strain of Burkholderia ( Burkholderia orbicola FZHZ-CPCA1 was deposited on September 4, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC No: 66914, and classified as Burkholderia (…). Burkholderia orbicola The address for storage is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0016] This invention also claims protection for the following applications: Burkholderia (the aforementioned Burkholderia) Burkholderia orbicola Application of FZHZ-CPCA1 in the adsorption and / or removal of heavy metals, namely copper, lead and / or cadmium.

[0017] Burkholderia (the aforementioned Burkholderia) Burkholderia orbicola The application of FZHZ-CPCA1 in mitigating heavy metal pollution, wherein the heavy metals are copper, lead and / or cadmium.

[0018] Burkholderia (the aforementioned Burkholderia) Burkholderia orbicola The application of FZHZ-CPCA1 in the treatment of water bodies polluted by heavy metals, wherein the heavy metals are copper, lead and / or cadmium.

[0019] Burkholderia (the aforementioned Burkholderia) Burkholderia orbicola Application of FZHZ-CPCA1 in the remediation of soil contaminated with heavy metals, wherein the heavy metals are copper, lead and / or cadmium.

[0020] Burkholderia (the aforementioned Burkholderia) Burkholderia orbicola Application of FZHZ-CPCA1 in the preparation of heavy metal removal agents, wherein the heavy metals are copper, lead and / or cadmium.

[0021] And a heavy metal removal agent or bactericidal agent containing the aforementioned Burkholderia (…Burkholderia orbicola FZHZ-CPCA1, wherein the heavy metal is copper, lead and / or cadmium.

[0022] A method for adsorbing and / or removing heavy metals, comprising applying the aforementioned heavy metal removal agent or bacterial agent, or the aforementioned Burkholderia (… Burkholderia orbicola FZHZ-CPCA1, wherein the heavy metal is copper, lead and / or cadmium.

[0023] A method for mitigating heavy metal pollution, comprising applying the aforementioned heavy metal removal agent or bacterial agent, or the aforementioned Burkholderia (… Burkholderia orbicola FZHZ-CPCA1, wherein the heavy metal is copper, lead and / or cadmium.

[0024] A method for treating heavy metal-contaminated water or soil, comprising applying the aforementioned heavy metal removal agent or bacterial agent, or the aforementioned Burkholderia (… Burkholderia orbicola FZHZ-CPCA1, wherein the heavy metal is copper, lead and / or cadmium.

[0025] Compared with the prior art, the present invention has the following beneficial effects: This invention relates to Burkholderia strains screened from environments contaminated with heavy metals. Burkholderia orbicola FZHZ-CPCA1 is suitable for the removal and remediation of copper, lead, and cadmium pollution; the CAS-Cu(II) plate fading zone phenomenon confirms Burkholderia (… Burkholderia orbicola FZHZ-CPCA1 can bind to copper, thereby reducing the concentration of free toxic copper in the environment; Burkholderia ( Burkholderia orbicola FZHZ-CPCA1 exhibits high tolerance to copper, lead, and cadmium, with an adsorption rate of 44.59% for copper, 98.71% for lead, and 48.39% for cadmium after 24 hours. This invention relates to Burkholderia (…). Burkholderia orbicola FZHZ-CPCA1 plays an important role in resisting and adsorbing copper, lead, and cadmium. Therefore, it can be used to resist heavy metal stress and to adsorb and remove copper, lead, and cadmium pollution. Attached Figure Description

[0026] Figure 1 The image shows the chelating ability of strain FZHZ-CPCA1 for copper.

[0027] Figure 2 Colony morphology observation for strain FZHZ-CPCA1.

[0028] Figure 3The results of Gram staining microscopy of strain FZHZ-CPCA1 are shown.

[0029] Figure 4 A phylogenetic tree of FZHZ-CPCA1 was constructed based on the 16S rRNA gene sequence.

[0030] Figure 5 The growth of strain FZHZ-CPCA1 in LB agar medium containing Cu is shown.

[0031] Figure 6 The growth of strain FZHZ-CPCA1 in LB agar medium containing Pb is shown.

[0032] Figure 7 The growth of strain FZHZ-CPCA1 in LB agar medium containing Cd is shown.

[0033] Figure 8 The growth of strain FZHZ-CPCA1 in LB broth medium containing Cu is shown.

[0034] Figure 9 The growth of strain FZHZ-CPCA1 in LB broth medium containing Pb is shown.

[0035] Figure 10 The growth of strain FZHZ-CPCA1 in LB broth containing Cd is shown. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0037] LB broth medium: 10.0 g peptone, 5.0 g yeast extract, 10.0 g sodium chloride, 1 L deionized water, pH adjusted to 6.8–7.2.

[0038] LB agar medium: 10.0 g peptone, 5.0 g yeast extract, 10.0 g sodium chloride, 1 L deionized water, 15 g / L agar, pH 6.9–7.3.

[0039] CAS-Cu(II) detection solution: Take 6 mL of 10 mmol / L copper sulfate solution (solvent: 10 mmol / L HCl), 6 mL of 2 mmol / L chromaine S (CAS) solution, 12 mL of 10 mmol / L hexadecyltrimethylammonium bromide (CTAB) solution, 1 mL of sodium borate buffer solution (pH 6.0–6.3), and 2 mL of 5% Tween 80 solution (v / v). Mix thoroughly, and adjust the pH to 4.0–4.3 with 0.1 mol / L NaOH and 0.1 mol / L HCl to finally prepare the purple detection solution.

[0040] Example 1: Isolation and Purification of Strains I. Experimental Methods Soil samples were collected from the environment surrounding a heavy metal processing plant in Zhoukou City, Henan Province. 10 g of soil sample was weighed and placed in 90 mL of sterile physiological saline. The mixture was shaken at 37℃ and 130 r / min for 30 min, and the supernatant was collected after standing. The supernatant was added to LB broth medium containing 0.5 mmol / L each of copper, cadmium, and lead for enrichment, and incubated at 37℃ and 130 r / min for 24 h. The supernatant was then collected and diluted 10 times. 2 10 3 10 4 and 10 5 Then, take 50 μL of each sample and spread it onto LB agar medium. Incubate at 37°C upside down for 2–3 days to isolate single colonies. Pick single colonies for streak purification. Inoculate the purified single colonies into LB broth medium for amplification to obtain multiple test strains.

[0041] Each test strain was individually inoculated onto LB agar medium and cultured at 37°C for 1 day. After the strains secreted sufficient extracellular polymers, an appropriate amount of CAS-Cu(II) detection solution was added to cover the colonies. The reaction was allowed to stand for 1 day, and then the detection solution was discarded. The plates were rinsed with deionized water, and the colony diameter and bleaching zone diameter were measured three times. The ratio (R1) of the bleaching zone diameter (D1) to the colony diameter (d1) was calculated. A larger R1 indicates a better ability of the strain to chelate copper with its extracellular active substances.

[0042] II. Experimental Results The results are as follows Figure 1 As shown, the R1 ratio determines the strength of the strain's copper chelating ability. A D1 / d1 = R1 value of 1 indicates that the strain's secreted extracellular material has no copper chelating activity. The CAS-copper(II) plate results show that the strain numbered FZHZ-CPCA1 has an R1 value of 3.95, indicating that the highly active extracellular polymer secreted by strain FZHZ-CPCA1 has a strong copper chelating ability. Example 2: Strain Identification I. Experimental Methods 1. The strain FZHZ-CPCA1 isolated and purified in Example 1 was streaked on LB agar medium and cultured for 24 h. The colonies were observed and Gram staining and microscopic examination and physiological and biochemical tests were performed.

[0043] 2. Using a bacterial genomic DNA preparation kit, total DNA was extracted from strain FZHZ-CPCA1 isolated and purified in Example 1. Using this DNA as a template, PCR amplification was performed using 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) as upper and lower primers.

[0044] The total gene amplification system consisted of 20 µL: 0.8 µL each of upper and lower primers, 2 µL of 10×PCR buffer (without MgCl2), 1.2 µL of MgCl2, 0.4 µL of dNTP (10 mM), 0.4 µL of Taq polymerase, 14.4 µL of ddH2O, and 1 µL of template.

[0045] PCR reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 90 s, 35 cycles; 72℃ extension for 10 min. DNA extraction kits and PCR product sequencing services were provided by Sangon Biotech, a Shanghai-based biotechnology company.

[0046] After purification, the amplified products were sequenced and analyzed. Then, the EzBioCloud program was used to obtain the possible similar groups of the sequencing groups. Using MEGA 5 software, a phylogenetic tree was constructed using the neighbor-joining method with a bootstrap value of 1000.

[0047] The species to which the target strain FZHZ-CPCA1 belongs was determined by a combination of phylogenetic tree analysis, colony morphology analysis, and physiological and biochemical tests.

[0048] II. Experimental Results 1. Figure 2 The results showed that when strain FZHZ-CPCA1 was streaked onto LB solid medium, its colony surface was moist and smooth, with small protrusions, semi-transparent appearance, and neat edges. Figure 3 The microscopic examination revealed that strain FZHZ-CPCA1 exhibited rod-shaped morphology and was a Gram-negative bacterium.

[0049] 2. The physiological and biochemical test results of strain FZHZ-CPCA1 are shown in Table 1.

[0050] Table 1: Physiological and biochemical test results of strain FZHZ-CPCA1

[0051] 3. The nucleotide sequence of the 16S rDNA gene of strain FZHZ-CPCA1 is shown in SEQ ID NO: 1. Upon comparison, it is similar to that of Burkholderia (…). Burkholderia orbicola The sequence similarity of the TAtl-371(T) gene was 99.63%. A phylogenetic tree was constructed using neighbor-joining analysis. Figure 4 The isolated strain FZHZ-CPCA1 belongs to Burkholderia ( ). Burkholderia orbicola Burkholderia ( ), that is, Burkholderia ( Burkholderia orbicola FZHZ-CPCA1. It was deposited on September 4, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), accession number GDMCCNo: 66914, and classified as: Burkholderia (…). Burkholderia orbicola The address for storage is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0052] Example 3: Strain FZHZ-CPCA1 exhibits high tolerance to copper, lead, and cadmium. I. Experimental Methods The strain FZHZ-CPCA1 isolated and purified in Example 1 was inoculated into LB broth medium and cultured at 37°C and 130 r / min for 24 h to obtain a bacterial suspension containing Burkholderia (…). Burkholderia orbicola Fermentation products of FZHZ-CPCA1.

[0053] Dilute the bacterial suspension to OD using sterile water. 600 After setting it to 1.000, it was then diluted sequentially to 0, 10, and so on. 1 10 2 10 3 and 10 4 The concentration of the bacterial suspension was increased by 10 times.

[0054] Take 5 μL of the five bacterial suspensions mentioned above and inoculate them sequentially onto LB agar medium containing Cu (0 mmol / L, 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L), Pb (0 mmol / L, 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L), and Cd (0 mmol / L, 0.5 mmol / L, 1 mmol / L). Incubate at 37℃ for 24 h and take pictures for analysis using a gel imaging system.

[0055] In addition, the five bacterial suspensions of the above concentrations were inoculated at a 1% (v / v) in LB broth medium containing Cu (2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L), Pb (0 mmol / L, 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L), and Cd (0 mmol / L, 0.5 mmol / L, 1.0 mmol / L), respectively, and incubated at 37℃ for 24 h at 130 r / min. The absorbance of the bacterial suspension at 600 nm was measured every 12 h to evaluate the growth of the strain.

[0056] II. Experimental Results (1) The results are as follows Figure 5 The results showed that strain FZHZ-CPCA1 grew well in LB agar medium at Cu concentrations of 0–3 mmol / L, indicating that 3 mmol / L Cu did not affect the growth of strain FZHZ-CPCA1; at a Cu concentration of 4 mmol / L, the growth of strain FZHZ-CPCA1 was inhibited, but it could still grow normally; at a Cu concentration of 5 mmol / L, the growth of strain FZHZ-CPCA1 was obviously greatly inhibited and could not grow normally.

[0057] The results are as follows Figure 6 The results showed that in LB agar medium, the growth of strain FZHZ-CPCA1 was not inhibited by Pb concentrations of 0–4 mmol / L and could grow normally; the growth of strain FZHZ-CPCA1 was significantly inhibited at a Pb concentration of 5 mmol / L; and strain FZHZ-CPCA1 basically did not grow at a Pb concentration of 6 mmol / L.

[0058] The results are as follows Figure 7 The results showed that in LB agar medium, strain FZHZ-CPCA1 grew normally without being inhibited by Cd at a concentration of 0–0.5 mmol / L; at a Cd concentration of 1 mmol / L, strain FZHZ-CPCA1 barely grew.

[0059] (2) The results are as follows Figures 8 to 10 The results showed that, without metal treatment, Burkholderia orbicola FZHZ-CPCA1 grew rapidly after 24 h of culture, with OD... 600 nm It can reach 1.41 or higher.

[0060] In LB broth medium, at Cu concentrations of 3 mmol / L and 4 mmol / L, strain FZHZ-CPCA1 showed an OD value of [missing value] after 24 h.600 nm The concentrations reached 1.13 and 0.16, respectively; at Pb concentrations of 4 mmol / L and 5 mmol / L, the OD values ​​of strain FZHZ-CPCA1 at 24 h were... 600 nm The OD values ​​reached 1.29 and 0.97, respectively; at Cd concentrations of 0.5 mmol / L and 1 mmol / L, strain FZHZ-CPCA1 showed OD values ​​of [missing value] over 24 hours. 600 nm The values ​​can reach 0.64 and 0.064 respectively.

[0061] The above results indicate that strain FZHZ-CPCA1 has a high tolerance to copper, lead, and cadmium.

[0062] Example 4: Strain FZHZ-CPCA1 exhibits high adsorption rates for copper, lead, and cadmium. I. Experimental Methods The strain FZHZ-CPCA1 isolated and purified in Example 1 was inoculated into LB broth medium and cultured at 37°C and 130 r / min for recovery to the optical density OD. 600 nm =0.6.

[0063] After resuscitation, strain FZHZ-CPCA1 was inoculated at a 2% inoculum in LB broth containing Cu (1 mmol / L), Pb (1 mmol / L), and Cd (0.5 mmol / L), respectively. The culture was then carried out at 37°C and 130 r / min. At 24 h, the supernatant was collected after centrifugation at 8000 rpm for 15 min, and the concentrations of residual copper, lead, and cadmium in the supernatant were determined by inductively coupled plasma mass spectrometry (ICP-MS). The adsorption rate of strain FZHZ-CPCA1 was calculated according to the following formula: , Where R is the adsorption rate; C0 is the initial concentration (mmol / L) of Cu, Pb, or Cd; C e The concentration of Cu, Pb, or Cd in the supernatant at 24 h.

[0064] II. Experimental Results The results are shown in Table 2. After 24 h, strain FZHZ-CPCA1 achieved an adsorption rate of 44.59% for copper, a high adsorption rate of 98.71% for lead, and an adsorption rate of 48.39% for cadmium. This indicates that it exhibits high adsorption rates for copper, lead, and cadmium solutions. Heavy metal removal agents prepared using strain FZHZ-CPCA1 can be applied on a large scale in the remediation of copper, lead, and cadmium contaminated soil or water.

[0065] Table 2 Burkholderia orbicola Adsorption rates of FZHZ-CPCA1 for Cu, Pb and Cd

[0066] Example 5: Strain FZHZ-CPCA1 exhibits high adsorption rates for lead and cadmium. I. Experimental Methods The strain FZHZ-CPCA1 isolated and purified in Example 1 was inoculated into LB liquid medium and cultured at 37°C and 130 r / min for recovery to the optical density OD. 600 nm =0.6.

[0067] After resuscitation, strain FZHZ-CPCA1 was inoculated at a 1% inoculum into LB liquid medium containing Cd (0.2 mmol / L) and Pb (0.596 mmol / L), respectively. The medium was incubated at 35°C with a shaking incubator at 180 rpm. 5 mL of the bacterial culture was collected daily, centrifuged at 8000 rpm for 15 min, and the supernatant was collected. The concentrations of residual lead and cadmium in the supernatant were determined using inductively coupled plasma mass spectrometry (ICP-MS). The adsorption rate of strain FZHZ-CPCA1 was calculated using the following formula: , Where R is the adsorption rate; C0 is the initial concentration of Pb or Cd (mmol / L); C e The concentration of Pb or Cd in the supernatant at 24 h.

[0068] II. Experimental Results The results are shown in Table 3. On the fifth day, strain FZHZ-CPCA1 achieved an adsorption rate of 98.84% for lead and 74.82% for cadmium.

[0069] Table 3: Adsorption rates of Pb and Cd by strain FZHZ-CPCA1

[0070] The results show that strain FZHZ-CPCA1 exhibits a high adsorption rate for high concentrations of lead and cadmium-containing solutions. Preparing strain FZHZ-CPCA1 into a heavy metal removal agent could enable its large-scale application in the remediation of lead- and cadmium-contaminated soil or water.

Claims

1. A strain of Burkholderia ( Burkholderia orbicola FZHZ-CPCA1, characterized in that, On September 4, 2025, it was deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66914, and classified as Burkholderia (…). Burkholderia orbicola ).

2. Burkholderia as described in claim 1 ( Burkholderia orbicola The application of FZHZ-CPCA1 in the adsorption and / or removal of heavy metals is characterized by, The heavy metals are copper, lead, and / or cadmium.

3. Burkholderia as described in claim 1 ( Burkholderia orbicola The application of FZHZ-CPCA1 in mitigating heavy metal pollution is characterized by, The heavy metals are copper, lead, and / or cadmium.

4. Burkholderia as described in claim 1 ( Burkholderia orbicola The application of FZHZ-CPCA1 in the treatment of heavy metal polluted water is characterized by, The heavy metals are copper, lead, and / or cadmium.

5. Burkholderia as described in claim 1 ( Burkholderia orbicola The application of FZHZ-CPCA1 in the remediation of heavy metal contaminated soil is characterized by, The heavy metals are copper, lead, and / or cadmium.

6. Burkholderia as described in claim 1 ( Burkholderia orbicola The application of FZHZ-CPCA1 in the preparation of heavy metal removal agents is characterized by, The heavy metals are copper, lead, and / or cadmium.

7. A heavy metal removal agent or bactericidal agent, characterized in that, Contains Burkholderia as described in claim 1 ( Burkholderia orbicola FZHZ-CPCA1, wherein the heavy metal is copper, lead and / or cadmium.

8. A method for adsorbing and / or removing heavy metals, characterized in that, Apply the heavy metal removal agent or bacterial agent according to claim 7, or Burkholderia according to claim 1. Burkholderia orbicola FZHZ-CPCA1, wherein the heavy metal is copper, lead and / or cadmium.

9. A method for mitigating heavy metal pollution, characterized in that, Apply the heavy metal removal agent or bacterial agent according to claim 7, or Burkholderia according to claim 1. Burkholderia orbicola FZHZ-CPCA1, wherein the heavy metal is copper, lead and / or cadmium.

10. A method for treating heavy metal-polluted water or soil, characterized in that, Apply the heavy metal removal agent or bacterial agent according to claim 7, or Burkholderia according to claim 1. Burkholderia orbicola FZHZ-CPCA1, wherein the heavy metal is copper, lead and / or cadmium.

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