Ochrobactrum and application thereof
By screening out the *Aureobacterium tumefaciens* QLM-2, which can grow using MTBE as the sole carbon source, and applying this strain in a biofilm reactor, the problem of low biodegradation efficiency of existing MTBE strains was solved, achieving efficient degradation of MTBE and maintaining stable performance in complex environments.
Patent Information
- Application Number
- CN202411447426.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-16
- Publication Date
- 2026-04-17
AI Technical Summary
Existing MTBE biodegrading strains suffer from slow microbial growth, low degradation rate, and low cell yield. Furthermore, the presence of benzene compounds in MTBE-contaminated environments inhibits their degradation effect.
A strain of Ochrobactrum sp., with accession number GDMCC No: 63825, was provided. It can grow with MTBE as the sole carbon source and form a biofilm in a biofilm reactor using polyurethane as the packing material to treat MTBE-contaminated wastewater.
It achieves efficient degradation of MTBE, with a degradation rate of 71.2%, and can maintain a high degradation rate even in the presence of benzene series compounds. It is adaptable to complex pollution scenarios and has a wide range of applications and competitive advantages.
Smart Images
Figure CN121874022A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of microbiology and environmental protection technology, specifically to a strain of *Bacillus anthracis* and its applications. Background Technology
[0002] Methyl tert-Buty Ether (MTBE) is currently the most commonly used oxygenated gasoline additive to increase the octane rating of gasoline. During production, sales, use, and storage, MTBE is released into the environment, causing pollution problems in groundwater, surface water, soil, and sediments. The U.S. Environmental Protection Agency has listed MTBE as a potential human carcinogen. Currently, some companies are building new MTBE production facilities or expanding existing ones. Given the current situation in China, alternative oxide additive technologies are not yet mature, and banning MTBE is not realistic. Therefore, it is crucial and urgent to research technologies for degrading MTBE-contaminated groundwater and soil in the environment.
[0003] Due to its stable chemical structure, high water solubility, low organic carbon fractionation coefficient, and resistance to soil adsorption and migration, conventional physical treatment methods such as adsorption and aeration are ineffective in removing MTBE. Biodegradation technology has become the recognized effective and green degradation method. However, because MTBE is a synthetic xenobiotic compound, natural microbial communities lack the genetic information for degradation, leading to problems such as low degradation efficiency, long adaptation periods, and low cell yield in microbial degradation technologies, limiting their industrial application. Furthermore, in real-world environments contaminated with MTBE, especially in oil refineries, other organic pollutants often coexist with MTBE. Benzene series compounds (benzene, toluene, ethylbenzene, and xylene, abbreviated as BTEX), as a major component of crude oil, are the most significant coexisting pollutants with MTBE. Some studies have shown that the presence of BTEX inhibits MTBE degradation, further complicating the practical application of biodegradable MTBE. Summary of the Invention
[0004] The purpose of this invention is to overcome the problems of slow microbial growth, low degradation rate and low cell yield of existing MTBE biodegrading strains, and to provide a strain of *Bacillus cereus* and its application, which can be used for the harmless bioremediation of MTBE-contaminated wastewater.
[0005] To achieve the above objectives, the present invention provides a bacterium called Ochrobactrum sp., which has the accession number GDMCC No: 63825.
[0006] A second aspect of the present invention provides a bacterial agent containing *Bacillus cereus* as described above.
[0007] Preferably, the bacterial agent is a liquid bacterial agent.
[0008] A third aspect of the present invention provides a method for preparing a bacterial agent, the method comprising: inoculating the above-described Aureobacterium tumefaciens into a liquid culture medium for cultivation.
[0009] Preferably, the method further includes: inoculating the obtained bacterial suspension into a fermentation medium for fermentation.
[0010] Preferably, the fermentation medium comprises glucose, corn steep liquor powder, soybean meal, ammonium sulfate, magnesium sulfate, sodium chloride, ferrous sulfate, calcium chloride, and polyether defoamer.
[0011] Preferably, the fermentation medium contains 1.2–1.8 wt% glucose, 0.85–1 wt% corn steep liquor powder, 3–4 wt% soybean meal, 0.01–0.02 wt% ammonium sulfate, 0.03–0.05 wt% magnesium sulfate, 0.01–0.03 wt% sodium chloride, 0.002–0.005 wt% ferrous sulfate, 0.01–0.02 wt% calcium chloride, and 0.05–0.2 wt% polyether defoamer.
[0012] A fourth aspect of the present invention provides a bacterial agent prepared by the method described above.
[0013] The fifth aspect of the present invention provides the use of the *Aureobacillus* or the bacterial agent described above in the degradation of methyl tert-butyl ether.
[0014] The sixth aspect of the present invention provides the application of the above-described Aureobacterium or the above-described bacterial agent in the treatment of MTBE contaminated wastewater.
[0015] The seventh aspect of the present invention provides a method for treating MTBE-contaminated wastewater, the method comprising: contacting the MTBE-contaminated wastewater with the *Bacillus cereus* or the bacterial agent described above.
[0016] Preferably, the treatment method is carried out in a biofilm reactor.
[0017] Preferably, the treatment method uses polyurethane as a filler and the *Bacillus cereus* as a biofilm-forming organism, and inoculates the biofilm through wastewater containing MTBE.
[0018] This invention provides a novel strain of *Bacillus anthocyanin*, with accession number GDMCC No: 63825. This strain can grow using MTBE as the sole carbon source and has the ability to efficiently degrade MTBE. Using this strain as a biofilm-forming strain and polyurethane as a packing material, a biofilm can be cultivated in a biofilm reactor to treat MTBE-contaminated wastewater, achieving efficient degradation of MTBE. Therefore, this strain can be used for the industrial treatment of MTBE-contaminated wastewater.
[0019] Furthermore, the presence of benzene compounds does not significantly inhibit the degradation rate of MTBE by this strain. Therefore, this strain has a wider range of applications and a clear competitive advantage.
[0020] Biological Preservation
[0021] The strain provided by this invention was deposited on September 21, 2023, with accession number GDMCC No: 63825, classified and named as Ochrobactrum sp. QLM-2, and deposited at Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China. Attached Figure Description
[0022] Figure 1 This is a scanning electron microscope image of *Bacillus anthracis* QLM-2 provided by this invention;
[0023] Figure 2 This is a colony morphology diagram of *Bacillus anserinae* QLM-2 provided by this invention;
[0024] Figure 3 This is a phylogenetic tree diagram of the 16S rDNA of Bacillus cereus QLM-2 provided by the present invention;
[0025] Figure 4 This is a graph showing the degradation performance of Bacillus angulans QLM-2 on MTBE at different temperatures provided by this invention;
[0026] Figure 5 This is a graph showing the effect of Bacillus angulans QLM-2 on the degradation performance of MTBE in the presence of BTEX concomitant organisms, provided by this invention.
[0027] Figure 6 This is a process flow diagram of the simulated MTBE wastewater treatment in Embodiment 7 of the present invention. Detailed Implementation
[0028] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.
[0029] The endpoints and any values of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of the various ranges, the endpoint values of the various ranges and individual point values, and individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.
[0030] Existing MTBE biodegrading strains often suffer from slow microbial growth, low degradation rates, and low cell yields, which have become bottlenecks for the further industrial application of biodegradation reactors. Furthermore, in real-world environments contaminated with MTBE, especially in refineries, other organic pollutants often coexist with MTBE. Benzene series compounds (benzene, toluene, ethylbenzene, and xylene, abbreviated as BTEX), as a major component of crude oil, are the most significant coexisting pollutants with MTBE. Some studies have shown that the presence of BTEX inhibits MTBE degradation, further complicating the practical application of biodegradable MTBE.
[0031] In view of this, the present invention provides a strain of Ochrobactrum sp., with accession number GDMCCNo: 63825 and accession date of September 21, 2023.
[0032] Using *Bacillus cereus* as the biofilm-forming strain and polyurethane as the packing material, a biofilm is formed in a biofilm reactor. Within 7 days of biofilm formation, the degradation rate of MTBE reaches 71.2%, achieving highly efficient MTBE degradation and laying the foundation for further industrial application of biofilm reactors for degrading bacteria. Furthermore, the presence of benzene compounds does not significantly inhibit the degradation rate of MTBE by this strain, enabling it to efficiently degrade MTBE in complex real-world pollution scenarios. Therefore, the *Bacillus cereus* provided by this invention can not only be used for efficient industrial treatment of MTBE wastewater but also has a wide range of applications and a significant competitive advantage.
[0033] In this invention, the *Bacillus cereus* is isolated and screened from MTBE-contaminated soil. The *Bacillus cereus* can grow using MTBE as the sole carbon source and has the ability to efficiently degrade MTBE.
[0034] In a specific implementation, the screening method for *Ailuropoda spp.* includes the following steps:
[0035] (1) Add MTBE-contaminated soil to sterile water, shake for 10-20 hours, centrifuge, and take the supernatant containing bacteria;
[0036] (2) Spread the bacterial supernatant onto LB solid medium and incubate for 24-50 h to obtain colonies;
[0037] (3) The colonies were inoculated into an enrichment medium and cultured with shaking for 10-30 h to obtain a culture solution;
[0038] (4) The culture medium is separated and purified until purified colonies with a single morphology are obtained, thereby obtaining the target strain;
[0039] In step (3), the enrichment medium is an inorganic salt medium with MTBE as the sole carbon source.
[0040] In one specific embodiment, in step (1), the MTBE-contaminated soil is collected from a depth of 30 to 50 cm.
[0041] In one specific embodiment, in step (2), the LB solid culture medium formula includes: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl and 10 g / L agar, pH 7.0.
[0042] In a preferred embodiment, in step (3), the enrichment culture medium contains 50-150 mg / L MTBE and 100-200 mg / L yeast extract.
[0043] In the most preferred embodiment, in step (3), the enrichment culture medium comprises: 100 mg / L MTBE, 200 mg / L yeast extract, 0.5 g / L potassium dihydrogen phosphate (KH2PO4), 0.5 g / L dipotassium hydrogen phosphate (K2HPO4), 0.1 g / L calcium chloride (CaCl2), 0.2 g / L magnesium sulfate (MgSO4), 0.1 g / L sodium chloride (NaCl), and 2 g / L ammonium sulfate ((NH4)2SO4), pH 7.0.
[0044] More preferably, each 1L of the enrichment medium also contains 1mL of trace element solution. The trace element solution is formulated as follows: 0.022g ZnSO4·4H2O, 0.039g Na2MoO4·2H2O, 0.186g MnCl2·4H2O, 0.005g Co(NO3)2·6H2O, and 0.008g CuSO4·5H2O dissolved in each 100mL of deionized water.
[0045] In specific implementation, step (4) includes: subjecting the culture medium to 10... -5 10 -6 and 10 -7Three dilutions were performed, and then 100 μL of the bacterial suspension was diluted and spread onto fresh LB solid medium. The culture was incubated in a constant temperature incubator. The above steps were repeated until single colonies grew on the plate. Colonies that grew faster and had different colors and morphologies were selected and inoculated onto new LB solid medium using the streak plate method. The above streak isolation process was repeated until purified colonies with a single morphology were formed.
[0046] In the screening method described in this invention, a target strain capable of efficiently degrading MTBE was finally screened and named QLM-2.
[0047] In this invention, the strain QLM-2 has a rod-shaped morphology.
[0048] In this invention, the colony characteristics of strain QLM-2 are as follows: the colony shape is round, the color is milky white, the edges are neat, and it is not sticky.
[0049] Furthermore, the 16S rDNA gene sequencing results of the strain QLM-2 are shown in SEQ ID NO: 1.
[0050] Based on comprehensive morphological observation and molecular biological characteristics, strain QLM-2 was identified as belonging to Ochrobactrum sp. and named Ochrobactrum QLM-2.
[0051] The present invention also proposes a bacterial agent containing *Aureobacillus cereus* as described above.
[0052] This invention does not limit the specific components of the bacterial agent, as long as it contains *Bacillus cereus* as described above. It can be a *Bacillus cereus* bacterial suspension, a *Bacillus cereus* fermentation broth, or a concentrated culture of *Bacillus cereus*. It is understood that the bacterial agent may also contain other bacterial strains used in combination with *Bacillus cereus* to enhance the degradation effect on MTBE.
[0053] In a preferred embodiment, the bacterial agent is a liquid bacterial agent.
[0054] The present invention also proposes a method for preparing a bacterial agent, the method comprising: inoculating the above-described Aureobacterium valerate into a liquid culture medium for cultivation.
[0055] In a first specific embodiment, the bacterial agent is a suspension of Bacillus anthracis, and its preparation method includes the following steps:
[0056] A1. Spread the Aleucobacterium leucosus onto LB solid medium and incubate at a constant temperature to activate the bacterial strain;
[0057] A2. The activated *Bacillus anguishii* was inoculated into LB liquid medium and cultured with shaking at 25–35°C and 100–300 rpm for 16–48 h to obtain the seed culture solution.
[0058] A3. The seed culture solution is inoculated into 1L of LB liquid medium at a volume ratio of 1-10%, and cultured with shaking at 25-35℃ and 100-300rpm, with aeration at a rate of 0.2-0.6L / min, for 16-48h to obtain a suspension of Bacillus cereus.
[0059] In a second specific embodiment, the bacterial agent is a fermentation broth of *Bacillus anthracis*, and the method includes: inoculating the *Bacillus anthracis* as described above into a liquid culture medium for cultivation, and inoculating the obtained bacterial suspension into a fermentation culture medium for fermentation. More specifically, the method includes the following steps:
[0060] S1. Activation of bacterial strain: The *Bacillus cereus* was spread on LB solid medium and incubated at a constant temperature to activate the bacterial strain.
[0061] S2, Primary seed culture: The activated *Bacillus cereus* was inoculated into LB liquid medium and cultured with shaking at 25–35°C and 100–300 rpm for 16–48 h to obtain the primary seed culture.
[0062] S3. Secondary seed culture: The primary seed culture is inoculated into liquid LB medium at an inoculation rate of 1-10% by volume, and cultured with shaking at 25-35℃ and 100-300 rpm for 16-48 h to obtain the secondary seed culture.
[0063] S4. Fermentation: The secondary seed culture solution is inoculated into the fermentation medium at an inoculation rate of 5-10% by volume for fermentation culture. The fermentation conditions are controlled as follows: temperature 27-35℃, rotation speed 120-300rpm, gas-liquid ratio (aeration ratio) of 1:(1-2). Fermentation is stopped when dissolved oxygen rises.
[0064] In a preferred embodiment, the LB liquid culture medium is formulated with: 10 g / L peptone, 5 g / L yeast extract and 10 g / L NaCl, pH 7.0.
[0065] In a preferred embodiment, the LB solid culture medium is formulated with: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl and 10 g / L agar, pH 7.0.
[0066] In a preferred embodiment, in step S4, the fermentation medium contains glucose, corn steep liquor powder, soybean meal, ammonium sulfate, magnesium sulfate, sodium chloride, ferrous sulfate, calcium chloride, and polyether defoamer.
[0067] More preferably, the fermentation medium contains 1.2–1.8 wt% glucose, 0.85–1 wt% corn steep liquor powder, 3–4 wt% soybean meal, 0.01–0.02 wt% ammonium sulfate, 0.03–0.05 wt% magnesium sulfate, 0.01–0.03 wt% sodium chloride, 0.002–0.005 wt% ferrous sulfate, 0.01–0.02 wt% calcium chloride, and 0.05–0.2 wt% polyether defoamer.
[0068] The present invention also proposes a bacterial agent prepared by the method described above.
[0069] This invention also proposes the application of *Bacillus cereus* or its agent as described above in the degradation of methyl tert-butyl ether (MTBE). This invention does not limit the specific scenarios in which *Bacillus cereus* or its agent degrades MTBE; it can be used as long as MTBE is present. Specifically, this could be in MTBE-contaminated soil or MTBE-contaminated wastewater.
[0070] The present invention also proposes the application of the above-mentioned *Bacillus angustifolius* or bacterial agent in the treatment of MTBE contaminated wastewater.
[0071] The present invention also proposes a method for treating MTBE-contaminated wastewater, the method comprising: contacting the MTBE-contaminated wastewater with the above-mentioned Bacillus cereus or bacterial agent.
[0072] To facilitate large-scale industrial treatment of MTBE wastewater, in a preferred embodiment, the treatment method is carried out in a biofilm reactor.
[0073] More preferably, the treatment method uses polyurethane as a filler, adopts an inoculation and biofilm formation method, uses the *Ailuropoda spp.* as the biofilm formation strain, and utilizes wastewater containing MTBE for biofilm formation.
[0074] In a specific implementation, the treatment method includes the following steps: using polyurethane microspheres as packing material, a 10L upflow plexiglass reactor is used as a biofilm reactor. Simulated wastewater containing 50-150 mg / L MTBE is injected into the bottom of the reactor through the inlet. The concentration of MTBE in the simulated wastewater is gradually increased to acclimate the microorganisms within the system. The liquid flows upward through the polyurethane packing layer. The reactor contains 6L of packing material. The fermentation broth of *Bacillus cereus* is inoculated into the reactor to achieve a bacterial concentration of OD0.05. 600=0.5~1; Aeration is carried out using an aeration device with a gas flow rate of 10~20mL / min. During the biodegradation process, yeast powder is added every 48 hours to a final concentration of 50~200mg / L to improve the metabolic efficiency of the degrading bacteria.
[0075] The biofilm reactor consists of an organic glass body with an inlet, an outlet, a sampling port, a biological packing layer, a nutrient supplementation system, and an aeration device. The nutrient supplementation system is a nutrient storage bottle connected to the nutrient input pipe via a peristaltic pump.
[0076] The present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods in the art. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available.
[0077] In the following examples, the reagents and culture media used include:
[0078] Trace element solution: Each 100 mL of deionized water contains 0.022 g of ZnSO4·4H2O, 0.039 g of Na2MoO4·2H2O, 0.186 g of MnCl2·4H2O, 0.005 g of Co(NO3)2·6H2O, and 0.008 g of CuSO4·5H2O.
[0079] Enrichment medium: 100 mg / L MTBE, 200 mg / L yeast extract, 0.5 g / L potassium dihydrogen phosphate (KH2PO4), 0.5 g / L dipotassium hydrogen phosphate (K2HPO4), 0.1 g / L calcium chloride (CaCl2), 0.2 g / L magnesium sulfate (MgSO4), 0.1 g / L sodium chloride (NaCl), 2 g / L ammonium sulfate ((NH4)2SO4), and 0.1% volumetric element solution, pH 7.0.
[0080] LB liquid medium: 10 g / L peptone, 5 g / L yeast extract and 10 g / L NaCl, pH 7.0.
[0081] LB solid medium: Add agar to LB liquid medium at a rate of 10 g / L.
[0082] Fermentation medium: 1.2 wt% glucose, 1 wt% corn steep liquor powder, 3 wt% soybean meal, 0.02 wt% ammonium sulfate, 0.05 wt% magnesium sulfate, 0.03 wt% sodium chloride, 0.005 wt% ferrous sulfate, 0.02 wt% calcium chloride, 0.2 wt% polyether defoamer, and the balance being water.
[0083] Example 1
[0084] This embodiment is used to illustrate the screening of Aphthous spp. QLM-2 according to the present invention.
[0085] (1) Collect MTBE-contaminated soil samples from a petrochemical plant in Linzi District, Zibo City. Take 15g of the contaminated soil and add it to a 250mL Erlenmeyer flask containing 100mL of sterile water. Shake and incubate at 27℃ and 120rpm for 16h. Centrifuge at 2000rpm and take the supernatant containing bacteria.
[0086] (2) Spread the bacterial supernatant obtained in step (1) onto LB solid medium and incubate for 48 h to obtain colonies;
[0087] (3) Inoculate the colonies obtained in step (2) into enrichment medium and culture them at 27°C and 120 rpm for 24 h with shaking to obtain the culture solution;
[0088] (4) The culture medium obtained in step (3) is subjected to 10 -5 10 -6 and 10 -7 The bacterial suspension was diluted in three gradients, and then 100 μL of the diluted suspension was spread onto fresh LB solid medium and incubated in a constant temperature incubator. The above steps were repeated until single colonies grew on the plate. Colonies with faster growth and different colors and morphologies were selected and inoculated onto new LB solid medium using the streak plate method. The above streak isolation process was repeated until purified colonies with a single morphology were formed, thus obtaining strain QLM-2, which can efficiently degrade MTBE.
[0089] Example 2
[0090] This embodiment is used to illustrate the identification of the *Aleucobacterium tumefaciens* QLM-2 described in this invention.
[0091] 1. Morphological observation
[0092] (1) The *Bacillus anguishii* QLM-2 obtained in Example 1 was observed under a scanning electron microscope, and the results are as follows: Figure 1 As shown.
[0093] Depend on Figure 1 It can be seen that strain QLM-2 is rod-shaped.
[0094] (2) Figure 2 This is a schematic diagram of the colony morphology of strain QLM-2 obtained in Example 1.
[0095] Depend on Figure 2 It can be seen that the colonies of strain QLM-2 are round, milky white in color, with neat edges, smooth surface, and are not sticky.
[0096] 2. Molecular biological identification
[0097] Identification method: The strain QLM-2 obtained in Example 1 was expanded and cultured, then sent to Shanghai Sangon Biotech Co., Ltd. for bacterial genome extraction, PCR amplification, and sequencing. The primers used for PCR amplification were the universal bacterial primers 27F and 1492R.
[0098] Test results: The 16S rDNA gene sequencing results of strain QLM-2 are shown in SEQ ID NO: 1, and its DNA sequence length is 1419bp.
[0099] Sequencing sequences were aligned using NCBI-Nucleotide BLAST, and a phylogenetic tree was constructed. Figure 3 The study found that strain QLM-2 was highly homologous to *Ochrobactrum*. Therefore, strain QLM-2 was identified as belonging to the genus *Ochrobactrum*, named *Ochrobactrum* sp., and deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on September 21, 2023, with accession number GDMCC No: 63825.
[0100] The sequence of SEQ ID NO: 1 is as follows:
[0101] GGCTCAGAACGAACGCTGGCGGCAGGCTTAACACATGCAAGTCG
[0102] AGCGCCCCGCAAGGGGAGCGGCAGACGGGTGAGTAACGCGTGGGAA
[0103] CGTACCTTTTGCTACGGAATAACTCAGGGAAACTTGTGCTAATACCGT
[0104] ATGTGCCCGAAAGGGGAAAGATTTATCGGCAAAGGATCGGCCCGCGT
[0105] TGGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCCA
[0106] TAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGC
[0107] CCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCG
[0108] CAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTT
[0109] GTAAAGCTCTTTCACCGGTGAAGATAATGACGGTAACCGGAGAAGAA
[0110] GCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCT
[0111] AGCGTTGTTCGGATTTACTGGGCGTAAAGCGCACGTAGGCGGACTAA
[0112] TAAGTCAGGGGTGAAATCCCGGGGCTCAACCCCGGAACTGCCTTTGA
[0113] TACTGTTAGTCTTGAGTATGGTAGAGGTGAGTGGAATTCCGAGTGTAG
[0114] AGGTGAAATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGC
[0115] TCACTGGACCATTACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAA
[0116] CAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGTTAG
[0117] CCGTTGGGGAGTTTACTCTTCGGTGGCGCAGCTAACGCATTAAACATT
[0118] CCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACG
[0119] GGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGC
[0120] GCAGAACCTTACCAGCCCTTGACATACCGGTCGCGGACACAGAGATG
[0121] TGTCTTTCAGTTAGGCTGGACCGGATACAGGTGCTGCATGGCTGTCGT
[0122] CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAAC
[0123] CCTCGCCCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGGGACTGC
[0124] CGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCCTCATGG
[0125] CCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGG
[0126] GCAGCGAGCACGCGAGTGTGAGCTAATCTCCAAAAGCCATCTCAGTT
[0127] CGGATTGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAA
[0128] TCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACA
[0129] CCGCCCGTCACACCATGGGAGTTGGTTTTACCCGAAGGCCGCTGTGCTA
[0130] ACCGCAAGGAGGCAGGCGACCACGGTAGGGTCAGCGACTGGGGTGA
[0131] AGTCGT.
[0132] Example 3
[0133] This embodiment is used to illustrate the bacterial agent (bacterial suspension) and its preparation method described in this invention.
[0134] (1) The *Bacillus cereus* QLM-2 obtained in Example 1 was spread on LB solid medium and placed in a constant temperature and humidity incubator and cultured at 27°C for 24 h to activate the colonies;
[0135] (2) Inoculate the activated colony QLM-2 from step (1) into LB liquid medium and culture it with shaking at 27°C and 120 rpm for 24 h to obtain seed liquid QLM-2.
[0136] (3) The seed culture QLM-2 obtained in step (2) was inoculated into 1L of LB liquid medium at a volume ratio of 5%. The medium was shaken and cultured at 27℃ and 120rpm with aeration at a rate of 0.5L / min for 24h to obtain an enriched bacterial culture of Bacillus cereus QLM-2. The concentration was adjusted to make the OD of the bacterial culture... 600 =1, and the bacterial solution of Bacillus cereus QLM-2 was obtained, which was named bacterial agent a.
[0137] Example 4
[0138] This embodiment illustrates the preparation method of the microbial agent (fermentation broth) described in this invention.
[0139] (1) The *Bacillus cereus* QLM-2 obtained in Example 1 was spread on LB solid medium and placed in a constant temperature and humidity incubator. It was cultured at 27°C for 24 h to activate the colony. The activated *Bacillus cereus* QLM-2 was inoculated into LB liquid medium and cultured at 27°C and 120 rpm for 24 h to obtain the primary seed culture QLM-2.
[0140] (2) The primary seed culture QLM-2 obtained in step (1) was inoculated into 1L of LB liquid medium at an inoculation rate of 5%. The medium was shaken and cultured at 27℃ and 120rpm with aeration at a rate of 0.5L / min for 24h to obtain an enriched bacterial culture of Bacillus cereus QLM-2. The concentration was adjusted to make the OD of the bacterial culture... 600 =1, resulting in secondary seed solution;
[0141] (3) Add fermentation culture medium to the fermenter, sterilize it, and inoculate the secondary seed liquid obtained in step (2) at a volume ratio of 5%. The fermentation process is controlled at a temperature of 27℃, a tank pressure of 0.05MPa, an initial rotation speed of 200rpm, dissolved oxygen ≥20%, and a gas-liquid ratio of 1:1. When the dissolved oxygen drops to 20% and the pH rises to 8.8, the fermentation is complete and the fermentation liquid is obtained. Name it as inoculant b.
[0142] Example 5
[0143] This embodiment is used to illustrate the degradation performance of MTBE by the *Aureobacterium tumefaciens* QLM-2 described in this invention.
[0144] (1) Effect of temperature on the degradation of MTBE by Aureobacterium tumefaciens QLM-2
[0145] Add 50 mL of inorganic salt culture medium to a 500 mL sealed serum bottle, add 100 mg / L MTBE as the sole carbon source, and inoculate with bacterial agent a obtained in Example 3 at a volume ratio of 5%. Place each sample in a constant temperature shaker at 15℃, 20℃, 25℃, 30℃, and 35℃ with a rotation speed of 120 rpm. Set up three parallel samples and one blank control group at each temperature. Take samples after 5 days to detect the remaining amount of MTBE in the serum bottle, and calculate the degradation rate under different temperature conditions by taking the average value.
[0146] Test results as follows Figure 4 As shown, the strain grows well at temperatures of 20–30°C, with an MTBE degradation rate of 64.5–72%. The degradation rate is lower at lower or higher temperatures, which may be due to the influence of high and low temperatures on the QLM-2 enzyme activity of Bacillus cereus, resulting in a lower degradation rate.
[0147] (2) Degradation efficiency of Aureobacterium tumefaciens QLM-2 for MTBE at different initial concentrations
[0148] Add 50 mL of inorganic salt culture medium to a 500 mL sealed serum bottle, and add MTBE at different concentrations of 50 mg / L, 100 mg / L, 150 mg / L, and 200 mg / L as the sole carbon source. Inoculate with bacterial agent a obtained in Example 3 at a volume ratio of 5%. Place each sample in a constant temperature shaker at 25 °C and a rotation speed of 120 rpm. Set up three parallel samples and one blank control group for each concentration. Take samples after 48 h to detect the remaining amount of MTBE in the serum bottle. Take the average value to calculate the degradation rate and degradation rate at different initial concentrations. The test results are shown in Table 1 below.
[0149] Table 1
[0150]
[0151] As shown in Table 1, the degradation effect was best when the initial MTBE concentration was 100 mg / L. At this initial concentration, the strain grew well, achieving a degradation rate of 76.3% and a degradation rate of 0.092 mg / L. MTBE / (mg dw •d) When the MTBE concentration is 200 mg / L, the degradation effect is poor. This may be because the high concentration of MTBE has a toxic effect on the strain and inhibits the growth and reproduction of the strain.
[0152] Example 6
[0153] This example illustrates the effect of the presence of benzene compounds on the degradation performance of MTBE by Aureobacterium tumefaciens QLM-2.
[0154] BTEX (benzene, toluene, ethylbenzene, and xylene) are common substances in petroleum, and MTBE contaminated sites are often accompanied by these substances, which may affect the biodegradation of MTBE. The concentration levels of each BTEX component in contaminated sites are mostly around 20 mg / L. Therefore, this experiment investigated the degradation of MTBE under the condition that 100 mg / L of BTEX (benzene, toluene, ethylbenzene, and xylene in a volume ratio of 1:1:1:1) coexisted with 100 mg / L of MTBE.
[0155] The results are as follows Figure 5 As shown, within a 7-day degradation cycle, the degradation rate of MTBE was 68% when BTEX was present and 71% when MTBE was present alone, indicating that BTEX had no effect on MTBE degradation.
[0156] Example 7
[0157] This embodiment illustrates the method for treating MTBE-contaminated wastewater according to the present invention.
[0158] The process flow for treating simulated MTBE wastewater using a biofilm reactor is as follows: Figure 6 As shown: 1. Simulated MTBE wastewater storage tank, 2. Peristaltic pump, 3. Biofilm bioreactor, 4. Aeration device, 5. Sampling port, 6. Nutrient solution storage tank, 7. Air pump, 8. Purified wastewater storage tank, 9. High-concentration MTBE storage tank, 10. Microporous aeration disc, 11. LZB-glass rotor flow meter.
[0159] The biofilm reactor 3 is an integrated reaction device, using *Bacillus cereus* QLM-2 as the biofilm inoculum. Tap water was used to simulate contaminated groundwater, prepared with an inorganic salt culture medium and an appropriate amount of MTBE, with the pH adjusted to 7-8. *Bacillus cereus* QLM-2 was enriched and cultured in the simulated wastewater in a constant-temperature shaker. Polyurethane was used as the packing material. A peristaltic pump 2 pumped the simulated wastewater from the simulated MTBE wastewater storage tank 1 into the bottom of the biofilm reactor 3, with the liquid flowing upwards through the packing layer. The reactor volume was 10L, containing 6L of polyurethane microsphere adsorbent material. A microporous aeration disc 10 was installed at the bottom. An air pump 7 controlled the dissolved oxygen in the gas flow regulation system via an LZB-glass rotor flowmeter 11, with a gas flow rate of 15mL / min. Biofilm formation was initiated by inoculating the bacterial agent b obtained in Example 4 into the reactor for biofilm formation on the packing material. The cell density OD in the reactor was... 600 =0.5.
[0160] The microorganisms in the system were gradually acclimatized by increasing the MTBE concentration in the simulated wastewater in a gradient manner. Four MTBE concentration gradients were set, increasing from 50 mg / L to 150 mg / L, with gradients of 50 mg / L, 80 mg / L, 100 mg / L, and 150 mg / L respectively. Each gradient incubation period lasted 24 hours. During the acclimatization process, 50 mg / L yeast powder was added through nutrient solution storage tank 6 to ensure the growth of the system's microorganisms and improve the growth and reproduction rate of *Aureobacterium tumefaciens* QLM-2. MTBE was intermittently added through high-concentration MTBE storage tank 9 to compensate for volatilization and ensure a relatively stable initial MTBE concentration. Biofilm formation was completed after 4 days of reactor operation. After biofilm formation, continuous operation was initiated, maintaining an MTBE concentration of 100 mg / L. Samples were taken every day from sampling port 5 to measure the MTBE concentration in the liquid phase and calculate the degradation rate.
[0161] The results showed that biofilm formation was completed after 4 days of operation in the biofilm reactor. When the stable concentration of MTBE was 100 mg / L, the degradation rate of MTBE was 71.2% within 7 days after biofilm formation.
[0162] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combining the various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.
Claims
1. A strain of Ochrobactrum sp., characterized in that, The preservation number of the *Aureobacterium pallida* is GDMCC No: 63825.
2. A microbial agent, characterized in that, The bacterial agent contains the *Alanium* bacillus as described in claim 1.
3. The microbial agent according to claim 2, characterized in that, The bacterial agent is a liquid bacterial agent.
4. A method for preparing a microbial agent, characterized in that, The method includes: inoculating the *Ailuropoda spp.* of claim 1 into a liquid culture medium for cultivation.
5. The method according to claim 4, characterized in that, The method also includes: inoculating the obtained bacterial suspension into a fermentation medium for fermentation.
6. The method according to claim 5, characterized in that, The fermentation medium includes glucose, corn steep liquor powder, soybean meal, ammonium sulfate, magnesium sulfate, sodium chloride, ferrous sulfate, calcium chloride, and polyether defoamer.
7. The method according to claim 6, characterized in that, The fermentation medium contains 1.2–1.8 wt% glucose, 0.85–1 wt% corn steep liquor powder, 3–4 wt% soybean meal, 0.01–0.02 wt% ammonium sulfate, 0.03–0.05 wt% magnesium sulfate, 0.01–0.03 wt% sodium chloride, 0.002–0.005 wt% ferrous sulfate, 0.01–0.02 wt% calcium chloride, and 0.05–0.2 wt% polyether defoamer.
8. The microbial agent prepared by the method according to any one of claims 4 to 7.
9. The use of the *Bacillus cereus* according to claim 1 or the bacterial agent according to any one of claims 2-3 and 8 in the degradation of methyl tert-butyl ether.
10. The use of the *Bacillus cereus* according to claim 1 or the bacterial agent according to any one of claims 2-3 and 8 in the treatment of MTBE-contaminated wastewater.
11. A method for treating MTBE-contaminated wastewater, characterized in that, The treatment method includes contacting the *Bacillus cereus* of claim 1 or the bacterial agent of any one of claims 2-3 and 8 with MTBE-contaminated wastewater.
12. The processing method according to claim 11, characterized in that, This treatment method is carried out in a biofilm reactor.
13. The processing method according to claim 12, characterized in that, This treatment method uses polyurethane as a filler and employs an inoculation and biofilm formation method, using the aforementioned *Bacillus cereus* as the biofilm-forming bacteria, and inoculates and forms a biofilm through wastewater containing MTBE.