Cupriavidus sp. And application of Cupriavidus sp. In degradation of benzohydroxamic acid

Abstract

The invention discloses a Cupriavidus sp. HY21 strain of BHA (benzohydroxamic acid) is separated from soil in a mining area, the Cupriavidus sp. HY21 strain is preserved in Guangdong Microbial Culture Collection Center on September 11, 2024, the preservation number of the Cupriavidus sp. HY21 strain is GDMCC No: 65117, and the optimal degradation conditions of the Cupriavidus sp. HY21 strain are that the pH is 5.5, the temperature is 30.5 DEG C, and the inoculation amount is 1.0 (OD 600nm). Under the condition, the degradation rate of 100 mg / L of BHA can reach 97% or above within 24 h. In addition, the hydroxamic acid collecting agent, benzoic acid, salicylic acid, phenol and / or catechol can be well degraded, and the method can be used for pollution remediation of the flotation reagent BuX.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more specifically, to a copper-greedy bacteria strain and application thereof in degrading benzohydroxamic acid. Background Art

[0002] Mining is one of the important pillar industries of the national economy and provides indispensable material resources for the development of human society. Flotation refers to the mineral processing process of floating solid minerals from a water suspension (ore pulp) based on the differences in the physical and chemical properties of the mineral surface. Flotation has been widely used in the mineral processing industry.

[0003] Flotation is a process of adding chemical agents to adsorb on the surface of minerals to change their wettability. Air is introduced into the flotation tank while the flotation tank is fully stirred to form a large number of bubbles. Hydrophobic particles will adhere to the bubbles and rise to the top with the bubbles to form foam. The foam layer continues to increase until the foam overflows or is directly scraped out. The hydrophilic particles settle to the bottom of the flotation tank, thereby achieving the purpose of sorting. The chemical agents used in the flotation process are called flotation agents, and their main function is to adjust the physical and chemical properties of the mineral surface and improve the floatability of the mineral. There are currently hundreds of flotation agents that have been developed, among which collectors can be selectively adsorbed on the surface of minerals and form a hydrophobic film on their surface, so that mineral particles can be more firmly attached to the bubbles and float up with them. This is the basis of the flotation process based on the principle of selective separation of minerals.

[0004] Benzohydroxamic acid (BHA) is the earliest used type of hydroxamic acid collector (also known as isohydroxamic acid or oxyhydroxamic acid). It is a white crystal at room temperature with a melting point of 126-130°C and a solubility in water of about 22 g / L (6°C).

[0005] The structural formula of BHA includes a benzene ring and a hydroxylamine group. The benzene ring structure makes BHA difficult to be degraded and utilized, and its chemical properties are stable. The N and O atoms in the hydroxylamine group have lone pairs of electrons and are close to each other, which makes BHA have a strong chelating ability and is easy to chelate with metal ions to form a stable insoluble complex. BHA has high selectivity for metals such as Cu, Fe, Pd, and Ni, and is currently commonly used in the flotation of oxide ores and rare earth ores.

[0006] BHA has physiological toxicity due to the presence of benzene rings and oxime groups, making it difficult to be degraded and utilized. Once released into the water environment, its residual time can be up to several years or even longer. The presence of BHA will increase the COD content in flotation wastewater, and due to the continuous accumulation of N elements, it is easy to cause eutrophication of water bodies, and the deterioration of water quality makes it difficult for aquatic organisms to survive, ultimately destroying the ecological balance. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a copper-greedy bacteria strain and its application in degrading benzohydroxamic acid.

[0008] The first object of the present invention is to provide a copper-greedy bacteria.

[0009] The second object of the present invention is to provide the use of the copper-greedy bacteria in degrading benzohydroxamic acid or in preparing products degrading benzohydroxamic acid.

[0010] The third object of the present invention is to provide the use of the copper-greedy bacteria for degrading benzoic acid, salicylic acid, phenol, catechol, salicylic hydroxamic acid and / or N-hydroxyphthalimide, or for preparing products for degrading benzoic acid, salicylic acid, phenol, catechol, salicylic hydroxamic acid and / or N-hydroxyphthalimide.

[0011] The fourth object of the present invention is to provide the use of the copper-greedy bacteria in degrading hydroxamic acid collectors, or in preparing products for degrading hydroxamic acid collectors.

[0012] A fifth object of the present invention is to provide a method for degrading mehydroxamic acid.

[0013] The sixth object of the present invention is to provide a method for degrading benzoic acid, salicylic acid, phenol, catechol, salicylic hydroxamic acid and / or N-hydroxyphthalimide.

[0014] A seventh object of the present invention is to provide a method for degrading hydroxamic acid collectors.

[0015] The eighth object of the present invention is to provide a product for degrading mehydroxamic acid.

[0016] The ninth object of the present invention is to provide a product for degrading benzoic acid, salicylic acid, phenol, catechol, salicylic hydroxamic acid and / or N-hydroxyphthalimide.

[0017] The tenth object of the present invention is to provide a product for degrading hydroxamic acid collectors.

[0018] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0019] The present invention claims protection for a strain of Cupriavidus sp. HY21, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 11, 2024, and its deposit number is: GDMCC No: 65117.

[0020] Protection is also claimed for the following applications:

[0021] The Cupricobacterium HY21 is used to degrade benzohydroxamic acid, or to prepare products that degrade benzohydroxamic acid.

[0022] The copper-greedy bacteria HY21 is used in degrading benzoic acid, salicylic acid, phenol, catechol, salicylic hydroxamic acid and / or N-hydroxyphthalimide, or in preparing products for degrading benzoic acid, salicylic acid, phenol, catechol, salicylic hydroxamic acid and / or N-hydroxyphthalimide.

[0023] The copper-greedy bacteria HY21 is used in the degradation of hydroxamic acid collectors, or in the preparation of products for the degradation of hydroxamic acid collectors.

[0024] Preferably, the hydroxamic acid collector is one or both of salicylic hydroxamic acid and N-hydroxyphthalimide.

[0025] Preferably, the degradation conditions are: pH 5-7, temperature 30-35°C, inoculation amount 1.5-2.5% volume fraction OD600 = 0.8-1.2.

[0026] More preferably, the degradation conditions are: pH 5.5, temperature 30.5°C, inoculation amount 2% volume fraction OD600 = 1.0.

[0027] The following methods are also claimed:

[0028] A method for degrading mehydroxamic acid, utilizing the Cupribotium HY21 to degrade mehydroxamic acid.

[0029] A method for degrading benzoic acid, salicylic acid, phenol, catechol, salicylic hydroxamic acid and / or N-hydroxyphthalimide, which utilizes the Cupric bacteria HY21 to degrade benzoic acid, salicylic acid, phenol, catechol, salicylic hydroxamic acid and / or N-hydroxyphthalimide.

[0030] A method for degrading hydroxamic acid collectors, utilizing the Cuproplast HY21 to degrade hydroxamic acid collectors.

[0031] Preferably, the hydroxamic acid collector is one or more of salicylic hydroxamic acid, N-hydroxyphthalimide or mehydroxamic acid.

[0032] Preferably, the degradation conditions are: pH 5-7, temperature 30-35°C, inoculation volume 1.5-2.5% OD 600 =0.8~1.2.

[0033] More preferably, the degradation conditions are: pH 5.5, temperature 30.5°C, inoculation volume 2% OD 600 =1.0.

[0034] The following methods are also claimed:

[0035] A product for degrading mehydroxamic acid contains the Cuproplast HY21.

[0036] A product for degrading benzoic acid, salicylic acid, phenol and / or catechol, comprising the Cuproplast HY21.

[0037] A product for degrading hydroxamic acid collectors, comprising the copper-greedy bacteria HY21.

[0038] Preferably, the hydroxamic acid collector is one or more of salicylic hydroxamic acid, N-hydroxyphthalimide or mehydroxamic acid.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The present invention isolated a BHA-degrading bacterium from the soil of the mining area: Cupriavidus sp. HY21. The optimal degradation conditions of strain HY21 are: pH 5.5, temperature 30.5°C, inoculation amount 1.0 (OD 600nm ), under this condition, the degradation rate of 100mg / L BHA can reach more than 97% in just 24h. In addition, it also has good degradation ability for hydroxamic acid collectors and benzoic acid, salicylic acid, phenol and / or catechol, and can be used for flotation agent BuX pollution remediation. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is the colony morphology characteristics of the strain on nutrient agar plate.

[0042] Figure 2 The morphological characteristics of the strains under SEM.

[0043] Figure 3 This is the phylogenetic tree of strain HY21 based on 16S rDNA sequence.

[0044] Figure 4 The chromatograms of quantitative ion (A) and qualitative ion (B) of BHA.

[0045] Figure 5 This is the effect of pH on the degradation of BHA by strain HY21.

[0046] Figure 6 This is the effect of temperature on the degradation of BHA by strain HY21.

[0047] Figure 7 This is the effect of inoculum dosage on the degradation of BHA by strain HY21.

[0048] Figure 8 pH(X 1 ) and temperature (X 2) Response surface diagram (A) and contour diagram (B) of the interactive effects of strain HY21 on BHA degradation.

[0049] Fig. 9 The degradation kinetic curve (A) and fitting curve (B) of strain HY21 for different initial concentrations of BHA.

[0050] Fig.10 These are the mass spectra of the intermediate degradation products of BHA (A. primary and secondary mass spectra of benzoic acid; B. primary and secondary mass spectra of salicylic acid; C. primary and secondary mass spectra of catechol; D. primary and secondary mass spectra of phenol).

[0051] Fig.11 This is the biochemical pathway for strain HY21 to degrade BHA. DETAILED DESCRIPTION

[0052] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0053] Benzohydroxamic acid (BHA) was purchased from Sigma-Aldrich, USA, with a purity of >99%; chromatographic grade methanol was purchased from Sigma-Aldrich, USA; hydrochloric acid was purchased from Guangzhou Chemical Reagent Co., Ltd. with an analytical purity; the remaining reagents were all analytically pure and purchased from Tianjin Damao Chemical Reagent Co., Ltd.

[0054] Liquid basic salt medium (MSM): K 2 HPO 4 1.5g / L, KH 2 PO 4 0.5g / L, (NH 4 ) 2 SO 4 0.5g / L, MgSO 4 7H 2 O 0.2g / L and NaCl 1.0g / L, pH value is about 7.0.

[0055] LB broth medium: peptone 10 g / L, yeast extract 5 g / L and sodium chloride 5 g / L, pH value is about 7.0.

[0056] Nutrient agar medium: peptone 10g / L, yeast extract 5g / L, sodium chloride 5g / L, agar 15g / L, pH value is about 7.0.

[0057] The calculation method of degradation rate is: degradation rate (%) = 100% × (C 0 -C 1 / C 0 ),

[0058] In the formula, C 1 is the residual concentration of BHA after treatment with degradation bacteria, C 0 is the initial concentration of BHA without treatment.

[0059] Example 1 Isolation and identification of strain HY21

[0060] 1. Experimental Methods

[0061] 1. Enrichment, separation and purification of strains

[0062] 100mL of MSM culture medium was placed in a 250mL conical flask for sterilization. After cooling, BHA was added to make its initial concentration reach 10mg / L. After adding 5g of the collected mining area contaminated soil (taken from Dabaoshan mining area in Shaoguan, Guangzhou), it was cultured in a shaker at 35℃ and 150rpm for 7 days in the dark. On the 7th day of culture, it was transferred to a new MSM culture medium with an inoculum of 10% (volume fraction), and the pollutant concentration was gradually increased for acclimatization, and the transfer was continuous for 5 times. Take the last culture solution and evenly spread it on the nutrient agar plate. After constant temperature culture for 48h, pick the colony and repeatedly streak it until a single colony is obtained. The single bacteria was inoculated into the MSM culture medium with 100mg / L BHA, and the shaker was cultured at 35℃ and 150rpm for 72h to detect its degradation effect, and the strain was inoculated into the test tube slant culture medium and stored in a refrigerator at 4℃ for use.

[0063] 2. Observe the characteristics of colonies

[0064] The screened strains were inoculated onto nutrient agar plates, inverted and cultured in a 35°C incubator overnight, and the shape, size, transparency, edge and color of the strains were observed.

[0065] 3. SEM observation of bacterial morphology

[0066] The strains screened were inoculated into LB broth medium for cultivation. 800 μL of the bacterial solution after 24 hours of cultivation was taken into a 1.5 mL centrifuge tube, and the supernatant was discarded after centrifugation at 8000 rpm for 5 minutes. The obtained bacterial precipitate was washed with sterile saline, centrifuged and the supernatant was discarded, and this operation was repeated 3 times. 1 mL of 2.5% glutaraldehyde electron microscope fixative was added to the washed bacteria, vortexed and mixed, and allowed to stand overnight in a 4°C refrigerator. After fixation, centrifuged at 8000 rpm for 5 minutes, the supernatant was discarded, the bacteria were collected and washed 3 times with sterile saline. Then, gradient dehydration was performed, that is, 30%, 50%, 70%, 80%, and 90% ethanol were added once, and 100% ethanol was added twice. Each dehydration treatment required standing for 15 minutes. After the last dehydration treatment, the bacteria were dropped on a cover glass and placed in a freeze dryer for freeze drying. The dried samples were sprayed with gold and then observed on the machine.

[0067] 4. Physiological and biochemical identification of strains

[0068] Physiological and biochemical experiments of strains were carried out with reference to the Manual of Common Bacterial Identification and Bergey's Manual of Determinative Bacteriology.

[0069] 5. Molecular biological identification of strains

[0070] PCR amplification: The 16S rDNA fragment of the strain was amplified using bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3').

[0071] PCR reaction system: Taq Mixture, 12.5 μL; ddH 2 O, 9.5 μL; 27F, 1 μL; 1492R, 1 μL; DNA template, 1 μL, the total reaction system is 25 μL.

[0072] PCR reaction conditions: 95℃, initial preheating for 5min; melting at 94℃, 45s, annealing at 55℃, 45s, extension at 72℃, 1min15s, 32 cycles, and finally 72℃ for 10min. After PCR amplification, the size and specificity of the amplified fragments were detected by 1.5% agarose gel electrophoresis, and photographed in a gel imaging system. The PCR amplification products were purified, recovered and sequenced by Shanghai Bioengineering Company.

[0073] Construction of phylogenetic tree: The 16S rDNA sequences of the two strains were obtained by sequencing by Sangon Company and submitted to the GenBank database. BLAST was used for comparison analysis, and ClustalX software was used for homology analysis of related sequences with high matching degree. Neighborhood-joining method in MEGA 11.0 software was used for relationship analysis to construct the phylogenetic tree of the strains.

[0074] 2. Experimental Results

[0075] After enrichment, separation and purification, a strain that can use BHA as the sole carbon source was named HY21. It can completely degrade BHA with an initial concentration of 100 mg / L in a short period of time. It was inoculated on a nutrient agar plate and its colony morphology was observed after overnight culture. On the nutrient agar plate, strain HY21 showed a milky white, irregular, opaque, and rough surface ( Figure 1 )

[0076] SEM observations showed that strain HY21 was short rod-shaped (0.8-2.0×0.5 μm) with a rough surface ( Figure 2 ).

[0077] Gram staining results showed that strain HY21 was a Gram-negative bacterium. Its methyl red test, nitrate reduction, catalase, starch hydrolysis, hydrogen sulfide, and acetylmethylcarbinol tests were all negative, while oxidation reaction, ornithine decarboxylase, lysine decarboxylase test, and urease production were positive. The strain could not utilize lactose and glucose, but could utilize malonate (Table 1).

[0078] Table 1 Physiological and biochemical characteristics of strain HY21

[0079]

[0080]

[0081] Note: “+” indicates that it can be used or is positive; “-” indicates that it cannot be used or is negative.

[0082] The genomic DNA of strain HY21 was used as a template and PCR amplification was performed using 16S rDNA bacterial universal primers. The product obtained after amplification was electrophoresed and a single band was found. The amplified product was sent to Shanghai Shenggong Biotechnology Co., Ltd. for purification, and the product length was detected to be 1161bp.

[0083] The 16S rDNA sequencing results of the strain were submitted to the NCBI Genbank database for BLAST comparison. The sequences with high homology to the target strain in the BLAST comparison analysis results were used for multiple sequence alignment, and the phylogenetic tree of strain HY21 was constructed by the neighbor-joining method ( Figure 3 HY21 has a high homology with the strains of Cupriavidus sp., so it can be judged that the strain HY21 is a strain of Cupriavidus sp.

[0084] Strain HY21 was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 11, 2024, with the deposit number: GDMCC No: 65117, and the classification name: Cupriavidus sp.

[0085] Example 3 A detection method for BHA based on LC-MS / MS

[0086] 1. Sample pretreatment method

[0087] 1mL of the culture medium of the strain HY21 to be tested was added to a 1.5mL centrifuge tube and centrifuged at 8000rpm for 5min. After centrifugation, 300μL of the supernatant was diluted 10 times. To prevent BuX degradation, the BuX content was immediately determined by UV-Vis. Another 100μL of the supernatant was diluted 100 times with methanol-water (50 / 50, v / v) as the solvent. Finally, it was filtered through a 0.22μm microporous membrane, and the filtrate was collected into a sample injection bottle. The BHA residue was determined by LC-MS / MS.

[0088] 2. LC-MS / MS detection method of BHA

[0089] A LC-MS / MS detection method for BHA was established using AB Sciex 5500MD high performance liquid chromatography-tandem mass spectrometer.

[0090] Chromatographic conditions: the chromatographic column is Water Xbridge C18 (3.0mm×50mm, 5μm); the mobile phase A is water; the mobile phase B is methanol; the column flow rate is 0.40mL / min; the column temperature is 40°C; the injection volume is 5μL; the mobile phase gradient elution program is as shown in Table 2.

[0091] Table 2 Mobile phase gradient elution program

[0092]

[0093] Mass spectrometry conditions: Considering that BHA is a weakly acidic compound, the negative ion mode was selected. Ion source: Electrospray ionization source negative ion (ESI- ); electrospray voltage: -4500V; nebulizer gas temperature: 500°C; nebulizer gas pressure: 55psi; auxiliary gas pressure: 50psi; curtain gas pressure: 35psi; collider pressure: 6psi; scanning mode: multiple reaction monitoring mode (MRM). The mass spectrometry parameters of BHA are shown in Table 3, and the quantitative ion and qualitative ion chromatograms are shown in Table 3. Figure 4 shown.

[0094] Table 3

[0095]

[0096] 4. BHA standard curve

[0097] Take 10 μL of BHA standard solution (1000 mg / L) and add it to the injection bottle, dilute it with 990 μL of chromatographic grade methanol, and obtain a BHA standard stock solution with a concentration of 10 mg / L. Prepare solutions of 50, 75, 150, 300, 600, and 1200 ng / mL according to the gradient dilution method, and the solvent is methanol-water (50 / 50, v / v) solution. According to the LC-MS / MS detection method of BHA above, the peak area corresponding to each concentration is determined, and the peak area determined is Y, and the corresponding standard solution concentration is X, and the standard curve "Y=a+bX" is drawn.

[0098] Example 4 Effects of different factors on the BHA degradation ability of strain HY21

[0099] 1. Effect of pH on the BHA degradation ability of strain HY21

[0100] 1. Experimental methods

[0101] The pH of the prepared MSM medium was adjusted to 3, 4, 5, 6, 7, and 8 using a Leiji precision pH meter, and sterilized after packaging. BHA was added after the medium was cooled to an initial concentration of 100 mg / L. HY21 bacterial suspension (OD600 = 1.0) was inoculated at a volume ratio of 2%, and the residual BHA content was measured after culturing at 35°C and 150 rpm for 24 hours (according to the method of Example 3). The control group was not inoculated, and three biological replicates were set for each group.

[0102] 2. Experimental results

[0103] from Figure 5It can be seen that the pH range of 3 to 8 has a significant effect on the degradation of BHA by strain HY21. When the pH is between 5 and 7, the degradation rate of strain HY21 can reach more than 97% after 24 hours of cultivation, and the highest degradation rate is 98.55% when the pH is 7; when the pH is <5, the degradation ability of strain HY21 to BHA decreases accordingly, and when the pH is 3, the degradation rate of strain HY21 to BHA is only 14.26%; when the pH is >7, the degradation ability of the strain decreases significantly, and the degradation rate of HY21 is only 22.03% when the pH is 8. The above results show that the degradation efficiency of strain HY21 will be significantly inhibited under strong acid and strong alkaline conditions, and the optimal pH range of the bacteria is between 5 and 7, that is, strain HY21 has a good degradation ability for BHA under weak acid and neutral conditions.

[0104] 2. Effect of temperature on the BHA degradation ability of strain HY21

[0105] 1. Experimental methods

[0106] At pH 7, 2% volume ratio of HY21 bacterial suspension (OD 600 =1.0), and cultured in a constant temperature incubator at 20, 25, 30, 35, and 40°C at 150 rpm. Samples were taken at 24 hours to determine the remaining BHA concentration in the culture solution (according to the method of Example 3). The control group was not inoculated, and three biological replicates were set for each group.

[0107] 2. Experimental results

[0108] result Figure 6 The results showed that temperature conditions had a significant effect on the degradation of BHA by strain HY21. When the temperature was between 30 and 35°C, the degradation rate of BHA by strain HY21 could reach more than 97%, but when the temperature exceeded this range, the degradation ability of strain HY21 on BHA was significantly reduced, especially when the temperature reached 20°C and 40°C, the degradation rate was only 6.6% and 15.14%. It can be seen that too high or too low temperature will have an adverse effect on the growth of strain HY21, significantly inhibiting its degradation of BHA.

[0109] 3. Effect of inoculum dose on the BHA degradation capacity of strain HY21

[0110] 1. Experimental methods

[0111] At pH 7 and temperature 35°C, the OD of HY21 bacterial suspension was first 600The concentrations of BHA were adjusted to 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6, respectively, and the bacterial suspension was inoculated into the MSM medium with an initial BHA concentration of 100 mg / L at a volume ratio of 2%, and then placed in an incubator for shaking culture at 150 rpm for 24 hours. After the culture was completed, samples were taken to determine the BHA concentration (according to the method of Example 3). The control group was not inoculated, and three biological replicates were set for each group.

[0112] 2. Experimental results

[0113] The results are as follows Figure 7 As shown in the figure, the effect of inoculum amount on the degradation of BHA by strain HY21 was not significant. 600nm ) range, the BHA degradation rate will slowly increase with the increase of inoculum amount. When the inoculum amount reaches 1.2 (OD 600nm ), the BHA degradation rate reached a maximum of 97.47%; when the inoculum amount was greater than 1.2 (OD 600nm ), the degradation ability of the strain will be weakened, which may be due to the high density of the bacterial community, competition among microorganisms, and relative lack of nutrients.

[0114] Example 5 Degradation conditions of strain HY21

[0115] 1. Experimental Methods

[0116] The effects of the above three factors (pH, temperature and inoculum size) on the degradation ability of strain HY21 were optimized and analyzed by response surface methodology to determine the optimal degradation conditions for strain HY21. The independent variables of this method are pH, temperature and inoculum size, which are numbered as X 1 , X 2 , X 3 , -1, 0, and 1 are used to represent the three levels of the independent variable, and the response value (Y) is the degradation rate of 100 mg / L BHA in the culture medium after the strain degraded for 24 h. The specific coding of each factor level is shown in Table 4.

[0117] Table 4 Experimental factor levels and coding based on Box-Behnken design

[0118]

[0119] Finally, the regression model is as follows:

[0120] Y i =b 0 +∑b i X i +∑b ij X i X j +∑b ii X i2 Among them, Yi is the predicted degradation rate of BHA. The reliability of the regression model was evaluated through model significance and variance analysis. Finally, the degradation strains were cultured according to the theoretical value of the optimal degradation conditions, and the degradation rate was used as the evaluation index to verify the reliability of the model conclusion.

[0121] 2. Experimental Results

[0122] Design expert 13.0 was used to obtain the optimized experimental design of degradation conditions for strain HY21 and the response value data (Table 5) by Box-Behnken design with BHA degradation rate as the response value (Y).

[0123] Table 5 Box-Behnken experimental design and BHA degradation results

[0124]

[0125] Note: The dependent variable results are the mean ± standard deviation after 3 repeated experiments. The same letter at the end indicates no significant difference (P>0.05).

[0126] The data in (Table 5) were used for polynomial regression analysis to establish a quadratic polynomial model for BHA degradation rate (Y), and the regression equation was as follows:

[0127] Y=96.45-13.8X 1 +2.54X 2 -27.07X 1 2 -26.58X 2 2 -7.28X 3 2 The quadratic polynomial model established based on the above data has a coefficient of determination (R 2 ) is 0.9932, indicating that the actual measured value is highly correlated with the predicted value, and the model can be used for the theoretical prediction of BHA by strain HY21. The calibration determination coefficient is 0.9844, indicating that the model well explains the variation of the response value of strain HY21. Finally, the coefficient of variation (CV) of the model is 4.24%, indicating that the model equation reflects the actual experimental measurement value.

[0128] The results of variance analysis (Table 6) showed that the model P < 0.0001, indicating that the model fit is highly significant. 1 ) had a significant effect on the degradation of BHA by strain HY21 (P<0.0001), and temperature (X 2 ) also had a significant effect on the degradation of BHA by strain HY21 (P<0.05), while the inoculum amount (X 3) had no significant effect on the degradation of the strain (P>0.05). 1 2 , X 2 2 The P values ​​of X were all less than 0.0001, indicating that the linear effect of X on the degradation rate of strain HY21 reached a very significant level. 3 2 The P value was less than 0.05, indicating that its linear effect on the degradation rate of strain HY21 reached a significant level.

[0129] Table 6 Quadratic polynomial model and its variance analysis

[0130]

[0131] Design expert 13.0 software was used to draw the response surface to study the effects of single factors and interactions between factors on BHA degradation. The variance analysis results showed that the effect of the inoculum amount on BHA degradation was not significant. Therefore, according to the existing fitting function, the inoculum amount was fixed at 1.0 (OD600nm, 2% volume fraction) and the pH (X 1 )、Temperature(X 2 ) on the degradation of BHA, and draw a three-dimensional response surface diagram and contour map ( Figure 8 )

[0132] The response surface plot shows that the model has a maximum point. The first-order partial derivative of the quadratic regression equation is used to obtain the optimal degradation conditions for the degradation of BHA by strain HY21: pH 5.5, temperature 30.5℃, inoculation amount 1.0 (i.e. OD 600 =1.0, 2% volume fraction), under which the model predicted a BHA degradation rate of 98.29%. To verify the accuracy of the model prediction results, strain HY21 was cultured under the predicted optimal degradation conditions, and the average value was taken after 3 repeated experiments. The degradation rate of strain HY21 for 100 mg / L BHA reached 97.83%, which is close to the predicted result, indicating the reliability of the prediction results using the model.

[0133] Example 6 Degradation effect of HY21 at different initial concentrations of BHA

[0134] 1. Experimental Methods

[0135] Under the optimal degradation conditions of HY21 (pH 5.5, temperature 30.5°C, inoculum 1.0 (OD 600=1.0, 2% volume fraction)) The HY21 bacterial suspension was inoculated into 50mL MSM medium. The BHA concentration in the medium was set to 50, 100, 150, and 200 mg / L, respectively, and it was placed in a constant temperature oscillating box with 150rpm shaking culture for 48h. Samples were taken when it was degraded to 0, 6, 12, 24, 30, 36, and 48h, and the residual concentration of BHA in the culture solution was determined (according to the method of Example 3). The degradation kinetic curve of strain HY21 for different initial concentrations of BHA was drawn, and a four-parameter Logistic model was used to fit it. The control group was not inoculated, and three parallels were set for each treatment group.

[0136] 2. Experimental Results

[0137] Fig. 9 The results in Figure A show that strain HY21 can tolerate a BHA concentration range of 50 to 200 mg / L. Fig. 9 Figure B is the fitting curve. A strain of Klebsiella BHA-degrading bacteria has been reported. After 12 days of culture, the degradation rate of BHA with an initial concentration of 100 mg / L was only 85.04% (Hu Chun, Wang Can, Gong Wenqi, et al. Study on the microbial degradation of three hydroxamic acid collectors [J]. Hubei Agricultural Science, 2013, 52(11): 2505-2507). In comparison, strain HY21 can tolerate higher concentrations of BHA and has a higher degradation efficiency for BHA. However, strain HY21 showed obvious hysteresis in the process of degrading high concentrations of BHA. This is because high concentrations of BHA have a toxic effect on strain HY21 and inhibit its growth.

[0138] Fig. 9 The degradation curve is "S" shaped and does not conform to the first-order kinetic model. The four-parameter Logistic model is further used to fit the BHA degradation rate (y) and time (t) at different BHA initial concentrations at different time points (Table 7). The fitting equation is as follows:

[0139]

[0140] Where y, y0 and ymax are the reaction time to t, t0 and the maximum BHA degradation rate respectively; b is the slope of the fitting curve; c is the degradation half-life.

[0141] From Table 7, we can see that R 2 All of them were greater than 0.99, indicating that the degradation of BHA by strain HY21 conformed to the four-parameter logistic model. The degradation half-life of strain HY21 for 50, 100, 150, and 200 mg / L BHA was 5.880, 8.846, 16.57, and 17.64 h, respectively, and the half-life would gradually prolong with the increase of concentration.

[0142] Table 7 Degradation kinetic equations of strain HY21 for different initial concentrations of BHA

[0143]

[0144] Example 7 Analysis of the broad spectrum of substrate utilization of strain HY21

[0145] 1. Experimental Methods

[0146] Two hydroxamic acid collectors, salicylic acid and N-hydroxyphthalimide, and possible intermediate degradation products of BHA, such as benzoic acid, salicylic acid, catechol, and phenol, were selected as test substrates. The above substances were added to 50 mL of sterile MSM culture medium to make their initial concentrations reach 100 mg / L, and then the HY21 bacterial suspension was inoculated and placed in a constant temperature oscillator under the optimal conditions (pH 5.5, temperature 30.5°C, inoculation amount 1.0 (OD 600 =1.0, 2% volume fraction)) and shake culture for 72h. Observe the color change of the culture medium and the turbidity of the bacterial solution to determine whether the strain HY21 can use these substrates for growth. The non-inoculated treatment was used as a blank control, and each group was treated with three replicates.

[0147] 2. Experimental Results

[0148] The results of substrate utilization of strain HY21 are shown in Table 8. Strain HY21 can not only utilize possible intermediate degradation products such as benzoic acid, salicylic acid, catechol and phenol, but also utilize other hydroxamic acid collectors such as salicylic acid and N-hydroxyphthaloyl as the only carbon source for its own growth, and has a wide range of substrate utilization capabilities.

[0149] Table 8 Substrate utilization of strain HY21

[0150]

[0151] Note: (++) indicates vigorous growth; (+) indicates possible growth; (-) indicates no growth.

[0152] Example 8 Metabolic pathway for degradation of BHA by strain HY21

[0153] 1. Experimental Methods

[0154] The HY21 bacterial suspension was inoculated into the sterilized MSM medium with pollutants added. The initial pH of the degradation system was 7.0, and the initial concentration of the pollutants was 100 mg / L. It was placed at 35°C and 150 rpm for shaking culture, and samples were taken at different periods. The culture solution was diluted ten times and scanned using UV-Vis in the wavelength range of 185-350 nm to detect the intermediate degradation products of BuX. The BHA intermediate metabolites were extracted using a C18 solid phase extraction column, and then the components to be analyzed adsorbed on the column filler were eluted with 10 mL of methanol. The eluted liquid nitrogen was blown to dryness, re-dissolved in 1 mL of methanol, and filtered through a 0.22 μm filter membrane. The filtrate was collected in a brown injection bottle for analysis. The intermediate metabolites of BHA were analyzed by HPLC-TOF-MS / MS (chromatographic and mass spectrometry conditions see: Zhao HM, Hu RW, Chen XX, et al. Biodegradation pathway of di-(2-ethylhexyl)phthalate by a novel Rhodococcus pyridinivorans XB and its bioaugmentation for remediation of DEHP contaminated soil[J]. Science of the total environment, 2018, 640: 1121-1131).

[0155] 2. Experimental Results

[0156] The culture fluids reacted for different time periods were enriched by C18 solid phase extraction columns and detected by HPLC-TOF-MS / MS technology. The intermediate metabolites in the process of BHA degradation by strain HY21 were analyzed and the specific data of the intermediate products are shown in Table 9.

[0157] Table 9 Identification of intermediate products in the process of BHA degradation by strain HY21 using HPLC-TOF-MS / MS

[0158]

[0159]

[0160] HPLC-TOF-MS / MS test results are as follows Fig.10 ,

[0161] BHA contains a benzene ring structure. The degradation of substances containing benzene rings often involves a ring-opening process. Referring to the biodegradation pathways of other hydroxamic acid collectors, the pathway for strain HY21 to degrade BHA is as follows: Fig.11 shown.

[0162] The biodegradation of BHA mainly occurs through the following two pathways: The first pathway is the benzoic acid pathway, where the amide bond in BHA breaks to generate benzoic acid and hydroxylamine, benzoic acid adds a hydroxyl group at its ortho position to generate salicylic acid, salicylic acid is hydroxylated again to generate catechol, which is further degraded by ring opening under the catalysis of dioxygenase and eventually enters the tricarboxylic acid cycle (TCA). The second pathway is to generate phenol first, then oxidize to generate catechol, and then enter the TCA cycle through the catechol pathway. The process of BHA converting to phenol is still unclear.

Claims

1. A Cupriavidus sp. HY21, characterized in that: It was deposited in Guangdong Provincial Microbiological Culture Collection Center on September 11, 2024, and its deposit number is: GDMCC No: 65117.

2. The use of the Cuproplast HY21 described in claim 1 in degrading benzohydroxamic acid or in preparing products degrading benzohydroxamic acid.

3. Use of the Cupric bacteria HY21 described in claim 1 in degrading benzoic acid, salicylic acid, phenol, catechol, salicylic hydroxamic acid and / or N-hydroxyphthalimide, or in preparing products that degrade benzoic acid, salicylic acid, phenol, catechol, salicylic hydroxamic acid and / or N-hydroxyphthalimide.

4. Use of the Cupriavidus sp.HY21 described in claim 1 in degrading hydroxamic acid collectors, or in preparing products for degrading hydroxamic acid collectors.

5. A method for degrading mehydroxamic acid, characterized in that: The Cupribotium HY21 described in claim 1 is used to degrade mehydroxamic acid.

6. A method for degrading benzoic acid, salicylic acid, phenol, catechol, salicylic hydroxamic acid and / or N-hydroxyphthalimide, characterized in that: The Cupric bacteria HY21 described in claim 1 is used to degrade benzoic acid, salicylic acid, phenol, catechol, salicylic hydroxamic acid and / or N-hydroxyphthalimide.

7. A method for degrading a hydroxamic acid collector, characterized in that: The Cupric bacteria HY21 described in claim 1 is used to degrade hydroxamic acid collectors.

8. A product for degrading mehydroxamic acid, characterized in that: Contains the Cuproplastis HY21 described in claim 1.

9. A product for degrading benzoic acid, salicylic acid, phenol, catechol, salicylic hydroxamic acid and / or N-hydroxyphthalimide, characterized in that: Contains the Cuproplastis HY21 described in claim 1.

10. A product for degrading hydroxamic acid collectors, characterized in that: Contains the Cuproplastis HY21 described in claim 1.

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