Cupriavidus necator and application thereof in degrading benzalkonium hydroxamic acid

Abstract

The application discloses a strain of Cupriavidus sp. and application thereof in degrading benzohydroxamic acid. The application separates a BHA degrading bacterium, Cupriavidus sp. HY21, from soil in a mining area, and the bacterium is preserved in the Guangdong Provincial Microbial Culture Collection Center on September 11, 2024, with a preservation number of GDMCC No: 65117. The optimal degrading condition is that the pH is 5.5, the temperature is 30.5 DEG C, and the inoculation amount is 1.0 (OD600nm). Under the condition, the degrading rate of 100 mg / L BHA can reach more than 97 % only in 24 hours. In addition, the bacterium also has good degrading capacity for hydroxamic acid collectors and benzoic acid, salicylic acid, phenol and / or o-diphenol, and can be used for BuX pollution remediation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, more particularly to a cupriavidus and its application in degrading benzohydroxamic acid. BACKGROUND

[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 is a mineral processing process that separates solid minerals from water suspension (ore pulp) according to 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 in which chemicals are added to change the wettability of the mineral surface. Air is introduced while the flotation tank is thoroughly stirred to form a large number of bubbles. Hydrophobic particles will adhere to the bubbles and rise to the top to form a foam layer. The foam layer is continuously raised until the foam overflows or the foam layer is scraped off directly. Hydrophilic particles settle at the bottom of the flotation tank, thereby achieving the purpose of separation. The chemicals used in the flotation process are called flotation reagents, which mainly adjust the physical and chemical properties of the mineral surface and improve the floatability of the mineral. Currently, there are hundreds of developed flotation reagents, among which collectors can selectively adsorb on the surface of minerals and form a hydrophobic film on the surface, allowing mineral particles to adhere more firmly to the bubbles and float with them. This is the basis of the flotation process based on selective separation of minerals.

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

[0005] The structure of BHA includes a benzene ring and a hydroxamic 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 hydroxamic group have lone pair electrons and are close together, giving BHA strong chelating ability and making it easy to chelate with metal ions to form stable insoluble complexes. BHA has high selectivity for Cu, Fe, Pd, Ni, etc., and is currently commonly used in the flotation of oxidized ores and rare earth ores.

[0006] BHA has physiological toxicity due to the presence of benzene ring and oxime group, making it difficult to be degraded and utilized. Once released into the water environment, its residual time can be several years or even longer. The presence of BHA increases the COD content in flotation wastewater, and due to the accumulation of N elements, it is easy to cause eutrophication of water bodies, leading to a decrease in water quality and making it difficult for aquatic organisms to survive, ultimately disrupting the ecological balance. SUMMARY

[0007] The present application aims to overcome the deficiencies of the prior art and provide a strain of Cupriavidus and its application in degrading benzohydroxamic acid.

[0008] A first object of the present application is to provide a strain of Cupriavidus.

[0009] A second object of the present application is to provide the application of the Cupriavidus in degrading benzohydroxamic acid or in preparing a product for degrading benzohydroxamic acid.

[0010] A third object of the present application is to provide the application of the Cupriavidus in degrading benzoic acid, salicylic acid, phenol, catechol, salicylhydroxamic acid and / or N-hydroxyphthalimide or in preparing a product for degrading benzoic acid, salicylic acid, phenol, catechol, salicylhydroxamic acid and / or N-hydroxyphthalimide.

[0011] A fourth object of the present application is to provide the application of the Cupriavidus in degrading hydroxamic acid collectors or in preparing a product for degrading hydroxamic acid collectors.

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

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

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

[0015] An eighth object of the present application is to provide a product for degrading hydroxamic acid.

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

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

[0018] To achieve the above objects, the present application is implemented by the following technical solutions:

[0019] The present application claims a strain of Cupriavidus (Cupriavidus sp.) HY21, which was preserved in the Guangdong Microbial Culture Collection Center on September 11, 2024, and the preservation number is GDMCC No: 65117.

[0020] The following applications are also claimed:

[0021] The application of the Cupriavidus HY21 in degrading benzohydroxamic acid or in preparing a product for degrading benzohydroxamic acid.

[0022] Use of the Chalmydomonas HY21 in degrading benzoic acid, salicylic acid, phenol, o-diphenol, salicylhydroxamic acid and / or N-hydroxyphthalimide, or in preparing products for degrading benzoic acid, salicylic acid, phenol, o-diphenol, salicylhydroxamic acid and / or N-hydroxyphthalimide.

[0023] Use of the Chalmydomonas HY21 in degrading hydroxamic acid collectors, or in preparing products for degrading hydroxamic acid collectors.

[0024] Preferably, the hydroxamic acid collectors are one or both of salicylhydroxamic acid and N-hydroxyphthalimide.

[0025] Preferably, the degrading 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 degrading conditions are pH 5.5, temperature 30.5°C, inoculation amount 2% volume fraction OD600=1.0.

[0027] Also claimed is the following method:

[0028] A method for degrading hydroxamic acid, using the Chalmydomonas HY21 to degrade the hydroxamic acid.

[0029] A method for degrading benzoic acid, salicylic acid, phenol, o-diphenol, salicylhydroxamic acid and / or N-hydroxyphthalimide, using the Chalmydomonas HY21 to degrade the benzoic acid, salicylic acid, phenol, o-diphenol, salicylhydroxamic acid and / or N-hydroxyphthalimide.

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

[0031] Preferably, the hydroxamic acid collectors are one or more of salicylhydroxamic acid, N-hydroxyphthalimide or hydroxamic acid.

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

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

[0034] Also claimed is the following method:

[0035] A product for degrading hydroxamic acid, comprising the Cupriavidus sp. HY21.

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

[0037] A product for degrading hydroxamic acid collector, comprising the Cupriavidus sp. HY21.

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

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

[0040] The present application isolates a BHA degrading bacterium, Cupriavidus sp. HY21, from the soil of a mining area. The optimal degrading conditions of the strain HY21 are: pH 5.5, temperature 30.5℃, inoculation amount 1.0 (OD 600nm ), under which the degrading rate of 100 mg / L BHA can reach more than 97% in only 24 h. In addition, the strain HY21 also has good degrading ability for hydroxamic acid collector and benzoic acid, salicylic acid, phenol and / or catechol, and can be used for BuX pollution remediation. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The colony morphological characteristics of the strain on nutrient agar plate.

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

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

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

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

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

[0047] Figure 7 The effect of inoculation amount on the degradation of BHA by the strain HY21.

[0048] Figure 8 The response surface graph (A) and the contour graph (B) of the interactive effect of pH (X1) and temperature (X2) on the degradation of BHA by the strain HY21.

[0049] Figure 9 Degradation kinetics curve of strain HY21 to different initial concentration of BHA (A) and its fitting curve (B).

[0050] Figure 10 Mass spectrum of BHA intermediate degradation products (A. Benzohydroxamic acid, B. Salicylic acid, C. o-Dihydroxybenzene, D. Phenol).

[0051] Figure 11 Biochemical pathway of strain HY21 degrading BHA. DETAILED DESCRIPTION

[0052] The application will be further described in conjunction with the accompanying drawings and specific examples, which are only used to explain the application and are not used to limit the scope of the application. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

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

[0054] Liquid minimal salt medium (MSM): K2HPO4 1.5 g / L, KH2PO4 0.5 g / L, (NH4)2SO4 0.5 g / L, MgSO4·7H2O 0.2 g / L and NaCl 1.0 g / L, with a pH value of about 7.0.

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

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

[0057] The degradation rate calculation method is: degradation rate (%) = 100% × (C0-C1 / C0),

[0058] In the formula, C1 is the residual concentration of BHA after treatment with degrading bacteria, and C0 is the initial concentration of BHA without treatment.

[0059] Isolation and identification of strain HY21

[0060] I. Experimental methods

[0061] 1. Enrichment and isolation of the strain

[0062] A 250 mL conical flask was filled with 100 mL MSM medium and sterilized. After cooling, BHA was added to achieve an initial concentration of 10 mg / L. 5 g of contaminated soil collected from the Dabaoshan mine in Guangzhou was added to the medium, and the mixture was cultured in a 35°C, 150 rpm shaker in the dark for 7 days. On the 7th day, 10% (v / v) of the culture was transferred to new MSM medium, and the concentration of the contaminant was gradually increased for acclimation. This process was repeated 5 times. The last culture was evenly spread on a nutrient agar plate, and after 48 h of incubation, single colonies were selected by repeated streaking. The single colonies were inoculated into MSM medium containing 100 mg / L BHA, and the mixture was cultured in a 35°C, 150 rpm shaker for 72 h. The degradation effect was detected, and the strain was inoculated into a test tube slant medium and stored in a 4°C refrigerator for future use.

[0063] 2. Observation of colony characteristics

[0064] The selected strain was inoculated onto a nutrient agar plate and incubated overnight in a 35°C incubator. The shape, size, transparency, edge, and color of the strain were observed.

[0065] 3. SEM observation of bacterial morphology

[0066] The selected strain was inoculated into LB broth medium and cultured for 24 h. 800 μL of the bacterial solution was transferred to a 1.5 mL centrifuge tube and centrifuged at 8000 rpm for 5 min. The supernatant was discarded, and the bacterial pellet was washed with sterile saline. This process was repeated 3 times. 1 mL of 2.5% glutaraldehyde fixative was added to the washed bacterial pellet, and the mixture was vortexed and incubated overnight at 4°C. After centrifugation at 8000 rpm for 5 min, the supernatant was discarded, and the bacterial pellet was washed with sterile saline 3 times. Subsequent dehydration was performed by adding 30%, 50%, 70%, 80%, and 90% ethanol, respectively, twice with 100% ethanol. Each dehydration step required 15 min of incubation. Finally, the bacterial pellet was dropped onto a cover glass and placed in a freeze-drying machine for drying. The dried sample was treated with gold spraying and observed under a scanning electron microscope.

[0067] 4. Physiological and biochemical identification of the strain

[0068] The physiological and biochemical experiments of the strains refer to the Common Bacteria Identification Manual and Bergey's Manual of Determinative Bacteriology

[0069] 5. Molecular biological identification of the strains

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

[0071] PCR reaction system: Taq Mixture, 12.5 μL; ddH2O, 9.5 μL; 27F, 1 μL; 1492R, 1 μL; DNA template, 1 μL, and the total reaction system was 25 μL.

[0072] PCR reaction conditions: 95°C, initial preheating for 5 min; melting 94°C, 45 s, annealing 55°C, 45 s, extension 72°C, 1 min 15 s, 32 cycles, and finally 72°C for 10 min. After the end of PCR amplification, 1.5% agarose gel electrophoresis was performed to detect the size and specificity of the amplified fragments, and the gel imaging system was photographed, and the PCR amplification products were purified and recovered by Shanghai Shengong Company and sequenced.

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

[0074] II. Experimental results

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

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

[0077] ​The results of gram staining showed that the strain HY21 was gram-negative bacteria, and the methyl red test, nitrate reduction, contact enzyme, starch hydrolysis, hydrogen sulfide, acetyl methyl methanol test were all negative, the oxidation reaction, ornithine decarboxylase, lysine decarboxylase test, 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: "+" means can be utilized or positive; "-" means can not be utilized or negative.

[0082] The genomic DNA of the strain HY21 was used as a template, and 16S rDNA bacterial universal primers were used for PCR amplification. After amplification, the product was electrophoresed, and the band was single. The amplification product was sent to Shanghai Shengong Biological Technology Co., Ltd. for purification. It was detected that the length of the product was 1161 bp.

[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 subjected to multiple sequence alignment, and the phylogenetic tree of the strain HY21 was constructed by the neighborhood-joining method (Neighborhood-joining). Figure 3 ) HY21 had high homology with Cupriavidus sp. strain, so it could be judged that the strain HY21 was Cupriavidus sp. strain.

[0084] The strain HY21 was preserved in Guangdong Microbial Culture Collection Center on September 11, 2024, and the preservation number was GDMCC No: 65117, and the classification and naming was Cupriavidus sp.

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

[0086] 1. Sample pretreatment method

[0087] 1 mL of the test strain HY21 culture solution was taken into a 1.5 mL centrifuge tube, and centrifuged at 8000 rpm for 5 min. After centrifugation, 300 μL of the supernatant was diluted 10 times. To prevent BuX degradation, the BuX content was immediately determined by UV-Vis. Another 100 μL of the supernatant was diluted 100 times with methanol-water (50 / 50, v / v) as the solvent. Finally, the filtrate was collected in a sample bottle after being filtered through a 0.22 μm microporous filter, and the BHA residual amount was determined by LC-MS / MS.

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

[0089] An LC-MS / MS detection method of BHA was established by using an AB Sciex 5500 MD high performance liquid chromatograph-tandem mass spectrometer.

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

[0091] Table 2. Mobile phase gradient elution program

[0092]

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

[0094] Table 3. Mass spectrometric parameters of BHA

[0095]

[0096] 4. BHA standard curve

[0097] Take 10 μL BHA standard solution (1000 mg / L) into the injection bottle, dilute with 990 μL chromatographic grade methanol to obtain BHA standard stock solution with a concentration of 10 mg / L. Prepare solutions with concentrations of 50, 75, 150, 300, 600, 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 determined peak area is Y, and the corresponding standard solution concentration is X, and the standard curve "Y=a+bX" is drawn.

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

[0099] I. Influence of pH on the BHA degradation ability of strain HY21

[0100] 1. Experimental method

[0101] The pH of the prepared MSM medium was adjusted to 3, 4, 5, 6, 7, and 8 respectively using a precise pH meter from Raydist, and then sterilized after aliquoting. After the medium cooled down, BHA was added to make the initial concentration reach 100 mg / L. HY21 bacterial suspension (OD600=1.0) was inoculated at a volume ratio of 2%, and the culture was incubated at 35°C with 150 rpm shaking for 24 h. The residual BHA content was determined (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 5 It can be seen that the pH in the range of 3-8 has a significant effect on the degradation of BHA by strain HY21. When the pH is 5-7, the degradation rate of strain HY21 after 24 h of culture can reach more than 97%, and the highest degradation rate is 98.55% at pH 7. When the pH is less than 5, the degradation ability of strain HY21 for BHA decreases, and the degradation rate of strain HY21 for BHA is only 14.26% at pH 3. When the pH is greater than 7, the degradation ability of the strain decreases significantly, and the degradation rate of HY21 is only 22.03% at pH 8. The above results show that the degradation efficiency of strain HY21 is significantly inhibited under strong acid and strong alkali conditions, and the optimal pH range of the strain is between 5 and 7, i.e. the strain HY21 has good degradation ability for BHA under weakly acidic and neutral conditions.

[0104] II. Influence of temperature on the BHA degradation ability of strain HY21

[0105] 1. Experimental method

[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 after 24 hours to determine the remaining BHA concentration in the culture medium (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 significantly affected the degradation of BHA by strain HY21. At temperatures between 30°C and 35°C, strain HY21 achieved a BHA degradation rate exceeding 97%. However, when the temperature exceeded this range, strain HY21's BHA degradation ability decreased significantly, particularly at temperatures of 20°C and 40°C, where the degradation rates were only 6.6% and 15.14%, respectively. This indicates that both excessively high and low temperatures adversely affected the growth of strain HY21, significantly inhibiting its BHA degradation.

[0109] 3. Effect of inoculation amount on the BHA degradation ability of strain HY21

[0110] 1. Experimental methods

[0111] At pH 7 and temperature 35°C, the OD value of HY21 bacterial suspension was first 600 The concentrations of the bacterial suspension were adjusted to 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6, respectively. The suspension was inoculated into MSM medium with an initial BHA concentration of 100 mg / L at a 2% volume ratio. The suspension was then shaken in an incubator at 150 rpm for 24 hours. After the incubation period, samples were taken and the BHA concentration was measured (according to the method in Example 3). A control group was not inoculated. Three biological replicates were set up 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 the inoculation amount. When the inoculation amount reaches 1.2 (OD 600nm ), the highest BHA degradation rate reached 97.47%; when the inoculation 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 population, competition among microorganisms, and relative lack of nutrients.

[0114] Example 5 Degradation conditions of strain HY21

[0115] I. 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 of strain HY21. The independent variables were pH, temperature, and inoculum size, numbered as X1, X2, and X3, respectively, and represented by -1, 0, and 1, respectively. The response value (Y) was the degradation rate of 100 mg / L BHA in the culture medium after 24 h of degradation by the strain. The specific factor level coding is shown in Table 4.

[0117] Table 4 Factor level and coding based on Box-Behnken design

[0118]

[0119] The final regression model is as follows:

[0120] Y i = b0+∑b i X i +∑b ij X i X j +∑b ii X i 2 wherein Yi is the predicted BHA degradation rate. The reliability of the regression model was evaluated by model significance and variance analysis. Finally, the degradation strain was 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] II. Experimental results

[0122] Using Design expert 13.0, the BHA degradation rate was used as the response value (Y), and the Box-Behnken design was used to obtain the strain HY21 degradation condition optimization experimental design and response value data (Table 5).

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

[0124]

[0125] Note: The dependent variable result is the average value ± standard deviation of 3 repeated experiments, and the same letter at the end indicates that the difference is not significant (P>0.05).

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

[0127] Y = 96.45 - 13.8X1 + 2.54X2 - 27.07X1 2 - 26.58X2 2 - 7.28X3 2 The coefficient of determination (R 2 ) of the quadratic polynomial model based on the above data was 0.9932, indicating that the actual measured values and predicted values had a high degree of correlation, and the model could be used for theoretical prediction of BHA by strain HY21. The adjusted coefficient of determination was 0.9844, indicating that the model explained the changes in the response values of strain HY21 well. Finally, the coefficient of variation (CV) of the model was 4.24%, indicating that the model equation reflected the actual experimental measurements.

[0128] The results of the analysis of variance (Table 6) showed that the model P < 0.0001, indicating that the model fitting was highly significant. Among the three selected independent variables, pH (X1) had a very significant effect on the degradation of BHA by strain HY21 (P < 0.0001), temperature (X2) also had a significant effect on the degradation of BHA by strain HY21 (P < 0.05), and the inoculum size (X3) had no significant effect on the degradation of BHA by strain HY21 (P > 0.05). At the same time, the P values of the coefficients of the quadratic terms X1 2 , X2 2 were less than 0.0001, indicating that the linear effects of the coefficients on the degradation rate of strain HY21 reached a very significant level; the P value of X3 2 was less than 0.05, indicating that the linear effect of the coefficient on the degradation rate of strain HY21 reached a significant level.

[0129] Table 6 Quadratic polynomial model and variance analysis of each item

[0130]

[0131] The response surface was drawn using Design expert 13.0 software to study the effects of single factors and the interactions between factors on the degradation of BHA. The results of the analysis of variance showed that the inoculum size had no significant effect on the degradation of BHA, so according to the fitted function, the inoculum size was fixed at 1.0 (OD600nm, 2% by volume), and the effects of pH (X1) and temperature (X2) on the degradation of BHA were analyzed, and three-dimensional response surface graphs and contour graphs were drawn. Figure 8

[0132] The response surface graph showed that the model had a maximum point, and the first-order partial derivative of the quadratic regression equation gave the optimal conditions for the degradation of BHA by strain HY21: pH 5.5, temperature 30.5°C, and inoculum size 1.0 (i.e. OD 600 ​= 1.0, 2% volume fraction), under which condition the model predicted the BHA degradation rate of 98.29%. To verify the accuracy of the model prediction, the 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 to 100 mg / L BHA reached 97.83%, which was close to the predicted result, indicating that the reliability of the model prediction result.

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

[0134] I. Experimental method

[0135] Under the optimal degradation conditions of HY21 (pH 5.5, temperature 30.5°C, inoculation amount 1.0 (i.e. OD 600 = 1.0, 2% volume fraction), under which condition the model predicted the BHA degradation rate of 98.29%. To verify the accuracy of the model prediction, the 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 to 100 mg / L BHA reached 97.83%, which was close to the predicted result, indicating that the reliability of the model prediction result.

[0136] II. Experimental results

[0137] Figure 9 The results in Fig. A show that the strain HY21 can tolerate BHA concentration range of 50-200 mg / L, Figure 9 Fig. B is the fitting curve. A strain of Klebsiella reported so far, after 12 days of culture, the degradation rate of BHA with initial concentration of 100 mg / L was only 85.04% (Hu C, Wang C, Gong WQ, et al. Microbial degradation of three hydroxamic acid collectors [J]. Hubei Agricultural Sciences, 2013, 52(11): 2505-2507). Compared with it, the strain HY21 can tolerate higher concentration of BHA and has higher degradation efficiency of BHA. However, the strain HY21 showed obvious lag phenomenon in the process of degrading high concentration of BHA, which is due to the toxic effect of high concentration of BHA on the strain HY21, inhibiting its growth.

[0138] Figure 9The degradation curve showed an "S" shape and did not conform to the first-order kinetic model. Further, the four-parameter Logistic model was used to fit the BHA degradation rate (y) and time (t) at different time points at different initial concentrations of BHA (Table 7), and the fitting equation was as follows:

[0139]

[0140] wherein y, y0 and ymax were the reaction rates at t, t0 and the highest BHA degradation rate, respectively; b was the slope of the fitting curve; and c was the degradation half-life.

[0141] As shown in Table 7, R 2 were all greater than 0.99, indicating that the degradation of BHA by the strain HY21 conformed to the four-parameter Logistic model. The degradation half-life of the 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 gradually increased with the increase of the concentration.

[0142] Table 7 Kinetic equation for the degradation of BHA at different initial concentrations by the strain HY21

[0143]

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

[0145] I. Experimental method

[0146] Salicylhydroxamic acid, 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 sterile MSM medium to achieve an initial concentration of 100 mg / L, and the HY21 bacterial suspension was inoculated into the medium, which was then placed in a constant temperature shaker under the optimal conditions (pH 5.5, temperature 30.5°C, inoculation amount 1.0 (OD 600 =1.0, 2% volume fraction)) and shaken for 72 h. The color change of the medium and the turbidity of the bacterial solution were observed to determine whether the strain HY21 could utilize these substrates for growth. The un-inoculated treatment was used as a blank control, and each group was repeated three times.

[0147] II. Experimental results

[0148] As shown in Table 8, the strain HY21 could not only utilize benzoic acid, salicylic acid, catechol and phenol as possible intermediate degradation products, but also utilize salicylhydroxamic acid, N-hydroxyphthalimide and other hydroxamic acid collectors as the sole carbon source for its own growth, and had a broad substrate utilization ability.

[0149] Table 8 Substrate utilization of strain HY21

[0150]

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

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

[0153] I. Experimental methods

[0154] HY21 bacterial suspension was inoculated into sterilized MSM medium with pollutants, the initial pH of the degradation system was 7.0, the initial concentration of the pollutants was 100 mg / L, and the system was cultured at 35°C and 150 rpm. Samples were taken at different time periods. The culture solution was diluted ten times and scanned at a wavelength of 185-350 nm using UV-Vis to detect the intermediate degradation products of BuX. The intermediate metabolic products of BHA were extracted using a C18 solid phase extraction column, and then eluted with 10 mL of methanol to elute the adsorbed components on the column filler. The eluent was blown to dryness with nitrogen, 1 mL of methanol was added for re-dissolution, and then passed through a 0.22 μm filter membrane. The filtrate was collected in a brown sample bottle for analysis. HPLC-TOF-MS / MS was used to analyze the intermediate metabolic products of BHA. (Chromatographic and mass spectrometric conditions are described in: Zhao H M, Hu R W, Chen X X, 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] II. Experimental results

[0156] The culture solution reacted for different time periods was enriched by a C18 solid phase extraction column, and then detected using HPLC-TOF-MS / MS technology. The analysis showed that the intermediate metabolic products in the process of strain HY21 degrading BHA were obtained. The specific data of the intermediate products are shown in Table 9.

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

[0158]

[0159]

[0160] The HPLC-TOF-MS / MS detection results are as follows Figure 10 ,

[0161] The benzene ring structure exists in BHA, and the degradation process of the benzene ring-containing substance often involves ring-opening process. Referring to the biodegradation pathways of other hydroxamic acid collectors, the pathway of strain HY21 degrading BHA is as shown in Figure 11 .

[0162] The biodegradation of BHA mainly passes through the following two pathways: the first pathway is benzoic acid pathway, the amide bond in BHA is broken to generate benzoic acid and hydroxylamine, the benzoic acid is hydroxylated at the ortho position to generate salicylic acid, the salicylic acid is hydroxylated again to generate o-diphenol, the o-diphenol is ring-opened under the catalysis of dioxygenase to be further degraded, and finally enters the tricarboxylic acid cycle (TCA). The second pathway is to generate phenol first, then oxidize to generate o-diphenol, and then enter the TCA cycle through the o-diphenol pathway, and the process of BHA converting into phenol is not clear.

Claims

1. A strain of Cupriavidus basing on the characteristics of, It was preserved in Guangdong Microbial Culture Collection Center on September 11, 2024, and the preservation number is: GDMCC No: 65117.

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

3. Use of Chryseomonas lindanora HY21 according to claim 1 for the degradation of hydroxamic acid collectors or for the preparation of a product for the degradation of hydroxamic acid collectors, characterized in that, The hydroxamic acid collector is benzohydroxamic acid collector.

4. A method of degrading methohydroxamic acid, characterized by, The Chalmydia HY21 in claim 1 is used to degrade hydroxamic acid.

5. A method of degrading hydroxamic acid-based collectors, characterized by, The Chalmydia HY21 in claim 1 is used to degrade hydroxamic acid collector, and the hydroxamic acid collector is benzohydroxamic acid collector.

6. A product for degrading methohydroxamic acid, characterized in that, The Chalmydia HY21 in claim 1 is contained.

7. A product for degrading hydroxamic acid collectors, characterized by The Chalmydia HY21 in claim 1 is contained, and the hydroxamic acid collector is benzohydroxamic acid collector.

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