Rhodococcus aetherivorans strain capable of degrading dimethylacetamide, culture method and application thereof

Abstract

The application discloses a Rhodococcus qingshengii strain capable of degrading dimethylacetamide as well as a culture method and application of the strain, and belongs to the technical field of biotechnology.The Rhodococcus qingshengii strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) on April 20, 2023, and the address of the CGMCC is No. 1, Xili, Beichen West Road, Chaoyang District, Beijing, and the Institute of Microbiology, Chinese Academy of Sciences; the preservation number is CGMCC No. 27163.The Rhodococcus qingshengii strain provided by the application has the ability to degrade dimethylacetamide, and under the condition of 28 DEG C and pH 7.0, the strain can degrade a certain concentration of DMAc in wastewater to more than 90% within 7 days, thereby providing a new feasible scheme for solving the problem of difficult treatment of wastewater containing DMAc in chemical production and providing technical support for application of biotechnology in sewage treatment.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to a strain of Rhodococcus qingshengii that degrades dimethylacetamide, its culture method, and its application. Background Technology

[0002] N,N-Dimethylacetamide (DMAc), as an important chemical raw material and a high-performance solvent, is mainly used in various chemical industries such as coatings, acrylamide, pharmaceuticals, petrochemicals, and polymer synthesis. In the coatings industry, it is used as a solvent. In the acrylamide industry, DMAc is mainly used as a solvent in the organic wet process production of acrylamide, showing significant advantages in terms of production technology, environmental energy consumption, product quality, and economic benefits. In the pharmaceutical industry, DMAc can be used as an intermediate in the synthesis of drugs (such as ethylproxil fumarate and ranolazine), and also as a catalyst or solvent in the production of antibiotics, increasing product quality and yield; the demand for DMAc in the pharmaceutical industry continues to grow. In the petrochemical industry, DMAc, as a catalyst, can significantly improve the reaction rate of dehydrogenation, cyclization, halogenation, and alkylation; as a solvent, it is used in the process of extracting butadiene from C4 gas from ethylene cracking. In the field of polymer materials, DMAc has good solubility for many polymers and is widely used in the production of soluble resin materials and composite films. In addition to the applications mentioned above, DMAc, due to its high boiling point and good solubility, can also be used as an extractant in liquid-liquid extraction and extractive distillation processes. Therefore, DMAc is widely used as a solvent in most industrial production processes and can be discharged with production wastewater without being consumed.

[0003] Dimethylacetamide (DMAc) is chemically stable, difficult to degrade, and exhibits significant physiological toxicity. Its high organic nitrogen content is a major factor contributing to the difficulty in treating and discharging DMAc-containing wastewater to meet standards. Domestic and international methods for DMAc degradation mainly include iron-carbon internal electrolysis, Fenton oxidation, ozone oxidation, adsorption, and photocatalytic oxidation. While these methods show good removal efficiency for DMAc wastewater, they are complex, costly, and prone to generating secondary pollution. Therefore, it is necessary to explore simpler, more convenient, and economical treatment methods.

[0004] Currently, biochemical methods are widely used in water treatment processes due to their advantages of low cost, high treatment efficiency, and no secondary pollution. Studies have reported that microorganisms can effectively degrade organic pollutants in wastewater, and biochemical methods have shown good results in degrading DMAc. However, the types of dimethylacetamide-degrading bacteria screened domestically and internationally are still relatively few. For example, few microorganisms such as Rhodococcus and Paracoccus have been isolated for treating DMAc-containing wastewater. Therefore, isolating and screening a highly efficient dimethylacetamide-degrading strain is of great significance for the treatment of DMAc-containing wastewater. Summary of the Invention

[0005] This invention discloses a strain of Rhodococcus qingshengii that degrades dimethylacetamide, its cultivation method, and its application. This Rhodococcus qingshengii strain can reduce the residual amount of dimethylacetamide in wastewater to more than 90%, thereby effectively solving at least one of the technical problems mentioned in the background art.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: A strain of Rhodococcus qingshengii that degrades dimethylacetamide was deposited on April 20, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27163. The 16S rDNA sequence of the strain is shown in SEQ ID NO. 1.

[0007] SEQ ID NO.1 16S rDNA sequence of Rhodococcus qingsheng:

[0008] The *Rhodococcus qingshengensis* is a Gram-positive aerobic rod-shaped bacterium with short rod-shaped cells, no flagella, and a size of approximately 1-3 μm in length and 0.4-0.9 μm in width. Its colonies are small, round in shape, with rough edges and raised surfaces, and are opaque apricot in color. It can grow using dimethylacetamide as both a carbon and nitrogen source.

[0009] This invention also provides a method for culturing a strain of Rhodococcus qingshengii that degrades dimethylacetamide, comprising the following steps: Step 1: Collect 10 g of activated sludge sample, add it to 100 mL of sterile water, shake it in a 30℃ constant temperature shaker for 1 h to mix it well, and then let it stand for 2 h before use. Step 2: Take 10 mL of the first supernatant obtained in Step 1 and add it to 100 mL of sterile water. Shake well and let stand for 2 hours. Step 3: Take 1 mL of the second supernatant obtained in Step 2 and drop it into 50 mL of LB liquid medium containing 2% dimethylacetamide (v / v). Incubate at 30°C for 72 h in a constant temperature shaker. Then, transfer 1 mL of the supernatant to 50 mL of LB liquid medium containing 3% dimethylacetamide (v / v). Incubate under the same conditions for 72 h. Repeat this transfer process until the volume ratio of dimethylacetamide in the LB liquid medium is 6%. Step 4: Take the bacterial suspension from the last culture step in Step 3, i.e., when the volume ratio of dimethylacetamide in LB liquid medium is 6%, and dilute it to 10 with sterile water. -4 After dilution, 100 μL of the diluted solution was added dropwise to LB solid medium containing 6% dimethylacetamide by volume, and incubated at 30°C for 72 h in a constant temperature incubator. Step 5: Select single colonies with obvious differences on the culture medium in Step 4 and streak them for isolation until a purified single strain is obtained. Step Six: Prepare OD from the strains obtained in Step Five. 600 The seed culture with a concentration of 1.5 was inoculated into an inorganic salt medium containing dimethylacetamide at a rate of 10%. After culturing for 168 h, the residual dimethylacetamide content in the medium was determined by high performance liquid chromatography. The strain with the highest dimethylacetamide degradation efficiency was selected for identification and preservation.

[0010] As a preferred improvement of the present invention, in step three, the LB liquid culture medium is prepared by mixing and dissolving 0.5 g NaCl, 0.5 g peptone, 0.25 g yeast extract, 1-3 mL dimethylacetamide and 47-49 mL sterile water, and sterilizing in a high-temperature sterilizer at 121°C for 20 min.

[0011] As a preferred improvement of the present invention, in step four, the LB solid culture medium is prepared as follows: 0.5 g NaCl, 0.5 g peptone, 0.25 g yeast extract, 1.1 g agar, 3 mL dimethylacetamide and 47 mL sterile water are mixed and dissolved, and sterilized in a high-temperature sterilizer at 121°C for 20 min.

[0012] As a preferred improvement of the present invention, in step five, four colonies are selected.

[0013] As a preferred improvement of the present invention, in step six, the seed solution is prepared as follows: the four isolated and purified single strains are inoculated into 100 mL of nutrient broth medium and cultured at 30°C and 150 rpm / min for 24 h to obtain OD. 600 The seed culture medium has a value of 1.5; the nutrient broth culture medium is prepared as follows: take 1 g broth powder, 0.5 g peptone, 0.2 g yeast extract, 0.5 g NaCl, 0.2 g potassium dihydrogen phosphate, 0.1 g maltodextrin, 0.1 g glucose and 100 mL sterile water, mix and dissolve, and sterilize in a high-temperature sterilizer at 121℃ for 20 min.

[0014] As a preferred improvement of the present invention, in step six, the inorganic salt culture medium is prepared by mixing and dissolving 1500 mg / L dipotassium hydrogen phosphate, 500 mg / L potassium dihydrogen phosphate, 200 mg / L MgSO4·7H2O, 1000 mg / L NaCl, 500 mg / L dimethylacetamide, and 100 mL sterile water, and then sterilizing it in a high-temperature sterilizer at 121°C for 20 min.

[0015] The present invention also provides the application of the aforementioned Rhodococcus qingsheng in the degradation of dimethylacetamide. The seed culture of Rhodococcus qingsheng is added to an inorganic salt culture medium containing 500 mg / L DMAc. After a period of cultivation, the residual DMAc content in the culture medium is determined by high performance liquid chromatography (HPLC), that is, the degradation ability of the strain of DMAc.

[0016] As a preferred improvement of the present invention, the inoculum amount of Rhodococcus qingshengi seed liquid is 1~20% when degrading DMAc.

[0017] As a preferred improvement of the present invention, the pH of the inorganic salt culture medium is 5-9 during the degradation of DMAc.

[0018] As a preferred improvement of the present invention, the culture temperature is 20~40℃ when degrading DMAc.

[0019] The beneficial effects of this invention are as follows: 1. The dimethylacetamide degrading bacteria obtained by using the present invention have the advantages of being easy to use and having a good removal effect, and are suitable for widespread use in the treatment of wastewater containing low concentrations of DMAc; 2. The *Rhodococcus qingshengensis* provided by this invention can grow in LB medium containing dimethylacetamide and has good tolerance to DMAc. When *Rhodococcus qingshengensis* seed culture is inoculated at a rate of 10% in wastewater containing dimethylacetamide and cultured at 28°C and pH 7.0 for 7 days with shaking, the residual DMAc in wastewater containing 500 mg / L DMAc can be reduced to more than 90%, demonstrating a very good degradation effect. The discovery of this degrading bacterium is of great significance for further research on the biochemical degradation of DMAc. Attached Figure Description

[0020] Figure 1 A schematic diagram of Rhodococcus qingshengense colonies; Figure 2 The image shows the cell morphology of *Rhodococcus qingshengensis* under a laser confocal microscope. Figure 3 A scanning electron microscope image of Rhodococcus qingshengensis; Figure 4 This is a schematic diagram illustrating the evaluation of the degradation effect of four bacterial strains on DMAc. Figure 5 This is a schematic diagram illustrating the effect of temperature on the degradation of DMAc by Rhodococcus qingshengense. Figure 6 A schematic diagram illustrating the effect of the inoculum amount of *Rhodococcus qingshengensis* on the degradation of DMAc; Figure 7 This is a schematic diagram illustrating the effect of pH on the degradation of DMAc by Rhodococcus qingshengensis. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] It should be noted that all directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of the present invention are only used to explain the relative positional relationship and movement of each component in a certain specific posture (as shown in the figure). If the specific posture changes, the directional indication will also change accordingly.

[0023] In this invention, the use of terms such as "first," "second," etc., is for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0024] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0025] This invention provides a strain of Rhodococcus qingshengii that degrades dimethylacetamide. It was microbially classified as Rhodococcus qingshengii and deposited on April 20, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27163. The 16S rDNA sequence of Rhodococcus qingshengii is shown in SEQ ID NO. 1.

[0026] SEQ ID NO.1 16S rDNA sequence of Rhodococcus qingsheng:

[0027] This invention also provides a method for culturing a strain of Rhodococcus qingshengii that degrades dimethylacetamide, comprising the following steps: Step 1: Sampling. Collect 10g of activated sludge sample from Maijia Wastewater Treatment Plant in Baiyun District, Guiyang City, Guizhou Province, add it to 100 mL of sterile water, shake it in a constant temperature shaker at 30℃ for 1 h to mix it, and then let it stand for 2 h before use. Step 2: Take 10 mL of the first supernatant obtained after standing in Step 1 and add it to 100 mL of sterile water. Shake well and let stand for 2 hours. Step 3: Take 1 mL of the second supernatant obtained in Step 2 and drop it into 50 mL of LB liquid medium containing 2% DMAc (v / v). Incubate at 30°C for 72 h in a constant temperature shaker. Then, transfer 1 mL of the supernatant to 50 mL of LB liquid medium containing 3% DMAc (v / v) and incubate under the same conditions for 72 h. Repeat this transfer process until the volume ratio of dimethylacetamide in the LB liquid medium is 6%. The preparation method of LB liquid culture medium is as follows: take 0.5 g NaCl, 0.5 g peptone, 0.25 g yeast extract, 1~3 mL dimethylacetamide and 47~49 mL sterile water, mix and dissolve them, and sterilize in a high temperature sterilizer at 121℃ for 20 min.

[0028] Step 4: Take the bacterial suspension from the last culture step in Step 3 and dilute it to 10 with sterile water. -4 After dilution, 100 μL of the diluted solution was added dropwise to LB solid medium containing 6% DMAc by volume, and incubated at 30°C for 72 h in a constant temperature incubator. The preparation method of LB solid culture medium is as follows: take 0.5 g NaCl, 0.5 g peptone, 0.25 g yeast extract, 1.1 g agar, 3 mL dimethylacetamide and 47 mL sterile water, mix and dissolve them, and sterilize in a high temperature sterilizer at 121℃ for 20 min.

[0029] Step 5: Select single colonies with obvious differences on the culture medium in Step 4 and streak them for isolation until a purified single strain is obtained. Step Six: Prepare OD from the strains obtained in Step Five. 600 The seed culture with a value of 1.5 was inoculated into an inorganic salt medium containing DMAc at an inoculation rate of 10%. After culturing for 168 h, the residual DMAc content in the medium was determined by high performance liquid chromatography (HPLC), which is the ability of the strain to degrade DMAc. The strain with the highest DMAc degradation efficiency was selected for identification and preservation.

[0030] When degrading DMAc, the inoculum size of *Rhodococcus qingshengensis* seed culture is 1-20%, preferably 10%, the culture temperature is 20-40℃, and the pH of the inorganic salt culture medium is 5-9.

[0031] Specifically, in step six, the seed culture preparation method is as follows: the four isolated and purified single strains are inoculated into 100 mL of nutrient broth medium and cultured at 30°C and 150 rpm / min for 24 h to obtain OD. 600 The seed culture medium has a pH of 1.5. The nutrient broth culture medium is prepared as follows: 1 g of broth powder, 0.5 g of peptone, 0.2 g of yeast extract, 0.5 g of NaCl, 0.2 g of potassium dihydrogen phosphate, 0.1 g of maltodextrin, 0.1 g of glucose, and 100 mL of sterile water are mixed and dissolved, and then sterilized in an autoclave at 121°C for 20 min.

[0032] The preparation method of inorganic salt culture medium is as follows: take 1500 mg / L of dipotassium hydrogen phosphate, 500 mg / L of potassium dihydrogen phosphate, 200 mg / L of MgSO4·7H2O, 1000 mg / L of NaCl, 500 mg / L of dimethylacetamide and 100 mL of sterile water, mix and dissolve them, and sterilize in a high-temperature sterilizer at 121℃ for 20 min.

[0033] The present invention also provides the application of the aforementioned Rhodococcus qingsheng in the degradation of dimethylacetamide. Under the conditions of 28°C and pH 7.0, the Rhodococcus qingsheng can degrade a certain concentration of DMAc in wastewater to more than 90% within 7 days.

[0034] The present invention will be further described below with reference to specific embodiments.

[0035] Example 1 Culture methods, identification and preservation of Rhodococcus qingshengensis (1) Enrichment and domestication of strains 10 g of activated sludge sample was collected from Maijia Sewage Treatment Plant in Baiyun District, Guiyang City, Guizhou Province. It was added to 100 mL of sterile water and shaken in a constant temperature shaker at 30℃ for 1 h to mix. After standing for 2 h, 10 mL of supernatant was added to 100 mL of sterile water and shaken again and stood for 2 h. Then, 1 mL of the supernatant was dropped into 50 mL of LB liquid medium containing 2% N,N-dimethylacetamide (DMAc) by volume, and cultured at 30°C for 72 h in a constant temperature shaker. Take 1 mL of the bacterial suspension from the above steps and transfer it to 50 mL of LB liquid medium containing 3% DMAc by volume. Incubate under the same conditions for 72 h. Repeat this transfer process until the volume ratio of DMAc in the LB liquid medium is 6%.

[0036] The preparation method of LB liquid culture medium is as follows: Take 0.5 g NaCl, 0.5 g peptone, 0.25 g yeast extract, 1~3 mL DMAc and 47~49 mL sterile water, mix and dissolve them, and sterilize in a high temperature autoclave at 121℃ for 20 min.

[0037] (2) Screening and isolation of strains Take the bacterial suspension from the last culture step in (1), i.e., when the volume ratio of DMAc in LB liquid medium is 6%, and dilute it with sterile water to 10. -4 After dilution, 100 μL of the diluted solution was added dropwise to LB solid medium containing 6% DMAc and incubated at 30°C for 72 h. Then, four single colonies showing significant differences were selected for streak isolation until a purified single strain was obtained. The obtained single strain was then prepared into an OD medium. 600 The seed culture with a value of 1.5 was inoculated into an inorganic salt medium containing DMAc at an inoculation rate of 10%. After culturing for 168 h, the residual DMAc content in the medium was determined by high performance liquid chromatography (HPLC). The strain with the highest DMAc degradation efficiency was selected for identification and preservation.

[0038] The preparation method of LB solid medium is as follows: Take 0.5 g NaCl, 0.5 g peptone, 0.25 g yeast extract, 1.1 g agar, 3 mL DMAc and 47 mL sterile water, mix and dissolve them, and sterilize in a high temperature autoclave at 121℃ for 20 min.

[0039] The seed culture preparation method is as follows: Four isolated and purified single strains were inoculated into 100 mL of nutrient broth medium and cultured at 30℃ and 150 rpm / min for 24 h to obtain OD. 600 Seed liquid with a value of 1.5.

[0040] The method for preparing the nutrient broth culture medium is as follows: take 1 g broth powder, 0.5 g peptone, 0.2 g yeast extract, 0.5 g NaCl, 0.2 g potassium dihydrogen phosphate, 0.1 g maltodextrin, 0.1 g glucose and 100 mL sterile water, mix and dissolve, and sterilize in a high-temperature autoclave at 121℃ for 20 min.

[0041] The inorganic salt culture medium is prepared as follows: 1500 mg / L dipotassium hydrogen phosphate, 500 mg / L potassium dihydrogen phosphate, 200 mg / L MgSO4·7H2O, 1000 mg / L NaCl, 500 mg / L DMAc and 100 mL sterile water are mixed and dissolved, and then sterilized in a high-temperature autoclave at 121℃ for 20 min.

[0042] (3) Identification of strains The strain obtained in (2) was inoculated onto slant culture medium and sent to Sangon Biotech (Shanghai) Co., Ltd. for strain identification. Molecular biological identification methods were used, and PCR amplification of 16S rDNA was performed using the universal primer 1492r for 16S rDNA. PCR reaction conditions: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 90 s, 30 cycles followed by 72℃ extension for 10 min. The PCR products were subjected to 1.5% agarose gel electrophoresis. After agarose gel electrophoresis, the PCR products were recovered, purified, and sequenced. Homologous sequences were searched in GenBank using Blast based on the obtained 16S rDNA sequence, and homologous sequence analysis and comparison were performed.

[0043] The preparation method of slant culture medium is as follows: Take 0.5 g NaCl, 0.5 g peptone, 0.25 g yeast extract, 1.1 g agar and 50 mL sterile water, mix and dissolve them, and sterilize in a high-temperature sterilizer at 121℃ for 20 min; when the temperature drops to 60℃, pour the culture medium into the sterilized test tube to 1 / 3 of the height, seal it with a rubber stopper, place it on a 1 cm thick wooden strip, and wait for it to cool to obtain the slant culture medium.

[0044] 16S rDNA gene sequence alignment analysis showed that the strain belongs to the genus Rhodococcus and has a sequence identity of 100.0% with Rhodococcus qingshengii.

[0045] (4) Preservation of strains After the obtained strain was inoculated onto an agar slant culture medium, it was sent to the China General Microbiological Culture Collection Center for preservation, with the preservation number CGMCC No. 27163.

[0046] like Figures 1 to 3 As shown, the strain obtained in Example 1 of this application has small colonies on LB solid medium, which are round in shape, with rough edges and raised surfaces. The colonies are opaque apricot in color, and the cells are short rods without flagella, with a size of about 1~3 μm in length and 0.4~0.9 μm in width.

[0047] Example 2: Effects of different strains on DMAc degradation The seed culture of the four strains prepared in Example 1 was inoculated at a volume concentration of 10% into an inorganic salt medium containing 500 mg / L DMAc at pH 7.0. The medium was continuously shaken at 30°C and 150 r / min for 168 h (7 days) in a constant temperature shaker. The concentration of residual DMAc in the medium was then measured.

[0048] like Figure 4 As shown, the results indicate that all strains screened in this experiment possessed the ability to degrade DMAc after 168 h of culture. Among them, strain 4 showed the best degradation effect on DMAc, with a degradation efficiency of 90.1% under aerobic conditions. Strain 3 was the second best, with a DMAc degradation efficiency of 68.6%. The remaining two strains had lower DMAc degradation efficiencies, which may be because the degradation conditions did not meet the physiological needs of the microorganisms, thus inhibiting their ability to degrade DMAc.

[0049] Example 3: Effect of culture temperature on the degradation of DMAc by Rhodococcus qingshengense The Rhodococcus qingshengensis seed culture prepared in Example 1 was inoculated at a volume concentration of 10% into an inorganic salt culture medium containing 500 mg / L DMAc at pH 7.0. The medium was continuously shaken and cultured in a constant temperature shaker at 20-40°C and 150 r / min for 168 h (7 days). The concentration of residual DMAc in the culture medium was then measured.

[0050] The results are as follows Figure 5 As shown in the figure, the degradation efficiency of DMAc by the strain increases within the temperature range of 20-28℃. This is because the increased temperature within this range is conducive to strain growth, thus accelerating the degradation of DMAc. The degradation efficiency is optimal at 28℃, reaching 95% after 168 h, indicating that 28℃ is the most suitable temperature for the growth of *Rhodococcus qingshengense*. The strain exhibits good degradation effects on DMAc between 24-32℃, but the degradation efficiency drops rapidly at 36-40℃, indicating that higher temperatures inactivate the strain and are detrimental to its growth and DMAc degradation. Therefore, this experiment suggests that the temperature range of 24-32℃ is suitable for culturing *Rhodococcus qingshengense*, within which the strain can effectively perform degradation, with 28℃ being the optimal growth temperature.

[0051] Example 4: Effect of Rhodococcus qingsheng inoculum amount on DMAc degradation efficiency The Rhodococcus qingshengensis seed culture prepared in Example 1 was inoculated into an inorganic salt medium containing 500 mg / L DMAc at a volume concentration of 1%, 3%, 5%, 10%, 15% and 20%, respectively. The medium was continuously shaken in a constant temperature shaker at 28°C and 150 r / min for 168 h (7 days). After that, the concentration of residual DMAc in the medium was measured.

[0052] The results are as follows Figure 6 As shown, when the inoculum size increases from 1% to 10%, the degradation effect of DMAc also increases. This is because when the inoculum size is small, the substrate concentration is relatively high, which has a certain inhibitory effect on bacteria, causing them to remain in the lag phase for a long time. Simultaneously, the low strain concentration also leads to a low DMAc removal rate. When the inoculum size continues to increase to 15-20%, the DMAc removal effect slightly decreases. This is because when the inoculum size is too large, competition between strains occurs due to nutrient limitations in the culture medium, inhibiting strain growth and resulting in no significant improvement in the strains' ability to degrade DMAc; in fact, it decreases. Therefore, a 10% inoculum size is selected as the optimal inoculum size for DMAc-degrading bacteria. The inoculum size indicates the proportional relationship between microbial biomass and substrate biomass, as well as the interaction between substrate and microorganisms.

[0053] Example 5: Effect of culture medium pH on the degradation performance of DMAc by Rhodococcus qingshengense The Rhodococcus qingshengensis seed culture prepared in Example 1 was inoculated into an inorganic salt medium containing 500 mg / L DMAc at a volume concentration of 10%. The pH of the medium was adjusted to 5.0, 6.0, 7.0, 8.0 and 9.0 respectively using 2 mol / L HCl and NaOH. The medium was continuously shaken in a constant temperature shaker at 28°C and 150 r / min for 168 h (7 days). The concentration of residual DMAc in the medium was then measured.

[0054] The results are as follows Figure 7 As shown, *Rhodococcus qingshengensis* degrades DMAc under different pH conditions. The highest DMAc removal rate (92.4%) was observed at pH 7.0. Both acidic and alkaline pH levels reduced the degradation efficiency of DMAc. This is because the final metabolic product of DMAc is NH3. A high initial pH increases the concentration of free NH3 in the culture medium, leading to microbial poisoning and reduced DMAc degradation efficiency. Conversely, an initial pH below 7.0 inhibits normal physiological activities of the microorganisms, causing acidic damage and thus decreasing DMAc degradation efficiency. Therefore, pH 7.0 was selected as the optimal growth pH for *Rhodococcus qingshengensis*.

[0055] The beneficial effects of this invention are as follows: 1. The dimethylacetamide degrading bacteria obtained by using the present invention have the advantages of being easy to use and having a good removal effect, and are suitable for widespread use in the treatment of wastewater containing low concentrations of DMAc; 2. The *Rhodococcus qingshengensis* provided by this invention can grow in LB medium containing dimethylacetamide and has good tolerance to DMAc. When *Rhodococcus qingshengensis* seed culture is inoculated at a rate of 10% in wastewater containing dimethylacetamide and cultured at 28°C and pH 7.0 for 7 days with shaking, the residual DMAc in wastewater containing 500 mg / L DMAc can be reduced to more than 90%, demonstrating a very good degradation effect. The discovery of this degrading bacterium is of great significance for further research on the biochemical degradation of DMAc.

[0056] The embodiments of this application have been described above with reference to the accompanying drawings. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A strain of Rhodococcus qingshengii that degrades dimethylacetamide ( Rhodococcus qingshengii ), characterized in that, The *Rhodococcus qingshengensis* was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 20, 2023, with accession number CGMCC No. 27163.

2. The application of Rhodococcus qingshengense as described in claim 1 in the degradation of dimethylacetamide.

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