CN114045238B - Rhodococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide and application thereof - Google Patents
Rhodococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide and application thereof Download PDFInfo
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Abstract
The invention discloses a rhodococcus ruber (Rhodococcus ruber) HJM-8 for efficiently degrading Dimethylacetamide (DMAC) and degradation application thereof, wherein the rhodococcus ruber HJM-8 is preserved in China center for type culture collection, and addresses: chinese, university of armed chinese, postal code: 430072, deposit number: cctccc NO: m2021654, storage date 2021, 6, 01; the rhodococcus ruber HJM-8 with DMAC degradation performance provided by the invention has the initial concentration of 500 mg.L in 24 hours ‑1 The degradation rate of DMAC reaches 100 percent, and the discovery of the degradation bacteria has important significance for the efficient purification of industrial wastewater containing DMAC.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to rhodococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide and application thereof.
Background
Dimethylacetamide (DMAC) is an important chemical raw material and a solvent with excellent performance, and is widely applied to industries such as polyurethane, acrylon, medicine, pesticide, dye, electronics and the like. DMAC has a certain toxicity, and is also relatively difficult to biodegrade, and can be discharged into the environment to have toxic effects on the environment and human beings.
DMAC is an important solvent in synthetic fibers such as acrylonitrile, polyurethane textile and polyurethane resin synthesis industry, and can be used as a C8 fraction separation styrene extraction solvent, and is widely used in the fields of medicine, polymer films and dye paint synthesis due to extremely high intersolubility. The main sources of DMAC wastewater include industries such as dye, leather manufacturing, chemical synthesis and the like, and the hazard is high concentration, strong toxicity and difficult degradation. The biochemical method is a method for biodegrading pollutants in the wastewater by utilizing bacteria, and has the technical characteristics of low energy consumption, high treatment efficiency, low treatment cost and the like. In the water treatment industry today, biochemical processes are one of the most widely used processes and are recognized in water treatment processes in many industries. In different wastewater treatment methods, the biochemical treatment has the advantages of low cost, high treatment efficiency and no secondary pollution, so the method is widely used and is the water treatment process with the greatest application. And how to improve the biochemical treatment efficiency is receiving research and attention. The biological enhancement technology, namely the biological enhancement technology, is a method for screening some dominant strains from sludge or soil and putting the dominant strains into wastewater, so that one or more pollutants in the wastewater can be removed in a targeted manner, and the biological treatment effect of the wastewater is improved. This technology resulted from the mid seventies of the last century, after which great attention was paid and developed. The main mechanisms of action of biological enhancement include direct action of highly efficient degrading bacteria and co-metabolism of microorganisms. The direct action of the high-efficiency degrading bacteria is the most widely applied and common action mode of the microbial degradation technology, and the high-efficiency degrading bacterial strain which takes target pollutants as the only carbon source or nitrogen source and the like is put into the wastewater after enrichment and domestication, so that the microorganisms can directly degrade the target pollutants in the water body, and the purpose of removing the pollutants is achieved. Microbial co-metabolism refers to the process by which certain microorganisms can degrade contaminants only in conjunction with the presence of primary energy source substances.
Disclosure of Invention
In order to solve the technical problems, the invention provides rhodococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide and application thereof in degradation of dimethylacetamide.
The specific technical scheme of the invention is as follows:
in a first aspect, the invention provides a strain of rhodococcus ruber which efficiently degrades dimethylacetamide, the microorganism class is named rhodococcus ruber (Rhodococcus ruber) HJM-8, and the rhodococcus ruber is preserved in China center for type culture Collection (China) at the following address: the university of martial arts in China, postal code 430072; the preservation number is CCTCC NO: M2021654; the sequence of the 16S rRNA of HJM-8 is shown in SEQ ID NO. 1.
The rhodococcus ruber HJM-8 is obtained from sludge in an aerobic tank of a medical wastewater treatment plant in Zhejiang through separation and purification, and can use dimethylacetamide as a sole carbon source for growth.
The rhodococcus ruber HJM-8 is characterized in that: the bacterial colony is white in color, is a small single bacterial colony, is free of spores, is opaque, has smooth surfaces and regular edges, and is observed to be bacillus under a scanning electron microscope.
In a second aspect, the invention provides a bacterial suspension and a preparation method of the bacterial suspension, wherein the bacterial suspension takes rhodococcus ruber HJM-8 which efficiently degrades dimethylacetamide as an active ingredient.
The preparation method of the bacterial suspension comprises the following specific steps:
(1) Slant culture: inoculating Rhodococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide onto slant culture medium, and culturing at 30-38deg.C for 1-3 days to obtain slant thallus; the final concentration composition of the slant culture medium is as follows: k (K) 2 HPO 4 1500mg·L -1 ，KH 2 PO 4 500mg·L -1 ，NaCl 1000mg·L -1 ，DMAC 500mg·L -1 ，MgSO 4 ·7H 2 O 200mg·L -1 The solvent is water, the pH value is 7.0-8.0, and the agar is 18-20 g.L -1 ；
(2) Seed culture: selecting bacterial colony from the inclined plane thallus, inoculating to a seed culture medium, and culturing at 30-38deg.C for 18-24 hr to obtain seed solution; the final concentration composition of the seed culture medium is as follows: naCl 10 g.L -1 Yeast extract powder 5 g.L -1 Peptone 10 g.L -1 The solvent is water, and the pH value is 7.0-8.0;
(3) Fermentation: inoculating the seed solution to a fermentation culture medium according to an inoculum size with the volume concentration of 1%, and culturing for 1-3 days at the temperature of 30-38 ℃ to obtain a fermentation culture solution, namely a bacteria-containing suspension; the final concentration composition of the fermentation medium is as follows: k (K) 2 HPO 4 1500mg·L -1 ，KH 2 PO 4 500mg·L -1 ，NaCl 1000mg·L -1 ，DMAC 500mg·L -1 ，MgSO 4 ·7H 2 O 200mg·L -1 The solvent is water, and the pH value is 7.0-8.0.
In a third aspect, the invention provides an application of a bacterial suspension containing rhodococcus ruber HJM-8 for efficiently degrading dimethylacetamide as an active ingredient in degradation of dimethylacetamide and a specific method thereof.
The specific method comprises the following steps: inoculating the bacterial suspension to a strain containing the strain with a final concentration of 500 mg.L -1 In the liquid selection culture medium of the dimethylacetamide, the dimethylacetamide is used as the sole carbon source, and the culture medium is obtained by shaking culture on a constant temperature shaking table at the temperature of 30-40 ℃ and at the speed of 160-300 rpm; the final concentration composition of the liquid selection medium is as follows: k (K) 2 HPO 4 1500mg·L -1 ，KH 2 PO 4 500mg·L -1 ，NaCl 1000mg·L -1 ，DMAC 500mg·L -1 ，MgSO 4 ·7H 2 O 200mg·L -1 The solvent is water, and the pH value is 8.0-9.0.
The beneficial effects of the invention are as follows: the invention provides a rhodococcus ruber HJM-8 for efficiently degrading dimethylacetamide and an application for removing the dimethylacetamide in wastewater, and the strain can achieve the initial concentration of 500 mg.L in 24 hours -1 The degradation rate of DMAC reaches 100 percent, and the discovery of the degradation bacteria has important significance for further research on DMAC biodegradation.
Drawings
FIG. 1 is a sample view and a scanning electron microscope view of strain HJM-8, wherein FIG. 1 (a) is a sample view of strain HJM-8, and FIG. 1 (b) is a scanning electron microscope view of strain HJM-8;
FIG. 2 is a phylogenetic tree of strain HJM-8;
FIG. 3 is a comparison of DMAC removal performance of strain HJM-8 versus control;
FIG. 4 is a graph showing the results of the test at different initial DMAC concentrations, wherein FIG. 4 (a) is a graph showing the concentration of Dimethylacetamide (DMAC) at different initial DMAC concentrations, and FIG. 4 (b) is a graph showing the bacterial density (OD) at different initial DMAC concentrations 600 ) A variation map;
FIG. 5 dimethyl at different temperaturesVariations in the concentration of phenylacetamide (DMAC) and the bacterial density (OD) 600 ) A variation map;
FIG. 6 changes in Dimethylacetamide (DMAC) concentration and bacterial density (OD) at various pH values 600 ) A variation map;
FIG. 7 changes in Dimethylacetamide (DMAC) concentration and bacterial density (OD) at various inoculum sizes 600 ) A variation graph.
Detailed Description
The invention provides a rhodococcus ruber for efficiently degrading dimethylacetamide, which is named as rhodococcus ruber (Rhodococcus ruber) HJM-8 in the classification of microorganisms, and is preserved in China center for type culture collection (China center for type culture collection) in 6 months of 2021, and the preservation number is CCTCC NO: M2021654; the sequence of the 16S rRNA of HJM-8 is shown in SEQ ID NO. 1.
The invention provides a bacteria-containing suspension taking rhodococcus ruber HJM-8 for efficiently degrading dimethylacetamide as an active ingredient, wherein the bacteria-containing suspension is prepared by performing slant culture, seed culture and fermentation on rhodococcus ruber HJM-8 for efficiently degrading dimethylacetamide.
The preparation method of the bacterial suspension comprises the following specific steps:
(1) Slant culture: inoculating Rhodococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide onto slant culture medium, and culturing at 30-38deg.C for 1-3 days to obtain slant thallus; the final concentration composition of the slant culture medium is as follows: k (K) 2 HPO 4 1500mg·L -1 ，KH 2 PO 4 500mg·L -1 ，NaCl 1000mg·L -1 ，DMAC 500mg·L -1 ，MgSO 4 ·7H 2 O 200mg·L -1 The solvent is water, the pH value is 7.0-8.0, and the agar is 18-20 g.L -1 ；
(2) Seed culture: selecting bacterial colony from the inclined plane thallus, inoculating to a seed culture medium, and culturing at 30-38deg.C for 18-24 hr to obtain seed solution; the final concentration composition of the seed culture medium is as follows: naCl 10 g.L -1 Yeast extract powder 5 g.L -1 Peptone 10 g.L -1 The solvent is water, and the pH value is 7.0-8.0;
(3) Fermentation: inoculating the seed solution into fermentation medium at 1% by volume, and culturing at 30-38deg.C for 1-3Obtaining fermentation culture solution which is a bacteria-containing suspension; the final concentration composition of the fermentation medium is as follows: k (K) 2 HPO 4 1500mg·L -1 ，KH 2 PO 4 500mg·L -1 ，NaCl 1000mg·L -1 ，DMAC 500mg·L -1 ，MgSO 4 ·7H 2 O 200mg·L -1 The solvent is water, and the pH value is 7.0-8.0.
The invention provides an application of a bacteria-containing suspension taking rhodococcus ruber HJM-8 for efficiently degrading dimethylacetamide as an active ingredient in degradation of dimethylacetamide.
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
The final concentration composition of the liquid selection medium used in the present invention is: k (K) 2 HPO 4 1500mg·L -1 ，KH 2 PO 4 500mg·L -1 ，NaCl 1000mg·L -1 ，DMAC 500mg·L -1 ，MgSO 4 ·7H 2 O 200mg·L -1 The solvent is water.
Example 1: isolation, purification and identification of rhodococcus ruber (Rhodococcus ruber) HJM-8
1. Separation and purification of rhodococcus ruber (Rhodococcus ruber) HJM-8
Rhodococcus ruber (Rhodococcus ruber) HJM-8 is screened from sludge in an aerobic tank of a medical wastewater treatment plant in Zhejiang, and comprises the following specific steps:
(1) Sampling: respectively carrying out multi-point sampling from sludge in an aerobic tank of a medical wastewater treatment plant in Zhejiang as a raw material for screening HJM-8 rhodococcus ruber (Rhodococcus ruber) for efficiently degrading dimethylacetamide;
(2) Isolation of strains: taking an appropriate amount of activated sludge in an aerobic tank, standing for 2 hours, taking 10mL of supernatant, inoculating the supernatant into a 250mL culture bottle containing 100mL of enrichment culture solution, and carrying out shaking culture on a constant-temperature shaking table at 30 ℃ and 160rpm for 24 hours, wherein the final concentration components of the enrichment culture solution are as follows: tryptone 2.5 g.L -1 Yeast paste 1.25 g.L -1 500 mg.L of agar powder -1 ，K 2 HPO 4 500mg·L -1 ，KH 2 PO 4 500mg·L -1 ，NaCl 1000mg·L -1 The method comprises the steps of carrying out a first treatment on the surface of the Diluting the bacterial liquid with sterile water 10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 Doubling; the obtained bacterial liquid is separated and purified by a liquid selection culture medium through a plurality of flat plate streaks, and a single colony is obtained and is marked as a strain HJM-8.
2. Identification of Strain HJM-8
a. Physiological and biochemical characteristics of Strain HJM-8
The obtained strain HJM-8 is subjected to morphological observation and physiological and biochemical identification, and the result shows that the strain HJM-8 can be used in a common bacterial LB culture medium with the final concentration of 500 mg.L -1 The bacterial colony is white, is a small single bacterial colony, has no spores, is opaque, has smooth surface and regular edges, the bacterial strain HJM-8 is shown in the figure 1 (a), the bacterial strain is observed to be bacillus under a scanning electron microscope, the optimal pH value for growth is 8.0, and the optimal temperature is 35 ℃ as shown in the figure 1 (b).
b. 16S rRNA sequence analysis of Strain HJM-8
Strain HJM-8 was identified as Rhodococcus ruber by 16S rRNA sequence analysis and physiological and biochemical experiments.
The sequencing results were:
cgatgaagccagcttgctgggtggattagtggcgaacgggtgagtaacacgtgggtgatctgccctgcacttcgggataagcctgggaaactgggtctaataccggataggacctcgggatgcatgttccggggtggaaaggttttccggtgcaggatgggcccgcggcctatcagcttgttggtggggtaacggcccaccaaggcgacgacgggtagccggcctgagagggcgaccggccacactgggactgagacacggcccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgatgcagcgacgccgcgtgagggatgacggccttcgggttgtaaacctctttcagtaccgacgaagcgcaagtgacggtaggtacagaagaagcaccggccaactacgtgccagcagccgcggtaatacgtagggtgcgagcgttgtccggaattactgggcgtaaagagctcgtaggcggtttgtcgcgtcgtctgtgaaaacccgcagctcaactgcgggcttgcaggcgatacgggcagacttgagtactgcaggggagactggaattcctggtgtagcggtgaaatgcgcagatatcaggaggaacaccggtggcgaaggcgggtctctgggcagtaactgacgctgaggagcgaaagcgtgggtagcgaacaggattagataccctggtagtccacgccgtaaacggtgggcgctaggtgtgggtttccttccacgggatccgtgccgtagctaacgcattaagcgccccgcctggggagtacggccgcaaggctaaaactcaaaggaattgacgggggcccgcacaagcggcggagcatgtggattaattcgatgcaacgcgaagaaccttacctgggtttgacatacaccggaccgccccagagatggggtttcccttgtggtcggtgtacaggtggtgcatggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgtcctgtgttgccagcacgtaatggtggggactcgcaggagactgccggggtcaactcggaggaaggtggggacgacgtcaagtcatcatgccccttatgtccagggcttcacacatgctacaatggccggtacagagggctgcgataccgcgaggtggagcgaatcccttaaagccggtctcagttcggatcggggtctgcaactcgaccccgtgaagtcggagtcgctagtaatcgcagatcagcaacgctgcggtgaatacgttcccgggccttgtacacaccgcccgtcacgtcatgaaagtcgg。
the 16S rDNA sequence of HJM-8 was uploaded to Genbank, accession number MZ144119 of Genbank was obtained, homology comparison was made with the gene sequence in Genbank, and FIG. 2 is a phylogenetic tree diagram of the strain. To further determine the reliability of the identification results, it was finally determined through physiological and biochemical experiments that strain HJM-8 belongs to Rhodococcus ruber, and therefore, this strain was named rhodococcus ruber (Rhodococcus ruber) HJM-8.
EXAMPLE 2 preparation of bacterial suspension containing Rhodococcus ruber HJM-8 efficiently degrading dimethylacetamide as active ingredient
The preparation process of the bacterial suspension comprises the following steps:
(1) Slant culture: inoculating rhodococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide onto a slant culture medium, and culturing at 30 ℃ for 3 days to obtain slant thalli; the final concentration composition of the slant culture medium is as follows: k (K) 2 HPO 4 1500mg·L -1 ，KH 2 PO 4 500mg·L -1 ，NaCl 1000mg·L -1 ，DMAC 500mg·L -1 ，MgSO 4 ·7H 2 O 200mg·L -1 The solvent is water, the pH value is 7.0, and the agar is 18 g.L -1 ；
(2) Seed culture: selecting bacterial colony from the inclined plane thallus, inoculating to a seed culture medium, and culturing at 30 ℃ for 18h to obtain seed liquid; the final concentration composition of the seed culture medium is as follows: naCl 10 g.L -1 Yeast extract powder 5 g.L -1 Peptone 10 g.L -1 The solvent is water, and the pH value is 7.0;
(3) Fermentation: the seed liquid is prepared into a volumeInoculating the inoculum size with the concentration of 1% to a fermentation culture medium, and culturing at 30 ℃ for 24 hours to obtain a fermentation culture solution which is a bacteria-containing suspension; the final concentration composition of the fermentation medium is as follows: k (K) 2 HPO 4 1500mg·L -1 ，KH 2 PO 4 500mg·L -1 ，NaCl 1000mg·L -1 ，DMAC 500mg·L -1 ，MgSO 4 ·7H 2 O 200mg·L -1 The solvent was water, pH 7.0.
Example 3: detection of degradation performance of rhodococcus ruber (Rhodococcus ruber) HJM-8 on dimethylacetamide and screening of degradation conditions
1. Detection of degradation Property of Rhodococcus ruber (Rhodococcus ruber) HJM-8 on Dimethylacetamide (DMAC)
The bacterial suspension prepared in example 2 and containing rhodococcus ruber HJM-8 as active ingredient for efficiently degrading dimethylacetamide was inoculated to a final concentration of 500 mg.L -1 In the liquid selection culture medium of the dimethylacetamide, dimethylacetamide is used as the only carbon source, so that the initial pH value of the culture medium is 8.0, 100mL is taken and placed in a 250mL triangular flask, and shaking culture is carried out on a constant temperature shaking table at 35 ℃ and 160rpm to obtain culture solution (used as an experimental group);
at the same time, 100mL of the solution is filled with the solution with the final concentration of 500 mg.L -1 A250 mL triangular flask of the medium was selected for the dimethylacetamide liquid, the initial pH of the medium was set to 8.0, and the medium was shake-cultured on a constant temperature shaker at 35℃and 160rpm as a blank.
During the culture, culture solutions of the experimental group and the blank group were extracted at 8 hours intervals, respectively, and the concentration of dimethylacetamide was measured and the degradation rate of DMAC was plotted as shown in FIG. 3. The detection method comprises the following steps:
the concentration of dimethylacetamide was measured by High Performance Liquid Chromatography (HPLC): sample treatment, taking 1ml of culture solution at intervals of 8 hours, centrifuging the culture solution at 10000rpm for 5min to remove microorganisms and obtain supernatant, and then carrying out HPLC quantitative analysis to measure the concentration of the dimethylacetamide.
As can be seen from FIG. 3, rhodococcus ruber (Rhodococcus ruber) HJM-8 has good degradation effect on Dimethylacetamide (DMAC) and initial concentration of 500 mg.L in 24 hours -1 The degradation rate of the dimethylacetamide reaches 100 percent.
2. Effect of initial concentration of Dimethylacetamide (DMAC) on degradation of dimethylacetamide by Rhodococcus ruber (Rhodococcus ruber) HJM-8
The bacterial suspensions prepared in example 2 and containing rhodococcus ruber HJM-8 as active ingredient for efficiently degrading dimethylacetamide are inoculated to different final concentrations (1 g.L) -1 、5g·L -1 、10g·L -1 、15g·L -1 、20g·L -1 ) In the liquid selection medium of (2), dimethylacetamide is used as the sole carbon source, the initial pH value of the medium is 8.0, 100mL of the medium is respectively taken and respectively put into 5 triangular flasks of 250mL, the medium is subjected to shaking culture on a constant temperature shaking table at 35 ℃ and 160rpm, and the concentration and the bacterial density (OD) of the dimethylacetamide are measured at intervals of 24 hours 600 ) The measurement results are shown in FIG. 4. Bacterial density (OD) 600 ) The detection method of (2) is as follows: the absorbance of the reaction solution was measured at a wavelength of 600nm by using an Shimadzu UV2401 type ultraviolet-visible spectrophotometer.
FIG. 4 (a) is a graph showing the concentration change of Dimethylacetamide (DMAC) at various initial DMAC concentrations, and FIG. 4 (b) is a graph showing the bacterial density (OD) at various initial DMAC concentrations 600 ) A variation graph.
As shown in FIG. 4, rhodococcus ruber (Rhodococcus ruber) HJM-8 has a good degradation effect on Dimethylacetamide (DMAC), and the DMAC removal efficiency decreases with increasing initial DMAC concentration, while the bacterial density (OD) of each group 600 ) With increasing concentration, the initial concentration is 1 g.L -1 The degradation rate reaches 100% at 72h, and the initial concentration is 5 g.L -1 Degradation rate reached 100% at 72 h.
In summary, the rhodococcus ruber (Rhodococcus ruber) HJM-8 has better degradation effect on Dimethylacetamide (DMAC), has strong degradation capability on high-concentration dimethylacetamide wastewater, and can basically reduce the dimethylacetamide to a standard capable of being discharged in a short time.
3. Screening of Rhodococcus ruber (Rhodococcus ruber) HJM-8 for optimal temperature conditions for Dimethylacetamide (DMAC) degradation
Preparation of example 2The obtained bacterial suspension with rhodococcus ruber HJM-8 as active ingredient for efficiently degrading dimethylacetamide is inoculated to final concentration of dimethylacetamide of 500 mg.L -1 In the liquid selection culture medium of (2), the initial pH value of the culture medium is 8.0, 100mL of the culture medium is respectively placed in 5 triangular flasks of 250mL, and the triangular flasks are respectively placed on a constant temperature shaking table at 25 ℃, 30 ℃, 35 ℃, 40 ℃ or 45 ℃ and 160rpm for shaking culture; after shaking culture for 24 hours, the concentration and bacterial density (OD) of dimethylacetamide were measured 600 ) The measurement results are shown in FIG. 5.
The results are shown in FIG. 5: strain HJM-8 is very tolerant to 30 ℃ and 35 ℃; with 35 ℃ as the best, the DMAC degradation rate of the strain HJM-8 reaches 55% in 24 hours; under the experimental condition, the optimal growth and propagation temperature of the strain HJM-8 is 30-35 ℃, and the strain can better exert degradation performance within the temperature range.
4. Screening of Rhodococcus ruber (Rhodococcus ruber) HJM-8 for optimal pH conditions for Dimethylacetamide (DMAC) degradation
The bacterial suspensions prepared in example 2 and containing rhodococcus ruber HJM-8 with dimethylacetamide degradation capacity as active ingredients are respectively inoculated into the bacterial suspensions containing dimethylacetamide with the final concentration of 500 mg.L -1 Respectively placing 100mL into 5 triangular flasks of 250mL, respectively adjusting the pH value of the culture medium to 5.0, 6.0, 7.0, 8.0 or 9.0, and culturing in a shaking table at a constant temperature of 30 ℃ and 160 rpm; after shaking culture for 24 hours, the concentration and bacterial density (OD) of dimethylacetamide were measured 600 ) The measured results are shown in FIG. 6;
the results are shown in FIG. 6: strain HJM-8 was very tolerant to pH values of 8.0 and 9.0; with the pH value of 8.0 as the best, the DMAC degradation rate of the strain HJM-8 reaches 55% in 24 hours; under the experimental conditions, the optimal growth and propagation pH value of the strain HJM-8 is 8.0-9.0, and the strain can better exert degradation performance within the pH value range.
5. Screening of optimal inoculum size for Rhodococcus ruber (Rhodococcus ruber) HJM-8 against Dimethylacetamide (DMAC) degradation
Seed solutions in the step (3) of example 2 were inoculated to a fermentation medium in respective inoculum sizes of 1%, 2%, 3%, 4% and 5% by volume, and bacterial suspensions containing rhodococcus ruber HJM-8 having dimethylacetamide degrading ability as an active ingredient were prepared in the other steps of example 2 without changing the inoculum sizes.
Inoculating bacterial suspension containing rhodococcus ruber HJM-8 with dimethylacetamide degradation capability as active ingredient with inoculum size of 1%, 2%, 3%, 4%, 5% respectively to final concentration of dimethylacetamide of 500 mg.L -1 Respectively placing 100mL into 5 triangular flasks of 250mL, and shake-culturing on a constant-temperature shaking table at 30 ℃ and 160 rpm; after shaking culture for 24 hours, the concentration and bacterial density (OD) of dimethylacetamide were measured 600 ) The measured results are shown in FIG. 7;
the results are shown in FIG. 7: as the inoculum size increased, the DMAC degradation rate measured at 24h increased; at an inoculum size of 5%, the DMAC degradation rate of strain HJM-8 reached 68% at 24 hours.
Sequence listing
<110> Zhejiang tree college (Zhejiang tree university)
<120> rhodococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1081
<212> RNA
<213> Rhodococcus ruber (Rhodococcus ruber)
<400> 1
cgagaagcca gcgcggggga agggcgaacg gggagaacac gggggacgcc cgcaccggga 60
aagccgggaa acgggcaaac cggaaggacc cgggagcagc cggggggaaa ggccgggcag 120
gagggcccgc ggccacagcg ggggggaacg gcccaccaag gcgacgacgg gagccggccg 180
agagggcgac cggccacacg ggacgagaca cggcccagac ccacgggagg cagcaggggg 240
aaagcacaag ggcgcaagcc gagcagcgac gccgcggagg gagacggccc ggggaaaccc 300
cagaccgacg aagcgcaagg acggaggaca gaagaagcac cggccaacac ggccagcagc 360
cgcggaaacg aggggcgagc ggccggaaac gggcgaaaga gccgaggcgg gcgcgcgcgg 420
aaaacccgca gccaacgcgg gcgcaggcga acgggcagac gagacgcagg ggagacggaa 480
ccgggagcgg gaaagcgcag aacaggagga acaccggggc gaaggcgggc cgggcagaac 540
gacgcgagga gcgaaagcgg ggagcgaaca ggaagaaccc ggagccacgc cgaaacgggg 600
gcgcaggggg gccccacggg accggccgag caacgcaaag cgccccgccg gggagacggc 660
cgcaaggcaa aaccaaagga agacgggggc ccgcacaagc ggcggagcag ggaaacgagc 720
aacgcgaaga accaccgggg acaacaccgg accgccccag agaggggccc gggcgggaca 780
gggggcaggc gcgcagccgg cggagagggg aagcccgcaa cgagcgcaac ccgccgggcc 840
agcacgaagg ggggaccgca ggagacgccg gggcaaccgg aggaaggggg gacgacgcaa 900
gcacagcccc agccagggcc acacagcaca aggccggaca gagggcgcga accgcgaggg 960
gagcgaaccc aaagccggcc agcggacggg gcgcaaccga ccccggaagc ggagcgcaga 1020
acgcagacag caacgcgcgg gaaacgcccg ggccgacaca ccgcccgcac gcagaaagcg 1080
g 1081
Claims (5)
1. Rhodococcus ruber capable of efficiently degrading dimethylacetamide is characterized in that the microorganism classification is named as Rhodococcus ruber (Rhodococcus ruber) HJM-8, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2021654 in the year 2021 and month 1; the sequence of the 16S rRNA of HJM-8 is shown in SEQ ID NO. 1.
2. A bacterial suspension containing the rhodococcus ruber as an active ingredient, which is characterized in that the bacterial suspension is prepared by performing slant culture, seed culture and fermentation on rhodococcus ruber HJM-8 with high-efficiency degradation of dimethylacetamide.
3. A method of preparing a bacterial suspension according to claim 2, comprising the specific steps of:
(1) Slant culture: inoculating Rhodococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide onto slant culture medium, and culturing at 30-38deg.C for 1-3 days to obtain slant thallus; the final concentration composition of the slant culture medium is as follows: k (K) 2 HPO 4 1500mg·L -1 ，KH 2 PO 4 500mg·L -1 ，NaCl 1000mg·L -1 ，DMAC 500mg·L -1 ，MgSO 4 ·7H 2 O 200mg·L -1 The solvent is water, the pH value is 7.0-8.0, and the agar is 18-20 g.L -1 ；
(2) Seed culture: selecting bacterial colony from the inclined plane thallus, inoculating to a seed culture medium, and culturing at 30-38deg.C for 18-24 hr to obtain seed solution; the final concentration composition of the seed culture medium is as follows: naCl 10 g.L -1 Yeast extract powder 5 g.L -1 Peptone 10 g.L -1 The solvent is water, and the pH value is 7.0-8.0;
(3) Fermentation: inoculating the seed solution to a fermentation culture medium according to an inoculum size with the volume concentration of 1%, and culturing for 1-3 days at the temperature of 30-38 ℃ to obtain a fermentation culture solution, namely a bacteria-containing suspension; the final concentration composition of the fermentation medium is as follows: k (K) 2 HPO 4 1500mg·L -1 ，KH 2 PO 4 500mg·L -1 ，NaCl 1000mg·L -1 ，DMAC 500mg·L -1 ，MgSO 4 ·7H 2 O 200mg·L -1 The solvent is water, and the pH value is 7.0-8.0.
4. Use of the bacterial suspension of claim 2 for degrading dimethylacetamide.
5. The use according to claim 4, characterized in that it is in particular: inoculating the bacterial suspension to a strain containing the strain with a final concentration of 500 mg.L -1 In the liquid selection culture medium of the dimethylacetamide, the dimethylacetamide is used as the sole carbon source, and the culture medium is obtained by shaking culture on a constant temperature shaking table at the temperature of 30-40 ℃ and at the speed of 160-300 rpm; the final concentration composition of the liquid selection medium is as follows: k (K) 2 HPO 4 1500mg·L -1 ，KH 2 PO 4 500mg·L -1 ，NaCl 1000mg·L -1 ，DMAC 500mg·L -1 ，MgSO 4 ·7H 2 O 200mg·L -1 The solvent is water, and the pH value is 8.0-9.0.
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