CN114045238A - 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 Rhodococcus ruber HJM-8 capable of efficiently degrading Dimethylacetamide (DMAC) and degradation application thereof, wherein Rhodococcus ruber HJM-8 is deposited in China center for type culture Collection with the address: china, wuhan university, zip code: 430072, deposit number: CCTCC NO: m2021654, date of deposit 2021, 6 months, 01 days; the Rhodococcus ruber HJM-8 with DMAC degradation performance can react for an initial concentration of 500 mg.L within 24h‑1The degradation rate of the DMAC reaches 100%, and the discovery of the degrading bacteria has important significance for the efficient purification of the industrial wastewater containing the DMAC.
Description
Technical Field
The invention relates to the field of microorganisms, and particularly relates 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 the industries of polyurethane, acrylon, medicine, pesticide, dye, electronics and the like. DMAC has certain toxicity, can have toxic action on the environment and human beings when being discharged into the environment, and is difficult to biodegrade.
DMAC is synthetic fiber like the important solvent in acrylonitrile, polyurethane weaving and polyurethane resin synthesis industry, also can regard as C8 fraction separation styrene extraction solvent to be used simultaneously, because its extremely strong intersolubility, DMAC also receives extensive use in medicine, polymer film, the synthetic field of dyestuff coating. The DMAC wastewater mainly comes from industries such as dye, leather manufacturing, chemical synthesis and the like, and is high in concentration, strong in toxicity and difficult to degrade. The biochemical method is a method for biologically degrading pollutants in wastewater by using bacteria, and has the process characteristics of low energy consumption, high treatment efficiency, low treatment cost and the like. In the water treatment industry today, biochemical methods are the most widely used process flow and are recognized in water treatment processes in many industries. Among different wastewater treatment methods, biochemical treatment has the advantages of low cost, high treatment efficiency and no secondary pollution, so that the method is widely used and is a water treatment process with the greatest application. How to improve the efficiency of biochemical treatment is followed by research and attention of people. The biological strengthening technology, namely the biological strengthening technology, is a method for screening some dominant bacteria from sludge or soil and putting the dominant bacteria into wastewater, and can be used for specifically removing one or more pollutants in the wastewater so as to improve the biological treatment effect of the wastewater. This technology has been generated in the mid seventies of the last century and has gained considerable attention and development in the future. The main action mechanisms of bioaugmentation include direct action of highly effective degrading bacteria and co-metabolism of microorganisms. The direct action of the high-efficiency degrading bacteria is the most widely and most common action mode of the application of the microbial degradation technology, and the high-efficiency degrading strains which take target pollutants as unique carbon sources or nitrogen sources and other energy sources are thrown into wastewater after enrichment and domestication, so that the microorganisms can directly degrade the target pollutants in water to achieve the purpose of removing the pollutants. The microbial co-metabolism refers to a process that certain microbes and primary energy substances can degrade pollutants together only when the microbes exist along with the primary energy 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 degrading dimethylacetamide.
The specific technical scheme of the invention is as follows:
in a first aspect, the invention provides a Rhodococcus ruber capable of efficiently degrading dimethylacetamide, which is classified and named Rhodococcus ruber HJM-8 and has been deposited in China center for type culture Collection on 6.1.2021 at the address: wuhan university in Wuhan, China, the postal code is 430072; the preservation number is CCTCC NO: M2021654; the 16S rRNA sequence of HJM-8 is shown in SEQ ID NO. 1.
The erythrococcus ruber HJM-8 is derived from sludge in an aerobic tank of a certain medical wastewater treatment plant in Zhejiang, is obtained by separation and purification, and can use dimethylacetamide as the only carbon source for growth.
The erythrococcus ruber HJM-8 is characterized in that: the colony color is white, the colony is small-size single colony, no spore, opaque, the surface is smooth, the edge is neat, the form of observing this thallus under the scanning electron microscope is the bacillus.
In a second aspect, the invention provides a bacterial suspension taking rhodococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide as an active ingredient and a preparation method of the bacterial suspension.
The preparation method of the bacteria-containing suspension comprises the following specific steps:
(1) slant culture: inoculating Rhodococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide to a slant culture medium, and culturing at 30-38 ℃ for 1-3 days to obtain slant thallus; the final concentration of the slant culture medium is as follows: k2HPO4 1500mg·L-1，KH2PO4 500mg·L-1，NaCl 1000mg·L-1，DMAC 500mg·L-1，MgSO4·7H2O 200mg·L-1The solvent is water, the pH value is 7.0-8.0, and the agar is 18-20 g.L-1；
(2) Seed culture: selecting colony from the slant thallus, inoculating to seed culture medium, and culturing at 30-38 deg.C for 18-24 hr to obtain seed solution; the final concentration of the seed culture medium is as follows: NaCl 10 g.L-15 g.L of yeast extract powder-1Peptone 10 g. L-1The solvent is water, and the pH value is 7.0-8.0;
(3) fermentation: inoculating the seed solution to a fermentation culture medium in an inoculation amount of 1% of volume concentration, and culturing at 30-38 ℃ for 1-3 days to obtain a fermentation culture solution, namely a bacterium-containing suspension; the final concentration of the fermentation medium is as follows: k2HPO4 1500mg·L-1，KH2PO4 500mg·L-1，NaCl 1000mg·L-1，DMAC 500mg·L-1，MgSO4·7H2O 200mg·L-1The 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 taking erythrococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide as an active ingredient in degrading dimethylacetamide and a specific method thereof.
The specific method comprises the following steps: inoculating the bacteria-containing suspension to a solution containing the bacteria at a final concentration of 500 mg-L-1In the liquid selective culture medium of the dimethylacetamide, the dimethylacetamide is taken as the only carbon source, and the shaking culture is carried out on a constant temperature shaking table at the temperature of 30-40 ℃ and the rotation speed of 160-300rpm, so as to obtain a culture solution; the final concentration composition of the liquid selection medium is as follows: k2HPO4 1500mg·L-1，KH2PO4500mg·L-1，NaCl 1000mg·L-1，DMAC 500mg·L-1，MgSO4·7H2O 200mg·L-1The solvent is water, and the pH value is 8.0-9.0.
The invention has the beneficial effects that: the invention provides Rhodococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide and application of the Rhodococcus ruber HJM-8 in removing dimethylacetamide in wastewater, wherein the Rhodococcus ruber can degrade dimethylacetamide in an initial concentration of 500 mg.L within 24 hours-1The degradation rate of the DMAC reaches 100%, and the discovery of the degrading bacteria has important significance for further research on the biodegradation of the DMAC.
Drawings
FIG. 1 is a sample diagram and a scanning electron micrograph of strain HJM-8, wherein FIG. 1(a) is a sample diagram of strain HJM-8, and FIG. 1(b) is a scanning electron micrograph 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 with a control;
FIG. 4 is a graph of test results of different DMAC initial concentrations, wherein FIG. 4(a) is a graph of the change in Dimethylacetamide (DMAC) concentration at different DMAC initial concentrations, and FIG. 4(b) is a graph of the bacterial density (OD) at different DMAC initial concentrations600) A variation graph;
FIG. 5 variation of Dimethylacetamide (DMAC) concentration and bacterial density (OD) at different temperatures600) A variation graph;
FIG. 6 variation of Dimethylacetamide (DMAC) concentration and bacterial density (OD) at different pH600) A variation graph;
FIG. 7 variation of Dimethylacetamide (DMAC) concentration and bacterial density (OD) at different inoculum sizes600) And (5) a variation graph.
Detailed Description
The invention provides a Rhodococcus ruber for efficiently degrading dimethylacetamide, which is classified and named Rhodococcus ruber HJM-8 by microorganisms, is preserved in China Center for Type Culture Collection (CCTCC) at 6 months and 1 day 2021, and has a preservation number of M2021654; the 16S rRNA sequence of HJM-8 is shown in SEQ ID NO. 1.
The invention provides a bacterium-containing suspension taking erythrococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide as an active ingredient, which is prepared by performing slant culture, seed culture and fermentation on erythrococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide.
The preparation method of the bacteria-containing suspension comprises the following specific steps:
(1) slant culture: inoculating Rhodococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide to a slant culture medium, and culturing at 30-38 ℃ for 1-3 days to obtain slant thallus; the final concentration of the slant culture medium is as follows: k2HPO4 1500mg·L-1，KH2PO4 500mg·L-1，NaCl 1000mg·L-1，DMAC 500mg·L-1，MgSO4·7H2O 200mg·L-1The solvent is water, the pH value is 7.0-8.0, and the agar is 18-20 g.L-1；
(2) Seed culture: selecting colony from the slant thallus, inoculating to seed culture medium, and culturing at 30-38 deg.C for 18-24 hr to obtain seed solution; the final concentration of the seed culture medium is as follows: NaCl 10 g.L-15 g.L of yeast extract powder-1Peptone 10 g. L-1The solvent is water, and the pH value is 7.0-8.0;
(3) fermentation: inoculating the seed solution to a fermentation culture medium in an inoculation amount of 1% of volume concentration, and culturing at 30-38 ℃ for 1-3 days to obtain a fermentation culture solution, namely a bacterium-containing suspension; the final concentration of the fermentation medium is as follows: k2HPO4 1500mg·L-1，KH2PO4 500mg·L-1，NaCl 1000mg·L-1，DMAC 500mg·L-1，MgSO4·7H2O 200mg·L-1The solvent is water, and the pH value is 7.0-8.0.
The invention provides an application of a bacterium-containing suspension taking erythrococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide as an active ingredient in degrading 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: k2HPO4 1500mg·L-1，KH2PO4500mg·L-1，NaCl 1000mg·L-1，DMAC 500mg·L-1，MgSO4·7H2O 200mg·L-1The solvent is water.
Example 1: isolation, purification and characterization of Rhodococcus ruber HJM-8
1. Isolation and purification of Rhodococcus ruber HJM-8
Rhodococcus ruber HJM-8 is screened from aerobic tank sludge of a medical wastewater treatment plant in Taizhou, Zhejiang, and comprises the following specific steps:
(1) sampling: sampling sludge from an aerobic tank of a certain medical wastewater treatment plant in Zhejiang at multiple points respectively to be used as a raw material for screening Rhodococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide;
(2) strain separation: taking a proper amount of activated sludge in an aerobic pool, standing for 2 hours, taking 10mL of supernatant, inoculating the supernatant into a 250mL culture bottle containing 100mL of enrichment culture solution, carrying out shake culture on a constant-temperature shaking table at 30 ℃ and 160rpm for 24 hours, and repeating for 3 times, wherein the final concentration components of the enrichment culture solution are as follows: tryptone 2.5 g.L-11.25 g.L yeast extract -1500 mg.L of agar powder-1，K2HPO4500mg·L-1，KH2PO4 500mg·L-1，NaCl 1000mg·L-1(ii) a Diluting the bacterial liquid with sterile water 10-2、10-3、10-4、10-5、10-6、10-7Doubling; and (3) separating and purifying the obtained bacterial liquid by using a liquid selective culture medium through multiple flat plate streaking to obtain a single bacterial colony which is marked as a bacterial strain HJM-8.
2. Identification of Strain HJM-8
a. Physiological and biochemical characteristics of Strain HJM-8
Morphological observation and physiological and biochemical identification are carried out on the obtained strain HJM-8, and the result shows that the strain HJM-8 can be cultured in an ordinary bacterium LB culture medium with the final concentration of 500 mg.L-1The bacterial colony grows on a liquid selective culture medium of the dimethylacetamide, the color of the bacterial colony is white, the bacterial colony is a small single bacterial colony, has no spores, is opaque, has a smooth surface and is neat in edge, the bacterial strain HJM-8 is shown in figure 1(a), the shape of the thallus observed under a scanning electron microscope is bacillus shown in figure 1(b), the optimal pH value for growth is 8.0, and the optimal temperature is 35 ℃.
b. 16S rRNA sequence analysis of Strain HJM-8
16S rRNA sequence analysis and physiological and biochemical experiment identification confirm that the strain HJM-8 is Rhodococcus ruber.
The sequencing result is as follows:
cgatgaagccagcttgctgggtggattagtggcgaacgggtgagtaacacgtgggtgatctgccctgcacttcgggataagcctgggaaactgggtctaataccggataggacctcgggatgcatgttccggggtggaaaggttttccggtgcaggatgggcccgcggcctatcagcttgttggtggggtaacggcccaccaaggcgacgacgggtagccggcctgagagggcgaccggccacactgggactgagacacggcccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgatgcagcgacgccgcgtgagggatgacggccttcgggttgtaaacctctttcagtaccgacgaagcgcaagtgacggtaggtacagaagaagcaccggccaactacgtgccagcagccgcggtaatacgtagggtgcgagcgttgtccggaattactgggcgtaaagagctcgtaggcggtttgtcgcgtcgtctgtgaaaacccgcagctcaactgcgggcttgcaggcgatacgggcagacttgagtactgcaggggagactggaattcctggtgtagcggtgaaatgcgcagatatcaggaggaacaccggtggcgaaggcgggtctctgggcagtaactgacgctgaggagcgaaagcgtgggtagcgaacaggattagataccctggtagtccacgccgtaaacggtgggcgctaggtgtgggtttccttccacgggatccgtgccgtagctaacgcattaagcgccccgcctggggagtacggccgcaaggctaaaactcaaaggaattgacgggggcccgcacaagcggcggagcatgtggattaattcgatgcaacgcgaagaaccttacctgggtttgacatacaccggaccgccccagagatggggtttcccttgtggtcggtgtacaggtggtgcatggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgtcctgtgttgccagcacgtaatggtggggactcgcaggagactgccggggtcaactcggaggaaggtggggacgacgtcaagtcatcatgccccttatgtccagggcttcacacatgctacaatggccggtacagagggctgcgataccgcgaggtggagcgaatcccttaaagccggtctcagttcggatcggggtctgcaactcgaccccgtgaagtcggagtcgctagtaatcgcagatcagcaacgctgcggtgaatacgttcccgggccttgtacacaccgcccgtcacgtcatgaaagtcgg。
the 16S rDNA sequence of HJM-8 was uploaded to Genbank to obtain Genbank accession number MZ144119, which was subjected to homology comparison with the gene sequence in Genbank, and FIG. 2 is a phylogenetic tree diagram of the strain. To further confirm the reliability of the identification result, it was finally confirmed through physiological and biochemical experiments that the strain HJM-8 belongs to Rhodococcus ruber, and thus, the strain was named Rhodococcus ruber HJM-8.
EXAMPLE 2 preparation of a suspension containing Rhodococcus ruber HJM-8 as an active ingredient which efficiently degrades dimethylacetamide
The preparation process of the bacteria-containing suspension comprises the following steps:
(1) slant culture: inoculating Rhodococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide to a slant culture medium, and culturing at 30 ℃ for 3 days to obtain slant thallus; the final concentration of the slant culture medium is as follows: k2HPO4 1500mg·L-1，KH2PO4500mg·L-1，NaCl 1000mg·L-1，DMAC 500mg·L-1，MgSO4·7H2O 200mg·L-1The solvent is water, the pH value is 7.0, and the agar is 18 g.L-1；
(2) Seed culture: selecting a bacterial colony from the inclined-plane thallus, inoculating the bacterial colony to a seed culture medium, and culturing for 18h at 30 ℃ to obtain a seed solution; the final concentration of the seed culture medium is as follows: NaCl 10 g.L-15 g.L of yeast extract powder-1Peptone 10 g. L-1The solvent is water, and the pH value is 7.0;
(3) fermentation: inoculating the seed solution to a fermentation culture medium by an inoculation amount with the volume concentration of 1%, and culturing at 30 ℃ for 24h to obtain a fermentation culture solution, namely a bacterium-containing suspension; the final concentration of the fermentation medium is as follows: k2HPO4 1500mg·L-1，KH2PO4500mg·L-1，NaCl 1000mg·L-1，DMAC 500mg·L-1，MgSO4·7H2O 200mg·L-1The solvent is water, and the pH value is 7.0.
Example 3: rhodococcus ruber HJM-8 for detecting degradation performance of dimethylacetamide and screening degradation conditions
1. Detection of Rhodococcus ruber HJM-8 for Dimethylacetamide (DMAC) degradation Performance
The bacterial suspension containing Rhodococcus erythropolis HJM-8 capable of efficiently degrading dimethylacetamide prepared in example 2 as an active ingredient was inoculated to a final concentration of 500 mg. L-1In the liquid selective culture medium of dimethylacetamide, dimethylacetamide is used as a unique carbon source, the initial pH value of the culture medium is 8.0, 100mL of the culture medium is placed in a 250mL triangular flask, and shaking culture is carried out on a constant temperature shaking table at 35 ℃ and 160rpm, so as to obtain a culture solution (used as an experimental group);
at the same time, 100mL of the solution is filled to contain the final concentration of 500 mg.L-1A250 mL Erlenmeyer flask of the culture medium was selected as a liquid of dimethylacetamide so that the initial pH of the culture medium was 8.0, and the culture was performed with shaking on a constant temperature shaker at 35 ℃ and 160rpm as a blank control.
During the culture, the culture solutions of the experimental group and the blank control group were extracted at intervals of 8 hours, and the concentration of dimethylacetamide was measured and a DMAC degradation rate graph was plotted, as shown in fig. 3. The detection method comprises the following steps:
the detection of the concentration of dimethylacetamide adopts High Performance Liquid Chromatography (HPLC): treating the sample, taking 1ml of culture solution at intervals of 8 hours, centrifuging the culture solution at 10000rpm for 5min to remove microorganisms to obtain supernatant, and then carrying out HPLC quantitative analysis to measure the concentration of the dimethylacetamide.
It can be seen from FIG. 3 that Rhodococcus ruber HJM-8 has a better effect on the degradation of Dimethylacetamide (DMAC) at an initial concentration of 500 mg.L at 24h-1The degradation rate of the dimethylacetamide reaches 100 percent.
2. Detection of Effect of initial concentration of Dimethylacetamide (DMAC) on Dimethylacetamide degradation by Rhodococcus ruber HJM-8
The bacterial suspensions containing Rhodococcus erythropolis HJM-8 which was prepared in example 2 and efficiently degraded in dimethylacetamide as an active ingredient were inoculated to final concentrations of dimethylacetamide (1 g. L)-1、5g·L-1、10g·L-1、15g·L-1、20g·L-1) In the liquid selective medium (2), dimethylacetamide is used as a sole carbon source, the initial pH value of the medium is 8.0, 100mL of the medium is respectively placed in 5 triangular flasks of 250mL, the medium is subjected to shaking culture on a constant temperature shaking table of 160rpm at 35 ℃ for 24h, and the concentration of dimethylacetamide and the bacterial density (OD) are measured600) The results are shown in FIG. 4. Bacterial density (OD)600) The detection method comprises the following steps: the absorbance of the reaction mixture was measured at a wavelength of 600nm using Shimadzu UV2401 UV-visible spectrophotometer.
FIG. 4(a) is a graph showing the change in Dimethylacetamide (DMAC) concentration at different initial DMAC concentrations, and FIG. 4(b) is a graph showing the bacterial density (OD) at different initial DMAC concentrations600) And (5) a variation graph.
As shown in FIG. 4, Rhodococcus ruber HJM-8 showed a better degradation effect on Dimethylacetamide (DMAC), and the DMAC removal efficiency decreased with the increase of the initial concentration of DMAC, and the bacterial density (OD) of each group was also lower600) With increasing concentration, the initial concentration is 1 g.L-1The degradation rate reaches 100 percent at 72h, and the initial concentration is 5 g.L-1The degradation rate reaches 100% at 72 h.
In view of the above, Rhodococcus ruber HJM-8 has a good effect of degrading Dimethylacetamide (DMAC), has a strong ability of degrading high-concentration dimethylacetamide wastewater, and can basically reduce dimethylacetamide to a standard capable of being discharged in a short time.
3. Rhodococcus ruber HJM-8 optimal temperature condition screening for Dimethylacetamide (DMAC) degradation
The bacterial suspensions containing Rhodococcus erythropolis HJM-8 which was prepared in example 2 and efficiently degraded in dimethylacetamide as an active ingredient were inoculated to a final concentration of dimethylacetamide of 500 mg. L-1In the liquid selective culture medium, 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 of 25 ℃, 30 ℃, 35 ℃, 40 ℃ or 45 ℃ and 160rpm for shaking culture; the concentration and bacterial density (OD) of dimethylacetamide was measured after 24h of shaking culture600) The results are shown in FIG. 5.
The results are shown in FIG. 5: the strain HJM-8 has good tolerance to 30 ℃ and 35 ℃; the optimal temperature is 35 ℃, and the DMAC degradation rate of the strain HJM-8 reaches 55% in 24 h; 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 in the temperature range.
4. Rhodococcus ruber HJM-8 optimal pH condition screening for Dimethylacetamide (DMAC) degradation
The bacterial suspensions containing Rhodococcus erythropolis HJM-8 having dimethylacetamide-degrading ability prepared in example 2 as an active ingredient were each inoculated to a final concentration of 500 mg. L of dimethylacetamide-1In the liquid selective culture medium of (1), 100mL of the culture medium is respectively placed in 5 triangular flasks of 250mL, the pH values of the culture medium are respectively adjusted to 5.0, 6.0, 7.0, 8.0 or 9.0, and shaking culture is carried out on a constant temperature shaking bed of 160rpm at 30 ℃; the concentration and bacterial density (OD) of dimethylacetamide was measured after 24h of shaking culture600) The measured results are shown in fig. 6;
the results are shown in FIG. 6: the strain HJM-8 has good tolerance to pH values of 8.0 and 9.0; the optimal pH value is 8.0, and the DMAC degradation rate of the strain HJM-8 reaches 55% in 24 h; under the experimental condition, the optimum growth and reproduction 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 Rhodococcus ruber HJM-8 for optimal inoculum size for Dimethylacetamide (DMAC) degradation
The seed solution in the step (3) of example 2 was inoculated into the fermentation medium in the amounts of 1%, 2%, 3%, 4% and 5% by volume, respectively, and bacterial suspensions containing 1%, 2%, 3%, 4% and 5% by volume of Rhodococcus ruber HJM-8 having dimethylacetamide-degrading ability as an active ingredient were prepared without changing the other steps of example 2.
Respectively inoculating 1%, 2%, 3%, 4%, 5% of bacterial suspension containing Rhodococcus ruber HJM-8 with dimethylacetamide degradability to dimethylacetamide with a final concentration of 500 mg.L-1In the liquid selective culture medium, 100mL of the culture medium is respectively put into 5 triangular flasks with 250mL, and shake culture is carried out on a constant temperature shaking table with 30 ℃ and 160 rpm; the concentration and bacterial density (OD) of dimethylacetamide was measured after 24h of shaking culture600) The measured results are shown in fig. 7;
the results are shown in FIG. 7: the DMAC degradation rate measured in 24h is increased along with the increase of the inoculation amount; when the inoculation amount is 5%, the DMAC degradation rate of the strain HJM-8 reaches 68% in 24 h.
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. A Rhodococcus ruber for efficiently degrading dimethylacetamide is characterized in that the Rhodococcus ruber is classified and named as Rhodococcus ruber HJM-8, is preserved in China Center for Type Culture Collection (CCTCC) at 6 months and 1 day 2021, and has a preservation number of M2021654; the 16S rRNA sequence of HJM-8 is shown in SEQ ID NO. 1.
2. A suspension containing the Rhodococcus ruber as claimed in claim 1 as an active ingredient, wherein said suspension is prepared by slant culturing, seed culturing and fermenting Rhodococcus ruber HJM-8 which is highly effective in degrading dimethylacetamide.
3. A method for preparing a suspension containing bacteria according to claim 2, which comprises the following steps:
(1) slant culture: inoculating Rhodococcus ruber HJM-8 capable of efficiently degrading dimethylacetamide to a slant culture medium, and culturing at 30-38 ℃ for 1-3 days to obtain slant thallus; the final concentration of the slant culture medium is as follows: k2HPO4 1500mg·L-1，KH2PO4 500mg·L-1，NaCl 1000mg·L-1，DMAC 500mg·L-1，MgSO4·7H2O 200mg·L-1The 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 colonies from the inclined plane thallus, inoculating the bacterial colonies to a seed culture medium, and culturing for 18-24h at 30-38 ℃ to obtain a seed solution; the final concentration of the seed culture medium is as follows: NaCl 10 g.L-15 g.L of yeast extract powder-1Peptone 10 g. L-1The solvent is water, and the pH value is 7.0-8.0;
(3) fermentation: inoculating the seed solution to a fermentation culture medium in an inoculation amount of 1% of volume concentration, and culturing at 30-38 ℃ for 1-3 days to obtain a fermentation culture solution, namely a bacterium-containing suspension; the final concentration of the fermentation medium is as follows: k2HPO4 1500mg·L-1，KH2PO4 500mg·L-1，NaCl 1000mg·L-1，DMAC 500mg·L-1，MgSO4·7H2O 200mg·L-1The solvent is water, and the pH value is 7.0-8.0.
4. Use of the bacterial-containing suspension of claim 2 for the degradation of dimethylacetamide.
5. The use according to claim 4, characterized in that it is in particular: inoculating the bacteria-containing suspension to a solution containing the bacteria at a final concentration of 500 mg-L-1In the liquid selective culture medium of the dimethylacetamide, the dimethylacetamide is taken as the only carbon source, and the shaking culture is carried out on a constant temperature shaking table at the temperature of 30-40 ℃ and the rotation speed of 160-300rpm, so as to obtain a culture solution; the final concentration composition of the liquid selection medium is as follows: k2HPO4 1500mg·L-1，KH2PO4 500mg·L-1，NaCl 1000mg·L-1，DMAC 500mg·L-1，MgSO4·7H2O 200mg·L-1The solvent is water, and the pH value is 8.0-9.0.
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