CN115806918B - Rhodococcus and application thereof - Google Patents
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- CN115806918B CN115806918B CN202211653913.4A CN202211653913A CN115806918B CN 115806918 B CN115806918 B CN 115806918B CN 202211653913 A CN202211653913 A CN 202211653913A CN 115806918 B CN115806918 B CN 115806918B
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- 241000316848 Rhodococcus <scale insect> Species 0.000 title claims abstract description 40
- OQEBBZSWEGYTPG-UHFFFAOYSA-N 3-aminobutanoic acid Chemical compound CC(N)CC(O)=O OQEBBZSWEGYTPG-UHFFFAOYSA-N 0.000 claims abstract description 22
- 238000009629 microbiological culture Methods 0.000 claims abstract description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 25
- 229910052757 nitrogen Inorganic materials 0.000 claims description 16
- 238000000855 fermentation Methods 0.000 claims description 12
- 230000004151 fermentation Effects 0.000 claims description 12
- 230000000813 microbial effect Effects 0.000 claims description 9
- 230000000593 degrading effect Effects 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 3
- 235000016698 Nigella sativa Nutrition 0.000 claims 2
- NVNLLIYOARQCIX-MSHCCFNRSA-N Nisin Chemical compound N1C(=O)[C@@H](CC(C)C)NC(=O)C(=C)NC(=O)[C@@H]([C@H](C)CC)NC(=O)[C@@H](NC(=O)C(=C/C)/NC(=O)[C@H](N)[C@H](C)CC)CSC[C@@H]1C(=O)N[C@@H]1C(=O)N2CCC[C@@H]2C(=O)NCC(=O)N[C@@H](C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(NCC(=O)N[C@H](C)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCSC)C(=O)NCC(=O)N[C@H](CS[C@@H]2C)C(=O)N[C@H](CC(N)=O)C(=O)N[C@H](CCSC)C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(N[C@H](C)C(=O)N[C@@H]3C(=O)N[C@@H](C(N[C@H](CC=4NC=NC=4)C(=O)N[C@H](CS[C@@H]3C)C(=O)N[C@H](CO)C(=O)N[C@H]([C@H](C)CC)C(=O)N[C@H](CC=3NC=NC=3)C(=O)N[C@H](C(C)C)C(=O)NC(=C)C(=O)N[C@H](CCCCN)C(O)=O)=O)CS[C@@H]2C)=O)=O)CS[C@@H]1C NVNLLIYOARQCIX-MSHCCFNRSA-N 0.000 claims 2
- 108010053775 Nisin Proteins 0.000 claims 2
- 239000004309 nisin Substances 0.000 claims 2
- 235000010297 nisin Nutrition 0.000 claims 2
- 241000218204 Nigella Species 0.000 claims 1
- 235000007413 Nigella arvensis Nutrition 0.000 claims 1
- 244000090896 Nigella sativa Species 0.000 claims 1
- 239000001711 nigella sativa Substances 0.000 claims 1
- 241000187562 Rhodococcus sp. Species 0.000 abstract description 22
- 239000000463 material Substances 0.000 abstract description 3
- 230000002906 microbiologic effect Effects 0.000 abstract description 3
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 abstract description 3
- 239000002351 wastewater Substances 0.000 abstract description 2
- XTFIVUDBNACUBN-UHFFFAOYSA-N 1,3,5-trinitro-1,3,5-triazinane Chemical compound [O-][N+](=O)N1CN([N+]([O-])=O)CN([N+]([O-])=O)C1 XTFIVUDBNACUBN-UHFFFAOYSA-N 0.000 description 61
- 239000000243 solution Substances 0.000 description 16
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 15
- 230000015556 catabolic process Effects 0.000 description 14
- 238000006731 degradation reaction Methods 0.000 description 14
- 230000012010 growth Effects 0.000 description 12
- 239000002609 medium Substances 0.000 description 11
- 239000001963 growth medium Substances 0.000 description 7
- 239000002699 waste material Substances 0.000 description 7
- 239000007788 liquid Substances 0.000 description 6
- 239000003344 environmental pollutant Substances 0.000 description 5
- 229910017053 inorganic salt Inorganic materials 0.000 description 5
- 239000002207 metabolite Substances 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 4
- 231100000719 pollutant Toxicity 0.000 description 4
- 239000002028 Biomass Substances 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 3
- 241000122973 Stenotrophomonas maltophilia Species 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- GQPLMRYTRLFLPF-UHFFFAOYSA-N Nitrous Oxide Chemical compound [O-][N+]#N GQPLMRYTRLFLPF-UHFFFAOYSA-N 0.000 description 2
- 241001104012 Rhodococcus sp. DN22 Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000006065 biodegradation reaction Methods 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000037353 metabolic pathway Effects 0.000 description 2
- 238000000053 physical method Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- OCKGFTQIICXDQW-ZEQRLZLVSA-N 5-[(1r)-1-hydroxy-2-[4-[(2r)-2-hydroxy-2-(4-methyl-1-oxo-3h-2-benzofuran-5-yl)ethyl]piperazin-1-yl]ethyl]-4-methyl-3h-2-benzofuran-1-one Chemical compound C1=C2C(=O)OCC2=C(C)C([C@@H](O)CN2CCN(CC2)C[C@H](O)C2=CC=C3C(=O)OCC3=C2C)=C1 OCKGFTQIICXDQW-ZEQRLZLVSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 239000012028 Fenton's reagent Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- 241000222393 Phanerochaete chrysosporium Species 0.000 description 1
- 241000187693 Rhodococcus rhodochrous Species 0.000 description 1
- 241000187563 Rhodococcus ruber Species 0.000 description 1
- 241001533030 Rhodococcus sp. 3M9 Species 0.000 description 1
- 241001666091 Rhodococcus sp. BFWQ7 Species 0.000 description 1
- 241000294645 Rhodococcus sp. SD3 Species 0.000 description 1
- 241000074610 actinobacterium K15 Species 0.000 description 1
- 241000001894 actinobacterium ZXY030 Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000012378 ammonium molybdate tetrahydrate Substances 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- FIXLYHHVMHXSCP-UHFFFAOYSA-H azane;dihydroxy(dioxo)molybdenum;trioxomolybdenum;tetrahydrate Chemical compound N.N.N.N.N.N.O.O.O.O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O[Mo](O)(=O)=O.O[Mo](O)(=O)=O.O[Mo](O)(=O)=O FIXLYHHVMHXSCP-UHFFFAOYSA-H 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 description 1
- 239000006184 cosolvent Substances 0.000 description 1
- -1 cyclic nitro compound Chemical class 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000002360 explosive Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000005189 flocculation Methods 0.000 description 1
- 230000016615 flocculation Effects 0.000 description 1
- 108091008053 gene clusters Proteins 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- CNFDGXZLMLFIJV-UHFFFAOYSA-L manganese(II) chloride tetrahydrate Chemical compound O.O.O.O.[Cl-].[Cl-].[Mn+2] CNFDGXZLMLFIJV-UHFFFAOYSA-L 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 239000001272 nitrous oxide Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 239000010802 sludge Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000009284 supercritical water oxidation Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 125000003258 trimethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])[*:1] 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 241001456058 uncultured Rhodococcus sp. Species 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The invention discloses rhodococcus and application thereof, and belongs to the technical field of energy-containing material microbiological treatment. A rhodococcus strain is provided which has the ability to degrade RDX and to utilize RDX to produce 3-aminobutyric acid. The invention provides Rhodococcus (Rhodococcus sp.) LMD, which is deposited in China general microbiological culture Collection center (China center), and has the deposit number: CGMCC No.24210. The ability of Rhodococcus sp.LMD to convert RDX into nitrogen-containing organic matters with high added value, which is used in the invention, suggests that the Rhodococcus sp.LMD has a certain application prospect in the resource utilization of RDX wastewater.
Description
Technical Field
The invention belongs to the technical field of energy-containing material microbiological treatment, and particularly relates to rhodococcus and application thereof.
Background
Environmental pollution problems with black soljin (RDX, trimethylene trinitro) have attracted considerable attention over many years of production, manufacture, use and destruction. RDX belongs to a cyclic nitro compound, is mainly used as a high-energy explosive and is widely applied worldwide. RDX has significant biotoxicity, is a toxic risk to both human and animals and plants, and is classified as a priority for controlling pollutants by the United states environmental protection agency. Therefore, it is particularly important to find a method for reasonably treating RDX waste liquid.
The treatment method of the RDX-containing waste liquid mainly comprises a physical method, a chemical method and a biological method. The physical method mainly comprises an active carbon adsorption method, an extraction method, a membrane separation method, a flocculation method and the like. The method is simple to operate, but has higher cost, and the treated residues are still pollutants or dangerous matters and need to be further treated. RDX-containing waste liquid is treated by a common chemical method (such as Fenton reagent oxidation method, supercritical water oxidation method, ozone method, electrolytic method and the like), and the method has high cost and incomplete treatment. The biodegradation method is a treatment method for converting and stabilizing waste RDX pollutants by utilizing the metabolism of organisms to make the waste RDX pollutants harmless. The biological degradation method becomes a relatively promising treatment method because the waste RDX material treatment process has the advantages of low energy consumption, no pollution, being universal with the existing biochemical tank, low operation cost and the like.
A plurality of strains are used for microbial degradation of RDX waste liquid, such as stenotrophomonas maltophilia, rhodococcus and white rot fungi. However, the stenotrophomonas maltophilia only has a degrading effect on RDX under aerobic conditions (Binks PR, nicklin S, bruce NC. Degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Stenotrophomonas maltophilia PB1.applied and Environmental Microbiology,1995, 61:1318-1322.); white rot fungi degrade RDX only under anaerobic conditions with degradation periods of up to 25 days or more (Aust sd. Degradation of environmental pollutants by Phanerochaete chrysosporium. Microbiological Ecology,1990, 20:197-209.); the rhodococcus can degrade RDX under aerobic/anaerobic conditions, has high RDX-tolerant concentration, and is more suitable for the existing sewage treatment system. Such as Rhodococcus sp.DN22 (Fournier D, halasz A, spain J, et al determination of Key Metabolites during Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine with Rhodococcus DN22.applied and Environmental Microbiology,2002, 68:166-172.), rhodococcus sp.11Y (Seth-Smith HM, rosser SJ, basran A, et al cloning, sequencing, and Characterization of the Hexahydro-1,3,5-Trinitro-1,3,5-Triazine Degradation Gene Cluster from Rhodococcus rhodochrous.applied and Environmental Microbiology,2002, 68:4764-4771.) can tolerate and degrade high concentrations of RDX, such as Rhodococcus sp.DN22 can tolerate 175. Mu.M (39 mg/L), rhodococcus sp.11Y can tolerate 250. Mu.M. However, all the reported rhodococcus strains degrade RDX into nitrite, nitrous oxide, ammonia, formaldehyde and carbon dioxide, and no high added value nitrogen-containing organic matters are generated.
Disclosure of Invention
The invention aims to provide a rhodococcus strain which can degrade RDX and produce 3-aminobutyric acid by using RDX.
The invention provides Rhodococcus (Rhodococcus sp.) named as Rhodococcus (Rhodococcus sp.) LMD which is deposited in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a deposition date of 2021, 12 months, 29, and a deposition number of: CGMCC No.24210.
The invention provides a microbial preparation containing the rhodococcus.
The invention provides an application of the rhodococcus or the microbial preparation in resisting the thioxogold.
The invention provides an application of the rhodococcus or the microbial preparation in degrading the Hemsleyaite.
The invention provides an application of the rhodococcus or the microbial preparation in producing 3-aminobutyric acid.
The invention provides a method for preparing 3-aminobutyric acid, which is characterized in that the rhodococcus is fermented for at least 48 hours at 30-37 ℃.
Further defined, the nitrogen source of the fermentation medium is nigelgold.
Further defined, the carbon source of the fermentation medium is glucose.
The invention provides a method for degrading black solgold, which is characterized in that rhodococcus mentioned above is added into a sample containing black solgold, and the reaction is carried out for at least 48 hours at 30-37 ℃.
Further defined, the highest concentration of nigeltin is 58 mg/l.
The beneficial effects are that: compared with the disclosed Rhodococcus strain, the Rhodococcus sp.LMD tolerance concentration is slightly higher than the highest concentration (55 mg/L) reported in the literature, and can tolerate supersaturated RDX wastewater (58 mg/L) and grow and reproduce in an inorganic salt culture medium by taking RDX as the sole nitrogen source. The strain degrades RDX in a saturated state in a culture medium by 68% in a 48-hour period under the conditions of the temperature of 30-37 ℃ and the pH value of about 7 and the rotating speed of 220r/min, and the degradation period and the degradation rate are similar to those reported in the literature.
Compared with the disclosed Rhodococcus strain and other RDX degradation strains, the Rhodococcus sp.LMD provided by the invention has the own metabolic characteristics, and can convert RDX into other high-added-value nitrogen-containing products, such as 3-aminobutyric acid found in the metabolites thereof, but the metabolites are not found in the strains reported in the literature.
[ biological preservation information ]: rhodococcus (Rhodococcus sp.) LMD, deposited in the China general microbiological culture Collection center with the deposit address: the collection date is 2021, 12 months and 29 days, and the collection number is: CGMCC No.24210.
Drawings
FIG. 1 is a colony chart of Rhodococcus sp.LMD;
FIG. 2 is a graph of Rhodococcus sp.LMD growth in RDX solution, where the abscissa is time and the ordinate is biomass;
FIG. 3 is a degradation curve of Rhodococcus sp.LMD in RDX solution, wherein the abscissa is degradation time and the ordinate is RDX concentration;
FIG. 4 is a graph of the tolerance of Rhodococcus sp.LMD in different concentrations of RDX, wherein the abscissa is time and the ordinate is biomass;
FIG. 5 is a partial metabolic pathway diagram of Rhodococcus sp.LMD with RDX as sole nitrogen source.
Detailed Description
Example 1 methods for isolation and identification of rhodococcus
Fermentation (inorganic salts) medium: 1.55g/L potassium dihydrogen phosphate, 4.95g/L dipotassium hydrogen phosphate, 10mM glycerol, 5mM glucose, 5mM sodium citrate, 200L trace elements, and RDX as the sole nitrogen source was added thereto after adjusting the pH to 7.2.
LB medium: 5g/L of yeast extract, 10g/L of tryptone and 10g/L of sodium chloride; 2% agar was added to the solid medium. The pH of the two media was about 7. Autoclaving at 115℃for 30min before inoculation.
Trace elements: 3.7g/L ammonium molybdate tetrahydrate, 2.9g/L zinc sulfate heptahydrate, 24.7g/L boric acid, 2.5g/L copper sulfate pentahydrate, 15.8g/L manganese chloride tetrahydrate.
TABLE 1 Medium composition Table
Screening, separating and purifying degradation bacteria: and (3) diluting the enriched activated sludge serving as mother liquor by distilled water in a series of times, namely 10 times, 20 times, 50 times and 100 times respectively.
The limiting solid plate surface is drawn with grid lines so as to mark the positions of colonies, 100L of mother solution is respectively sucked by a liquid-transferring gun, 10 times of solution, 20 times of solution, 50 times of solution and 100 times of solution are diluted by a liquid-transferring gun, and the solution is dripped on the limiting solid culture medium with the drawn grid lines, and the culture is carried out for 24 hours at the temperature of 30 ℃. Single colony plates with better growth vigor and better dispersity are selected, and the shape, the size, the color, the transparency, the wettability, the edge characteristics, the bulge degree and the like of the colonies are observed.
The test results show that: the strain is a gram-positive bacillus without forming spores, and fig. 2 shows colonies formed by the strain in LB solid medium, and the colonies can be seen to be round arched, orange yellow, opaque, smooth and moist in surface and complete in edge.
The physiological and biochemical characteristics of the strain are as follows: gram positive, polymorphic bacteria, which may form embryonic mycelia, break immediately into irregular cells, typically light orange, pink, orange or red, with colorless variants.
Identification of degrading bacteria: the invention adopts a 16S rDNA means to carry out homology analysis on the gene sequences of the screened strains, and carries out sequence comparison through a NCBI (National Center for Biotechnology Information) database to obtain a chart.
Results: the 16S rDNA (GTGGCGGTGCTTACCATGCAAGTCGAACGATGAAGCCC AGCTTGCTGGGTGGATTAGTGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTTCGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATAGGACCTCGGGATGCATGTTCCGGGGTGGAAAGGTTTTCCGGTGCAGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAACGGCCCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGTACCGACGAAGCGCAAGTGACGGTAGGTACAGAAGAAGCACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGTCTGTGAAAACCCGCAGCTCAACTGCGGGCTTGCAGGCGATACGGGCAGACTTGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCGCTAGGTGTGGGTTTCCTTCCACGGGATCCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATACACCGGACCGCCCCAGAGATGGGGTTTCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTGTGTTGCCAGCACGTAATGGTGGGGACTCGCAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCGGTACAGAGGGCTGCGATACCGCGAGGTGGAGCGAATCCCTTAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCCTCGTGGGAGGAGCCGTCGAAGGTAATCCAAT, SEQ ID No. 1) gene molecular biological identification of the strain showed 100% homology with Rhodococcus sp. The identification results of the strains are shown in Table 2. The Rhodococcus obtained was designated Rhodococcus sp.LMD. FIG. 1 is a colony chart of Rhodococcus sp.LMD.
Table 2Rhodococcus sp.LMD results at NCBI
| Numbering device | Name of the name | NCBI contrast |
| KX099297.1 | Rhodococcus sp. | 100％ |
| JN896994.1 | Rhodococcus sp.SD3 | 100％ |
| JF895525.1 | Rhodococcus ruber | 99.93％ |
| FJ594459.1 | Rhodococcus sp.BFWQ7 | 99.93％ |
| FJ863098.1 | uncultured Rhodococcus sp. | 99.86％ |
| JF820114.1 | Rhodococcus sp.PG-3-14 | 99.79％ |
| KC887934.1 | actinobacterium K15 | 99.79％ |
| KC119169.1 | Rhodococcus sp.3M9 | 99.79％ |
| LT630357.3 | Rhodococcus electrodiphilus | 99.79％ |
| JN049479.1 | actinobacterium ZXY030 | 99.79％ |
Example 2A method of degrading Heixojin
RDX degradation test: the Rhodococcus sp.LMD obtained in example 1 was inoculated with degrading bacteria in an amount of 5% in an inorganic salt medium containing RDX at different concentrations (54 mg/L;36mg/L;18mg/L, respectively), and cultured in a shaking table at a constant temperature of 30-37℃for 220r/min, the change in the bacterial solution was observed, and the culture solution was taken every 12 hours to determine the growth status of the strain and the residual amount of RDX in the solution.
As shown in FIG. 2, the growth of the strain containing 36mg/L RDX and 54mg/L RDX was better than that of the strain containing 18mg/L RDX, and the biomass was 2.5 or more. After 84 hours of culture, the strain enters a logarithmic growth phase, and the growth speed of the strain is slowed down and the activity is gradually reduced along with the continuous consumption of nutrient substances in the culture medium. As shown in FIG. 3, the RDX content in the solution is changed, when RDX is used as the sole nitrogen source, the growth condition of the rhodococcus is good, the RDX concentration is reduced from 18mg/L to 5.8mg/L in the third day, and the degradation rate reaches 68%.
Example 3A method for producing 3-aminobutyric acid
1. Resistance studies of rhodococcus LMD to RDX:
under the condition that the acetone content is ensured not to influence the growth of the strain (the acetone content is less than 10%), respectively preparing 10mg/L,20mg/L,30mg/L,40mg/L,50mg/L,70mg/L and 100mg/L RDX-acetone aqueous solutions, and when the RDX concentration exceeds 40mg/L, white precipitation appears; the supernatant of RDX solutions with different concentrations was filtered and quantitatively analyzed by liquid phase, and the saturated concentration of RDX acetone solution in water was found to be 36mg/L.
RDX is insoluble in water and soluble in acetone, so acetone is selected as a cosolvent, 1.0g of RDX solid is weighed and dissolved in 50ml of acetone solution to prepare an RDX-acetone solution with the concentration of 20 g/L. Filtering RDX-acetone solution with concentration of 20g/L with a filter membrane, adding into fermentation broth as unique nitrogen source, and preparing fermentation broth containing low concentration RDX, medium concentration RDX and high concentration RDX, and judging tolerance of the cover strain to RDX according to growth condition of rhodococcus.
Low concentration RDX: fermentation broth containing 20mg/L RDX as the sole nitrogen source;
medium concentration RDX: fermentation broth containing 36mg/L RDX as the sole nitrogen source;
high concentration RDX: fermentation broth containing 58mg/L RDX as the sole nitrogen source;
obtaining secondary seeds: the Rhodococcus sp.LMD selected monoclonal strain obtained in example 1 was inoculated into 10mL LB liquid medium and cultured at 37℃and 220rpm for 24 hours; the cells in the culture broth were inoculated into a new LB liquid medium at an inoculum size of 5%, and cultured at 37℃and 220rpm for 24 hours.
10ml of secondary seeds were added to the fermentation broth at an inoculum size of 5%, and the OD at that time was measured 600 The strain growth was observed every 12 h.
As shown in FIG. 4, when RDX with different concentrations is used as the sole nitrogen source, the growth condition of the rhodococcus is good, 36mg/LRDX is used as the fermentation broth of the sole nitrogen source, the nutrition is enough, the growth time is longest, and under the condition of lower concentration, the growth condition of the strain is slightly inferior to the saturation concentration.
2. In an experiment of exploring RDX metabolites, an inorganic salt culture medium is selected to ferment rhodococcus, and a control experiment is set;
control group: inorganic salt culture medium with 18mg/L nitrite as unique nitrogen source;
experimental group: 18mg/LRDX is the only nitrogen source of inorganic salt culture medium;
through metabonomics study, a control group (18 mg/L nitrite is taken as a nitrogen source) is compared, the rhodococcus takes RDX as a sole nitrogen source to produce an organic nitrogen product 3-aminobutyric acid, products in the control group do not contain 3-aminobutyric acid, predicted metabolic pathways of RDX to 3-aminobutyric acid are shown in figure 5, and metabonomics difference metabolites are shown in table 3.
TABLE 3 metabonomics partial differential metabolites
Claims (10)
1. Rhodococcus sp, characterized in that the Rhodococcus is named Rhodococcus LMD and is deposited in the China general microbiological culture Collection center with the date of deposit being 2021, 12 months 29, and the deposit number is: CGMCC No.24210.
2.A microbial preparation comprising the rhodococcus of claim 1.
3. Use of the rhodococcus of claim 1 or the microbial agent of claim 2 for combating nigella.
4. Use of the rhodococcus of claim 1 or the microbial agent of claim 2 for degrading thioxogold.
5. Use of the rhodococcus of claim 1 or the microbial agent of claim 2 for producing 3-aminobutyric acid.
6. A process for preparing 3-aminobutyric acid, comprising fermenting the rhodococcus of claim 1 at 30-37℃for at least 48 hours.
7. The method of claim 6, wherein the nitrogen source of the fermentation medium is nigelgold.
8. The method of claim 6, wherein the carbon source of the fermentation medium is glucose.
9. A method for degrading nisin, comprising adding the rhodococcus of claim 1 to a sample containing nisin, and reacting at 30-37 ℃ for a period of at least 48 hours.
10. The method of claim 9, wherein the maximum concentration of nigella sativa is 58 mg/l.
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| Cloning,Sequencing,and Characterization of the Hexahydro-1,3,5-Trinitro-1,3,5-Triazine Degradation Gene Cluster from Rhodococcus rhodochrous.Cloning,Sequencing,and Characterization of the Hexahydro-1,3,5-Trinitro-1,3,5-Triazine Degradation Gene Cluster from Rhodococcus rhodochrous.Applied and Environmental Microbiology.2002,第68卷(第10期),全文. * |
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