CN116656535A - Bacterial strain with chlorohydrocarbon degradation capability and application thereof - Google Patents
Bacterial strain with chlorohydrocarbon degradation capability and application thereof Download PDFInfo
- Publication number
- CN116656535A CN116656535A CN202310372902.7A CN202310372902A CN116656535A CN 116656535 A CN116656535 A CN 116656535A CN 202310372902 A CN202310372902 A CN 202310372902A CN 116656535 A CN116656535 A CN 116656535A
- Authority
- CN
- China
- Prior art keywords
- chlorinated hydrocarbon
- ept
- degradation
- degrading
- strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000001580 bacterial effect Effects 0.000 title claims description 35
- 230000015556 catabolic process Effects 0.000 title abstract description 57
- 238000006731 degradation reaction Methods 0.000 title abstract description 57
- 150000008280 chlorinated hydrocarbons Chemical class 0.000 claims abstract description 53
- 230000000593 degrading effect Effects 0.000 claims abstract description 34
- 241000894006 Bacteria Species 0.000 claims abstract description 23
- 241000670472 Exiguobacterium mexicanum Species 0.000 claims abstract description 5
- 241001467578 Microbacterium Species 0.000 claims abstract description 5
- 241000589774 Pseudomonas sp. Species 0.000 claims abstract description 5
- 241000193388 Bacillus thuringiensis Species 0.000 claims abstract description 3
- 229940097012 bacillus thuringiensis Drugs 0.000 claims abstract description 3
- 238000004321 preservation Methods 0.000 claims description 8
- 238000009629 microbiological culture Methods 0.000 claims description 6
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 4
- 238000011065 in-situ storage Methods 0.000 claims description 3
- 238000005067 remediation Methods 0.000 claims description 3
- 241000589516 Pseudomonas Species 0.000 claims 2
- 230000001788 irregular Effects 0.000 claims 1
- 239000003344 environmental pollutant Substances 0.000 abstract description 25
- 231100000719 pollutant Toxicity 0.000 abstract description 25
- 238000012216 screening Methods 0.000 abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 12
- 239000002689 soil Substances 0.000 abstract description 8
- 238000000926 separation method Methods 0.000 abstract description 6
- 238000002474 experimental method Methods 0.000 abstract description 5
- 239000003638 chemical reducing agent Substances 0.000 abstract description 3
- 239000010842 industrial wastewater Substances 0.000 abstract description 3
- XSTXAVWGXDQKEL-UHFFFAOYSA-N Trichloroethylene Chemical compound ClC=C(Cl)Cl XSTXAVWGXDQKEL-UHFFFAOYSA-N 0.000 description 29
- UBOXGVDOUJQMTN-UHFFFAOYSA-N trichloroethylene Natural products ClCC(Cl)Cl UBOXGVDOUJQMTN-UHFFFAOYSA-N 0.000 description 29
- 239000007788 liquid Substances 0.000 description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 13
- 239000001963 growth medium Substances 0.000 description 13
- 238000000034 method Methods 0.000 description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 230000012010 growth Effects 0.000 description 10
- 244000005700 microbiome Species 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 238000010790 dilution Methods 0.000 description 8
- 239000012895 dilution Substances 0.000 description 8
- 235000019441 ethanol Nutrition 0.000 description 8
- 230000000813 microbial effect Effects 0.000 description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 7
- 229910052799 carbon Inorganic materials 0.000 description 7
- 238000012258 culturing Methods 0.000 description 7
- 238000001179 sorption measurement Methods 0.000 description 7
- 239000012528 membrane Substances 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 238000003794 Gram staining Methods 0.000 description 5
- 241001052560 Thallis Species 0.000 description 5
- 239000000356 contaminant Substances 0.000 description 5
- 238000004043 dyeing Methods 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 238000004817 gas chromatography Methods 0.000 description 5
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 5
- 239000002351 wastewater Substances 0.000 description 5
- 238000009395 breeding Methods 0.000 description 4
- 230000001488 breeding effect Effects 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 239000002699 waste material Substances 0.000 description 4
- 241000775876 Bacillus luciferensis Species 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 229910017053 inorganic salt Inorganic materials 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 239000010802 sludge Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000589776 Pseudomonas putida Species 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- MVPPADPHJFYWMZ-UHFFFAOYSA-N chlorobenzene Chemical compound ClC1=CC=CC=C1 MVPPADPHJFYWMZ-UHFFFAOYSA-N 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000003113 dilution method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000037353 metabolic pathway Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 239000012487 rinsing solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- CYTYCFOTNPOANT-UHFFFAOYSA-N Perchloroethylene Chemical group ClC(Cl)=C(Cl)Cl CYTYCFOTNPOANT-UHFFFAOYSA-N 0.000 description 1
- BZHJMEDXRYGGRV-UHFFFAOYSA-N Vinyl chloride Chemical compound ClC=C BZHJMEDXRYGGRV-UHFFFAOYSA-N 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- -1 amine oxalate Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 108700043460 bacteria ModE Proteins 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 239000010627 cedar oil Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 239000003673 groundwater Substances 0.000 description 1
- 150000008282 halocarbons Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000001011 safranin dye Substances 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000005211 surface analysis Methods 0.000 description 1
- 231100000378 teratogenic Toxicity 0.000 description 1
- 230000003390 teratogenic effect Effects 0.000 description 1
- 229950011008 tetrachloroethylene Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/36—Adaptation or attenuation of cells
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/36—Organic compounds containing halogen
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
-
- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters
Abstract
The invention relates to domestication screening and application of three wild strains with high-efficiency chlorinated hydrocarbon pollutant degrading capability, wherein three chlorinated hydrocarbon degrading bacteria are all from soil of a field with chlorinated hydrocarbon pollutant for a long time, and are obtained through gradient increasing pollutant concentration domestication screening, and have stronger high-concentration chlorinated hydrocarbon environment adapting capability. Through separation and identification, the three strains are respectively: pseudomonas sp., CGMCC No.: 24871; microbacterium mexico, exiguobacterium mexicanum, CGMCC number: 24872; bacillus thuringiensis, CGMCC number: 24874. the degradation experiment results show that the three chlorinated hydrocarbon degrading bacteria have the capability of efficiently degrading chlorinated hydrocarbon, and the strain shows excellent chlorinated hydrocarbon degradation capability when being put into actual industrial wastewater of a water reducing agent factory, thereby having great significance for repairing chlorinated hydrocarbon pollution.
Description
Technical Field
The invention belongs to the fields of microbial degradation and microbial breeding, and particularly relates to domestication screening and application of three wild strains with high-efficiency chlorinated hydrocarbon pollutant degradation capability.
Background
Along with the rapid development of the economy in China, the urban process is accelerated, and the field pollution problem is also aggravated. Chlorinated hydrocarbons are one of the common types of pollution. The detection ratio of the halogenated hydrocarbon in the organic polluted groundwater in China is more than 80%, wherein chlorinated hydrocarbon is the most prominent. Most chlorinated hydrocarbons have cancerogenic, teratogenic and mutagenic 'three-cause' effects, cause great harm to human health and ecological environment, and especially Trichloroethylene (TCE), tetrachloroethylene (PCE) and the like are listed in 68 kinds of preferential control pollutants 'blacklists' by China.
The microbial degradation means that the microbial life activity is utilized to realize the conversion of pollutants from substances with complex and high toxicity into substances with simple structure, low toxicity and even no toxicity, and the method is an environment restoration technology with low cost, no secondary pollution and thorough restoration. The bioremediation of chlorinated hydrocarbon mainly depends on toxic and harmful chlorinated hydrocarbon pollutants in the microbial degradation environment, and finally is decomposed into nontoxic and harmless CO 2 And H 2 O. Since microorganisms themselves may possess metabolic pathways for related contaminants and microorganisms are able to excite or evolve metabolic pathways for related contaminants in environments that are detrimental to their growth, many wild strains exist in the natural environment that possess the ability to degrade chlorinated hydrocarbons. However, microorganisms in natural environment exist in a mixed bacteria mode, single strains with the capability of efficiently degrading chlorinated hydrocarbon pollutants are separated and screened through a certain biotechnology means and cultured, and then the strains are applied to in-situ remediation of chlorinated hydrocarbon pollution sites, so that the bioremediation efficiency of chlorinated hydrocarbon pollutants can be remarkably improved.
Microbial domestication generally refers to a method for directionally breeding microorganisms by artificially increasing the concentration of pollutants to gradually adapt the microorganisms to a certain condition. The strain with higher tolerance and degradation capability can be obtained through domestication, and the domestication is often used for breeding high-efficiency degradation strain with higher degradation capability on certain pollutants. The microorganism species in the contaminated site are generally very abundant, and by gradually increasing the concentration of the contaminant, most microorganisms in the contaminated sample can be inhibited or killed, eventually leaving a small portion of dominant strains that can adapt themselves or mutate to the harsh environment. The microbial domestication technology is an important means for breeding strains capable of efficiently degrading pollutants in natural environment.
Disclosure of Invention
The invention aims to provide three wild strains with the capability of efficiently degrading chlorinated hydrocarbon pollutants and application examples thereof, enrich the types of chlorinated hydrocarbon degrading microorganisms and improve the bioremediation efficiency of application scenes such as in-situ remediation of chlorinated hydrocarbon polluted sites.
In order to achieve the above purpose, the technical scheme of the invention is realized as follows:
three wild strains HK-EPT-3, HK-EPT-4 and HK-EPT-6 (with preservation numbers of CGMCC No.24871, CGMCC No.24872 and CGMCC No. 24874) for efficiently degrading chlorinated hydrocarbon pollutants are characterized in that the wild strains adapt to and survive in a high-concentration chlorinated hydrocarbon polluted environment after the wild strains are naturally evolved in the chlorinated hydrocarbon polluted environment for a long time. The bacterial strain HK-EPT-3 is Pseudomonas sp, and the 16SrDNA sequence is shown as a nucleotide sequence of SEQ ID No.1 through separation and screening and 16SrDNA sequence identification; the strain HK-EPT-4 is a microbacterium mexico species (Exiguobacterium mexicanum), and the 16SrDNA sequence of the strain is shown as a nucleotide sequence of SEQ ID No. 2; the strain HK-EPT-6 is a Bacillus species (Bacillus luciferensis) of which 16SrDNA sequence is shown as a nucleotide sequence of SEQ ID No. 3.
The invention relates to a domestication screening and identifying method of wild strain for degrading chlorinated hydrocarbon pollutants, which comprises the following steps:
(1) Collecting soil polluted by chlorinated hydrocarbon for a long time, domesticating by using a soil sample, continuously culturing the domesticated culture solution by a dilution coating flat plate method, and separating single bacterial colonies;
(2) The isolated single colony is subjected to morphological observation, the species of the single colony is determined by 16SrDNA sequence analysis, and the growth curve and degradation dynamics curve of the strain are measured to verify the chlorohydrocarbon degradation capability of the strain.
The application of the wild degrading bacteria in degrading chlorinated hydrocarbon pollutants is specifically described as follows:
the invention provides three wild strains for degrading chlorinated hydrocarbon pollutants, wherein the three wild strains are obtained from soil samples of places polluted by chlorinated hydrocarbon throughout the year, and are obtained through domestication, separation and screening, and have the capability of efficiently degrading chlorinated hydrocarbon pollutants.
The application of the chlorohydrocarbon degradation strain in chlorohydrocarbon pollutant degradation is as follows:
(1) After the strain is activated, the bacterial liquid is inoculated into a culture medium containing chlorinated hydrocarbon pollutants for degradation.
(2) After 7 days of degradation, the strain was analyzed for degradation of chlorinated hydrocarbon contaminants.
The degradation capability of the wild degrading bacteria for chlorinated hydrocarbon is at a higher level compared with other researches: the degradation rate of three wild degrading bacteria on chlorinated hydrocarbon is measured by single bacteria in an inorganic salt culture medium with 300mg/L trichloroethylene as the only carbon source, the 7-day degradation rate of chlorinated hydrocarbon degrading strain HK-EPT-3 on trichloroethylene is 98.12%, the 7-day degradation rate of chlorinated hydrocarbon degrading strain HK-EPT-4 on trichloroethylene is 79.62%, and the 7-day degradation rate of HK-EPT-6 on trichloroethylene is 90.16%. Gia-Luen Guo et al used Pseudomonas putida F co-metabolically degrading trichloroethylene in a fiber bed bioreactor using toluene as the carbon source. When the concentration of trichloroethylene is 2.4 mg/L-100 mg/L, after 95mg/L toluene is additionally added as carbon source, the removal rate of trichloroethylene is 90% [ Gia-Luen Guo, dyi-Hwa Tseng1, shir-Ly Huang.Co-metabolic degradation of trichloroethylene by Pseudomonas putida in a fibrous bed bioreactor [ J ]. Biotechnology Letters,2001 (23): 1653-1657]. Toluene is additionally added to Sui H and the like as a carbon source, 80% of trichloroethylene is removed by microorganisms at a trichloroethylene concentration of 1.46mg/L, and less degradation of trichloroethylene is observed when the trichloroethylene concentration is higher than 48.8mg/L [ Sui H, xin-Gang L I, shi-Min X U.Cometauic microbial degradation of trichloroethylene in the presence of toluene [ J ]. Journal of Environmental Sciences,2004,16 (3): 3]. Zhang et al in an Upstream Anaerobic Sludge Blanket (UASB) reactor, trichloroethylene degrading anaerobic granular sludge can effectively degrade 80% of 36.5mg/L trichloroethylene wastewater [ Zhang, Y., hu, M, li, P., wang, X, and Meng, Q.analysis of trichloroethylene removal and bacterial community function based on pH-adjusted in an up flow anaerobic sludge blanket reactor.appl. Microbiol. Biotechnol.2015 (99), 9289-9297]. According to the report of the recent review article, 300mg/L belongs to the higher concentration of trichloroethylene [ Wu, zhineng; man, quanli; niu, hanyu; lyu, honghong. Percent advances and trends of trichloroethylene biodegradation: A technical reviews. Front in microbiology.2022 (13): 1053169], and the degradation capacity exhibited by the chlorohydrocarbon degrading bacterium of the present invention does not involve a method of further improving degradation efficiency by adding additional carbon sources, optimizing by means of a reactor, response surface analysis, etc., i.e., it is at a higher level.
Compared with the prior art, the invention has the following advantages:
the three chlorohydrocarbon degrading bacteria are obtained by gradient increasing pollutant concentration domestication and screening and have the following characteristics: the method has stronger environment adaptability of high-concentration chlorinated hydrocarbon, and degradation experiment results show that three chlorinated hydrocarbon degrading bacteria have high-efficiency chlorinated hydrocarbon degrading capability, and in addition, the strain shows excellent chlorinated hydrocarbon degrading capability when being put into actual industrial wastewater of a water reducing agent factory. The method provides a new degradation strain for degradation and repair of chlorinated hydrocarbon pollutants, improves degradation efficiency, enriches species abundance of chlorinated hydrocarbon degradation strains, and provides an experimental basis for further development and application of the strains.
Biological preservation description:
HK-EPT-3, pseudomonas sp, deposited in China general microbiological culture Collection center, CGMCC, at a deposit address: the preservation number is CGMCC No.24871, and the state is survival.
HK-EPT-4, the genus Microbacterium mexico (Exiguobacterium mexicanum), was deposited in China general microbiological culture Collection center, CGMCC, for short, at the deposit address: the preservation number is CGMCC No.24872, and the state is survival.
HK-EPT-6, bacillus thuringiensis (Bacillus luciferensis) was deposited in China general microbiological culture Collection center, CGMCC, with deposit address: the preservation number is CGMCC No.24874, and the state is survival.
Drawings
FIG. 1 is a plate-drawing of strains HK-EPT-3, HK-EPT-4 and HK-EPT-6, left: HK-EPT-3; in (a): HK-EPT-4; right: HK-EPT-6;
FIG. 2 is a gram of the strains HK-EPT-3, HK-EPT-4 and HK-EPT-6, left: HK-EPT-3, red; in (a): HK-EPT-4, purple; right: HK-EPT-6, purple;
FIG. 3 is a graph of growth curves of the strains HK-EPT-3, HK-EPT-4 and HK-EPT-6, left: HK-EPT-3; in (a): HK-EPT-4; right: HK-EPT-6;
FIG. 4 is a graph of degradation kinetics of the strains HK-EPT-3, HK-EPT-4 and HK-EPT-6, left: HK-EPT-3; in (a): HK-EPT-4; right: HK-EPT-6.
Detailed Description
The invention is further illustrated by the following examples:
the domestication screening and application processes of the chlorohydrocarbon degrading bacteria HK-EPT-3, HK-EPT-4 and HK-EPT-6 can be summarized as follows: (1) collecting soil samples of chlorinated hydrocarbon polluted sites; (2) gradually increasing the selection pressure to perform gradual domestication by taking typical chlorinated hydrocarbon trichloroethylene as a unique carbon source; (3) coating and screening to obtain single bacterial colony; (4) performing strain identification by 16SrDNA sequencing; (5) testing the growth capacity and degradation capacity; (6) and testing the repairing capability of the strain on actual wastewater.
Example 1: domestication, screening and separation of chlorohydrocarbon degradation strain
(1) Sampling: collecting soil polluted by chlorinated hydrocarbon for a long time, collecting about 100g of soil sample at a sampling point 6, and packaging and preserving the soil sample by using a sealing bag.
(2) Domestication: trichloroethylene is used as a single carbon source, the concentration of the trichloroethylene is gradually increased by 100mg/L, 300mg/L and 500mg/L respectively, and samples are collected at 30 ℃ and 220r/min in 250mL shake flasks for domestication culture of pesticide pollution places.
(3) And (3) switching: after acclimation for about one week, the bacterial cells in the shake flask are obviously grown, 1% of the acclimated chlorohydrocarbon is added into 100mL of culture medium, and the culture is continued at 30 ℃ and 220r/min in 250mL shake flasks and repeated for 2-3 times.
(4) Dilution: single strains were screened by dilution-spread plating. Taking 1mL of the degraded bacterial liquid after transfer in a 1.5mL pipette, and diluting into 10 by a ten-fold dilution method -5 、10 -6 、10 -7 、10 -8 、10 -9 And (5) standby application.
(5) And (3) pouring a plate: the solid culture medium used in the experimental process is placed into a water bath to be heated until the solid culture medium is melted, the culture medium is cooled to about 50 ℃, the culture dish is held by hand, a gap is opened near the flame by covering the dish, about 15mL of the culture medium is rapidly added, and the culture medium is solidified.
(6) Coating: 0.1ml of bacterial liquid was pipetted into agar plates of different dilution numbers (three replicates for each number). And uniformly smearing the solution on a flat plate by using a coater, and sterilizing the coater to an outer flame of an alcohol lamp when the dilution is replaced by using one coater for each dilution. Placing the smeared flat plate on a table for 20-30min, inverting the flat plate, and culturing overnight in a 30 ℃ incubator.
(7) Scribing: single colonies were obtained by plate streaking. Selecting a smooth inoculating loop, and picking a small amount of strains according to a sterile operation method. The flat plate is inverted beside the alcohol lamp, the bottom of the dish is held by the left hand, the flat plate is vertical to the tabletop as much as possible, and the side with the culture medium faces the alcohol lamp. The residual bacteria on the ring should be burnt immediately after each scratch is finished, so as to avoid influencing the separation effect of each area at the back due to excessive bacteria. When the seed ring is burnt, the bottom of the dish is held by the left hand and covered above the dish cover (without being placed in the dish cover), so as to prevent the contamination of the mixed bacteria. And so on, streaking 4-5 times to better separate out individual colonies.
(8) Culturing at constant temperature: the streak plate is inverted, cultured in a 30 ℃ incubator for about 24 hours, taken out and observed to judge whether single bacterial colonies are separated, and the strain morphology is observed, so that three single bacteria are finally obtained, and the streak plate diagram of the strain is shown in figure 1.
(9) And (3) bacterial storage: each strain pre-stores 2 glycerol pipes.
Operating in ultra-clean bench sterilized by ultraviolet. Holding a bacterial strain mother liquor conical flask, shaking uniformly, uncovering a sealing film, burning a bottle mouth by using an alcohol lamp external flame, holding a 1000 mu L pipettor, sucking 1mL of bacterial solution, adding into a frozen storage tube sterilized by high-pressure steam, adding 1mL of newly prepared 50% glycerol, and sealing and numbering for storage. 2 glycerol tubes are stored in each strain, and one part of glycerol tube is stored at-80 ℃ and-20 ℃ respectively.
Example 2: morphological observations of strains
Gram staining is a widely used differential staining method in bacteriology, the target strain obtained by separation is streaked on 6 solid culture medium plates to separate single colonies, and the single colonies are placed into a constant temperature incubator at 30 ℃ for culture, and the growth condition of the strain and the basic characteristics of the colonies such as colony morphology, colony size, colony color, colony edge and the like are observed. Gram staining of the strain is carried out, and the specific method for staining is as follows:
(1) preparation: wiping the new glass slide with mirror wiping paper, marking the unused side of the glass slide to determine the position of the thalli, and baking the position to be coated with the bacteria on the flame of the alcohol lamp to remove grease;
(2) smearing: the bacteria liquid shaking tube is held on the left, a tube plug is opened near an alcohol lamp of an ultra clean bench, a sterilized inoculating loop is used for dipping the bacteria liquid from the bacteria shaking tube, and a coating film with the diameter of about 2mm is uniformly coated on a clean and lipid-free glass slide.
(3) Fixing: allowing the smear to naturally dry in air, allowing the bacterial film to pass the slide glass upwards through flame for 2-3 times (preferably without scalding hands), and fixing the bacterial film;
(4) dyeing: dripping an amine oxalate crystal violet dye solution on the fixed glass-carrying smear, and dyeing for 1min;
(5) washing: slowly flushing the dye liquor on the smear with water, and sucking the dye liquor with water-absorbing paper;
(6) mordant dyeing: 1 drop of iodine solution is rapidly dripped into the bacterial membrane, the bacterial membrane is dyed for 1min, and the bacterial membrane is washed with water;
(7) decoloring: after the water is absorbed by the absorbent paper, 95% ethanol is continuously added dropwise for decolorization for 20-30s, and the effluent is immediately washed when no purple exists;
(8) counterstaining: dripping safranin dye liquor, counterstaining for 3-5min, washing with water, and finishing gram dyeing;
(9) and (3) observation: dripping cedar oil, and observing with an oil lens.
Dyeing result: purple is gram positive and red is gram negative.
The gram staining results of the strain of the invention are shown in figure 2: the bacterial strain HK-EPT-3 is round and light yellow, and is gram-negative; the bacterial strain HK-EPT-4 has short and small thalli, and the gram staining result is purple and is gram positive bacteria; the strain HK-EPT-5 has slender thallus and the gram staining result is purple, and is gram positive bacterium.
Example 3: strain 16SrDNA sequence analysis
(1) Extraction of genomic DNA
According to the extraction instruction of the bacterial genome DNA rapid extraction kit of Bomaide biotechnology Co., ltd, the genome DNA of the target strain is extracted. The specific operation steps are as follows:
the strain was inoculated into a test tube containing 5mL of LB liquid medium and cultured overnight at 30℃with shaking.
(1) Taking 0.5-2mL of culture bacterial liquid, centrifuging at 10000rpm for 30s, absorbing and discarding supernatant as much as possible, and collecting thalli;
(2) adding 200 mu L of lysate TL into the bacterial precipitate, and oscillating until the bacterial precipitate is thoroughly suspended;
(3) adding 20 mu L of proteinase K (20 mg/mL) solution, fully and uniformly mixing, adding 220 mu L of binding solution CB, immediately vortex oscillating and uniformly mixing, standing at 70 ℃ for 10min, keeping the solution clear, and centrifuging briefly to remove water drops in a tube cover;
(4) adding 220 mu L of absolute ethyl alcohol after cooling, immediately vortex oscillating and fully mixing uniformly, wherein flocculent precipitation can occur at the moment;
(5) adding the mixture obtained in the last step into an adsorption column AC, centrifuging at 13000rpm for 30-60s, and pouring out the waste liquid in the collection tube;
(6) adding 500 μl of inhibitor removing solution IR, centrifuging at 12000rpm for 30s, and discarding the waste liquid;
(7) adding 500 mu L of rinsing solution WB, centrifuging at 12000rpm for 30s, and discarding the waste liquid;
(8) adding 500 mu L of rinsing solution WB, centrifuging at 12000rpm for 30s, and discarding the waste liquid;
(9) placing the adsorption column AC back into an empty collecting pipe, centrifuging at 13000rpm for 2min, placing the adsorption column at room temperature for several minutes to thoroughly dry the residual rinsing liquid in the adsorption material, so as to prevent the residual ethanol in the rinsing liquid from inhibiting downstream reaction;
the adsorption column AC was taken out, placed in a clean centrifuge tube, 100. Mu.L of elution buffer EB was added to the middle portion of the adsorption membrane, and the mixture was left at room temperature for 3-5min and centrifuged at 12000rpm for 1min. The obtained solution was again put into a centrifugal adsorption column, and the solution was left at room temperature for 2min and centrifuged at 12000rpm for 1min to obtain genomic DNA.
(2) 16SrDNAPCR amplification and alignment
PCR amplification was performed using the above-mentioned extracted genomic DNA as a template and 16SrDNA universal primers 27F and 1492R. And (3) after agarose gel electrophoresis verification of the PCR reaction products, sending the PCR products to a gold-only sequencing company for sequencing, and searching homologous sequences for comparison and analysis of sequencing results by using BLAST programs in NCBI database to determine species. As a result of the comparison, the strain HK-EPT-3 was Pseudomonas sp, the strain HK-EPT-4 was Exiguobacterium mexicanum, and the strain HK-EPT-6 was Bacillus luciferensis.
Example 4: determination of Strain growth Curve
(1) Preparation: the liquid culture corresponding to 100mL of the screened strain is prepared based on a plurality of 250mL conical flasks, and the culture is sterilized by high-pressure steam for later use.
(2) Inoculating: each of the flasks was inoculated with 1mL of the bacterial solution in a 250mL shaking flask containing 100mL of an inorganic salt solution containing 300mg/L trichloroethylene.
(3) Culturing: culturing at 30deg.C and 220 r/min. Sampling and testing every 2-3 hours in the initial stage, adjusting the sampling interval time according to the specific conditions in the later stage according to different growth conditions of strains, and performing the experiment for 30 hours.
(4) And (3) sample measurement: and (3) sucking 1mL of bacterial liquid from a culture bottle by using a 1000 mu L pipettor, taking water as a standard sample to measure an absorbance OD600 value after a proper amount of dilution, and controlling the OD value to be in a range of 0.1-0.8 so as to meet the proportional relation between the OD value and the cell content. The dilution adopts a scientific dilution method, when the dilution is carried out for N times, m mu L of bacterial liquid is taken and added with (N-1) m mu L of water to be uniformly mixed, and the total amount of the added liquid in a cuvette is proper between 1.5 and 3 mL.
According to experimental data, taking the average value of 3 absorbance OD values (excluding data with obvious differences) measured at the same time t, and making a time t-absorbance OD curve graph to characterize the growth condition of the strain. The growth curves of the chlorohydrocarbon degrading strains HK-EPT-3, HK-EPT-4 and HK-EPT-6 are shown in FIG. 3.
Example 5: bacterial suspension preparation and trichloroethylene degradation experiment
(1) Preparing a bacterial suspension: inoculating the strains after screening into 100mL of LB liquid nutrient medium respectively, culturing at 220rpm until the logarithmic phase of the growth curve, taking 4-5mL of bacterial liquid in the logarithmic phase into a centrifuge tube, and setting the centrifugation parameters as follows: at 8000rpm for 10min, removing supernatant after centrifugation, washing the thalli for 3 times by using a sterile MSM culture medium, diluting the thalli to prepare a bacterial liquid with the OD600 of about 3, and storing at-4 ℃ for later use;
(2) Degradation experiment: inoculating the bacterial liquid into a 250mL shaking bottle filled with 100mL of inorganic salt degradation culture medium containing 300mg/L trichloroethylene according to an inoculation proportion of 1%, and culturing the shaking bottle in a shaking table at 220rpm and 30 ℃ for 7 days;
(3) Extraction of chlorohydrocarbon trichloroethylene: the substrate in the medium was extracted with n-hexane. Extracting for 2 times per bottle, taking the extract into a 50mL test tube, filtering with an organic filter membrane to remove fine impurities, then placing the sample into a 1.5mL clean centrifuge tube, and preserving at-20deg.C for gas chromatography;
(4) Degradation rate measurement: the trichloroethylene degradation rate was measured using gas chromatography, and the conditions for the gas chromatography were: gas chromatograph: GC-430; FID detector: the detection column was Rxi-1HT (30 m. Times.0.32 mm. Times.0.25 μm) nonpolar column. The method comprises the following steps: inlet temperature: 250 ℃, detector temperature: 260℃and nitrogen (99.999%) as carrier gas, flow rate 1mL/min, split ratio 10:1, 1. Mu.L of sample was introduced. Programming temperature: the temperature was kept at 40℃for 4min and raised to 250℃at 20℃per min. And (3) taking pure hexane with chromatography as a solvent, and quantifying according to the peak area. The degradation rate of the substrate is calculated: in each sample, a control group and an experimental group were included. After calculating the integral of the residual peak area of the substrate, the substrate degradation rate formula is as follows: wherein AF refers to the peak area of the residual substrate.
The degradation rate formula is as follows:
the degradation rate of the chlorohydrocarbon degradation strain HK-EPT-3 on trichloroethylene was 98.12%, the degradation rate of the chlorohydrocarbon degradation strain HK-EPT-4 on trichloroethylene on 7 days was 79.62%, the degradation rate of the chlorohydrocarbon degradation strain HK-EPT-6 on trichloroethylene on 7 days was 90.16%, and the degradation kinetics curves were shown in FIG. 4.
Example 6: test of actual wastewater restoration capability of chlorinated hydrocarbon degradation strain
(1) The test of the ability of the chlorohydrocarbon degrading strain to repair the actual wastewater was performed with reference to example 5, and the bacterial suspension was inoculated into a shake flask containing 100mL of actual industrial wastewater (the main chlorohydrocarbon contaminants are vinyl chloride, 1, 2-dichloroethane and chlorobenzene) from a water reducing agent plant at an inoculation ratio of 5%, and the shake flask was placed in a shaker at 220rpm and 30℃for 7 days;
(2) Extraction of chlorinated hydrocarbons: the substrate in the culture medium is extracted with an extractant. Extracting for 2 times per bottle, taking the extract into a 50mL test tube, filtering with an organic filter membrane to remove fine impurities, then placing the sample into a 1.5mL clean centrifuge tube, and preserving at-20deg.C for gas chromatography;
(3) The degradation rate of chlorinated hydrocarbons after 7 days of degradation of chlorinated hydrocarbons by chlorinated hydrocarbon degrading bacteria was measured by gas chromatography, and water samples before and after degradation were sent to Stansted detection Co., ltd. For further detection of other pollutants, and the obtained degradation rate measurement results are shown in Table 1.
TABLE 1 test results of the ability of chlorinated Hydrocarbon degrading Strain to repair actual wastewater
The degradation rate of the three strains on chlorinated hydrocarbon and other pollutants in 7 days is over 98.83 percent, and the content of the degraded pollutants reaches the national standard of building block development and construction (GB 36600-2018).
These results show that all three strains obtained by screening have the capability of efficiently degrading chlorinated hydrocarbon and have practical application value.
While the invention has been described in detail with reference to specific/preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and changes can be made without departing from the spirit of the invention, and it is intended that such modifications and changes be considered as the scope of the invention.
Claims (7)
1. Pseudomonas HK-EPT-3 with chlorohydrocarbon degrading capacity is deposited in China general microbiological culture Collection center and classified as Pseudomonas sp; the preservation number is CGMCCNO.24871.
2. The Pseudomonas HK-EPT-3 according to claim 1, wherein the bacterial colony is round, light yellow, smooth in surface and edge, rod-shaped under a scanning electron microscope, and is a gram-negative bacterium.
3. Microbacterium HK-EPT-4 with chlorohydrocarbon degrading capacity is deposited in China general microbiological culture Collection center and classified as Microbacterium mexico, exiguobacterium mexicanum; the preservation number is CGMCC NO.24872.
4. The micro-bacillus HK-EPT-4 of claim 3, wherein the micro-bacillus is short, long rod-shaped, and irregular in edge, and is a gram-positive bacterium.
5. Bacillus HK-EPT-6 with chlorohydrocarbon degrading capacity is deposited in China general microbiological culture Collection center and classified as Bacillus thuringiensis; the preservation number is CGMCC NO.24874.
6. The bacillus HK-EPT-6 of claim 5, wherein the bacillus is slender, smooth in surface and saw-tooth-shaped in edge, and is a gram positive bacterium.
7. The use of the three strains according to claims 1, 3 and 5 for in situ remediation of chlorinated hydrocarbon polluted environments.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310372902.7A CN116656535A (en) | 2023-04-10 | 2023-04-10 | Bacterial strain with chlorohydrocarbon degradation capability and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310372902.7A CN116656535A (en) | 2023-04-10 | 2023-04-10 | Bacterial strain with chlorohydrocarbon degradation capability and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116656535A true CN116656535A (en) | 2023-08-29 |
Family
ID=87714245
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310372902.7A Pending CN116656535A (en) | 2023-04-10 | 2023-04-10 | Bacterial strain with chlorohydrocarbon degradation capability and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116656535A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117397610A (en) * | 2023-12-05 | 2024-01-16 | 山东省海洋资源与环境研究院(山东省海洋环境监测中心、山东省水产品质量检验中心) | Method for repairing polluted bottom mud of cage culture |
-
2023
- 2023-04-10 CN CN202310372902.7A patent/CN116656535A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117397610A (en) * | 2023-12-05 | 2024-01-16 | 山东省海洋资源与环境研究院(山东省海洋环境监测中心、山东省水产品质量检验中心) | Method for repairing polluted bottom mud of cage culture |
| CN117397610B (en) * | 2023-12-05 | 2024-03-19 | 山东省海洋资源与环境研究院(山东省海洋环境监测中心、山东省水产品质量检验中心) | Method for repairing polluted bottom mud of cage culture |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110396488B (en) | Mixed bacterial system for degrading polycyclic aromatic hydrocarbon pollutants and application method | |
| CN111748483B (en) | Bacillus for degrading petroleum hydrocarbon and application thereof | |
| CN107893047B (en) | Petroleum aromatic hydrocarbon degrading bacterium and application thereof | |
| CN110938569B (en) | Microbial agent for hexavalent chromium pollution treatment and hexavalent chromium pollution treatment method | |
| CN106635909B (en) | Crude oil degradation mixed bacteria, microbial inoculum and application thereof | |
| CN116656535A (en) | Bacterial strain with chlorohydrocarbon degradation capability and application thereof | |
| CN114540226B (en) | Polycyclic aromatic hydrocarbon degrading strain LJB-25 in petroleum polluted soil, and microbial inoculum and application thereof | |
| WO2008062557A1 (en) | Novel microorganism capable of microbially decomposing chloro-, methylthio- and methoxy-triazine agrichemicals | |
| CN110734886B (en) | Bacterial strain capable of tolerating high-concentration chromium and application thereof | |
| CN104046580B (en) | Sphingobacterium bacterial strain and its application for degrading polycyclic aromatic hydrocarbons class organic pollution | |
| CN115725438A (en) | Hexavalent chromium reducing bacterium and application thereof | |
| AlGhazawi et al. | Selection of bacteria and plant seeds for potential use in the remediation of diesel contaminated soils | |
| CN104099266B (en) | Achromobacter bacterial strain and application thereof for degrading polycyclic aromatic hydrocarbons class organic pollution | |
| CN113755371B (en) | Strain A.seifertigii P52-1 and application thereof in degradation of polychlorinated biphenyl | |
| CN113621534B (en) | Polychlorinated biphenyl degrading strain P52-2 and application thereof | |
| Iyer et al. | A study on Feasibility of Bioremediation of Crude Oil contaminated soil from Kalol with Indigenous Mixed Culture | |
| CN113637602B (en) | Labrys portucalensis P49-1 and application thereof | |
| CN114134079B (en) | Tetracycline antibiotic degrading bacteria, method and application | |
| CN114058527B (en) | Oil-containing pollutant degradation strain XT2, microbial agent and application | |
| CN115820453A (en) | Bacterial strain capable of degrading dibenzothiophene, bacterial solution produced by bacterial strain and application of bacterial solution | |
| CN108130291B (en) | Yeast for degrading petroleum aromatic hydrocarbon and application thereof | |
| CN109082399B (en) | Rejuvenation method of high-temperature micro-aerobic composting nitrogen-retaining bacteria for reducing ammonia volatilization of livestock and poultry manure compost | |
| Vargas et al. | Isolation of microorganisms with capability to degrade polycyclic aromatic hydrocarbons (PAH's) | |
| CN117802003A (en) | Campylobacter BL0 and application thereof | |
| Abubakar et al. | Biodegradation of pyrene by a mixed culture isolated from hydrocarbon-polluted soil |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |