CN114381392B - High-efficiency ethyl chloride degrading bacterium and application thereof - Google Patents
High-efficiency ethyl chloride degrading bacterium and application thereof Download PDFInfo
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- HRYZWHHZPQKTII-UHFFFAOYSA-N chloroethane Chemical compound CCCl HRYZWHHZPQKTII-UHFFFAOYSA-N 0.000 title claims abstract description 126
- 229960003750 ethyl chloride Drugs 0.000 title claims abstract description 125
- 230000000593 degrading effect Effects 0.000 title claims abstract description 47
- 241000894006 Bacteria Species 0.000 title claims abstract description 39
- 239000002351 wastewater Substances 0.000 claims abstract description 25
- 241000589516 Pseudomonas Species 0.000 claims abstract description 9
- 238000000855 fermentation Methods 0.000 claims description 25
- 230000004151 fermentation Effects 0.000 claims description 25
- 230000001580 bacterial effect Effects 0.000 claims description 20
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 239000000306 component Substances 0.000 claims description 11
- 238000012258 culturing Methods 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 10
- 239000000575 pesticide Substances 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 229940041514 candida albicans extract Drugs 0.000 claims description 7
- 239000012138 yeast extract Substances 0.000 claims description 7
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
- 229910052760 oxygen Inorganic materials 0.000 claims description 6
- 239000001301 oxygen Substances 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 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 4
- 239000012880 LB liquid culture medium Substances 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 238000004806 packaging method and process Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 3
- 238000005516 engineering process Methods 0.000 claims description 2
- 238000009629 microbiological culture Methods 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 238000012546 transfer Methods 0.000 claims description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims 2
- 239000007836 KH2PO4 Substances 0.000 claims 1
- 235000010216 calcium carbonate Nutrition 0.000 claims 1
- 229910000019 calcium carbonate Inorganic materials 0.000 claims 1
- 239000002054 inoculum Substances 0.000 claims 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims 1
- 235000019341 magnesium sulphate Nutrition 0.000 claims 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims 1
- 235000019796 monopotassium phosphate Nutrition 0.000 claims 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 7
- 229910052799 carbon Inorganic materials 0.000 abstract description 7
- 238000002474 experimental method Methods 0.000 abstract description 3
- 238000010186 staining Methods 0.000 abstract description 2
- 230000035764 nutrition Effects 0.000 abstract 1
- 235000016709 nutrition Nutrition 0.000 abstract 1
- 230000015556 catabolic process Effects 0.000 description 21
- 238000006731 degradation reaction Methods 0.000 description 21
- 239000000243 solution Substances 0.000 description 12
- 229910052698 phosphorus Inorganic materials 0.000 description 11
- 239000011574 phosphorus Substances 0.000 description 11
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 10
- 238000000034 method Methods 0.000 description 7
- 230000012010 growth Effects 0.000 description 6
- 230000001276 controlling effect Effects 0.000 description 5
- 239000002068 microbial inoculum Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241000793855 Pseudomonas hunanensis Species 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- KMJJJTCKNZYTEY-UHFFFAOYSA-N chloro-diethoxy-sulfanylidene-$l^{5}-phosphane Chemical compound CCOP(Cl)(=S)OCC KMJJJTCKNZYTEY-UHFFFAOYSA-N 0.000 description 3
- 229910017053 inorganic salt Inorganic materials 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000010802 sludge Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000004065 wastewater treatment Methods 0.000 description 3
- 102000012440 Acetylcholinesterase Human genes 0.000 description 2
- 108010022752 Acetylcholinesterase Proteins 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 229940022698 acetylcholinesterase Drugs 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000003235 crystal violet staining Methods 0.000 description 2
- 239000003344 environmental pollutant Substances 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 231100000719 pollutant Toxicity 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000010865 sewage Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 241000269335 Ambystoma laterale x Ambystoma jeffersonianum Species 0.000 description 1
- 206010008479 Chest Pain Diseases 0.000 description 1
- 239000005944 Chlorpyrifos Substances 0.000 description 1
- 206010010071 Coma Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 208000008454 Hyperhidrosis Diseases 0.000 description 1
- 206010023644 Lacrimation increased Diseases 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 206010037423 Pulmonary oedema Diseases 0.000 description 1
- 206010039424 Salivary hypersecretion Diseases 0.000 description 1
- 208000005392 Spasm Diseases 0.000 description 1
- 206010046543 Urinary incontinence Diseases 0.000 description 1
- 206010047513 Vision blurred Diseases 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 241001148470 aerobic bacillus Species 0.000 description 1
- 230000009603 aerobic growth Effects 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- FFBHFFJDDLITSX-UHFFFAOYSA-N benzyl N-[2-hydroxy-4-(3-oxomorpholin-4-yl)phenyl]carbamate Chemical compound OC1=C(NC(=O)OCC2=CC=CC=C2)C=CC(=C1)N1CCOCC1=O FFBHFFJDDLITSX-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- SBPBAQFWLVIOKP-UHFFFAOYSA-N chlorpyrifos Chemical compound CCOP(=S)(OCC)OC1=NC(Cl)=C(Cl)C=C1Cl SBPBAQFWLVIOKP-UHFFFAOYSA-N 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000012851 eutrophication Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000037315 hyperhidrosis Effects 0.000 description 1
- 230000004317 lacrimation Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- LCCNCVORNKJIRZ-UHFFFAOYSA-N parathion Chemical compound CCOP(=S)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 LCCNCVORNKJIRZ-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- ATROHALUCMTWTB-OWBHPGMISA-N phoxim Chemical compound CCOP(=S)(OCC)O\N=C(\C#N)C1=CC=CC=C1 ATROHALUCMTWTB-OWBHPGMISA-N 0.000 description 1
- 229950001664 phoxim Drugs 0.000 description 1
- 238000011197 physicochemical method Methods 0.000 description 1
- 208000005333 pulmonary edema Diseases 0.000 description 1
- 210000001747 pupil Anatomy 0.000 description 1
- 230000004478 pupil constriction Effects 0.000 description 1
- -1 pyrimidyl phosphorus Chemical compound 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 208000026451 salivation Diseases 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 208000016255 tiredness Diseases 0.000 description 1
- 230000003867 tiredness Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- AMFGTOFWMRQMEM-UHFFFAOYSA-N triazophos Chemical compound N1=C(OP(=S)(OCC)OCC)N=CN1C1=CC=CC=C1 AMFGTOFWMRQMEM-UHFFFAOYSA-N 0.000 description 1
- 239000003403 water pollutant Substances 0.000 description 1
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
- A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
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- 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
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- 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
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/20—Organic substances
- A62D2101/22—Organic substances containing halogen
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- 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
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Abstract
The invention discloses an ethyl chloride high-efficiency degrading bacterium and application thereof, which screens out a strain YL-5 capable of degrading ethyl chloride efficiently, and is identified as pseudomonas hunanPseudomonas hunanensis) Gram-positive staining, heterotrophic aerobic nutrition, and can grow by using ethyl chloride as the only carbon source. Experiments prove that under the condition that the initial concentration of the ethyl chloride is 3g/L, the removal rate of the strain to the ethyl chloride can reach 100% within 48 hours. The invention can effectively solve the problem of poor biochemical effect of the high-concentration ethyl chloride wastewater, and provides a new effective way for biochemical treatment of the high-concentration ethyl chloride wastewater.
Description
Technical Field
The invention relates to the field of wastewater treatment, in particular to a bacterial strain capable of degrading organic phosphorus pollutant ethyl chloride and application thereof.
Background
Pesticides are chemical products for guaranteeing the sustainable development of agricultural production in China, various varieties are wide, most pesticides are seriously polluted in the production process, and organic phosphate pesticides are the most important types of pesticides. The main harm of organic phosphorus to human body is that it can combine with acetylcholinesterase in human body, so that acetylcholinesterase can not be hydrolyzed, resulting in blurred vision, headache, dizziness, tiredness, lacrimation, salivation, abdominal pain, emesis, chest distress, nausea, vexation, discomfort, hyperhidrosis, pupil constriction, general spasm, confusion, disappearance of pupil reaction or coma, pulmonary edema, urinary incontinence, respiratory central system failure, death, etc. Meanwhile, phosphorus is also one of important pollution elements for eutrophication of water body. Thus, the national requirements for the emission of organic phosphorus are extremely high: the first-level standard organophosphorus pesticides (calculated by phosphorus) in the integrated wastewater discharge standard (GB 8978-1996) cannot be detected, the first-level standard total phosphorus in the chemical synthesis type pharmaceutical industry water pollutant discharge standard (GB 21904-2008) is 1.0mg/L, and the first-level standard A standard total phosphorus in the pollutant discharge standard of urban wastewater treatment plants (GB 18918-2002) is 1.0mg/L. O, O-diethyl thiophosphoryl chloride (ethyl chloride for short) is an important intermediate of organophosphorus pesticides (triazophos, chlorpyrifos, phoxim, parathion, pyrimidyl phosphorus and the like) and widely exists in pesticide production wastewater.
Because the ethyl chloride has certain toxicity to common microorganisms, the biochemical treatment effect of the existing wastewater containing the ethyl chloride is not ideal, so that the wastewater containing the ethyl chloride is treated by adopting a plurality of physicochemical methods in practice, the cost is higher, and secondary pollution is easy to cause. Therefore, bacteria capable of efficiently degrading the ethyl chloride are screened, and organic phosphorus in the wastewater is converted into inorganic phosphorus under the condition of mild reaction conditions, so that the method is extremely important for solving the problem that the ethyl chloride wastewater is difficult to treat.
Disclosure of Invention
The invention aims to solve the technical problem of finding a microorganism which can not only endure high-concentration ethyl chloride but also rapidly realize degradation. In addition, the invention provides a fermentation process for rapidly culturing the high-efficiency degrading bacteria of the ethyl chloride.
The invention screens and separates the ethyl chloride high-efficiency degrading bacteria, can effectively and rapidly degrade the ethyl chloride in the sewage, and simultaneously provides a fermentation culture process of the ethyl chloride high-efficiency degrading bacteria, thereby realizing rapid enrichment and expansion culture of strains in a short time.
The invention provides ethyl chloride high-efficiency degrading bacteria YL-5, which is classified and named as pseudomonas hunan (Pseudomonas hunanensis) and is preserved in China general microbiological culture collection center, address: the preservation number of the Beijing city Chaoyang area North Chen Xili No.1 and 3 is CGMCC No.23934.
The physiological characteristics are as follows: the thallus is in a rod shape, and is used for producing spores, liquefying gelatin and heterotrophic aerobic bacteria and gram-positive staining. Obvious colonies appear on the agar solid plate for 24 hours, and the colonies are round or oval, neat in edge, opaque and moist and matt.
The strain obtained by screening can tolerate ethyl chloride solution with high concentration of not more than 4 g/L. The ethyl chloride degrading bacterium YL-5 provided by the invention can grow by taking ethyl chloride as the only carbon source. YL-5 achieved 100% completion of the initial concentration of 3g/L ethyl chloride within 48 hours under laboratory shaking conditions. Meanwhile, the practical treatment effect of the ethyl chloride high-efficiency degrading bacteria YL-5 on the wastewater containing the ethyl chloride is verified. The microbial inoculum obtained by fermenting YL-5 is added into the ethyl chloride wastewater containing 1g/L according to the volume ratio of 10%, and after aeration treatment for 24 hours, the degradation rate of the ethyl chloride can reach 98%, so that the treatment effect is very ideal.
The invention provides a simple, convenient and rapid fermentation process for degrading ethyl chloride by using high-efficiency bacteria, which comprises the following steps of activation, transfer, expansion culture and the like, wherein the specific process flow is as follows:
s1, activation: single colony of ethyl chloride high-efficiency degrading bacteria is selected from a solid flat plate and is transferred into LB liquid culture medium containing ethyl chloride, the rotation speed of a shaking table is 120-150 rpm, and shaking table culture is carried out at 30-37 ℃ for 8-16h until the logarithmic phase;
s2 switching: transferring the ethyl chloride degrading bacterial liquid in logarithmic phase in the process 1 into a 50L seed tank for culturing according to the access amount of 2-5%, controlling the temperature of the seed tank to be 30-37 ℃, controlling the rotating speed to be 220-250rpm, controlling the dissolved oxygen DO to be 4-6mg/L, and culturing for 24-48h; the strain can tolerate higher concentration of ethyl chloride and can obviously improve the complete degradation rate of the ethyl chloride.
S3, expanding culture: transferring the ethyl chloride degrading bacterial liquid cultured in the seed tank in the process 2 into a 1000L fermentation tank for expansion culture according to the inoculation amount of 2% -5%, wherein the culture medium components of the fermentation tank are the same as those of the seed tank, and the physical and chemical parameter indexes are as follows: the temperature is 37 ℃, the rotating speed is 250rpm, the dissolved oxygen is 8mg/L, and the fermentation time is 48-72h. After fermentation, the effective viable count of the tank bacterial liquid can reach 10 9 And (3) taking out the fermentation culture solution from the tank, and packaging the fermentation culture solution by a plastic barrel to obtain the ethyl chloride high-efficiency degradation microbial inoculum.
The strain of the invention can grow by using ethyl chloride as the only carbon source, and preferably, the LB medium comprises the following components: 1-3 g/L of ethyl chloride (O, O-diethyl thiophosphoryl chloride), 5-15g/L of nitrogen source and 2-10 g/L of inorganic salt, and the pH value is kept between 7.2 and 7.8; further preferably, the components of the LB medium are as follows: 1g/L ethyl chloride (O, O-diethyl thiophosphoryl chloride), 5g/L yeast extract, 10g/L peptone, 2g/L sodium chloride, 1g/L sodium bicarbonate, 0.2g/L sodium carbonate, and the pH is maintained between 7.2 and 7.8. The culture medium of the seed tank comprises the following components in percentage by mass: ethyl chloride 0.1-0.2%, glucose 0.4-0.6%, NH 4 Cl 0.1～0.2％，KH 2 PO 4 0.005～0.01％，MgSO 4 0.025～0.05％，NaCl 0.01～0.02％，CaCO 3 0.015～0.3％，NaHCO 3 0.1 to 0.2 percent, yeast extract 0.005 to 0.01 percent and the balance of water. The pH is maintained between 7.2 and 7.8 by implementing the pH self-control adjustment technology. More preferably, ethyl chloride 0.2%, glucose 0.6%, NH 4 Cl 0.2％，KH 2 PO 4 0.01％，MgSO 4 0.05％，NaCl 0.02％，CaCO 3 0.3％，NaHCO 3 0.2%, yeast extract 0.01% and pH maintained between 7.2-7.8.
The high-efficiency degrading bacteria for the ethyl chloride are applied to degrading the ethyl chloride.
A microbial inoculum containing ethyl chloride high-efficiency degrading bacteria, wherein the microbial inoculum is prepared by fermentationFermenting and culturing Pseudomonas Hunan (Pseudomonas hunanensis) YL-5, and packaging the fermentation culture solution after discharging from the tank to obtain the bacterial agent for efficiently degrading the ethyl chloride, wherein the effective viable count of the bacterial agent for efficiently degrading the ethyl chloride can reach 10 9 More than one per ml.
The ethyl chloride high-efficiency degrading bacteria or the application of the ethyl chloride high-efficiency degrading bacteria agent in degrading ethyl chloride.
The ethyl chloride high-efficiency degrading bacteria or the application of the ethyl chloride high-efficiency degrading bacteria agent in pesticide wastewater treatment.
After strain culture is carried out on pseudomonas hunan (Pseudomonas hunanensis) YL-5, the strain is added into wastewater to be treated containing ethyl chloride to degrade the ethyl chloride. The strain can resist high-concentration ethyl chloride wastewater and has high degradation rate.
The beneficial effects are that:
the high-efficiency degradation bacteria for the ethyl chloride can realize rapid and high-efficiency degradation of the ethyl chloride, and provides an effective solution to the problem of difficult biochemical treatment of wastewater containing the ethyl chloride. Has important practical significance for protecting ecological environment and human health. Meanwhile, the fermentation culture process of the high-efficiency degrading bacteria for the ethyl chloride is simple and convenient, is easy to operate, greatly reduces the risk of bacteria contamination in the middle link, has low production cost, short fermentation period time and rapid expansion culture, and has wide application prospect in the field of biological fermentation.
Drawings
FIG. 1 is a photograph showing crystal violet staining of ethyl chloride degrading bacterium YL-5 in accordance with the present invention under a x1000 magnification of an oil microscope.
FIG. 2 is a plot of initial amount of ethyl chloride of 3g/L versus time for strain YL-5 under laboratory shake flask conditions and a plot of growth of strain YL-5 versus time under such conditions.
FIG. 3 is a graph showing the concentration of ethyl chloride and degradation rate over time of strain YL-5 fermentation broth in an experiment for treating actual ethyl chloride-containing wastewater.
Detailed Description
The following examples are applicable to the present invention but are not intended to limit the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
EXAMPLE 1 isolation and purification of seed
The isolation and purification steps of the strain YL-5 are as follows:
(1) Domestication enrichment
50ml of activated sludge is taken and added into a 1L domestication device containing basic culture solution with ethyl chloride as the only carbon source, and the activated sludge is from a sewage treatment biochemical pond of a pesticide enterprise for treating wastewater containing the ethyl chloride for a long time in Jiangsu province. The basic culture solution comprises the following formula: ethyl chloride 0.5g/L, NH 4 Cl 0.5g/L、KH 2 PO 4 0.1g/L、Na 2 HPO 4 0.1g/L、MgCl 2 0.02g/L、CaCl 2 0.03g/L、NaHCO 3 0.5g/L. Introducing air, domesticating and culturing for about 15-20 days, and periodically monitoring the concentration of ethyl chloride and the growth condition of sludge. And after the complete degradation of the ethyl chloride is detected, timely supplementing, and gradually increasing the concentration of the ethyl chloride to 3g/L to obtain an ethyl chloride degrading bacteria enrichment liquid.
(2) Separation
Under aseptic conditions, transferring the domesticated enriched bacterial solution into an aseptic fresh basic culture solution with ethyl chloride as the sole carbon source according to the inoculation amount of 2%, wherein the basic culture solution has the same formula as that of the step 1. Shaking culture at 37deg.C to logarithmic phase, diluting and coating the culture broth with ethyl chloride as solid separation medium with ethyl chloride content of 1g/L, NH 4 Cl 0.5g/L、KH 2 PO 4 0.1g/L、MgCl 2 0.02g/L、CaCl 2 0.03g/L、NaHCO 3 1g/L, 2% of agar. Culturing in a constant temperature incubator at 37deg.C for 2-3 days.
(3) Purification
In an ultra-clean workbench, picking single colonies growing in the ethyl chloride separation solid culture medium by using an inoculating needle, and further streaking, purifying and culturing the ethyl chloride degrading bacteria by using a flat streak method until the single colonies grow. Repeating the operation for more than 3 times until the colony form growing on the plate is single, thus obtaining a strain of purified ethyl chloride high-efficiency degrading bacteria.
Example 2 identification of strains
The purified ethyl chloride degrading bacteria are subjected to 16s rDNA sequencing, the sequence is shown as SEQ.NO.1, and the sequencing comparison result is Pseudomonas hunan (Pseudomonas hunanensis) and is named YL-5. The main physiological characteristics are that gram staining is positive, aerobic growth can be carried out, and ethyl chloride can be used as the only carbon source and energy source for growth. A few bacterial samples are taken for crystal violet staining, the bacterial forms are observed to form rods under a multiplied by 1000 oil microscope, spores are present, physiological and biochemical identification of the bacterial strains is carried out according to the common bacterial system identification manual, and a microscopic photograph is shown in figure 1.
Example 3 growth curves of Strain YL-5 and degradation experiments on ethyl chloride
The purified ethyl chloride degrading bacteria YL-5 single colony in the invention is selected in an inorganic salt culture medium shake flask containing 3g/L ethyl chloride, and the formula of the inorganic salt culture medium comprises the following components: NH (NH) 4 Cl 0.5g/L、KH 2 PO 4 0.1g/L、Na 2 HPO 4 0.1g/L、MgCl 2 0.02g/L、CaCl 2 0.03g/L、NaHCO 3 1.5g/L、0.5g/L NaCO 3 . Shaking table rotational speed is 180rpm, and the temperature is 37 ℃, shaking table cultivation. The cell concentration OD600 was measured by sampling at 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, 48h, 60h, and 72h, respectively. OD600 was measured using a visible light spectrophotometer and ethyl chloride concentration was measured using liquid chromatography. The data is shown in figure 2, the strain YL-5 grows slowly in 12h at the initial concentration of ethyl chloride of 3g/L, so that the degradation of ethyl chloride is slow, the degradation speed of ethyl chloride is obviously improved along with the increase of the concentration of bacteria, and the removal rate of ethyl chloride reaches 50% after 48 h. When the culture is carried out for 72 hours, the effect of complete degradation of the ethyl chloride is achieved, and along with complete degradation of the ethyl chloride, the bacterial growth loses the carbon source and thus tends to be stable, and the OD600 value is stabilized to be about 1.3. The data sheetThe strain YL-5 disclosed by the invention has a good degradation effect on ethyl chloride degrading bacteria.
Example 4 fermentation culture Process of Strain YL-5
Single colony of ethyl chloride high-efficiency degrading bacteria is selected from the solid flat plate, and is transferred into LB liquid culture medium containing ethyl chloride, the rotation speed of a shaking table is 150rpm, and shaking table culture is carried out at 37 ℃ for 8-16 hours until the logarithmic phase. The LB culture medium comprises the following components: 1g/L ethyl chloride, 5g/L yeast extract, 10g/L peptone, 2g/L sodium chloride, 1g/L sodium bicarbonate, 0.2g/L sodium carbonate, and the pH is maintained between 7.2 and 7.8.
Transferring the activated ethyl chloride degrading bacteria liquid in logarithmic phase into a 50L seed tank according to the access amount of 5%, culturing for 36h, controlling the temperature of the seed tank to be 37 ℃ and the rotating speed to be 220-250rpm, and controlling dissolved oxygen DO to be 4-6 mg/L. The culture medium of the seed tank comprises the following components in percentage by mass: ethyl chloride 0.2%, glucose 0.6%, NH 4 Cl 0.2％，KH 2 PO 4 0.01％，MgSO 4 0.05％，NaCl0.02％，CaCO 3 0.3%, yeast extract 0.01%, naHCO 3 0.2%, the balance being water, the pH is maintained between 7.2 and 7.8.
And finally, transferring the ethyl chloride degrading bacterial liquid cultured in the seed tank into a 1000L fermentation tank for expansion culture according to the inoculation amount of 2% -5%, wherein the culture medium components of the fermentation tank are the same as those of the seed tank, and the physical and chemical parameter indexes are as follows: the temperature is 37 ℃, the rotating speed is 250rpm, the dissolved oxygen is 8mg/L, and the fermentation time is 72 hours. After fermentation, the effective viable count of the tank bacterial liquid can reach 10 9 And (3) taking out the fermentation culture solution from the tank, and packaging the fermentation culture solution by a plastic barrel to obtain the ethyl chloride high-efficiency degradation microbial inoculum.
Example 5 application of Strain YL-5 in actual Ethyl chloride wastewater
In order to further verify the application effect of the ethyl chloride high-efficiency degrading bacteria YL-5 used in the invention in actual ethyl chloride-containing wastewater, the ethyl chloride-containing wastewater was obtained from a Jiang Sumou farm wastewater station. The concentration of ethyl chloride in the wastewater is 2.2g/L through measurement, and the concentration is higher. We fermented ethyl chloride degrading bacteria YL-5Directly adding the mixed solution into the production wastewater of the ethyl chloride according to the inoculation amount of 10 percent, and supplementing a proper amount of nitrogen source NH 4 Cl and a small amount of phosphorus source KH 2 PO 4 And the pH of the system is regulated to be stabilized at about 7.5 so as to meet the requirement of normal growth of microorganisms. Under the condition of room temperature aeration, the change of ethyl chloride in the system is monitored by sampling at regular time according to 0h, 2h, 6h, 12h, 18h and 24 h. The ethyl chloride degradation data are shown in figure 3: the ethyl chloride in the wastewater is degraded within 2h, the concentration of the ethyl chloride is reduced from 2.2g/L to 1.9g/L, the degradation effect is 13.6%, the concentration of the ethyl chloride is reduced to 0.56g/L after 12h, and the degradation rate is 74.5%. After 24 hours, the concentration of the ethyl chloride is reduced to 0.05g/L, and the degradation rate is as high as 97.7%. The experimental data show that the ethyl chloride high-efficiency degrading bacteria YL-5 also has good treatment effect in the actual wastewater containing the ethyl chloride, and has wide application prospect.
Sequence listing
<110> Jiangsu Nanyan environmental protection technology Co., ltd
<120> an ethyl chloride high-efficiency degradation bacterium and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1438
<212> DNA
<213> Synthesis (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
ggcatggcgg cagctacaca tgcaagtcga gcggatgacg ggagcttgct ccttgattca 60
gcggcggacg ggtgagtaat gcctaggaat ctgcctggta gtgggggaca acgtttcgaa 120
aggaacgcta ataccgcata cgtcctacgg gagaaagcag gggaccttcg ggccttgcgc 180
tatcagatga gcctaggtcg gattagctag ttggtggggt aatggctcac caaggcgacg 240
atccgtaact ggtctgagag gatgatcagt cacactggaa ctgagacacg gtccagactc 300
ctacgggagg cagcagtggg gaatattgga caatgggcga aagcctgatc cagccatgcc 360
gcgtgtgtga agaaggtctt cggattgtaa agcactttaa gttgggagga agggcagtaa 420
gttaatacct tgctgttttg acgttaccga cagaataagc accggctaac tctgtgccag 480
cagccgcggt aatacagagg gtgcaagcgt taatcggaat tactgggcgt aaagcgcgcg 540
taggtggttt gttaagttgg atgtgaaagc cccgggctca acctgggaac tgcatccaaa 600
actggcaagc tagagtacgg tagagggtgg tggaatttcc tgtgtagcgg tgaaatgcgt 660
agatatagga aggaacacca gtggcgaagg cgaccacctg gactgatact gacactgagg 720
tgcgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc gtaaacgatg 780
tcaactagcc gttggaatcc ttgagatttt agtggcgcag ctaacgcatt aagttgaccg 840
cctggggagt acggccgcaa ggttaaaact caaatgaatt gacgggggcc cgcacaagcg 900
gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggcct tgacatgcag 960
agaactttcc agagatggat tggtgccttc gggaactctg acacaggtgc tgcatggctg 1020
tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgta acgagcgcaa cccttgtcct 1080
tagttaccag cacgtaatgg tgggcactct aaggagactg ccggtgacaa accggaggaa 1140
ggtggggatg acgtcaagtc atcatggccc ttacggcctg ggctacacac gtgctacaat 1200
ggtcggtaca gagggttgcc aagccgcgag gtggagctaa tctcacaaaa ccgatcgtag 1260
tccggatcgc agtctgcaac tcgactgcgt gaagtcggaa tcgctagtaa tcgcgaatca 1320
gaatgtcgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccatgggagt 1380
gggttgcacc agaagtagct agtctaacct tcgggaggac ggtaccacgg tgatctgc 1438
Claims (8)
1. An ethyl chloride degrading bacterium which is classified and named as Pseudomonas hunanPseudomonas hunanensis) YL-5 is preserved in China general microbiological culture collection center with the preservation number of CGMCC No.23934.
2. An ethyl chloride degrading bacterial agent is characterized in that the bacterial agent is prepared by fermenting and culturing pseudomonas hunanPseudomonas hunanensis) YL-5, and packaging after the fermentation culture solution is taken out of the tank to obtain the ethyl chloride degrading bacterial agent, wherein the effective viable count of the bacterial agent is more than or equal to 10 9 And each ml.
3. The use of the ethyl chloride-degrading bacterium according to claim 1 for degrading ethyl chloride.
4. The use of the ethyl chloride-degrading bacterium according to claim 1 in the treatment of pesticide wastewater containing ethyl chloride.
5. A use according to claim 3, characterized in that: the pseudomonas Hunan is treatedPseudomonas hunanensis) After the YL-5 is cultured, the strain is added into the wastewater to be treated containing the ethyl chloride to degrade the ethyl chloride.
6. The use according to claim 5, characterized in that: pseudomonas huona HunanPseudomonas hunanensis) YL-5 is used for culturing the strain, comprising the steps of activation, transfer and expansion culture,
s1, activation: selecting single colony of ethyl chloride degrading bacteria from a solid flat plate, transferring the single colony into LB liquid culture medium containing ethyl chloride, and performing shake culture for 8-16h at the rotation speed of a shaking table of 130~150 rpm,30~37 ℃ until the logarithmic phase;
s2 switching: transferring ethyl chloride degrading bacterial liquid in the logarithmic phase into a seed tank according to the access amount of 2-5%, culturing for 24-48h, controlling the temperature of the seed tank to be 30-37 ℃ and the rotating speed to be 220-250rpm, and controlling dissolved oxygen DO to be 4-6 mg/L;
s3, expanding culture: transferring the ethyl chloride degrading bacterial liquid cultured in the seed tank into a fermentation tank for expansion culture according to the inoculum size of 2% -5%, wherein the culture medium components of the fermentation tank are the same as those of the seed tank, and the physicochemical parameter indexes are as follows: the temperature is 30-37 ℃, the rotating speed is 200-250 rpm, the dissolved oxygen is 5-8 mg/L, and the fermentation time is 48-72h.
7. The use according to claim 6, characterized in that: the LB liquid culture medium comprises the following components: 1g/L ethyl chloride, 5g/L yeast extract, 10g/L peptone, 2g/L sodium chloride, 1g/L sodium bicarbonate, 0.2g/L sodium carbonate, and the pH is kept between 7.2 and 7.8.
8. The use according to claim 6, characterized in that: the mass ratio of the culture medium components of the seed tank is as follows: 0.1-0.2% of ethyl chloride, 0.4-0.6% of glucose and NH 4 Cl 0.1~0.2%，KH2PO4 0.005~0 .01％，MgSO4 0.025~0 .05％，NaCl 0.01~0 .02％，CaCO3 0.015~0 .3％，NaHCO 3 0.1-0.2%, yeast extract 0.005-0.01%, and water in balance, and maintaining the pH between 7.2-7.8 by implementing pH self-control adjustment technology.
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| CN111690562A (en) * | 2020-06-09 | 2020-09-22 | 北京农学院 | Pseudomonas laenaensis capable of degrading phenolic acid autotoxic substances and application thereof |
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| CN111690562A (en) * | 2020-06-09 | 2020-09-22 | 北京农学院 | Pseudomonas laenaensis capable of degrading phenolic acid autotoxic substances and application thereof |
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| 一株高效甲醛降解菌的分离筛选与降解特性研究;周雪媚;李玉光;陈霜玲;付慧群;姜思朋;王永阔;;生态环境学报(12);全文 * |
| 乙草胺降解菌系的分离鉴定及其生长降解特性研究;陈森;韦德萍;王欢;韩丽珍;;基因组学与应用生物学(11);全文 * |
| 设施土壤中解磷-反硝化复合功能细菌筛选及其活性研究;张广志等;土壤通报;第51卷(第6期);全文 * |
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