CN113755374B - 一种降解2,4-二氯酚的停滞棒杆菌及其应用 - Google Patents
一种降解2,4-二氯酚的停滞棒杆菌及其应用 Download PDFInfo
- Publication number
- CN113755374B CN113755374B CN202111052211.6A CN202111052211A CN113755374B CN 113755374 B CN113755374 B CN 113755374B CN 202111052211 A CN202111052211 A CN 202111052211A CN 113755374 B CN113755374 B CN 113755374B
- Authority
- CN
- China
- Prior art keywords
- dichlorophenol
- corynebacterium
- dichlorophen
- culture
- degrading
- 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.)
- Active
Links
- 241000186216 Corynebacterium Species 0.000 title claims abstract description 38
- 229960003887 dichlorophen Drugs 0.000 title claims abstract description 27
- 230000000593 degrading effect Effects 0.000 title claims abstract description 21
- 239000002689 soil Substances 0.000 claims abstract description 32
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 239000007788 liquid Substances 0.000 claims description 9
- HFZWRUODUSTPEG-UHFFFAOYSA-N 2,4-dichlorophenol Chemical compound OC1=CC=C(Cl)C=C1Cl HFZWRUODUSTPEG-UHFFFAOYSA-N 0.000 abstract description 84
- 230000015556 catabolic process Effects 0.000 abstract description 30
- 238000006731 degradation reaction Methods 0.000 abstract description 30
- 239000001963 growth medium Substances 0.000 abstract description 30
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 16
- 229910052799 carbon Inorganic materials 0.000 abstract description 16
- 230000006378 damage Effects 0.000 abstract description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 11
- 239000010802 sludge Substances 0.000 abstract description 10
- 241000894006 Bacteria Species 0.000 abstract description 5
- 238000012216 screening Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 241000186308 Corynebacterium stationis Species 0.000 description 11
- 229910017053 inorganic salt Inorganic materials 0.000 description 11
- 238000000034 method Methods 0.000 description 11
- 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 description 9
- 238000012258 culturing Methods 0.000 description 9
- 239000008103 glucose Substances 0.000 description 9
- 239000012880 LB liquid culture medium Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- 239000002028 Biomass Substances 0.000 description 6
- 229940121710 HMGCoA reductase inhibitor Drugs 0.000 description 6
- 239000002471 hydroxymethylglutaryl coenzyme A reductase inhibitor Substances 0.000 description 6
- 239000002609 medium Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000003213 activating effect Effects 0.000 description 4
- 238000009630 liquid culture Methods 0.000 description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 238000006065 biodegradation reaction Methods 0.000 description 3
- 208000014674 injury Diseases 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- JKFZMMGVYNLCRY-UHFFFAOYSA-N 2,4-dichlorophenol Chemical compound OC1=CC=C(Cl)C=C1Cl.OC1=CC=C(Cl)C=C1Cl JKFZMMGVYNLCRY-UHFFFAOYSA-N 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 238000013033 photocatalytic degradation reaction Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000010865 sewage Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 238000003911 water pollution Methods 0.000 description 2
- MNHVNIJQQRJYDH-UHFFFAOYSA-N 2-[2-(1-chlorocyclopropyl)-3-(2-chlorophenyl)-2-hydroxypropyl]-1,2-dihydro-1,2,4-triazole-3-thione Chemical compound N1=CNC(=S)N1CC(C1(Cl)CC1)(O)CC1=CC=CC=C1Cl MNHVNIJQQRJYDH-UHFFFAOYSA-N 0.000 description 1
- GTWMCYZXHLGFIS-UHFFFAOYSA-N 6,6-dichlorocyclohexa-2,4-dien-1-one Chemical compound ClC1(Cl)C=CC=CC1=O GTWMCYZXHLGFIS-UHFFFAOYSA-N 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 239000005825 Prothioconazole Substances 0.000 description 1
- OHVGNSMTLSKTGN-BTVCFUMJSA-N [C].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O Chemical compound [C].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O OHVGNSMTLSKTGN-BTVCFUMJSA-N 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- XCJYREBRNVKWGJ-UHFFFAOYSA-N copper(II) phthalocyanine Chemical compound [Cu+2].C12=CC=CC=C2C(N=C2[N-]C(C3=CC=CC=C32)=N2)=NC1=NC([C]1C=CC=CC1=1)=NC=1N=C1[C]3C=CC=CC3=C2[N-]1 XCJYREBRNVKWGJ-UHFFFAOYSA-N 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 229910052564 epsomite Inorganic materials 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 150000004820 halides Chemical class 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 208000020470 nervous system symptom Diseases 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000003986 organophosphate insecticide Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 238000003900 soil pollution Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
- B09C1/105—Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
-
- 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
- C02F2101/34—Organic compounds containing oxygen
- C02F2101/345—Phenols
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Environmental & Geological Engineering (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Mycology (AREA)
- Soil Sciences (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Biodiversity & Conservation Biology (AREA)
- Hydrology & Water Resources (AREA)
- Water Supply & Treatment (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及一种降解2,4‑二氯酚的停滞棒杆菌及其应用，该降解菌为停滞棒杆菌(Corynebacterium stationis)ZH1027，保藏于中国典型培养物保藏中心，保藏编号为CCTCC M 2021922，其由活性污泥经以2,4‑二氯酚作为唯一碳源的胁迫驯化培养基，逐步提高2,4‑二氯酚浓度对活性污泥进行逐级胁迫驯化，每级胁迫驯化培养之间进行一次间歇损伤修复培养，再经过筛选得到。本发明的菌株能够耐受水体、土壤环境中高浓度2,4‑二氯酚，对污染水体、土壤环境中2,4‑二氯酚具有较好的降解效果。
Description
技术领域
本发明涉及2,4-二氯酚降解领域，具体涉及一种降解2,4-二氯酚的停滞棒杆菌及其应用。
背景技术
2,4-二氯酚是一种有机化合物，分子式为C6H4Cl2O，主要用作有机磷杀虫剂丙硫磷等农药及医药合成的中间体。易挥发，腐蚀性强，能灼烧皮肤，刺激眼睛及皮肤，中毒严重者，可产生贫血及各种神经***症状，对皮肤过敏者，可引起皮炎而难治愈。2,4-二氯酚为白色针状结晶，能随水蒸气挥发，溶于乙醇、***、苯和四氯化碳等有机溶剂，微溶于水，对组织有强烈刺激性。
2,4-二氯酚污染物的主要来源是生产2,4-二氯酚的化工厂、石油化工厂等。土壤污染的主要情况有各种高浓度2,4-二氯酚废水直接污染土壤，固体2,4-二氯酚由于事故倾洒在土壤等。水体污染主要由水体沿岸上游污染源的事故排放，陆地事故(如交通运输过程中的翻车事故)发生后经土壤流入水体或槽罐直接翻入路边水体的情况等。这些污染会导致土壤或水体中形成高浓度的2,4-二氯酚污染。
针对2,4-二氯酚污染处理的方法很多,其中吸附法及光、化学催化降解的报道居多，如文献“林旭萌,宿程远,黄纯萍,等.污泥质生物炭对2,4-二氯酚的吸附性能[J].环境工程,2019(8).”报道污泥质生物炭对2,4-二氯酚的吸附去除率最高可达55.7％；又如“黄影,宋雄伟,吴忆,等.一种可吸附有机卤化物中2,4-二氯酚的降解方法:,CN109319917A[P].2019.”记载当污染物中2,4-二氯酚的浓度为20mg/L可有效被化学催化剂催化降解；文献“郭炎、李晶、郭超凡、丛珊珊、杨照地.CuPc/g-C_3N_4的制备和光催化降解2,4-二氯酚性能研究[J].化学工程师,2020,34；299(08):8-10.”报道了一种新型的2,4-二氯酚高性能光催化降解剂。上述方法虽然能有效或部分去除2,4-二氯酚，但生物降解法被认为是一种经济有效且无二次污染的方法【李碧,陈轶,孔殿超.2,4-二氯酚降解研究进展[J].广州化工,2013,41(005):40-]42】。针对高浓度的2,4-二氯酚生物降解的关键在于获取对高浓度2,4-二氯酚具有较好耐受性，且能高效降解能力的菌株。
发明内容
本发明要解决的技术问题是针对以上不足，提供一种降解2,4-二氯酚的停滞棒杆菌及其应用。
为解决以上技术问题，本发明采用以下技术方案：
一种降解2,4-二氯酚的停滞棒杆菌，为停滞棒杆菌(Corynebacteriumstationis)ZH1027，于2021年7月21日保藏于中国典型培养物保藏中心，保藏编号为CCTCCM 2021922。
进一步的，所述的降解2,4-二氯酚的停滞棒杆菌由活性污泥经唯一碳源培养基逐级胁迫驯化与LB培养基间歇损伤修复方法培养后，再经过筛选得到。
进一步的，唯一碳源培养基逐级胁迫驯化与LB培养基间歇损伤修复方法具体为，采用以2,4-二氯酚作为唯一碳源的胁迫驯化培养基，逐步提高2,4-二氯酚浓度对活性污泥进行逐级胁迫驯化，每级胁迫驯化培养之间进行一次间歇损伤修复培养。
所述的降解2,4-二氯酚的停滞棒杆菌在降解土壤环境中2,4-二氯酚的应用。
所述的降解2,4-二氯酚的停滞棒杆菌在降解液体环境中2,4-二氯酚的应用。
本发明的有益效果为：与现有的2,4-二氯酚降解菌相比，本发明的通过2,4-二氯酚唯一碳源培养基逐级胁迫驯化与LB培养基间歇损伤修复方法得到一株能够耐受水体、土壤环境中高浓度2,4-二氯酚的菌株，菌株命名为Corynebacterium stationis ZH1027，该菌株对污染水体、土壤环境中2,4-二氯酚具有较好的降解效果。
下面结合附图和实施例对本发明进行详细说明。
附图说明
图1Corynebacterium stationis ZH1027在显微镜下的细胞形态；
图2Corynebacterium stationis ZH1027进化树；
图3不同碳源条件Corynebacterium stationis ZH1027生长曲线；
图4本发明的菌株在液体环境中对2,4-二氯酚的降解曲线；
图5本发明的菌株在土壤环境中对2,4-二氯酚的降解曲线。
具体实施方式
以下结合附图对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。
为解决现有降解菌在高浓度2,4-二氯酚环境中的耐受性及降解效率低的问题，本发明提供一种降解2,4-二氯酚的停滞棒杆菌及其应用，具体地说，利用“2,4-二氯酚唯一碳源培养基逐级胁迫驯化与LB培养基间歇损伤修复”方法，从湖北省孝昌县污水处理厂曝气池中分离筛选出一株能够耐受高浓度2,4-二氯酚并进行高效降解的菌株，菌株命名为Corynebacterium stationis ZH1027，其保藏编号为CCTCC M 2021922，保藏日期为2021年7月21日；相比于现有生物降解菌株，Corynebacterium stationis ZH1027在高浓度2,4-二氯酚的水体、土壤环境中表现出更好的耐受性和更高的降解效率。
一、2,4-二氯酚高耐受及降解菌株的筛选
(一)材料
活性污泥样本取自湖北省孝感市孝昌县污水处理厂曝气池。
间歇损伤修复培养基(LB培养基)：酵母提取物5g/L、蛋白胨10g/L、NaCl 10g/L。
胁迫驯化培养基：NH4NO31 g，MgSO4·7H2O 0.2g，KH2PO4 0.5g，K2HPO4 0.5g，NaCl0.2g，FeSO4·7H2O 0.01g，2,4-二氯酚按胁迫驯化需要从50mg/L逐级提高浓度至1,500mg/L(2,4-二氯酚按50mg/L为一个提升浓度，灭菌后加入)
以上培养基均在121℃蒸汽灭菌30min。
(二)逐级胁迫驯化与间歇损伤修复培养
将活性污泥按占1％的比例接种到LB液体培养基中，37℃恒温摇床200rpm培养24小时活化。将活化培养的活性污泥培养物按10％的比例转接至含50mg/L 2,4-二氯酚为唯一碳源的胁迫驯化培养基，37℃恒温摇床200rpm培养48小时。上述培养物作为种子液，按占1％的比例接种到LB液体培养基中，37℃恒温摇床200rpm进行损伤修复培养。上述培养物作为种子液，按占10％的比例接种到含100mg/L 2,4-二氯酚为唯一碳源的二级胁迫驯化培养基中，37℃恒温摇床200rpm进行胁迫驯化。二级胁迫驯化培养物作为种子液，按占1％的比例接种到LB液体培养基中，37℃恒温摇床200rpm进行损伤修复培养。随后进行三级胁迫驯化，胁迫驯化培养基中2,4-二氯酚浓度提升至150mg/L，依次进行200mg/L、250mg/L、300mg/L等2,4-二氯酚浓度梯度的逐级胁迫驯化培养，直至2,4-二氯酚浓度到1,500mg/L，每级胁迫驯化培养之间进行一次间歇损伤修复培养。由于胁迫驯化培养基中2,4-二氯酚为唯一碳源，不能以2,4-二氯酚为碳源的微生物无法生长而被逐级淘汰。另外由于2,4-二氯酚，特别是高浓度的2,4-二氯酚对微生物细胞有毒性，逐级胁迫驯化培养之间进行间歇损伤修复培养，有利于细胞的损伤修复，从而使对2,4-二氯酚高耐受性的微生物得以有效胁迫驯化。
(三)高耐受、高降解效率的菌株筛选、分离及鉴定
将经逐级胁迫驯化培养的活性污泥培养物稀释涂布在含1000mg/L 2,4-二氯酚的无机盐培养基平板，37℃培养。在无机盐培养基上筛选得到约30个单菌落。挑选单菌落接种至含1000mg/L 2,4-二氯酚的无机盐液体培养基中在37℃恒温培养72小时后，按5％的接菌量接种至50mL含1000mg/L 2,4-二氯酚的无机盐液体培养基中，继续恒温培养72小时。经高浓度2,4-二氯酚耐受性测试，分离纯化得到5株能以2,4-二氯酚为唯一碳源生长且对2,4-二氯酚有较好耐受性的菌株。分别对5株菌株进行2,4-二氯酚降解效率的测定，苯酚降解效率的测定方法为：称取0.5g 2,4-二氯酚用100mL容量瓶定容，得到5g/L 2,4-二氯酚母液，取母液至培养基中使2,4-二氯酚的初始浓度1000mg/L，用LB液体培养基活化菌株，控制接种后培养基中的初始OD600＝0.1，于37℃，200rpm振荡培养，每隔3h取样1mL培养液至EP管中，12000rpm离心5min后取上清液，用双蒸水将上清液稀释50倍，经过0.22μm的有机相微孔滤膜过滤，滤液用HPLC分析测定其2,4-二氯酚含量，分析菌株对苯酚的降解效率，从5株菌株中选取2,4-二氯酚降解效率最高的一株菌株，命名为ZH1027。
(四)ZH1027形态观察及分子鉴定
菌株ZH1027革兰氏染色呈现阳性，16×100倍油镜观察，细胞呈现短棒状(如图1)。
PCR扩增ZH1027的16S rDNA序列，16SrDNA测序委托武汉擎科伟业生物科技有限公司进行。使用NCBI的Blast工具(https://blast.ncbi.nlm.nih.gov/Blast.cgi)与已知核酸序列进行比对，将同源性最高的物种判断为送检样品的种属，选择同源性较高的已知16SrDNA序列通过MEGA软件构建进化树。
待检菌株的16S rDNA序列在NCBI上进行blast比对，通过比对的结果在MEGA软件中进行进化树的构建如图2所示。结果显示该菌株属于棒状杆菌属，与Corynebacteriumstationis亲缘关系大于93％，命名为Corynebacterium stationis ZH1027(停滞棒杆菌ZH1027)。
二、Corynebacterium stationis ZH1027对2,4-二氯酚的耐受性
将Corynebacterium stationis ZH1027用LB液体培养基37℃培养活化24小时，取培养物按1％接种量转接至分别含300mg/L～1800mg/L的2,4-二氯酚的LB液体培养基中，培养体积为50ml，利用分光光度计测定培养液中OD600表征其生长生物量(表1)，根据生物量判断Corynebacterium stationis ZH1027在液体中对2,4-二氯酚的耐受性。液体环境中2,4-二氯酚浓度低于1600mg/L时，菌株表现出较强的生长能力。当2,4-二氯酚浓度达到1700mg/L时，菌株在培养基中的生物量明显减少，2,4-二氯酚浓度达到1800mg/L时，菌株无法生长。Corynebacterium stationis ZH1027在液体环境中对2,4-二氯酚的最高耐受浓度为1700mg/L。
表1.溶液环境中菌株ZH1027对2,4-二氯酚(2,4-二氯酚)的耐受性
将富含有机质的土壤烘干，50目过筛后灭菌分装，分别按每公斤土样添加2,4-二氯酚500mg～4900mg(500mg/Kg～4900mg/Kg)，均匀混合。将Corynebacterium stationisZH1027用LB液体培养基37℃培养活化24小时，取培养物按10％接种量转接至分别含500mg/Kg～4900mg/Kg的2,4-二氯酚的有机质土壤，均匀混合，所有操作在无菌条件下进行。37℃恒温培养1周后分别取样等量土样进行稀释涂布平板计数，根据平板计数情况判断Corynebacterium stationis ZH1027在土壤中对2,4-二氯酚的耐受性(表2)。土壤环境中2,4-二氯酚浓度低于4300mg/L时，菌株表现出较强的生长能力，当2,4-二氯酚浓度达到4900mg/L时，菌株在培养基中的生物量明显减少，仍能正常生长，具有很好的2,4-二氯酚耐受性。Corynebacterium stationis ZH1027在土壤环境中对2,4-二氯酚的耐受浓度显著高于液体环境中，是因为土壤中2,4-二氯酚部分以固形物形式存在，对菌体的毒害作用较小。
表2.土壤环境中菌株ZH1027对2,4-二氯酚(2,4-二氯酚)的耐受性
三、不同碳源组分对Corynebacterium stationis ZH1027生长特性的影响
分别配制含500mg/L 2,4-二氯酚(DCP)、1g/L葡萄糖、500mg/L 2,4-二氯酚(DCP)+1g/L葡萄糖的无机盐液体培养基，按1％接种量接入经液体培养活化的Corynebacteriumstationis ZH1027，初始OD600值保持一致，在平行条件下采用全自动生长曲线分析仪Bioscreen C测定生物量，50小时生长曲线如图3。在含葡萄糖的无机盐培养基中，Corynebacterium stationis ZH1027表现出最好生物趋势。以2,4-二氯酚作为唯一碳源时，与葡萄糖作为碳源相比菌株生长受到明显抑制，2,4-二氯酚毒性的作用明显。在含2,4-二氯酚的培养基中同步添加葡萄糖，可以使Corynebacterium stationis ZH1027生物量明显增加，说明培养体系中葡萄糖碳源可以部分消除2,4-二氯酚对Corynebacteriumstationis ZH1027毒性作用。
四、Corynebacterium stationis ZH1027对2,4-二氯酚的降解
(一)液体环境中对2,4-二氯酚的降解
分别配制无机盐培养基和无机盐+葡萄糖的培养基1000ml，灭菌后按500mg/L的浓度加入2,4-二氯酚。用LB培养基37℃恒温培养24小时活化Corynebacterium stationisZH1027，将活化后的菌液按10％的接种量分别接入含500mg/L的2,4-二氯酚无机盐培养基和无机盐+葡萄糖的培养基，37℃恒温，200rpm震荡培养，每24小时同步取样分析培养液中2,4-二氯酚的含量，绘制2,4-二氯酚(2,4-DCP)降解曲线，如图4所示。连续培养第8天后，在无机盐培养基中Corynebacterium stationis ZH1027对2,4-二氯酚的降解率为47.8％，在无机盐+葡萄糖培养基中对2,4-二氯酚的降解率可达到92.5％。葡萄糖作为其他有机碳源加入可提升菌株Corynebacterium stationis ZH1027对2,4-二氯酚的降解效率。
(二)土壤环境中对2,4-二氯酚的降解
为排除土壤中其他微生物对2,4-二氯酚的降解作用影响，准确测定Corynebacterium stationis ZH1027对土壤中2,4-二氯酚的降解效率，将富含有机质的土壤烘干，50目过筛后灭菌分装。每公斤土样添加2,4-二氯酚500mg，均匀混合。将Corynebacterium stationis ZH1027用LB液体培养基37℃培养活化24小时，取培养物按10％接种量转接至含500mg/Kg的2,4-二氯酚的有机质土壤，混匀37℃恒温培养箱避光培养。每24小时同步取样分析土壤中2,4-二氯酚的含量，绘制2,4-二氯酚(2,4-DCP)降解曲线，如图5所示。15天后，灭菌的土壤中Corynebacterium stationis ZH1027对2,4-二氯酚的降解率为61.4％。
为评估Corynebacterium stationis ZH1027对土壤中2,4-二氯酚实际应用的降解效果。对实验土壤不做灭菌处理，其他操作方式相同，15天后，未灭菌土壤中Corynebacterium stationis ZH1027对2,4-二氯酚的降解率达到89.3％。表明利用Corynebacterium stationis ZH1027进行生物强化可有效提高对2,4-二氯酚的降解效率。
以上所述为本发明最佳实施方式的举例，其中未详细述及的部分均为本领域普通技术人员的公知常识。本发明的保护范围以权利要求的内容为准，任何基于本发明的技术启示而进行的等效变换，也在本发明的保护范围之内。
Claims (3)
1.一种降解2,4-二氯酚的停滞棒杆菌，其特征在于，为停滞棒杆菌(Corynebacterium stationis)ZH1027，保藏于中国典型培养物保藏中心，保藏编号为CCTCC M 2021922。
2.根据权利要求1所述的降解2,4-二氯酚的停滞棒杆菌在降解土壤环境中2,4-二氯酚的应用。
3.根据权利要求1所述的降解2,4-二氯酚的停滞棒杆菌在降解液体环境中2,4-二氯酚的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111052211.6A CN113755374B (zh) | 2021-09-08 | 2021-09-08 | 一种降解2,4-二氯酚的停滞棒杆菌及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111052211.6A CN113755374B (zh) | 2021-09-08 | 2021-09-08 | 一种降解2,4-二氯酚的停滞棒杆菌及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113755374A CN113755374A (zh) | 2021-12-07 |
| CN113755374B true CN113755374B (zh) | 2022-05-03 |
Family
ID=78794130
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111052211.6A Active CN113755374B (zh) | 2021-09-08 | 2021-09-08 | 一种降解2,4-二氯酚的停滞棒杆菌及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113755374B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115433289B (zh) * | 2022-08-22 | 2024-02-27 | 广东省农业科学院蚕业与农产品加工研究所 | 一种益生元、提取方法及在富集肠道益生菌中的应用 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101929158B1 (ko) * | 2018-06-07 | 2018-12-13 | 씨제이제일제당 (주) | 5'-크산틸산을 생산하는 미생물 및 이를 이용한 5'-크산틸산의 제조방법 |
| CN111363694B (zh) * | 2020-01-21 | 2022-05-06 | 中国科学院城市环境研究所 | 一株停滞棒杆菌、筛选方法及其用途 |
| KR102269639B1 (ko) * | 2020-02-12 | 2021-06-25 | 대상 주식회사 | L-글루탐산 생산능이 향상된 코리네박테리움 글루타미쿰 변이주 및 이를 이용한 l-글루탐산의 생산 방법 |
| CN112430561A (zh) * | 2020-12-01 | 2021-03-02 | 广东肇庆星湖生物科技股份有限公司 | 一种停滞棒杆菌的高效转化方法 |
-
2021
- 2021-09-08 CN CN202111052211.6A patent/CN113755374B/zh active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN113755374A (zh) | 2021-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113215033B (zh) | 一种磺胺类抗生素降解菌及其应用 | |
| CN114890555B (zh) | 一种用于治理农村黑臭水体的固体微生物制剂及其制备方法与应用 | |
| CN113755374B (zh) | 一种降解2,4-二氯酚的停滞棒杆菌及其应用 | |
| CN113444661A (zh) | 一株新鞘氨醇杆菌及其在废水除磷中的应用 | |
| CN114045239B (zh) | 具有二甲基乙酰胺降解能力的泛养副球菌ybh-7及应用 | |
| CN115786179A (zh) | 降解邻二氯苯的菌株及其应用 | |
| CN114107092A (zh) | 一株降解邻苯二甲酸酯的植物内生菌戈登氏菌l191及其应用 | |
| CN115125178B (zh) | 一株具有四环素类抗生素降解功能的类芽孢杆菌、方法及应用 | |
| CN112980763A (zh) | 一种苯胺废水优势菌的驯化及筛选方法 | |
| CN115011514B (zh) | 一株腐植酸降解细菌及其应用 | |
| CN114045238B (zh) | 高效降解二甲基乙酰胺的赤红球菌hjm-8及应用 | |
| CN113980851B (zh) | 具有二甲基乙酰胺降解能力的副球菌ybh-x及应用 | |
| CN114621903A (zh) | 用于处理医药废水的降解菌株及其在生物强化处理中的应用 | |
| CN115960745A (zh) | 一株耐盐环氧氯丙烷降解菌及其在处理高盐水体中环氧氯丙烷的应用 | |
| CN113862177B (zh) | 一种同步降解混合酚的溶肌谷氨酸杆菌及其应用 | |
| CN109097296B (zh) | 一种具有降解一甲胺能力的蜡状芽孢杆菌及其应用 | |
| CN110669716A (zh) | 一种耐高浓度苯酚、重金属和耐低温红球菌分离方法 | |
| CN109207400B (zh) | 一种高效降解黑土中邻苯二甲酸酯的复合菌剂及降解方法 | |
| CN115216428B (zh) | 一种抗汞细菌及其在汞污染治理方面的应用 | |
| CN114703085B (zh) | 一种用于降解多环芳烃的复合微生物菌剂 | |
| CN117467583B (zh) | 一株喹诺酮类抗生素降解菌、菌剂及其制备方法和应用 | |
| CN113801828B (zh) | 一株硝基苯高效降解菌及其制备应用 | |
| CN110938561B (zh) | 一株伯克氏菌y-4及其在吸附重金属离子和磺胺类抗生素中的应用 | |
| CN115449498B (zh) | 寡养单胞菌、菌剂及其降解芳香胺废水的方法和处理*** | |
| CN114134079B (zh) | 一株四环素类抗生素降解菌、方法及应用 |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A Stagnation Corynebacterium for Degradation of 2,4-Dichlorophenol and Its Application Granted publication date: 20220503 Pledgee: Overpass Branch of Jinan Rural Commercial Bank Co.,Ltd. Pledgor: Shandong Shennong Ecological Technology Co.,Ltd. Registration number: Y2024980008028 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right |