CN106947710A - The screening and application of one plant of HBCD degradation bacteria - Google Patents

The screening and application of one plant of HBCD degradation bacteria Download PDF

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CN106947710A
CN106947710A CN201611191061.6A CN201611191061A CN106947710A CN 106947710 A CN106947710 A CN 106947710A CN 201611191061 A CN201611191061 A CN 201611191061A CN 106947710 A CN106947710 A CN 106947710A
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acinetobacter haemolyticus
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王莹莹
韩伟
韩敏
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Nankai University
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    • C12N1/20—Bacteria; Culture media therefor
    • 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 screening and its application of one plant of HBCD degradation bacteria, belong to environment pollutant biological treatment technical field.HBCD degradation bacteria strains provided by the present invention are derived from by HBCD contaminated soils, are obtained through artificial enrichment, separation and purifying.The bacterial strain is acinetobacter haemolyticus (being named as Acinetobacter haemolyticus strain HW 2), it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date is August in 2016 30, and deposit number is CGMCC No.13280.The bacterial strain is Gram-negative bacteria, faint yellow, in the opaque circular colonies of the milky of surface elevation.The bacterium is shaft-like, 12 μm of 0.5 0.7 μ m of diameter under ESEM.In liquid medium within, the bacterial strain has preferable degradation capability to HBCD.

Description

The screening and application of one plant of HBCD degradation bacteria
Technical field
The invention belongs to microorganism and its applied technical field of reparation HBCD HBCD pollutions.It is related to one plant The screening technique of HBCD degradation bacterias and its to HBCD biodegrading process and application.
Background technology
HBCD (HBCD) is a kind of new brominated flame-retardant, and because its flame retarding efficiency is high, heat endurance is good, Required addition is small, the advantages of small and cheap on material property influence, be widely used in electronics, electrical equipment, chemical industry, build Build, traffic, weaving, in the field such as oil and mining.HBCD is the third-largest brominated flame-retardant for being only second to PBDEs and bisphenol-A. HBCD mainly has three kinds of isomers, i.e. α-HBCD, β-HBCD and γ-HBCD.Wherein, in industrial products shared by γ-HBCD Ratio is up to that 75~89%, α-HBCD are at least generally for 10~13%, β-HBCD<0.5~12%.
HBCD has highly lipophilic, low aqueous solubility, low-vapor pressure, can step by step amplify along food chain, and can be remote in the environment Distance translation.HBCD a large amount of presence are detected in soil, water body, deposit, the varying environment medium such as air.HBCD is to interior Excretory system and reproductive system have toxicity, while having potential carcinogenicity afterwards to a certain degree reaching.It can be seen that, long-term life Production, using HBCD cause its amount in the environment constantly to accumulate, serious threat is caused to human health and Environmental security. Therefore, HBCD degraded has become the hot issue of global environmental worker's concern.And current, the research degraded for HBCD It is relatively fewer, it is concentrated mainly on chemical degradation, light degradation, microbial degradation and some combined degradation technologies.Wherein, microorganism Degraded is the important means for eliminating HBCD in environment, is had the advantages that efficient, cheap but current on HBCD microbial degradations Research is also less.Therefore, efficient HBCD degradation bacterias are screened, and physio-biochemical characteristics, the degradation characteristic to degradation bacteria strains etc. enters Row research, can provide theoretical foundation and technical support to be studied by HBCD contaminated soils and water resource reparation etc..
The content of the invention
Present invention aim to address the degradation problem on HBCD, there is provided a kind of HBCD efficient degrading bacterias Screening and application.
Technical solution of the present invention
Present invention firstly provides one plant of HBCD efficient degrading bacterial strains-acinetobacter haemolyticus (Acinetobacter Haemolyticus strain HW-2), the bacterial strain has been preserved in the common micro- life of China Committee for Culture Collection of Microorganisms Thing center (Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica), deposit number CGMCC No.12918, it is proposed that Classification And Nomenclature is Acinetobacter haemolyticus.
Described Acinetobacter haemolyticus strain HW-2 bacterial strains are Gram-negative bacteria, yellowish Color, in the opaque circular colonies of the milky of surface elevation；The bacterium is shaft-like, diameter 0.5-0.7 μ m 1-2 μ under ESEM m。
Above-mentioned bacterial strains are derived from the sub- tooth electronic waste Polluted area soil in Tianjin, are obtained by screening.
Screening technique of the present invention, is comprised the following steps that：
Weigh 10g and be added to 0.85% physiological saline of the 50-100mL through high-temperature sterilization by HBCD contaminated soil samples In, after being fully vortexed, 10-30 minutes are stood, takes 5mL soil supernatants to cross 0.45 μm of filter membrane, takes 1mL filtrates to be transferred to 10mg/L HBCD for sole carbon source minimal medium in, in 30 DEG C, 150r/min constant-temperature tables cultivate, transfer weekly once, make HBCD ultimate density reaches 30mg/L, and after cultivating 3 weeks, bacterial strain is separated with plate streak on LB solid mediums Purifying, selects the faster dominant colony of growth, purifies 3 times repeatedly, obtain single pure bacterium colony, be transferred in LB liquid medium and expand After big culture, the bacterial strain full genome is extracted, after PCR amplifications, 16rDNA sequencings are carried out, row will be sequenced and submit GenBank to carry out Analysis, it is Acinetobacter haemolyticus to identify the degradation bacteria strains.
Wherein, the component of described minimal medium includes：1) basic inorganic salt culture medium：Na2HPO4·12H2O 25.6g/L,KH2PO4 3g/L,(NH4)2SO41.77g/L；2) micro- culture medium：HCl 200μL/L,CaCO3 0.08g/ L,FeCl3·6H2O 0.0774g/L,MnCl2·4H2O 0.0115g/L,CuSO4·5H2O 0.00146g/L,CoCl2·6H2O 0.0013g/L,ZnO 0.004g/L,H3BO3 0.00124g/L,EDTA Na4·2H2O 0.792g/L,MgCl2·6H2O 0.1342g/L,Na2MoO4·2H2O 0.0104g/L.The pH of the minimal medium is 7.4.
Described LB culture mediums composition includes：10g/L peptones, 10g/L NaCl, and 5g/L yeast extracts.
Degraded invention also provides above-mentioned Acinetobacter haemolyticus strain HW-2 in HBCD In application.
Described application refers to Acinetobacter haemolyticus strain HW-2 bacterium in the degraded to HBCD In application；The bacterial strain has preferable degradation capability to HBCD, by experiment of single factor, show that the bacterial strain is degraded to HBCD Optimal conditions be：7,35 DEG C of pH, 500 μ g/L HBCD concentration.
Research shows in 7,35 DEG C of pH, 150mL culture volumes, and under 1mg/L HBCD concentration, more than 90% HBCD can Degraded in 38 days.
The detailed process and step of HBCD degradeds of the present invention are as follows：
First, HBCD degraded
HBCD degraded first, will be enlarged by the Acinetobacter haemolyticus after culture using batch experiment After strain HW-2 bacterium eccentric cleanings, suspended with minimal medium, with 105Cells/mL accesses 250mL contains In the brown serum bottle of 150mL minimal mediums.Then, presetting HBCD is added into 150mL inorganic salts nutrient solutions.With The processing of bacterium is not connect as experiment contrast.Degraded culture is carried out with 150rpm/min rotating speed in 35 ± 2 DEG C of incubator. Different reaction time samplings, determines bacterial growth amount and HBCD changes of contents.
2nd, the growing state of bacterium is determined in HBCD degradation processes
Laboratory apparatus is CyFlow Space (Partec companies, Germany) flow cytometer, and light source power is 50mW, transmitting Wavelength 488nm, microbial cell number is determined using absolute counting, and its range of linearity is 2 × 102~1 × 105Cells/mL, inspection Rising limit is 200cells/mL, and absolute counting error is less than 5%.Fluorescent dye is produced from Invitrogen companies of the U.S. SYBR Green I.Colouring method：Take nutrient solution 1mL in the special loading pipe of flow cytometer, add 10 μ L SYBR Green I, vibration is mixed, and lucifuge stands sample detection after 15min at room temperature.Instrument gain is set to FSC=700, SSC=250, FL1 =250, FL3=700, Speed=3.With the appropriate dilute sample of ultra-pure water to ensure in continuous mode Flow cytometry speed Degree is less than 500cells/s.
3rd, in liquid phase HBCD extracting process foundation
HBCD extraction uses liquid-liquid extraction method.1mL inorganic salt sample is taken from 250mL brown serum bottle in going out In the 5mL centrifuge tubes for crossing bacterium, 1mL dichloromethane is added, be acutely vortexed 5min, then 4 DEG C, 10000rpm centrifugations 10min.Centrifugation Afterwards, lower floor's dichloromethane comprising HBCD is crossed after 0.22 μm of organic filter membrane, is stored in 1.5mL brown makings bottles, -20 DEG C Refrigerator store is to be measured.This method rate of recovery is 96%~99%, average recovery rate>98%.
4th, HBCD detection
The high phase liquid chromatogram-mass spectrometry HPLC-MS of HBCD detection uses (Waters APGC/UPLC Xevo TQ-S, Waters, Milford, MA, USA), liquid-phase chromatographic column be Germany's Macherey-Nagel Series of Chiral chromatographic column (200mm × 4.0mm×5μm,Macherey-Nagel,German).Flow rate of mobile phase is 0.4ml/min, and column temperature is 25 degrees Celsius, taper hole electricity Press as 12V.Mobile phase A is acetonitrile/methanol (85/15), and Mobile phase B is ultra-pure water.Mobile phase A/Mobile phase B is 60/40.The party Method detection limit<5ppb.
Advantages and positive effects of the present invention：
It is of the invention to have following remarkable advantage and beneficial effect compared with existing restorative procedure or technology：
(1) present invention firstly discovers that acinetobacter haemolyticus (Acinetobacter haemolyticus strain HW-2) There is preferable degradation capability to HBCD.
(2) bacterial strain is not required to add any chemicals and other for the degraded of HBCD in water body in addition to strain Additive, will not produce secondary pollution.And operating process is simple, low is required to management condition, available for HBCD polluted-waters Repair.
(3) in view of the generality and diversity of HBCD pollutions, and current HBCD microbial degradations research shortage, utilize Efficient HBCDs degradation bacterias, which carry out HBCD degradeds, has very important realistic meaning and application value.
Brief description of the drawings
Fig. 1 is forms of the Acinetobacter haemolyticus strain HW-2 under ESEM.
Fig. 2 for Acinetobacter haemolyticus strain HW-2 cultivated in LB and HBCD degradation processes in FCM analysis figure.
Fig. 3 is Acinetobacter haemolyticus strain HW-2 16S rDNA gene order systematic growths Tree.
Fig. 4 is degradation curves (A) of the Acinetobacter haemolyticus strain HW-2 to 1mg/L HBCD With growth curve of bacteria (B).
Fig. 5 is the degradation rates of Acinetobacter haemolyticus strain HW-2 at different conditions.
Fig. 6 be Acinetobacter haemolyticus strain HW-2 in degradation process using organic carbon and Extracellular protein changes
Below by specific embodiment and with reference to accompanying drawing, technical scheme is described in further detail.
Embodiment
The screening of embodiment 1, Acinetobacter haemolyticus strain HW-2
Weigh 5-10g and be added to 50-100mL through high-temperature sterilization by HBCD (HBCD) contaminated soil sample In 0.85% physiological saline, after being fully vortexed, 10-30 minutes are stood, takes 5mL soil supernatants to cross 0.45 μm of filter membrane, takes 1mL Filtrate is transferred in the minimal medium that 10mg/L HBCD are sole carbon source, in training in 30 DEG C, 150r/min constant-temperature tables Support, transfer weekly once, HBCD ultimate density is reached 30mg/L, after cultivating 3 weeks, drawn on LB solid mediums with flat board Collimation method is isolated and purified to bacterial strain, is selected the faster dominant colony of growth, is purified 3 times repeatedly, obtain single pure bacterium colony, transfers Expand into LB liquid medium after culture, extract the bacterial strain full genome, after PCR amplifications, carry out 16rDNA sequencings, will be sequenced Row submit GenBank to be analyzed, and it is acinetobacter haemolyticus (Acinetobacter haemolyticus) to identify the bacterial strain.
Wherein, the component of the minimal medium, and the LB culture mediums composition referring to Summary.
The degraded of embodiment 2, Acinetobacter haemolyticus strain HW-2 to HBCD
35 DEG C of work in 100mL LB liquid medium by Acinetobacter haemolyticus strain HW-2 Change reaches logarithmic phase in 45 hours.Bacterial strain is carried out after eccentric cleaning, prepared in brown 250mL serum bottles using HBCD as only The 150mL minimal mediums of one carbon source.In pH 7, by Acinetobacter haemolyticus strain HW-2 bacterium with 1×105Cells/mL is transferred in the minimal medium that HBCD concentration is 1mg/L, and experiment is used as using the processing that does not connect bacterium Control.Degraded culture is carried out with 150rpm/min rotating speed in 35 DEG C of incubator.Sample, determine in the different reaction time The changes of contents of bacterial growth and HBCD.Test result indicate that, Acinetobacter haemolyticus strain HW-2 At 38 to HBCD degradation rate>90%, such as Fig. 4.
The optimal conditions that embodiment 3, Acinetobacter haemolyticus strain HW-2 degrade to HBCD
Using experiment of single factor, 3 factors are selected：PH, temperature, HBCD concentration, set 5 levels respectively, pH (5,6,7,8, 9), temperature (20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C), HBCD concentration (500 μ g/L, 1000 μ g/L, μ g/L, 2000 μ g/L, 5000 μ g/L, 10000ug/L), degraded bars of the optimization Acinetobacter haemolyticus strain HW-2 to HBCD Part.Each 3 repetitions of processing, degradation time is 15 days, and result of study is found, Acinetobacter haemolyticus The strain HW-2 bacterium degradation condition optimal to HBCD is that pH is 7,35 DEG C of temperature, and HBCD concentration is 500 μ g/L.Such as Fig. 5.
The metabolism of embodiment 4, Acinetobacter haemolyticus strain HW-2 in HBCD degradation processes Product changes
Acinetobacter haemolyticus strain HW-2 can utilize HBCD synthesis in HBCD degradation processes Various extracellular proteins, are changed into using carbon source while HBCD can be degraded.In no added HBCD acetone culture medium, dissolubility Albumen and using carbon source and with the addition of HBCD have significant change.Add after HBCD, deposited using carbon source and dissolubility albumen In time lengthening.Such as Fig. 6.
<110> Organization Name :Nankai University
<120> Title :The screening and application of one plant of HBCD degradation bacteria
<160> Number of Sequences : 3
<210> Information for SEQ ID No.1
<211> Length : 1422
<212> Type : DNA
<213> Organism Name : Acinetobacter haemolyticus strain HW-2
<223> Other Information : 16S rDNA sequence
<400> Sequence Description: 1
ctttaacaca ttgcagtcga gcggggaagt gtagcttgct acattaccta gcggcggacg 60
ggtgagtaat gcttaggaat ctgcctatta gtgggggaca acattccgaa aggaatgcta 120
ataccgcata cgtcctacgg gagaaagcag gggatcttcg gaccttgcgc taatagatga 180
gcctaagtcg gattagctag ttggtggggt aaaggcctac caaggcgacg atctgtagcg 240
ggtctgagag gatgatccgc cacactggga ctgagacacg gcccagactc ctacgggagg 300
cagcagtggg gaatattgga caatgggcgg aagcctgatc cagccatgcc gcgtgtgtga 360
agaaggcctt ttggttgtaa agcactttaa gcgaggagga ggctactcta gttaatacct 420
agagatagtg gacgttactc gcagaataag caccggctaa ctctgtgcca gcagccgcgg 480
taatacagag ggtgcgagcg ttaatcggat ttactgggcg taaagcgtgc gtaggcggct 540
gattaagtcg gatgtgaaat ccctgagctt aacttaggaa ttgcattcga tactggtcag 600
ctagagtatg ggagaggatg gtagaattcc aggtgtagcg gtgaaatgcg tagagatctg 660
gaggaatacc gatggcgaag gcagccatct ggcctaatac tgacgctgag gtacgaaagc 720
atggggagca aacaggatta gataccctgg tagtccatgc cgtaaacgat gtctactagc 780
cgttggggcc tttgaggctt tagtggcgca gctaacgcga taagtagacc gcctggggag 840
tacggtcgca agactaaaac tcaaatgaat tgacgggggc ccgcacaagc ggtggagcat 900
gtggtttaat tcgatgcaac gcgaagaacc ttacctggtc ttgacatagt aagaactttc 960
cagagatgga ttggtgcctt cgggaactta catacaggtg ctgcatggct gtcgtcagct 1020
cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttttcc ttatttgcca 1080
gcgggttaag ccgggaactt taaggatact gccagtgaca aactggagga aggcggggac 1140
gacgtcaagt catcatggcc cttacgacca gggctacaca cgtgctacaa tggtcggtac 1200
aaagggttgc tacctagcga taggatgcta atctcaaaaa gccgatcgta gtccggattg 1260
gagtctgcaa ctcgactcca tgaagtcgga atcgctagta atcgcggatc agaatgccgc 1320
ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accatgggag tttgttgcac 1380
cagaagtagg tagtctaacc gtaaggagga cgcttaccac gg 1422
<210> Information for SEQ ID No.2
<211> Length : 20
<212> Type : DNA
<213> Organism Name : 27F
<223> Other Information :Sense primer for expanding 16S rDNA
<400> Sequence Description : 2
agagtttgat cctggctca g
<210> Information for SEQ ID No.3
<211> Length : 22
<212> Type : DNA
<213> Organism Name : 1492R
<223> Other Information :Anti-sense primer for expanding 16S rDNA
<400> Sequence Description : 3
tacggctacc ttgttacgac tt

Claims (9)

1. a kind of HBCD degradation bacteria strains, the bacterial strain is acinetobacter haemolyticus (Acinetobacter Haemolyticus strain HW-2), China Committee for Culture Collection of Microorganisms's common micro-organisms center has been preserved in, Its preserving number is CGMCC No.13280.
2. a kind of microbial inoculum containing bacterial strain described in claim 1.
3. HBCD degradation bacteria strains Stenotrophomonas HW-2 screening technique described in claim 1, it is characterised in that should Method is comprised the following steps that：
Weigh 5-10g and be added to 0.85% lifes of the 50-100mL through high-temperature sterilization by HBCD contaminated soil sample Manage in salt solution, after being fully vortexed, stand 10-30 minutes, take 5mL soil supernatants to cross 0.45 μm of filter membrane, take 1mL filtrates to be transferred to 10mg/L HBCD for sole carbon source minimal medium in, in 30 DEG C, 150r/min constant-temperature tables cultivate, transfer weekly Once, HBCD ultimate density is made to reach 30mg/L, after cultivating 3 weeks, with plate streak to bacterial strain on LB solid mediums Isolated and purified, select the faster dominant colony of growth, purify 3 times repeatedly, obtain single pure bacterium colony, be transferred to liquid LB trainings Support in base and expand after culture, extract the bacterial strain full genome, after PCR amplifications, carry out 16rDNA sequencings, row will be sequenced and submit GenBank is analyzed, and identifies that the bacterial strain has highest phase with acinetobacter haemolyticus Acinetobacter haemolyticus Like degree.
4. HBCD degradation bacteria strains Stenotrophomonas HW-2 according to claim 3 screening technique, its feature The component of minimal medium described in being includes：1) basic inorganic salt culture medium：Na2HPO4·12H2O 25.6g/L, KH2PO4 3g/L,(NH4)2SO41.77g/L；2) micro- culture medium：HCl 200μL/L,CaCO3 0.08g/L, FeCl3·6H2O 0.0774g/L,MnCl2·4H2O 0.0115g/L,CuSO4·5H2O 0.00146g/L,CoCl2·6H2O 0.0013g/L,ZnO 0.004g/L,H3BO3 0.00124g/L,EDTA Na4·2H2O 0.792g/L,MgCl2·6H2O 0.1342g/L,Na2MoO4·2H2O 0.0104g/L；The pH of the minimal medium is 7.4.
5. HBCD degradation bacteria strains Stenotrophomonas HW-2 according to claim 3 screening technique, its feature It is that described LB culture mediums composition includes：10g/L peptones, 10g/L NaCl, and 5g/L yeast extracts.
6. HBCD degradation bacteria (the Acinetobacter haemolyticus strain HW- described in claim 1 2) application in HBCD degraded.
7. the application of HBCD degradation bacteria according to claim 6, it is characterised in that described application refers to Application of the Acinetobacter haemolyticus strain HW-2 bacterium in the degraded to HBCD.
8. the application of HBCD degradation bacteria strains according to claim 7, it is characterised in that described Acinetobacter haemolyticus strain HW-2 bacterium have preferable degradation capability to HBCD, optimal by exploring Condition, show that the bacterial strain is to the HBCD optimal conditions degraded：PH=7,35 DEG C, 500 μ g/L HBCD concentration.
9. the application of HBCD degradation bacteria according to claim 7, it is characterised in that the Stenotrophomonas pair HBCD has a preferable degradation capability, and research shows in 7,35 DEG C of pH, 150mL culture volumes, under 1mg/L HBCD concentration, 90% HBCD is degraded by Acinetobacter haemolyticus strain HW-2 after 38 days.
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CN109652343A (en) * 2019-02-15 2019-04-19 上海交通大学 It is a kind of degrade hexabromocyclododecane bacterial strain screening method and its application
CN109825453A (en) * 2019-03-05 2019-05-31 上海交通大学 One plant of hexabromocyclododecane efficient degrading bacterial strain and its application in environment remediation
CN109825453B (en) * 2019-03-05 2022-04-12 上海交通大学 Hexabromocyclododecane degrading strain and application thereof in environmental remediation

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