CN105039222B - Dell Ford bacterium LW26 and its application in degradation chlorobenzene - Google Patents
Dell Ford bacterium LW26 and its application in degradation chlorobenzene Download PDFInfo
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Abstract
The present invention provides the bacterial strain of a highly effective degrading chlorobenzene-Dell Ford bacterium (Delftiatsuruhatensis) LW26 and its applications in terms of microorganism decomposition handles chlorobenzene, Dell's Ford bacterium LW26, it is preserved in China typical culture collection center, address: China, Wuhan, Wuhan University, 430072, preservation date: on March 15th, 2015, deposit number: CCTCC NO:M 2015113；Chlorobenzene degradation bacteria provided by the invention can be bred and by its permineralization using chlorobenzene as sole carbon source and the energy at CO2And H2O；The bacterial strain can efficient degradation chlorobenzene in 23 DEG C~30 DEG C, the environment of pH6.0~9.0；The bacterial strain has stronger adaptive capacity to environment, and the present invention will play a significant role in chlorobenzene exhaust gas and waste water treatment practice.
Description
(1) technical field
The present invention relates to one plant of novel chlorobenzene efficient degrading bacterias --- Dell Ford bacterium (Delftia tsuruhatensis)
LW26 and its application.
(2) background technique
Chlorobenzene is the monocyclic aromatic compound for there was only hydrogen atom and chlorine atom on phenyl ring, the low, hydrophobicity with solubility
The features such as strong.In recent years, the intermediate as a kind of important organic solvent and Chemical Manufacture, chlorobenzene are widely used in moulding
The industries such as material, dyestuff, medicine, pesticide, organic synthesis.Since its water-soluble low and bio-toxicity is big, serious dirt is caused to environment
Dye.Chlorobenzene can inhibit nerve center after entering human body, easily in conjunction with enzyme system, there is teratogenesis carcinogenesis.Due to using extensively,
Chlorobenzene all has higher recall rate in a variety of surrounding mediums, and certain prestige is constituted to human health and ecology erroneous zone
The side of body, chlorobenzene are included in priority pollutants list by Environmental Protection Agency (EPA).
Currently, being directed to the emission control of this pollutant, domestic and international researcher is had conducted extensive research, various processing
Method is come into being, and mainly has absorption method, supercritical ultrasonics technology, membrane separation process, catalytic oxidation, photochemical oxidation method, electrochemical process
Deng.In recent years, bioanalysis is proved to have a good application prospect in the purification of chlorobenzene compound.Purification biotechnology is
Using microbiota metabolic activity, it converts pollutant to the energy, cell component and the innoxious small molecule of cell metabolism
Substance (such as H2O、CO2Deng), have many advantages, such as efficient, low consumption compared to other methods, reaction condition is mild, secondary pollution is small.
One of key using biotechnology degradation chlorobenzene is to obtain chlorobenzene efficient degrading bacteria.Currently, domestic and foreign scholars
Do numerous studies in this respect, but since chlorobenzene is volatile and the characteristic of difficult for biological degradation, separated obtained chlorobenzene
Degradation bacteria type is also than relatively limited, in addition, separated obtained strains for degrading efficiency need to be improved.Such as ox celestial being is to one plant
Chlorobenzene dominant degradation bacteria Lysinibacillus fusiformis LW13 carry out degradation condition optimization it was found that, working as chlorine
When benzene initial concentration is 100mg/L, the degradation rate of chlorobenzene reaches maximum, and when chlorobenzene concentration reaches 180mg/L, the drop of chlorobenzene
Solution is inhibited (the environmental project such as ox celestial being, 2013,31 (1): 43-46.) by obvious；2010, Zhang Lili etc., which has found one plant, to be had
The Ralstonia pickettii H2 of chlorobenzene degradation capability, when chlorobenzene concentration is lower than 250mg/L, H2 can rapidly degrade chlorine
Benzene, bacterial growth is good, and in 250mg/L, strain growth and degradation are by obvious inhibition (ZL101880642)；Li Mingtang
Etc. the chlorobenzene degradation bacteria acinetobacter calcoaceticus (Acinetobacter calcoaceticus) filtered out, it is with initial concentration
When the chlorobenzene of 50mg/L is sole carbon source and the energy, need to could degrade chlorobenzene by 120h (the microbiology such as Li Mingtang completely
Report, 2010,50 (5): 586-592.).
After retrieving the relevant literature, there is not yet with Dell Ford bacterium degradation chlorobenzene report.
(3) summary of the invention
The deficiency that the present invention is lower for chlorobenzene biodegrade efficiency, microorganism generation time is long, providing efficiently to drop
Solve Dell Ford bacterium LW26 and its application of chlorobenzene.
The technical solution adopted by the present invention is that:
One plant of new strains --- Dell Ford bacterium (Delftia tsuruhatensis) LW26 is preserved in for present invention offer
China typical culture collection center, the deposit date is on March 15th, 2015, deposit number CCTCC No:M 2015113, ground
Location: China, Wuhan, Wuhan University, postcode 430072.
Chlorobenzene efficient degrading bacteria provided by the present invention --- Dell Ford bacterium LW26 is by acquiring Zhejiang Polytechnical University
The biomembrane of bioconversion and biological cleaning use for laboratory in the bio-trickling filter of purification chlorobenzene exhaust gas, through separation, after purification
It obtains.Dell's Ford bacterium LW26 colony characteristics are as follows: thallus be in rod-short, size be (0.6~0.8) μ m (1.0~
1.5) μm, no gemma；In solid R2Bacterium colony is rounded on A culture medium, milky, and protrusion, flush edge, form is full, smooth
Wet, lawn is along scribing line growth；Contacting enzyme reaction is the positive, V-P reaction, clark and Lubsreaction, nitrate reduction reaction, indoles examination
It tests, citrate using test is feminine gender, Gram-negative.
The present invention relates to application of the Dell Ford bacterium LW26 in microbial degradation chlorobenzene.Specifically, described
Using are as follows: Dell Ford bacterium LW26 is seeded in the minimal medium containing chlorobenzene, at 23~40 DEG C of temperature, pH4.0~
(preferably 23~30 DEG C, pH 6.0~9.0) are cultivated under conditions of 10.0, realize the degradation to chlorobenzene.
Further, chlorobenzene initial concentration in minimal medium is 100~500mg/L；Dell's Ford bacterium
The OD that LW26 is obtained with the wet thallus after seed culture or fermented and cultured through minimal medium dilution600The bacterium of=0.1-0.5 is outstanding
Liquid form is added, and bacteria suspension volume inoculum concentration is 1-5%, preferably OD600=0.15 bacteria suspension volume inoculum concentration is 2%.Into one
Step, the minimal medium composition are as follows: CaCl2, 0.023g/L；MgSO4, 0.2g/L；(NH4)2SO4, 2.5g/L；KH2PO4,
1.0g/L；Na2HPO4, 4.5g/L；Microelement mother liquor 1mL/L；Solvent is water, pH 7.0~7.5.The microelement is female
Liquid composition are as follows: FeSO4·7H2O, 1.0g/L；CuSO4·5H2O, 0.02g/L；H3BO3, 0.014g/L；MnSO4·4H2O,
0.10g/L；ZnSO4·7H2O, 0.10g/L；Na2MoO4·2H2O, 0.02g/L；CoCl2·6H2O, 0.02g/L；Solvent is water.
Further, when bacterium solution dosage is smaller, Dell's Ford bacterium LW26 bacteria suspension can pass through inclined-plane culture, seed
Culture and bacteria suspension prepare three steps and obtain；When dosage is larger, inclined-plane culture, seed culture, fermentation liquid culture can be passed through
It prepares four steps with bacteria suspension to obtain, detailed process is as follows:
(1) Dell Ford bacterium LW26 inclined-plane culture: is inoculated in R2A solid medium, 28~32 DEG C of culture 48h are obtained
Inclined-plane thalline；The R2A solid medium composition are as follows: yeast powder, 0.50g/L；Tryptone, 0.50g/L；Casein,
0.50g/L；Glucose, 0.50g/L；Soluble starch, 0.50g/L；Sodium Pyruvate, 0.30g/L；KH2PO4, 0.45g/L；
MgSO4, 0.05g/L；Agar, 15~18g/L；Solvent is water, pH 7.2.
(2) seed culture: from step (1) inclined-plane, one oese colony inoculation of picking is into seed culture medium, and 28~32
DEG C culture 24~36h, obtain seed liquor.The seed culture medium is R2A fluid nutrient medium, in addition to no agar, remaining component
With R2A solid medium is identical.
(3) seed liquor that step (2) obtain fermented and cultured: is seeded to fermentation with the inoculum concentration of volumetric concentration 5~10%
Culture medium cultivates 24~36h under the conditions of 28~32 DEG C, pH 6.0~8.0, obtains fermentation liquid.The fermentation medium composition
Are as follows: yeast powder, 0.5g/L；CaCl2, 0.023g/L；MgSO4, 0.2g/L；(NH4)2SO4, 2.5g/L；KH2PO4, 1.0g/L；
Na2HPO4, 4.5g/L；Microelement mother liquor, 1mL/L；Solvent is water, and pH 7.0~7.5, microelement mother liquor composition is the same as inorganic
Microelement mother liquor in salt culture medium.
(4) prepared by bacteria suspension: seed liquor (or fermentation liquid) being centrifuged 10min in 6000rpm, supernatant is abandoned, with having sterilized
Minimal medium clean thallus, then 6000rpm be centrifuged 10min, abandon supernatant, wash repeatedly 2 times.By the wet of acquisition
Thallus dilutes the bacteria suspension for obtaining required concentration with sterile minimal medium.
Compared with prior art, the beneficial effects are mainly reflected as follows: at 23~30 DEG C, 6.0~9.0 condition of pH
Under, Dell Ford bacterium LW26 can be degradable by the chlorobenzene of 100mg/L in minimal medium in 16h, to the highest of chlorobenzene
Concentration of degrading is up to 500mg/L.And the bacterial strain H2 chlorobenzene tolerable concentration that patent application CN 101880642A is provided is only 250mg/
L.Chlorobenzene degradation bacteria acinetobacter calcoaceticus (Acinetobacter calcoaceticus) (Li Ming that Li Mingtang etc. is filtered out
Hall etc. microorganism journal, 2010,50 (5): 586-592.), using initial concentration for 50mg/L chlorobenzene as sole carbon source and the energy
When, chlorobenzene could need to be degraded by 120h complete.Compared to the prior art, Dell's Ford bacterium (Delftia provided by the invention
Tsuruhatensis) LW26 has the characteristics that degradation speed is fast, tolerable concentration is high, will practice in chlorobenzene exhaust gas and waste water treatment
In play a significant role.
(4) Detailed description of the invention
Fig. 1 is Dell's Ford bacterium LW26 transmission electron microscope photo.
Fig. 2 is the systematic growth tree graph of Dell's Ford bacterium LW26.
Fig. 3 is the influence that temperature grows Dell Ford bacterium LW26 and chlorobenzene is degraded.
Fig. 4 is the influence that pH grows Dell Ford bacterium LW26 and chlorobenzene is degraded.
Fig. 5 is degradation (a) and growth curve (b) of the Dell Ford bacterium LW26 to chlorobenzene under different chlorobenzene initial concentrations.
Fig. 6 is bio-trickling filter process flow chart, 1. air pumps, 2. mass flowmenters, 3. spinner flowmeters, 4. strippings
Bottle, 5. hybrid bottles, 6. liquid storage bottles, 7. lye bottles, 8. exhaust ports, 9. gas sampling mouths, 10. filler sample taps, 11. wriggle
Pump.
Fig. 7 is the inlet and outlet concentration and removal efficiency of bio-trickling filter processing simulation chlorobenzene exhaust gas.
Fig. 8 is sbr reactor device process flow chart, 1. blenders, 2.pH meter, 3. water inlet pipes, 4. dissolved oxygen instruments, 5. drainings
Mouthful, 6.pH probe, 7. dissolved oxygen instrument sensors, 8. temperature control instrument, 9. temperature sensors, 10. sludge pipes, 11. aerators, 12.
Flowmeter, 13. air pumps.
Fig. 9 is the chlorobenzene degradation curve of sbr reactor device processing simulation chlorobenzene waste water.
(5) specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This:
Minimal medium composition used in embodiment are as follows: CaCl2, 0.023g/L；MgSO4, 0.2g/L；(NH4)2SO4,
2.5g/L；KH2PO4, 1.0g/L；Na2HPO4, 4.5g/L；Microelement mother liquor, 1mL/L；Solvent is water, pH 7.0~7.5.Institute
The microelement mother liquor composition stated are as follows: FeSO4·7H2O, 1.0g/L；CuSO4·5H2O, 0.02g/L；H3BO3, 0.014g/L；
MnSO4·4H2O, 0.10g/L；ZnSO4·7H2O, 0.10g/L；Na2MoO4·2H2O, 0.02g/L；CoCl2·6H2O, 0.02g/
L；Solvent is water.
R used2A solid medium composition are as follows: yeast powder, 0.50g/L；Tryptone, 0.50g/L；Casein, 0.50g/
L；Glucose, 0.50g/L；Soluble starch, 0.50g/L；Sodium Pyruvate, 0.30g/L；KH2PO4, 0.45g/L；MgSO4,
0.05g/L；Agar, 15~18g/L；Solvent is water, pH 7.2.
R used2A fluid nutrient medium composition are as follows: yeast powder, 0.50g/L；Tryptone, 0.50g/L；Casein, 0.50g/
L；Glucose, 0.50g/L；Soluble starch, 0.50g/L；Sodium Pyruvate, 0.30g/L；KH2PO4, 0.45g/L；MgSO4,
0.05g/L；Solvent is water, pH 7.2.
Fermentation medium composition used are as follows: yeast powder, 0.5g/L；CaCl2, 0.023g/L；MgSO4, 0.2g/L；(NH4)2SO4, 2.5g/L；KH2PO4, 1.0g/L；Na2HPO4, 4.5g/L；Microelement mother liquor, 1mL/L；Solvent is water, pH 7.0~
7.5。
Embodiment 1: separation, purifying and its identification of Dell Ford bacterium LW26
(1) separation and purifying of Dell's Ford bacterium LW26
The biomembrane of bio-trickling filter filler surface is acquired, which is Zhejiang Polytechnical University's bioconversion and biological cleaning
The bio-trickling filter of laboratory treatment chlorobenzene exhaust gas, treatment tank of the inoculation of activated-sludge from Zhejiang pharmaceutical factory are stable
Operation 5 months.Biomembrane obtains the bacterial strain of one plant of energy efficient degradation chlorobenzene after isolating and purifying.Specific step is as follows:
5g filler is taken from bio-trickling filter, and biomembrane is washed in shaking flask with 30mL sterile water, is placed in shaking table 160rpm,
30 DEG C vibrate 1 hour.Then bacterial strain enrichment sieve is carried out using the minimal medium (chlorobenzene initial concentration 100mg/L) containing chlorobenzene
Choosing.
Mixed bacteria liquid in shaking flask is after 5 passages enrichment, in R2Dilution spread on A solid medium, in 30 DEG C of perseverances
It is cultivated 2~3 days in warm incubator, according to the otherness of phage populations, the single colonie grown on picking plate carries out scribing line separation,
Obtain 4 plants of pure bacterium colonies.Screen 4 plants of chlorobenzene degradation bacterias are seeded to using chlorobenzene as the inorganic salts culture of sole carbon source and the energy
In base, each bacterial strain is investigated to the treatment effect of chlorobenzene, one plant of bacterial strain LW26 with efficient chlorobenzene degrading activity of final acquisition.
(2) identification of bacterial strain LW26
Bacterial strain LW26 cell is in rod-short, and size is (0.6~0.8) μ m (1.0~1.5) μm, no gemma；Bacterium colony is in circle
Shape, milky, protrusion, flush edge, form is full, smooth wet, and lawn is shown in Fig. 1 along scribing line growth, electromicroscopic photograph.
The physiological and biochemical property of bacterial strain LW26 are as follows: contact enzyme reaction is the positive, and V-P reaction, clark and Lubsreaction, nitrate are also
Former reaction, indole test, citrate are feminine gender, Gram-negative using test.
Bacterial strain LW26 slant strains entrust Sangon Biotech (Shanghai) Co., Ltd. to carry out PCR amplification and sequencing, obtain
It is following (SEQ ID NO.1, GenBank accession number is KP966097) to obtain 16S rDNA sequence:
GAACAGGGGGACCGCCTTACAATGCTAGTCGAACGGTAACAGGTCTTCGGACGCTGACGAGTGGCGAA
CGGGTGAGTAATACATCGGAACGTGCCCAGTCGTGGGGGATAACTACTCGAAAGAGTAGCTAATACCGCATACGAT
CTGAGGATGAAAGCGGGGGACCTTCGGGCCTCGCGCGATTGGAGCGGCCGATGGCAGATTAGGTAGTTGGTGGGAT
AAAAGCTTACCAAGCCGACGATCTGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAG
ACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGCAGGATG
AAGGCCTTCGGGTTGTAAACTGCTTTTGTACGGAACGAAAAAGCTCCTTCTAATACAGGGGGCCCATGACGGTACC
GTAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACT
GGGCGTAAAGCGTGCGCAGGCGGTTATGTAAGACAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTTGTG
ACTGCATGGCTAGAGTACGGTAGAGGGGGATGGAATTCCGCGTGTAGCAGTGAAATGCGTAGATATGCGGAGGAAC
ACCGATGGCGAAGGCAATCCCCTGGACCTGTACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGAT
ACCCTGGTAGTCCACGCCCTAAACGATGTCAACTGGTTGTTGGGAATTAGTTTTCTCAGTAACGAAGCTAACGCGT
GAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGA
TGATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCCACCTTTGACATGGCAGGAAGTTTCCAGAGATGGATT
CGTGCTCGAAAGAGAACCTGCACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTC
CCGCAACGAGCGCAACCCTTGTCATTAGTTGCTACATTCAGTTGAGCACTCTAATGAGACTGCCGGTGACAAACCG
GAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATAGGTGGGGCTACACACGTCATACAATGGCTGGTACA
GAGGGTTGCCAACCCGCGAGGGGGAGCTAATCCCATAAAACCAGTCGTAGTCCGGATCGCAGTCTGCAACTCGACT
GCGTGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGC
CCGTCACACCATGGGAGCGGGTCTCGCCAGAAGTAGGTAGCCTAACCGCAAGGAGGGCGCTACCCACGGGCGGGTT
TGCCGG。
Sequencing result is uploaded into GenBank, acquisition GenBank accession number is KP966097, and in GenBank
Gene order carries out sequence analysis.It is as shown in Figure 2 that phylogenetic tree is established accordingly, thus it is speculated that bacterial strain LW26 may be Delftia
tsuruhatensis.By the analysis of 16S rRNA sequence and the identification of Biolog microbial identification system, in conjunction with bacterial strain LW26 physiology
Biochemical character determines that bacterial strain LW26 is Delftia tsuruhatensis.
Embodiment 2: the preparation of Dell's Ford bacterium LW26 bacteria suspension
When bacterium solution dosage is smaller, Dell's Ford bacterium LW26 bacteria suspension can by inclined-plane culture, seed culture and
Bacteria suspension prepares three steps and obtains；It, can be outstanding by inclined-plane culture, seed culture, fermentation liquid culture and bacterium when dosage is larger
Liquid prepares four steps and obtains, and detailed process is as follows:
(1) Dell Ford bacterium LW26 inclined-plane culture: is inoculated in R2A solid medium, 28~32 DEG C of culture 48h are obtained
Inclined-plane thalline；
(2) seed culture: from step (1) inclined-plane, one oese colony inoculation of picking is into seed culture medium, and 28~32
DEG C culture 24~36h, obtain seed liquor；
(3) seed liquor that step (2) obtain fermented and cultured: is seeded to fermentation with the inoculum concentration of volumetric concentration 5~10%
Culture medium cultivates 24~36h under the conditions of 28~32 DEG C, pH 6.0~8.0, obtains fermentation liquid；
(4) prepared by bacteria suspension: seed liquor (or fermentation liquid) being centrifuged 10min in 6000rpm, supernatant is abandoned, with having sterilized
Minimal medium clean thallus, then 6000rpm be centrifuged 10min, abandon supernatant, wash repeatedly 2 times, by the wet bacterium of acquisition
Body dilutes the bacteria suspension for obtaining required concentration with sterile minimal medium.
Embodiment 3: degradation characteristic of the Dell Ford bacterium LW26 to chlorobenzene
(1) influence that temperature grows Dell Ford bacterium LW26 and chlorobenzene is degraded
Implement Dell's Ford LW26 at different temperatures to the degradation experiment of chlorobenzene, it is found that it is higher it has at 23~30 DEG C
Degradation chlorobenzene ability, specific experiment scheme is as follows:
50mL minimal medium is added in the shaking flask of 300mL, after forty minutes, chlorine is added in 7.0,110 DEG C of pH value sterilizings
Benzene makees sole carbon source, makes the initial concentration 100mg/L of chlorobenzene, adds 2 method of 1mL embodiment through inclined-plane and seed culture extremely
The Dell Ford bacterium LW26 bacteria suspension (OD of logarithmic growth phase600=0.15).In experimentation, each temperature design three parallel
Sample and a blank control (LW26 bacteria suspension is not added).Each sample is individually positioned in 23 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C
Shaking table in constant temperature incubation (shaking speed be equal 160rpm), sampled respectively in 0h and for 24 hours, chlorobenzene concentration and bacterium in detection shaking flask
The strain density of strain.
As a result as shown in figure 3, in order, the removal rate of chlorobenzene is reachable for the strain growth when temperature is 23~30 DEG C
To 99%；Bacterial strain declines the removal rate of chlorobenzene at 35 DEG C of temperature, and only 76.2%.With further increasing for temperature,
The growth of bacterial strain is suppressed, chlorobenzene degradation capability sharp fall.
(2) influence that pH grows Dell Ford bacterium LW26 and chlorobenzene is degraded
Implement Dell's Ford LW26 at different pH to the degradation experiment of chlorobenzene, the results showed that LW26 has in pH6.0~9.0
There is the ability of higher degradation chlorobenzene, specific experiment scheme is as follows:
50mL minimal medium is added in the shaking flask of 300mL, with 1mol/L NaOH aqueous solution or 1mol/L HCl/water
The pH of minimal medium is adjusted to 4.0,5.0,6.0,7.0,8.0,9.0,10.0 by solution, and 110 DEG C sterilize for sterilizing 40 minutes
Afterwards, be added chlorobenzene make sole carbon source, make the initial concentration 100mg/L of chlorobenzene, add 2 method of 1mL embodiment through inclined-plane with
Seed culture to logarithmic growth phase Dell Ford bacterium LW26 bacteria suspension (OD600=0.15).In experimentation, each pH design
Three Duplicate Samples and a blank control (LW26 bacteria suspension is not added).Each sample is placed on to constant temperature incubation in 30 DEG C of shaking table
(shaking speed is equal 160rpm).It is sampled respectively in 0h and for 24 hours, detects the strain density of the chlorobenzene concentration and bacterial strain in shaking flask.
As a result as shown in figure 4, Dell's Ford bacterium LW26 upgrowth situation is good when pH is 6.0,7.0,8.0,9.0,
It is higher to chlorobenzene removal efficiency to the removal rate of chlorobenzene up to 90% or more in for 24 hours.And in the culture medium that pH is 4,10, warp
Culture for 24 hours, strain density is still very low, and the removal rate of chlorobenzene is 30% hereinafter, show to wear under conditions of peracid or alkali excessively
Your Ford bacterium LW26 is difficult to survive.
(3) degradation situation of Dell's Ford bacterium LW26 to the chlorobenzene of different initial concentrations
Under different chlorobenzene initial concentrations, implement degradation of Dell's Ford bacterium LW26 to chlorobenzene, as a result, it has been found that LW26 highest
Degradable 500mg/L chlorobenzene, specific experiment scheme are as follows:
50mL minimal medium is added in the shaking flask of 300mL, 7.0,110 DEG C of pH value sterilize after forty minutes, middle addition
Chlorobenzene makees sole carbon source, and making the initial concentration of chlorobenzene is respectively 100,200,300,400,500mg/L.It is separately added into 1mL reality again
Apply Dell Ford bacterium LW26 bacteria suspension (OD of 2 method of example through inclined-plane and seed culture to logarithmic growth phase600=0.15).Experiment
In the process, each initial concentration designs three Duplicate Samples and a blank control (LW26 bacteria suspension is not added).Each sample is put
Constant temperature incubation (shaking speed is equal 160rpm) is set in 30 DEG C of shaking table, is sampled every 2h, chlorobenzene concentration and bacterial strain are detected
Strain density draws Dell's Ford bacterium LW26 to the degradation curve figure and its growth curve chart of chlorobenzene.
As a result as shown in figure 5, when chlorobenzene initial concentration 400mg/L and it is following when, Dell Ford bacterium LW26 can fast prompt drop
Chlorobenzene is solved, bacterial growth is in order.When chlorobenzene initial concentration is in 400mg/L or more, due to the inhibiting effect of substrate, degradation
Ability is declined slightly, and the bacterial strain is to the highest degradation concentration of chlorobenzene up to 500mg/L.
Embodiment 4: Dell's Ford bacterium LW26 purification simulation chlorobenzene exhaust gas
Simulation chlorobenzene exhaust gas is handled using bio-trickling filter, process flow is as shown in Figure 6.
Two-way is divided by the air that air pump 1 bloats, enters the stripping bottle that chlorobenzene waste liquid is housed through mass flowmenter 2 all the way
In 4, it is used for stripping chlorobenzene, the chlorobenzene gas that stripping goes out to be mixed with another way by the air that spinner flowmeter 3 enters hybrid bottle 5
After uniformly, simulate to obtain the chlorobenzene exhaust gas of various concentration by control mass flowmenter and spinner flowmeter.Gas is from bottom to top
Flow through packed tower, in packed tower filler be Pall ring, recirculating nutrient solution tower top is then promoted to from peristaltic pump 11 after to lower spray, be
The microorganism adhered on filler provides its required nutrient, biomass is measured by sampling in filler sample tap 10, in the gas of tower top
Sampler body mouth determines gas concentration, and treated, and exhaust gas is flowed out through exhaust port 8, flows into liquid storage bottle 6 after nutrient solution spray, leads to
It crosses lye bottle 7 and adjusts liquid pH value in liquid storage bottle 6.The operation temperature of device is controlled by the heating wire and thermostat for being wound in tower body
At 30 DEG C or so.The spray flow of nutrient solution is 5~6L/h, replaces one time of nutrition liquid every 4d, pH is using 0.5mol/L's
NaOH aqueous solution is adjusted to maintain between 6.8~7.2.It will be prepared according to 2 method of embodiment through inclined-plane, seed and fermented and cultured
Dell Ford bacterium LW26 bacteria suspension (OD600=0.5) it is seeded in bio-trickling filter, inoculum concentration 2L.During start-up
Residence time is 90s, and initial chlorobenzene inlet gas concentration is 200mg/m3。
As shown in fig. 7, after filter tower operation 22d, start-up success, when chlorobenzene inlet concentration is 750~850mg/m3When,
The removal rate of chlorobenzene is still greater than 90%.
Embodiment 5: Dell Ford bacterium LW26 processing simulation chlorobenzene waste water
It is as shown in Figure 8 that simulation chlorobenzene waste water process flow is handled using sbr reactor device.
In the reactor according to 1: 1 ratio inoculation of activated-sludge and LW26 bacterium solution (according to 2 method of embodiment through inclined-plane,
Seed and fermented and cultured preparation).Reactor total measurement (volume) is 4L, and dischargeable capacity 3L, each periodic duty time is 8h, specific to transport
Row parameter is as follows: using instant water coming-in and instantaneous water outlet, aeration time and sedimentation time are respectively 7h and 1h in each period.Fortune
Between the departure date, DO is 2~4mg/L in reactor, and MLSS is 1.5~3g/L, pH 6.5~7.5, and temperature is controlled by temperature controller to exist
25±1℃。
As shown in figure 9, when chlorobenzene concentration of intaking is 110mg/L, chlorobenzene removal efficiency be can reach after SBR is stable
92% or more.
Claims (5)
1. Dell Ford bacterium (Delftia tsuruhatensis) LW26, is preserved in China typical culture collection center, preservation
Date is on March 15th, 2015, deposit number CCTCC No:M 2015113, address: China, Wuhan, Wuhan University, postcode
430072。
2. a kind of application of Dell's Ford bacterium LW26 described in claim 1 in microbial degradation chlorobenzene.
3. application as claimed in claim 2, it is characterised in that the application be Dell Ford bacterium LW26 is seeded to containing
It in the minimal medium of chlorobenzene, is cultivated under conditions of 23~40 DEG C, pH 4.0~10.0, realizes the degradation to chlorobenzene；Institute
The minimal medium composition stated are as follows: CaCl20.023g/L, MgSO40.2g/L, (NH4)2SO42.5g/L, KH2PO4
1.0g/L, Na2HPO44.5g/L, microelement mother liquor 1mL/L, solvent are water, pH 7.0~7.5；The microelement is female
Liquid composition are as follows: FeSO4·7H2O 1.0g/L, CuSO4·5H2O 0.02g/L, H3BO30.014g/L, MnSO4·4H2O
0.10g/L, ZnSO4·7H2O 0.10g/L, Na2MoO4·2H2O 0.02g/L, CoCl2·6H2O 0.02g/L, solvent are water.
4. application as claimed in claim 3, it is characterised in that chlorobenzene initial concentration in minimal medium is 100~
500mg/L；Dell's Ford bacterium LW26 is obtained with the wet thallus after seed culture or fermented and cultured through minimal medium dilution
The OD obtained600The bacteria suspension form of=0.1-0.5 is added, and bacteria suspension volume inoculum concentration is 1-5%.
5. application as claimed in claim 4, which is characterized in that the bacteria suspension obtains as follows:
(1) Dell Ford bacterium LW26 inclined-plane culture: is inoculated in R2A solid medium, 28~32 DEG C of culture 48h obtain inclined-plane bacterium
Body；The R2A solid medium composition are as follows: yeast powder 0.50g/L, tryptone 0.50g/L, casein 0.50g/L, glucose
0.50g/L, soluble starch 0.50g/L, Sodium Pyruvate 0.30g/L, KH2PO40.45g/L, MgSO40.05g/L, agar 15
~18g/L, solvent are water, pH 7.2；
(2) seed culture: from one oese colony inoculation of picking on step (1) inclined-plane into seed culture medium, 28~32 DEG C of trainings
24~36h is supported, seed liquor is obtained；The seed culture medium is R2A fluid nutrient medium, in addition to no agar, remaining component and R2A
Solid medium is identical；
(3) seed liquor that step (2) obtain fermented and cultured: is seeded to fermented and cultured with the inoculum concentration of volumetric concentration 5~10%
Base cultivates 24~36h under the conditions of 28~32 DEG C, pH 6.0~8.0, obtains fermentation liquid；The fermentation medium composition are as follows: ferment
Female powder 0.5g/L, CaCl20.023g/L, MgSO40.2g/L, (NH4)2SO42.5g/L, KH2PO41.0g/L, Na2HPO4
4.5g/L, microelement mother liquor 1mL/L, solvent are water, pH 7.0~7.5；
(4) prepared by bacteria suspension: fermentation liquid being centrifuged 10min in 6000rpm, supernatant is abandoned, with sterilized minimal medium
Thallus is cleaned, then 6000rpm is centrifuged 10min, abandons supernatant, washes repeatedly 2 times, by the sterile inorganic salts of the wet thallus of acquisition
Culture medium suspends, and obtains the bacteria suspension.
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| CN101016525A (en) * | 2006-10-13 | 2007-08-15 | 北京工商大学 | Delftia with aerobic denitrifying capability and method of treating waste water by the same |
| CN104531585A (en) * | 2014-12-31 | 2015-04-22 | 中国水稻研究所 | Delftia tsuruhatensis and application thereof |
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| CN101016525A (en) * | 2006-10-13 | 2007-08-15 | 北京工商大学 | Delftia with aerobic denitrifying capability and method of treating waste water by the same |
| CN104531585A (en) * | 2014-12-31 | 2015-04-22 | 中国水稻研究所 | Delftia tsuruhatensis and application thereof |
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| Title |
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| 联苯菊酯降解菌的筛选、鉴定及其降解特性;景岳龙 等;《西北农林科技大学学报（自然科学版）》;20101031;第38卷(第10期);第99页左栏，第100页右栏 * |
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