CN105039222A - Delftia tsuruhatensis LW26 and application thereof in chlorobenzene degradation - Google Patents

Delftia tsuruhatensis LW26 and application thereof in chlorobenzene degradation Download PDF

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CN105039222A
CN105039222A CN201510498197.0A CN201510498197A CN105039222A CN 105039222 A CN105039222 A CN 105039222A CN 201510498197 A CN201510498197 A CN 201510498197A CN 105039222 A CN105039222 A CN 105039222A
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chlorobenzene
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obtains
dell
culture
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CN105039222B (en
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陈建孟
叶杰旭
陈东之
李伟
林彤晖
诸葛蕾
江宁馨
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Zhejiang University of Technology ZJUT
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/59—Biological synthesis; Biological purification

Abstract

The invention provides a strain Delftia tsuruhatensis LW26 which is capable of efficiently degrading chlorobenzene and application thereof in microbial decomposition and treatment of chlorobenzene. The Delftia tsuruhatensis LW26 is preserved in China Center for Type Culture Collection (CCTCC): Wuhan University, Wuhan, China, 430072, the preservation date is March 15, 2015 and a preservation number is CCTCC NO. M2015113. The chlorobenzene degradation bacteria provided by the invention can use chlorobenzene as a unique carbon source and energy for propagation and completely mineralize the chlorobenzene into CO2 and H2O; the strain can effectively degrade chlorobenzene in an environment of 23-30 DEG C and pH of 6.0-9.0; the strain has higher environment suitability and can play an important role in practice of waste gas and waste water treatment.

Description

Dell Ford bacterium LW26 and the application in degraded chlorobenzene thereof
(1) technical field
The present invention relates to the novel chlorobenzene efficient degrading bacteria of a strain---Dell Ford bacterium (Delftiatsuruhatensis) LW26 and application thereof.
(2) background technology
Chlorobenzene is monocyclic aromatic compound phenyl ring only having hydrogen atom and chlorine atom, has the features such as solubleness is low, hydrophobicity is strong.In recent years, as the intermediate of a kind of important organic solvent and Chemical Manufacture, chlorobenzene is widely used in the industries such as plastics, dyestuff, medicine, agricultural chemicals, organic synthesis.Because its water-soluble low and bio-toxicity is large, severe contamination is caused to environment.Can nervous center be suppressed after chlorobenzene enters human body, easily be combined with enzyme system, have teratogenesis carcinogenesis.Owing to using extensively, chlorobenzene all has higher recall rate in multiple surrounding medium, constitutes certain threat to HUMAN HEALTH and ecology erroneous zone, and chlorobenzene is listed in priority pollutants list by Environmental Protection Agency (EPA).
At present, for the emission control of this pollutant, domestic and international researcher has carried out large quantity research, and various treatment process is arisen at the historic moment, and mainly contains absorption method, supersonic method, membrane separation process, catalytic oxidation, photochemical oxidation method, electrochemical process etc.In recent years, biological process is proved to be and has a good application prospect in the purification of chlorobenzene compound.Purification biotechnology utilizes microbiota metabolic activity, is that the energy of cellular metabolism, cell component and innoxious small-molecule substance are (as H by contamination transform 2o, CO 2deng), to have efficiently compared to other method, low consumption, the advantages such as reaction conditions is gentle, secondary pollution is little.
One of key of biotechnology degraded chlorobenzene is utilized to be obtain chlorobenzene efficient degrading bacteria.At present, Chinese scholars has done large quantity research in this respect, but due to the volatile and characteristic of difficult for biological degradation of chlorobenzene, has been separated the chlorobenzene degradation bacteria kind obtained also more limited, in addition, has been separated the strains for degrading efficiency obtained and has need to improve.As Niu Xian etc. is carrying out finding in the experiment of degradation condition optimization to a strain chlorobenzene dominant degradation bacteria LysinibacillusfusiformisLW13, when chlorobenzene starting point concentration is 100mg/L, the degradation rate of chlorobenzene reaches maximum, and when chlorobenzene concentration reaches 180mg/L, the degraded of chlorobenzene be subject to obvious suppression (Niu Xian etc. environmental engineering, 2013,31 (1): 43-46.); 2010, Zhang Lili etc. find that a strain has the RalstoniapickettiiH2 of chlorobenzene degradation capability, when chlorobenzene concentration is lower than 250mg/L, H2 can degrade chlorobenzene rapidly, bacterial growth is good, and when 250mg/L, strain growth and degraded are subject to obvious suppression (ZL101880642); The chlorobenzene degradation bacteria acinetobacter calcoaceticus (Acinetobactercalcoaceticus) that Li Mingtang etc. filter out, with starting point concentration be the chlorobenzene of 50mg/L for sole carbon source and the energy time, chlorobenzene could need be degraded completely through 120h (Li Mingtang etc. microorganism journal, 2010,50 (5): 586-592.).
Through retrieval related documents, there is not yet the report with Dell's Ford bacterium degraded chlorobenzene.
(3) summary of the invention
The present invention is directed to that chlorobenzene biological degradation efficiency is lower, deficiency that microorganism generation time is long, providing can the Dell Ford bacterium LW26 of efficient degradation chlorobenzene and application thereof.
The technical solution used in the present invention is:
The invention provides a strain new strains---Dell Ford bacterium (Delftiatsuruhatensis) LW26, be preserved in China typical culture collection center, preservation date is on March 15th, 2015, deposit number CCTCCNo:M2015113, address: China, Wuhan, Wuhan University, postcode 430072.
Chlorobenzene efficient degrading bacteria provided by the present invention---Dell Ford bacterium LW26 is by gathering Zhejiang Polytechnical University's bio-transformation and the microbial film of biopurification use for laboratory in the bio-trickling filter purifying chlorobenzene waste gas, obtains after separation, purifying.Described Dell's Ford bacterium LW26 colony characteristics is as follows: thalline is rod-short, and size is (0.6 ~ 0.8) μm × (1.0 ~ 1.5) μm, without gemma; At solid R 2on A substratum, bacterium colony is rounded, oyster white, protruding, flush edge, and form is full, smooth moistening, and lawn is along line growth; Catalase reaction is for positive, and V-P reaction, clark and Lubsreaction, nitrate reduction reaction, indole test, Citrate trianion utilize test to be feminine gender, Gram-negative.
The present invention relates to the described application of Dell Ford bacterium LW26 in microbiological deterioration chlorobenzene.Concrete, described is applied as: be seeded in the minimal medium containing chlorobenzene by Dell Ford bacterium LW26, temperature 23 ~ 40 DEG C, and under the condition of pH4.0 ~ 10.0 (preferably 23 ~ 30 DEG C, pH6.0 ~ 9.0) cultivate, realize the degraded to chlorobenzene.
Further, described chlorobenzene starting point concentration in minimal medium is 100 ~ 500mg/L; The OD that described Dell Ford bacterium LW26 obtains through minimal medium dilution with the wet thallus after seed culture or fermentation culture 600the bacteria suspension form of=0.1-0.5 adds, and bacteria suspension volume inoculum size is 1-5%, preferred OD 600the bacteria suspension volume inoculum size of=0.15 is 2%.Further, described minimal medium consists of: CaCl 2, 0.023g/L; MgSO 4, 0.2g/L; (NH 4) 2sO 4, 2.5g/L; KH 2pO 4, 1.0g/L; Na 2hPO 4, 4.5g/L; Trace element mother liquor 1mL/L; Solvent is water, pH7.0 ~ 7.5.Described micro-mother liquor consists of: FeSO 47H 2o, 1.0g/L; CuSO 45H 2o, 0.02g/L; H 3bO 3, 0.014g/L; MnSO 44H 2o, 0.10g/L; ZnSO 47H 2o, 0.10g/L; Na 2moO 42H 2o, 0.02g/L; CoCl 26H 2o, 0.02g/L; Solvent is water.
Further, when bacterium liquid consumption is less, described Dell Ford bacterium LW26 bacteria suspension obtains by slant culture, seed culture and collecting cells three steps; When large usage quantity, by slant culture, seed culture, fermented liquid cultivates and collecting cells four steps obtain, and detailed process is as follows:
(1) slant culture: Dell Ford bacterium LW26 is inoculated in R 2a solid medium, cultivates 48h for 28 ~ 32 DEG C, obtains inclined-plane thalline; Described R 2a solid medium consists of: yeast powder, 0.50g/L; Tryptones, 0.50g/L; Casein food grade, 0.50g/L; Glucose, 0.50g/L; Zulkovsky starch, 0.50g/L; Sodium.alpha.-ketopropionate, 0.30g/L; KH 2pO 4, 0.45g/L; MgSO 4, 0.05g/L; Agar, 15 ~ 18g/L; Solvent is water, pH7.2.
(2) seed culture: picking one transfering loop colony inoculation is in seed culture medium from step (1) inclined-plane, cultivates 24 ~ 36h for 28 ~ 32 DEG C, obtains seed liquor.Described seed culture medium is R 2a liquid nutrient medium, except without except agar, all the other components and R 2a solid medium is identical.
(3) fermentation culture: the seed liquor that step (2) obtains is seeded to fermention medium with the inoculum size of volumetric concentration 5 ~ 10%, in 28 ~ 32 DEG C, cultivates 24 ~ 36h under the condition of pH6.0 ~ 8.0, obtains fermented liquid.Described fermention medium consists of: yeast powder, 0.5g/L; CaCl 2, 0.023g/L; MgSO 4, 0.2g/L; (NH 4) 2sO 4, 2.5g/L; KH 2pO 4, 1.0g/L; Na 2hPO 4, 4.5g/L; Trace element mother liquor, 1mL/L; Solvent is water, pH7.0 ~ 7.5, and micro-mother liquor composition is with minimal medium medium trace element mother liquor.
(4) collecting cells: by seed liquor (or fermented liquid) at the centrifugal 10min of 6000rpm, abandon supernatant liquor, with sterilized minimal medium cleaning thalline, then at the centrifugal 10min of 6000rpm, abandons supernatant liquor, repeated washing 2 times.The wet thallus obtained is obtained the bacteria suspension of desired concn with aseptic minimal medium dilution.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: at 23 ~ 30 DEG C, under the condition of pH6.0 ~ 9.0, Dell Ford bacterium LW26 by degradable for the chlorobenzene of 100mg/L in minimal medium, can reach 500mg/L to the highest degraded concentration of chlorobenzene in 16h.And the bacterial strain H2 chlorobenzene tolerable concentration that patent application CN101880642A provides is only 250mg/L.Chlorobenzene degradation bacteria acinetobacter calcoaceticus (Acinetobactercalcoaceticus) that Li Mingtang etc. filter out (Li Mingtang etc. microorganism journal, 2010,50 (5): 586-592.), with starting point concentration be the chlorobenzene of 50mg/L for sole carbon source and the energy time, need could by chlorobenzene degraded completely through 120h.Compared to prior art, Dell Ford bacterium (Delftiatsuruhatensis) LW26 provided by the invention has the advantages that degradation speed is fast, tolerable concentration is high, will play a significant role in chlorobenzene waste gas and waste water treatment practice.
(4) accompanying drawing explanation
Fig. 1 is Dell Ford bacterium LW26 transmission electron microscope photo.
Fig. 2 is the phylogeny tree graph of Dell Ford bacterium LW26.
Fig. 3 is that temperature is on the impact that Dell Ford bacterium LW26 grows and chlorobenzene is degraded.
Fig. 4 is that pH is on the impact that Dell Ford bacterium LW26 grows and chlorobenzene is degraded.
Fig. 5 be under different chlorobenzene starting point concentration Dell Ford bacterium LW26 to the degraded (a) of chlorobenzene and growth curve (b).
Fig. 6 is bio-trickling filter process flow sheet, 1. pneumatic pump, 2. mass flowmeter, 3. spinner-type flowmeter, 4. stripping bottle, 5. hybrid bottle, 6. liquid storage bottle, 7. alkali lye bottle, 8. exhaust port, 9. gas sampling mouth, 10. filler thief hole, 11. peristaltic pumps.
Fig. 7 is import and export concentration and the removal efficiency of bio-trickling filter treatment of simulated chlorobenzene waste gas.
Fig. 8 is sbr reactor device process flow sheet, 1. agitator, and 2.pH counts, 3. water inlet pipe, 4. dissolved oxygen instrument, 5. water port, 6.pH pops one's head in, 7. dissolved oxygen instrument sensor, 8. temperature control instrument, 9. temperature sensor, 10. shore pipe, 11. aerators, 12. under meters, 13. pneumatic pumps.
Fig. 9 is the chlorobenzene degradation curve of sbr reactor device treatment of simulated chlorobenzene waste water.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
In embodiment, minimal medium used consists of: CaCl 2, 0.023g/L; MgSO 4, 0.2g/L; (NH 4) 2sO 4, 2.5g/L; KH 2pO 4, 1.0g/L; Na 2hPO 4, 4.5g/L; Trace element mother liquor, 1mL/L; Solvent is water, pH7.0 ~ 7.5.Described micro-mother liquor consists of: FeSO 47H 2o, 1.0g/L; CuSO 45H 2o, 0.02g/L; H 3bO 3, 0.014g/L; MnSO 44H 2o, 0.10g/L; ZnSO 47H 2o, 0.10g/L; Na 2moO 42H 2o, 0.02g/L; CoCl 26H 2o, 0.02g/L; Solvent is water.
R used 2a solid medium consists of: yeast powder, 0.50g/L; Tryptones, 0.50g/L; Casein food grade, 0.50g/L; Glucose, 0.50g/L; Zulkovsky starch, 0.50g/L; Sodium.alpha.-ketopropionate, 0.30g/L; KH 2pO 4, 0.45g/L; MgSO 4, 0.05g/L; Agar, 15 ~ 18g/L; Solvent is water, pH7.2.
R used 2a liquid nutrient medium consists of: yeast powder, 0.50g/L; Tryptones, 0.50g/L; Casein food grade, 0.50g/L; Glucose, 0.50g/L; Zulkovsky starch, 0.50g/L; Sodium.alpha.-ketopropionate, 0.30g/L; KH 2pO 4, 0.45g/L; MgSO 4, 0.05g/L; Solvent is water, pH7.2.
Fermention medium used consists of: yeast powder, 0.5g/L; CaCl 2, 0.023g/L; MgSO 4, 0.2g/L; (NH 4) 2sO 4, 2.5g/L; KH 2pO 4, 1.0g/L; Na 2hPO 4, 4.5g/L; Trace element mother liquor, 1mL/L; Solvent is water, pH7.0 ~ 7.5.
Embodiment 1: the separation of Dell Ford bacterium LW26, purifying and qualification thereof
(1) separation of Dell's Ford bacterium LW26 and purifying
Gather the microbial film of bio-trickling filter filling surface, this filter tower is the bio-trickling filter of Zhejiang Polytechnical University's bio-transformation and biopurification laboratory treatment chlorobenzene waste gas, inoculation of activated-sludge from the treatment tank in pharmaceutical factory, Zhejiang, steady running 5 months.Microbial film obtains the bacterial strain of a strain energy efficient degradation chlorobenzene after separation and purification.Concrete steps are as follows:
From bio-trickling filter, get 5g filler, wash with 30mL sterilized water in shaking flask by microbial film, be placed in shaking table 160rpm, 30 DEG C vibrate 1 hour.Then the minimal medium (chlorobenzene starting point concentration 100mg/L) containing chlorobenzene is utilized to carry out bacterial strain enrichment isolation.
Mixed bacteria liquid in shaking flask after 5 enrichments of going down to posterity, at R 2dilution spread on A solid medium, cultivates 2 ~ 3 days in 30 DEG C of constant incubators, and according to the otherness of phage populations, single bacterium colony that picking flat board grows carries out line and is separated, and obtains the pure bacterium colony of 4 strain.The 4 strain chlorobenzene degradation bacteria screened are seeded to chlorobenzene be sole carbon source and the energy minimal medium in, investigate the treatment effect of each bacterial strain to chlorobenzene, final obtain the bacterial strain LW26 that a strain has efficient chlorobenzene degrading activity.
(2) qualification of bacterial strain LW26
Bacterial strain LW26 cell is rod-short, and size is (0.6 ~ 0.8) μm × (1.0 ~ 1.5) μm, without gemma; Bacterium colony is rounded, oyster white, protruding, flush edge, and form is full, smooth moistening, and lawn is along line growth, and electromicroscopic photograph is shown in Fig. 1.
The physiological and biochemical property of bacterial strain LW26 is: catalase reaction is for positive, and V-P reaction, clark and Lubsreaction, nitrate reduction reaction, indole test, Citrate trianion utilize test to be feminine gender, Gram-negative.
Bacterial strain LW26 slant strains entrusts Sangon Biotech (Shanghai) Co., Ltd. to carry out pcr amplification and order-checking, obtains 16SrDNA sequence following (SEQIDNO.1, GenBank accession number is KP966097):
GAACAGGGGGACCGCCTTACAATGCTAGTCGAACGGTAACAGGTCTTCGGACGCTGACGAGTGGCGAACGGGTGAGTAATACATCGGAACGTGCCCAGTCGTGGGGGATAACTACTCGAAAGAGTAGCTAATACCGCATACGATCTGAGGATGAAAGCGGGGGACCTTCGGGCCTCGCGCGATTGGAGCGGCCGATGGCAGATTAGGTAGTTGGTGGGATAAAAGCTTACCAAGCCGACGATCTGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGCAGGATGAAGGCCTTCGGGTTGTAAACTGCTTTTGTACGGAACGAAAAAGCTCCTTCTAATACAGGGGGCCCATGACGGTACCGTAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTATGTAAGACAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTTGTGACTGCATGGCTAGAGTACGGTAGAGGGGGATGGAATTCCGCGTGTAGCAGTGAAATGCGTAGATATGCGGAGGAACACCGATGGCGAAGGCAATCCCCTGGACCTGTACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTGGTTGTTGGGAATTAGTTTTCTCAGTAACGAAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCCACCTTTGACATGGCAGGAAGTTTCCAGAGATGGATTCGTGCTCGAAAGAGAACCTGCACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCTACATTCAGTTGAGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATAGGTGGGGCTACACACGTCATACAATGGCTGGTACAGAGGGTTGCCAACCCGCGAGGGGGAGCTAATCCCATAAAACCAGTCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGCGGGTCTCGCCAGAAGTAGGTAGCCTAACCGCAAGGAGGGCGCTACCCACGGGCGGGTTTGCCGG。
Sequencing result is uploaded to GenBank, and obtaining GenBank accession number is KP966097, and carries out sequence analysis with the gene order in GenBank.Set up phylogenetic tree accordingly as shown in Figure 2, infer that bacterial strain LW26 may be Delftiatsuruhatensis.By 16SrRNA sequential analysis and the qualification of Biolog microbial identification system, in conjunction with bacterial strain LW26 physiological and biochemical property, determine that this bacterial strain LW26 is Delftiatsuruhatensis.
Embodiment 2: the preparation of Dell Ford bacterium LW26 bacteria suspension
When bacterium liquid consumption is less, described Dell Ford bacterium LW26 bacteria suspension obtains by slant culture, seed culture and collecting cells three steps; When large usage quantity, by slant culture, seed culture, fermented liquid cultivates and collecting cells four steps obtain, and detailed process is as follows:
(1) slant culture: Dell Ford bacterium LW26 is inoculated in R 2a solid medium, cultivates 48h for 28 ~ 32 DEG C, obtains inclined-plane thalline;
(2) seed culture: picking one transfering loop colony inoculation is in seed culture medium from step (1) inclined-plane, cultivates 24 ~ 36h for 28 ~ 32 DEG C, obtains seed liquor;
(3) fermentation culture: the seed liquor that step (2) obtains is seeded to fermention medium with the inoculum size of volumetric concentration 5 ~ 10%, in 28 ~ 32 DEG C, cultivates 24 ~ 36h under the condition of pH6.0 ~ 8.0, obtains fermented liquid;
(4) collecting cells: by seed liquor (or fermented liquid) at the centrifugal 10min of 6000rpm, abandon supernatant liquor, with sterilized minimal medium cleaning thalline, then the centrifugal 10min of 6000rpm, abandon supernatant liquor, repeated washing 2 times, obtains the bacteria suspension of desired concn with aseptic minimal medium dilution by the wet thallus obtained.
Embodiment 3: Dell Ford bacterium LW26 is to the degradation characteristic of chlorobenzene
(1) temperature is on the impact that Dell Ford bacterium LW26 grows and chlorobenzene is degraded
Implement Dell Ford LW26 at different temperatures to the degradation experiment of chlorobenzene, find that it has the ability of higher degraded chlorobenzene at 23 ~ 30 DEG C, specific experiment scheme is as follows:
50mL minimal medium is added in the shaking flask of 300mL, pH value 7.0,110 DEG C of sterilizings are after 40 minutes, add chlorobenzene and make sole carbon source, the starting point concentration making chlorobenzene is 100mg/L, then adds 1mL embodiment 2 method through inclined-plane and seed culture to the Dell Ford bacterium LW26 bacteria suspension (OD of logarithmic phase 600=0.15).In experimentation, each temperature design three Duplicate Samples and a blank (not adding LW26 bacteria suspension).Each sample is placed on respectively constant temperature culture in the shaking table of 23 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C (shaking speed is equal 160rpm), respectively in 0h and 24h sampling, detects the strain density of chlorobenzene concentration and bacterial strain in shaking flask.
As shown in Figure 3, when temperature is 23 ~ 30 DEG C, this strain growth in order for result, and the clearance of chlorobenzene all can reach 99%; During temperature 35 DEG C, the clearance of bacterial strain to chlorobenzene declines to some extent, is only 76.2%.Along with the further raising of temperature, the growth of bacterial strain is suppressed, and chlorobenzene degradation capability significantly declines.
(2) pH is on the impact that Dell Ford bacterium LW26 grows and chlorobenzene is degraded
Under different pH, implement Dell Ford LW26 to the degradation experiment of chlorobenzene, result shows that LW26 has the ability of higher degraded chlorobenzene in pH6.0 ~ 9.0, and specific experiment scheme is as follows:
50mL minimal medium is added in the shaking flask of 300mL, with the 1mol/LNaOH aqueous solution or the 1mol/LHCl aqueous solution by the pH regulator to 4.0 of minimal medium, 5.0,6.0,7.0,8.0,9.0,10.0, after 110 DEG C of sterilizings sterilizing in 40 minutes, add chlorobenzene and make sole carbon source, the starting point concentration making chlorobenzene is 100mg/L, then adds 1mL embodiment 2 method through inclined-plane and seed culture to the Dell Ford bacterium LW26 bacteria suspension (OD of logarithmic phase 600=0.15).In experimentation, each pH designs three Duplicate Samples and a blank (not adding LW26 bacteria suspension).Each sample is placed on constant temperature culture in the shaking table of 30 DEG C (shaking speed is equal 160rpm).Respectively in 0h and 24h sampling, the chlorobenzene concentration in detection shaking flask and the strain density of bacterial strain.
As shown in Figure 4, when pH is 6.0,7.0,8.0,9.0, Dell Ford bacterium LW26 upgrowth situation is good, in 24h, all reach more than 90% to the clearance of chlorobenzene, higher to chlorobenzene removal efficiency for result.And be in the substratum of 4,10 at pH, through the cultivation of 24h, strain density is still very low, the clearance of chlorobenzene all below 30%, show peracid or cross alkali condition under, Dell Ford bacterium LW26 is difficult to existence.
(3) Dell's Ford bacterium LW26 is to the degraded situation of the chlorobenzene of different starting point concentration
Under different chlorobenzene starting point concentration, implement Dell Ford bacterium LW26 to the degraded of chlorobenzene, found that LW26 the highest degradable 500mg/L chlorobenzene, specific experiment scheme is as follows:
In the shaking flask of 300mL, add 50mL minimal medium, pH value 7.0,110 DEG C of sterilizings are after 40 minutes, in add chlorobenzene and make sole carbon source, make the starting point concentration of chlorobenzene be respectively 100,200,300,400,500mg/L.Add 1mL embodiment 2 method more respectively through inclined-plane and seed culture to the Dell Ford bacterium LW26 bacteria suspension (OD of logarithmic phase 600=0.15).In experimentation, each starting point concentration designs three Duplicate Samples and a blank (not adding LW26 bacteria suspension).Each sample is placed on constant temperature culture in the shaking table of 30 DEG C (shaking speed is equal 160rpm), every 2h sampling, the strain density of chlorine detection benzene concentration and bacterial strain, draws Dell Ford bacterium LW26 to the degradation curve figure of chlorobenzene and its growth curve chart.
Result as shown in Figure 5, when chlorobenzene starting point concentration 400mg/L and following time, Dell Ford bacterium LW26 chlorobenzene capable of being fast degraded, bacterial growth is in order.When chlorobenzene starting point concentration is at more than 400mg/L, due to the restraining effect of substrate, degradation capability slightly declines, and the highest degraded concentration of this bacterial strain to chlorobenzene can reach 500mg/L.
Embodiment 4: Dell Ford bacterium LW26 purification simulation chlorobenzene waste gas
Utilize bio-trickling filter treatment of simulated chlorobenzene waste gas, technical process as shown in Figure 6.
The air bloated by pneumatic pump 1 is divided into two-way, one tunnel enters through mass flowmeter 2 is equipped with in the stripping bottle 4 of chlorobenzene waste liquid, for stripping chlorobenzene, the air mixed that the chlorobenzene gas that stripping goes out and another road enter hybrid bottle 5 through spinner-type flowmeter 3 evenly after, obtained the chlorobenzene waste gas of different concns by Mass Control under meter and spinner-type flowmeter simulation.Gas flows through packing tower from bottom to top, in packing tower, filler is Pall ring, recirculating nutrient solution is then promoted to the backward lower spray of tower top by peristaltic pump 11, for the microorganism that filler adheres to provides its desired nutritional element, at filler thief hole 10 sampling and measuring biomass, measure at the gas sampling mouth of tower top concentration of giving vent to anger, the waste gas after process flows out through exhaust port 8, flow into liquid storage bottle 6 after nutritive medium spray, regulate liquid pH value in liquid storage bottle 6 by alkali lye bottle 7.The service temperature of device is controlled at about 30 DEG C by the nichrome wire and thermostat being wound in tower body.The spray flux of nutritive medium is 5 ~ 6L/h, changes one time of nutrition liquid every 4d, and its pH adopts the NaOH aqueous solution of 0.5mol/L to regulate to maintain between 6.8 ~ 7.2.By according to embodiment 2 method through inclined-plane, the Dell Ford bacterium LW26 bacteria suspension (OD for preparing of seed and fermentation culture 600=0.5) be seeded in bio-trickling filter, inoculum size is 2L.In start-up process, the residence time is 90s, and initial chlorobenzene inlet gas concentration is 200mg/m 3.
As shown in Figure 7, after filter tower runs 22d, start-up success, when chlorobenzene inlet concentration is 750 ~ 850mg/m 3time, the clearance of chlorobenzene is still greater than 90%.
Embodiment 5: Dell Ford bacterium LW26 treatment of simulated chlorobenzene waste water
Utilize the technical process of sbr reactor device treatment of simulated chlorobenzene waste water as shown in Figure 8.
In the reactor according to 1: 1 ratio inoculation of activated-sludge and LW26 bacterium liquid (according to embodiment 2 method through inclined-plane, seed and fermentation culture preparation).Reactor cubic capacity is 4L, and useful volume is 3L, and each periodic duty time is 8h, and carrying out practically parameter is as follows: adopt instant water coming-in and instantaneous water outlet, in each cycle, aeration time and sedimentation time are respectively 7h and 1h.Run duration, in reactor, DO is 2 ~ 4mg/L, MLSS is 1.5 ~ 3g/L, pH6.5 ~ 7.5, and temperature is controlled at 25 ± 1 DEG C by temperature controller.
As shown in Figure 9, after SBR is stable, when chlorobenzene concentration of intaking is 110mg/L, chlorobenzene removal efficiency can reach more than 92%.

Claims (6)

1. Dell's Ford bacterium (Delftiatsuruhatensis) LW26, is preserved in China typical culture collection center, and preservation date is on March 15th, 2015, deposit number CCTCCNo:M2015113, address: China, Wuhan, Wuhan University, postcode 430072.
2. the application of a Dell Ford bacterium LW26 according to claim 1 in microbiological deterioration chlorobenzene.
3. apply as claimed in claim 2, it is characterized in that described application is seeded in the minimal medium containing chlorobenzene by Dell Ford bacterium LW26, in 23 ~ 40 DEG C, cultivate under the condition of pH4.0 ~ 10.0, realize the degraded to chlorobenzene.
4. apply as claimed in claim 3, it is characterized in that described chlorobenzene starting point concentration in minimal medium is 100 ~ 500mg/L; The OD that described Dell Ford bacterium LW26 obtains through minimal medium dilution with the wet thallus after seed culture or fermentation culture 600the bacteria suspension form of=0.1-0.5 adds, and bacteria suspension volume inoculum size is 1-5%.
5. apply as claimed in claim 3, it is characterized in that described minimal medium consists of: CaCl 20.023g/L, MgSO 40.2g/L, (NH 4) 2sO 42.5g/L, KH 2pO 41.0g/L, Na 2hPO 44.5g/L, micro-mother liquor 1mL/L, solvent is water, pH7.0 ~ 7.5; Described micro-mother liquor consists of: FeSO 47H 2o1.0g/L, CuSO 45H 2o0.02g/L, H 3bO 30.014g/L, MnSO 44H 2o0.10g/L, ZnSO 47H 2o0.10g/L, Na 2moO 42H 2o0.02g/L, CoCl 26H 2o0.02g/L, solvent is water.
6. apply as claimed in claim 4, it is characterized in that, described bacteria suspension obtains as follows:
(1) slant culture: Dell Ford bacterium LW26 is inoculated in R 2a solid medium, cultivates 48h for 28 ~ 32 DEG C, obtains inclined-plane thalline; Described R 2a solid medium consists of: yeast powder 0.50g/L, Tryptones 0.50g/L, casein food grade 0.50g/L, glucose 0.50g/L, Zulkovsky starch 0.50g/L, Sodium.alpha.-ketopropionate 0.30g/L, KH 2pO 40.45g/L, MgSO 40.05g/L, agar 15 ~ 18g/L, solvent is water, pH7.2;
(2) seed culture: picking one transfering loop colony inoculation is in seed culture medium from step (1) inclined-plane, cultivates 24 ~ 36h for 28 ~ 32 DEG C, obtains seed liquor; Described seed culture medium is R 2a liquid nutrient medium, except without except agar, all the other components and R 2a solid medium is identical;
(3) fermentation culture: the seed liquor that step (2) obtains is seeded to fermention medium with the inoculum size of volumetric concentration 5 ~ 10%, in 28 ~ 32 DEG C, cultivates 24 ~ 36h under the condition of pH6.0 ~ 8.0, obtains fermented liquid; Described fermention medium consists of: yeast powder 0.5g/L, CaCl 20.023g/L, MgSO 40.2g/L, (NH 4) 2sO 42.5g/L, KH 2pO 41.0g/L, Na 2hPO 44.5g/L, micro-mother liquor 1mL/L, solvent is water, pH7.0 ~ 7.5;
(4) collecting cells: by fermented liquid at the centrifugal 10min of 6000rpm, abandon supernatant liquor, with sterilized minimal medium cleaning thalline, then the centrifugal 10min of 6000rpm, abandon supernatant liquor, repeated washing 2 times, suspends the aseptic minimal medium of the wet thallus of acquisition, the bacteria suspension described in acquisition.
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