CN115093982A - Pseudomonas ZB30 and application thereof in styrene waste gas degradation - Google Patents
Pseudomonas ZB30 and application thereof in styrene waste gas degradation Download PDFInfo
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- B01D—SEPARATION
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- B01D53/34—Chemical or biological purification of waste gases
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- B01D53/34—Chemical or biological purification of waste gases
- B01D53/46—Removing components of defined structure
- B01D53/72—Organic compounds not provided for in groups B01D53/48 - B01D53/70, e.g. hydrocarbons
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- B01D—SEPARATION
- B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
- B01D53/34—Chemical or biological purification of waste gases
- B01D53/74—General processes for purification of waste gases; Apparatus or devices specially adapted therefor
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Abstract
The invention discloses pseudomonas ZB30 and application thereof in styrene waste gas degradation. The Pseudomonas sp fungus "ZB 30" of the present invention has the deposit number: CCTCC NO: M2021371. Application of Pseudomonas fungus ZB30 in degrading styrene waste gas is provided. The abundance of ZB30 fungus in the bioactive filler is artificially increased, so that the efficiency of the bioactive filler in removing the styrene-containing waste gas can be improved. The reactor added with the pseudomonas ZB30 has obviously improved absorption and removal efficiency for the styrene-containing waste gas after entering a stabilization period. The active filler after the pseudomonas ZB30 is added can shorten the start-up time of the biological trickling filter reactor.
Description
Technical Field
The invention belongs to the field of environmental pollution treatment, and relates to a Pseudomonas sp fungus ZB30 and application thereof in styrene waste gas biodegradation.
Background
Atmospheric pollution is one of the main problems of environmental pollution and is also a hot point of concern currently. Along with the development of social economy, the industry is developing more and more rapidly. Because the condition of air pollution caused by industrial emission is more and more serious, a large amount of stink waste gas such as styrene and the like can be generated in the actual industrial production process. Styrene belongs to volatile organic compounds and is mainly produced by industries such as plastics, paints, coatings, fine chemicals and the like. GB14554-93 'discharge Standard of malodorous substances' formulated in China lists styrene as a characteristic factor of malodorous waste gas. The discharge of the styrene waste gas not only causes pollution to soil, atmosphere and water resources, but also causes great potential harm to the physical and mental health of human beings. Styrene can invade human body through various ways such as respiratory system, skin and gastrointestinal tract, and the like, and causes harm to respiratory tract mucous membrane, nervous system, liver, lung and the like of human body. The patients with mild styrene can cause sore throat, rhinorrhea, cough, etc., and the patients with severe styrene can cause acute poisoning, and symptoms such as emesis, nausea, debilitation, giddiness, anorexia, abdominal distention and melancholy, etc. appear. Therefore, the treatment of the styrene waste gas has great significance for the environment and human health. With the continuous improvement of living standard, the environmental protection consciousness of people is continuously improved, the requirements on the living quality and the environmental conditions are higher and higher, and how to efficiently treat the styrene waste gas becomes the focus of attention of people.
The treatment method of the styrene waste gas can be divided into three methods: physical, chemical and biological methods. Among them, physical methods include adsorption, membrane separation, absorption, and the like; chemical methods include photocatalytic methods, thermal destruction methods, and the like; the biological method mainly comprises a biological trickling filtration method, a biological washing method, a biological filter method and the like. Although the physical method and the chemical method have many advantages, they have many disadvantages, such as high investment cost, complicated operation, and easy generation of secondary pollution. Compared with the traditional treatment method, the biological method for treating the waste gas has the advantages of simple equipment requirement, low operating cost, difficult generation of secondary pollution, good safety, high treatment efficiency and the like. One of the important methods for treating styrene waste gas by adopting a biological method is to screen out bacterial strains with the capacity of efficiently degrading styrene waste gas, and the method becomes a main method for treating foul waste gas at home and abroad. At present, few researches on the microbial degradation of styrene waste gas are reported.
Disclosure of Invention
It is an object of the present invention to provide a Pseudomonas sp fungus "ZB 30".
The invention is realized by the following technical scheme:
the Pseudomonas sp fungus ZB30 provided by the invention is preserved in China center for type culture Collection with the address: china, wuhan university, 430072, accession number: CCTCC NO, M2021371, preservation date: year 2021, 4 months and 14 days.
The biological characteristics of the strain are as follows: nearly round, regular edge, pale yellowish white, smooth surface, gram negative.
The technical scheme for screening the strains comprises the following steps:
the provided strain is obtained by screening from the activated sludge of a biological trickling filter reactor of an industrial enterprise. Taking 10g of acclimatized bioreactor sludge, diluting the sludge to 5 times, 50 times and 500 times of the sludge with double distilled water, and respectively coating the sludge on PDA fungus solid culture media. Coating 2mL of diluted sludge solution on each culture dish, culturing for 2 days at 32 ℃ in a constant temperature box, picking mycelia from the edges of bacterial colonies, transferring to a fresh PDA plate, performing streaking separation culture, repeating until a pure culture is obtained, and transferring to a PDA inclined plane for storage.
The 16S rDNA sequence of the Pseudomonas sp fungus ZB30 is as follows, SEQ ID NO. 1. The highest homology of 100.0% was found to be the highest with the Pseudomonas fungus (GeneBank accession No.: MN410633) by BLAST search in NCBI, so that this strain was identified as Pseudomonas sp.
SEQ ID NO.1：
CAGTCGAGCGGCAGCGGGTCCTTCGGGATGCCGGCGAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGC CTGGTAGTGGGGGATAACTCGGGGAAACTCGAGCTAATACCGCATACGTCCTACGGGAGAAAGCGGGGGATCTTC GGACCTCGCGCTACCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCTACGATCC GTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGG GGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAaGAAGgTCTTCGGATTGTAAAGCA CTTTAAGTTGGGAGGAAGGGCAGTAAGCTAATACCtTGCTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAA CTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGG TGGTTTGATAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAATTGCATCCAAAACTGTCTGACTAGAGTATG GCAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGAC CACCTGGGCTAATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGC CGTAAACGATGTCGACTAGCCGTTGGGATCCTTGAGATCTTAGTGGCGCAGCTAACGCATTAAGTCGACCGCCTG GGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAAT TCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATGCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGA ACTCTGACACAGgTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCA ACCCTTGTCCTTAGTTACCAGCACGTTAAGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTG GGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGC CAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAG TCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAC ACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAGTCTAACCTC
Another object of the present invention is to provide the use of the above Pseudomonas sp fungus "ZB 30" for the degradation of styrene-containing exhaust gases.
Preferably, the use of the Pseudomonas sp fungus "ZB 30" for the degradation purification of styrene off-gas in a biotrickling filter reactor.
The biological trickling filter tower reactor has the tower height of 8000mm, the tower diameter of 1500mm, the filler height of 1200mm and the waste gas flow of 3000m 3 H is the ratio of the total weight of the catalyst to the total weight of the catalyst. The spraying density is 10m 3 /(m 2 H) the residence time was 12 s. Injecting Pseudomonas fungus "ZB 30" onto the substrate, wherein the concentration of the fungus "ZB 30" in the substrate is not less than 3.5 × 10 7 CFU/g. The matrix composition is as follows: the mixture of the activated carbon, the ceramsite and the wood chips comprises the following components in percentage by mass: 1: 2.
the invention also aims to provide a styrene waste gas degradation method, which is to introduce the styrene waste gas into a biological trickling filter reactor, wherein the filler in the biological trickling filter reactor contains not less than 3.5 multiplied by 10 per gram of matrix 7 CFU/g of the Pseudomonas sp.
Preservation description:
the pseudomonad (Pseudomonas sp.) fungus "ZB 30" provided by the invention is preserved in China center for type culture Collection, address: china, wuhan university, 430072, deposit number: CCTCC NO, M2021371, preservation date: 21/4/2021.
The invention has the beneficial effects that:
(1) the Pseudomonas fungus ZB30 can reduce the concentration of styrene in foul waste gas, and has excellent treatment effect.
(2) The acclimation time of the active filler added with the pseudomonas fungus strain ZB30 is obviously shortened, and the biological trickling filter reactor can be quickly started;
(3) the method for degrading the pseudomonas fungus ZB30 in the styrene waste gas, which is provided by the invention, has simple operation;
(4) the pseudomonas strain ZB30 provided by the invention can be stored for a long time, can be continuously used after being activated after being stored in an ultra-low temperature refrigerator at-80 ℃ for one year, has no obvious reduction on the removal efficiency of styrene-containing waste gas, and has better preservability.
Drawings
FIG. 1 is a schematic view of a bioreactor configuration;
FIG. 2 is a colony morphology of the strain;
FIG. 3 is a phylogenetic tree diagram.
Detailed Description
In order to make the objects, techniques and features of the present invention more apparent, the present invention is further described in detail below with reference to specific examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1: isolated culture of Pseudomonas fungus "ZB 30
The provided strain is obtained by screening activated sludge from a biological trickling filter reactor (shown in figure 1) of an industrial enterprise. Taking 10g of acclimatized bioreactor sludge, diluting the sludge to 5 times, 50 times and 500 times of the sludge by using double distilled water, and respectively coating the sludge on a PDA fungus solid culture medium. Coating 2mL of diluted sludge solution on each culture dish, culturing for 2 days in a constant temperature box at 32 ℃, picking mycelium from the edge of a colony, transferring to a fresh PDA flat plate, performing streak isolation culture, repeating to obtain a pure culture, and transferring to a PDA inclined plane for storage.
Example 2: morphological and molecular biological identification of Pseudomonas fungus "ZB 30
According to the manual of fungal identification, morphological characteristics of pseudomonas fungus ZB30 are observed under a microscope, and the morphological characteristics of the strain including colony morphology, color, size, edge characteristics, hypha characteristics, growth speed and the like are observed by inoculating the screened strain on a PDA plate by a point seeding method, culturing at a constant temperature of 32 ℃ and observing with naked eyes.
The morphological characteristics of the Pseudomonas fungus "ZB 30" of the invention are as follows:
as shown in FIG. 2, the Pseudomonas fungus "ZB 30" strain was near round, regular, pale yellow-white, smooth in surface, gram-negative on PDA medium.
Example 3: molecular biological characterization of Pseudomonas fungus "ZB 30
1. Genomic DNA extraction
The method for extracting the genome DNA comprises the following steps: selecting 2mL of bacterial liquid, centrifuging for 3 minutes at 4 ℃ and 12000rpm, and collecting to obtain thalli; adding 600 μ L of 2 × CTAB (containing 2% β -mercaptoethanol), rapidly freezing in liquid nitrogen for 1 min, transferring to 64 deg.C water bath for 1 min, repeating the above process for 3 times, shaking at high speed for 2min, and water bath at 64 deg.C for 30 min; equal volume of chloroform was added: isoamyl alcohol (volume ratio 24:1), turning upside down and mixing uniformly, standing on ice for 3 minutes, then centrifuging for 15 minutes at 4 ℃ and 12000rpm, and taking the supernatant to a new centrifuge tube; adding isopropanol with the same volume into the supernate, slightly reversing the mixture up and down, uniformly mixing, standing on ice for 30 minutes, and then centrifuging for 5 minutes at 4 ℃ and 12000 rpm; discarding the supernatant, washing the precipitate with 75% absolute ethanol, air-drying at room temperature to obtain fungal DNA, and adding ddH 2 And O, dissolving the DNA precipitate, and storing in a refrigerator for later use.
2. ITS-PCR amplification and molecular identification of Pseudomonas fungus ZB30
The rRNA gene Internal Transcribed Spacer (ITS) of the fungal genome is amplified by using the fungus universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') as PCR amplification primers through a PCR technology. PCR reaction (20. mu.L): 2 XTaq PCR MasterMix 10. mu.L, upstream primer (10. mu. mol/L) 1. mu.L, downstream primer (10. mu. mol/L) 1. mu.L, DNA template (50ng/L) 1. mu.L, using ddH 2 The volume of O is filled to 20. mu.L. Then, PCR amplification is carried out on a PCR instrument, and the amplification conditions are as follows: pre-denaturation at 94 ℃ for 5 min; 32 cycles of: denaturation at 94 ℃ for 50 seconds, annealing at 58 ℃ for 50 seconds, and extension at 72 ℃ for 1 minute for 30 seconds; further extension at 72 ℃ for 10 minutes. Finally, the PCR product is purified and then sent to a sample for sequencing.
The nucleotide sequence obtained by sequencing was subjected to BLAST alignment in NCBI, and the alignment showed that the homology with Pseudomonas fungus "ZB 30" (GenBank accession No. MN410633) was the highest and 100.0%, so that the strain was identified as Pseudomonas fungus.
Example 4: phylogenetic analysis of Pseudomonas bacterium "ZB 30
The sequence of Pseudomonas fungus "ZB 30" obtained as described above was subjected to BLAST search at NCBI, and ITS sequence of known species similar thereto was downloaded for use in construction of phylogenetic trees. Performing multi-sequence comparison by using clustalx1.83, setting a Bootstrap value as 1000 by using MEGA7.0 software and a Neighbor Joining statistical method according to the comparison result, and constructing a phylogenetic tree based on an rDNA ITS sequence by using a Tamura-Nei model base substitution mode. A specific phylogenetic tree is shown in fig. 3.
Example 5: fermentation of Pseudomonas fungus ZB30 and preparation of filler
After being cultured and purified by PDA solid medium, pseudomonas bacteria ZB30 is inoculated to the fermentation medium, and the amplification culture is carried out according to the volume ratio of the seed solution to the fermentation medium of 1: 9. The fermentation medium is 10g/L of Tryton, 5g/L of Yeast extract and 10g/L of sodium chloride, and ddH is utilized 2 And (O) preparation. The temperature of fermentation culture is 32-37 deg.C, dissolved oxygen is greater than 1.6mg/L, pressure is 0.08MPa, and culture time is 48 h. Placing the fermentation liquor in a refrigerated centrifuge, centrifuging at 4 ℃ and 4000rpm for 30 minutes, removing supernatant, collecting precipitated thalli, and re-suspending the thalli by using fresh sterile culture solution to obtain fungus suspension with the final concentration of 0.8-1.0 g/L. The "ZB 30" suspension is adsorbed on the packing to complete the preparation of packing rich in Pseudomonas sp "ZB 30" fungus.
Example 6: pseudomonas bacteria ZB30 for improving the efficiency of the bio-trickling filter in absorbing and removing styrene-containing waste gas
Two sets of parallel biological trickling filter reactors (a control group and a treatment group) are designed, original activated sludge and activated sludge obtained by artificially adding fungi obtained in the embodiment are respectively adopted, and the difference of the absorption and removal efficiency of the two groups of equipment on the ammonia-containing waste gas in a working day period is detected. After two sets of equipment are selected and started for 10 days, sampling is carried out to determine required parameters. The styrene-containing concentration of the waste gas at the inlet of the reactor of the biological trickling filtration tower is 150mg/m respectively 3 、300mg/m 3 、450mg/m 3 、600 mg/m 3 And 750mg/m 3 And measuring the degradation rate of the styrene. The measurement results are shown in table 1. As can be seen from the data results in Table 1, the removal efficiency of the biotrickling filter reactor of the treatment group on the styrene-containing waste gas is between 98.5 and 99.8 percent, which is obviously higher than that of the control group.
Example 7: pseudomonas bacteria ZB30 for shortening start-up time of biological trickling filter reactor
Two sets of parallel biological trickling filter reactors of a control group and a treatment group are designed, original activated sludge and activated sludge obtained after fungus inoculation obtained in the embodiment are respectively adopted, and the styrene-containing waste gas removal efficiency from the first day to the 10 th day after the start is detected. The sampling time is fixed at 8 am, waste gas samples of an input port and an output port are extracted, and the content of ammonia gas is measured, wherein the unit is mg/m 3 . The measured data are shown in Table 2.
The results in table 2 show that the biotrickling filter reactor of the treatment group reached an efficiency of 97.6% already from day 6 and 99.8% by day 10; whereas the control group of biotrickling reactors reached 86.5% efficiency from day 6 and then gradually stabilized. The statistical results of the data in table 2 show that the microorganism genus fungus "ZB 21" can significantly shorten the start-up time of the biotrickling reactor.
The above description is only a preferred embodiment of the present invention and should not be taken as limiting the invention, and any modifications, equivalents, improvements and the like that are made within the spirit and principle of the present invention should be included in the scope of the present invention.
Sequence listing
<110> university of teachers in Hangzhou
<120> pseudomonas ZB30 and application thereof in styrene waste gas degradation
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1388
<212> DNA
<213> ZB30
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cagtcgagcg gcagcgggtc cttcgggatg ccggcgagcg gcggacgggt gagtaatgcc 60
taggaatctg cctggtagtg ggggataact cggggaaact cgagctaata ccgcatacgt 120
cctacgggag aaagcggggg atcttcggac ctcgcgctac cagatgagcc taggtcggat 180
tagctagttg gtgaggtaaa ggctcaccaa ggctacgatc cgtagctggt ctgagaggat 240
gatcagccac actggaactg agacacggtc cagactccta cgggaggcag cagtggggaa 300
tattggacaa tgggcgaaag cctgatccag ccatgccgcg tgtgtgaaga aggtcttcgg 360
attgtaaagc actttaagtt gggaggaagg gcagtaagct aataccttgc tgttttgacg 420
ttaccgacag aataagcacc ggctaacttc gtgccagcag ccgcggtaat acgaagggtg 480
caagcgttaa tcggaattac tgggcgtaaa gcgcgcgtag gtggtttgat aagttggatg 540
tgaaagcccc gggctcaacc tgggaattgc atccaaaact gtctgactag agtatggcag 600
agggtggtgg aatttcctgt gtagcggtga aatgcgtaga tataggaagg aacaccagtg 660
gcgaaggcga ccacctgggc taatactgac actgaggtgc gaaagcgtgg ggagcaaaca 720
ggattagata ccctggtagt ccacgccgta aacgatgtcg actagccgtt gggatccttg 780
agatcttagt ggcgcagcta acgcattaag tcgaccgcct ggggagtacg gccgcaaggt 840
taaaactcaa atgaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga 900
agcaacgcga agaaccttac caggccttga catgcagaga actttccaga gatggattgg 960
tgccttcggg aactctgaca caggtgctgc atggctgtcg tcagctcgtg tcgtgagatg 1020
ttgggttaag tcccgtaacg agcgcaaccc ttgtccttag ttaccagcac gttaaggtgg 1080
gcactctaag gagactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaagtcatc 1140
atggccctta cggcctgggc tacacacgtg ctacaatggt cggtacaaag ggttgccaag 1200
ccgcgaggtg gagctaatcc cataaaaccg atcgtagtcc ggatcgcagt ctgcaactcg 1260
actgcgtgaa gtcggaatcg ctagtaatcg tgaatcagaa tgtcacggtg aatacgttcc 1320
cgggccttgt acacaccgcc cgtcacacca tgggagtggg ttgctccaga agtagctagt 1380
ctaacctc 1388
Claims (10)
1. A fungal strain "ZB 30", characterized by being classified as a pseudomonas pseudomonasp, deposited at the chinese type culture collection, address: china, wuhan university, 430072, deposit number: CCTCCNO M2021371, preservation date: 14 days 4 months 2021; the biological characteristics are as follows: nearly round, regular edge, pale yellowish white, smooth surface, gram negative.
2. A fungal strain "ZB 30" according to claim 1, wherein the rDNA ITS sequence is as shown in SEQ ID NO: 1 is shown.
3. Use of a fungal strain "ZB 30" according to claim 1 or 2 for the degradation of styrene containing exhaust gases.
4. Use according to claim 3, for the degradation purification of styrene-containing waste gases in a biotrickling filter reactor.
5. Use according to claim 4, characterised in that the bio-trickling filter reactor contains not less than 3.5 x 10 filler per gram matrix 7 CFU/g according to claim 1The Pseudomonas sp fungus "ZB 30".
6. The use of claim 5, wherein the matrix is a mixture of activated carbon, ceramsite and wood chips, and the mass ratio of the mixture is 2: 1: 2.
7. use according to claim 3, wherein the operating parameters of the biotrickling filter reactor are: the height of the tower is 8000mm, the diameter of the tower is 1500mm, the height of the packing is 1200mm, and the waste gas flow is 3000m 3 H is used as the reference value. The spraying density is 10m 3 /(m 2 H) residence time of 12 s.
8. A styrene waste gas degradation method is characterized in that styrene waste gas is introduced into a biological trickling filter reactor, wherein the filler in the biological trickling filter reactor contains not less than 3.5 multiplied by 10 per gram of matrix 7 CFU/g of the Pseudomonas sp.
9. The method of claim 8, wherein the matrix is a mixture of activated carbon, ceramsite and wood chips, and the mass ratio of the mixture is 2: 1: 2.
10. the method of claim 8, wherein the operating parameters of the bio-trickling filter reactor are: the height of the tower is 8000mm, the diameter of the tower is 1500mm, the height of the packing is 1200mm, and the waste gas flow is 3000m 3 H is used as the reference value. The spraying density is 10m 3 /(m 2 H) the residence time was 12 s.
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