CN114015582A - Microbacterium ZB21 and application thereof in degrading trimethylamine waste gas - Google Patents
Microbacterium ZB21 and application thereof in degrading trimethylamine waste gas Download PDFInfo
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- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
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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
- B01D53/46—Removing components of defined structure
- B01D53/54—Nitrogen compounds
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- 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
- B01D53/84—Biological processes
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Abstract
The invention relates to a microbacterium ZB21 and application thereof in degrading trimethylamine waste gas. The fungus ZB21 of the genus Microbacterium (Exiguobacterium sp.) of the present invention has a deposit number: CCTCC NO: M2021370. Application of fungus ZB21 in degrading trimethylamine waste gas. Artificially increasing the abundance of ZB21 fungi in the bioactive filler can improve the efficiency of the bioactive filler in removing trimethylamine-containing waste gas. After the reactor added with the microbacterium ZB21 enters a stable period, the absorption and removal efficiency of the trimethylamine-containing waste gas is remarkably improved. The start-up time of the bio-trickling filter reactor can be shortened by adding the active filler after the micro-bacillus ZB21 is added.
Description
Technical Field
The technology belongs to the field of environmental pollution treatment, and relates to a strain of Microbacterium sp ZB21 and application thereof in trimethylamine waste gas biodegradation.
Background
Air pollution is a topic which is relatively concerned by people at present and is one of the main problems of environmental pollution. Along with the development of social economy, the industry is developing more and more rapidly. Accordingly, the air pollution caused by the industrial exhaust gas emission is more and more serious, and a large amount of foul waste gas such as trimethylamine is generated in the actual industrial production process. Trimethylamine is one of the main representatives of organic amine waste gas pollutants, and causes serious odor pollution to the atmospheric environment due to large emission and low odor threshold value. The emission of trimethylamine type malodorous exhaust gas has caused serious harm to the health and ecological environment of residents. Therefore, the treatment of trimethylamine waste gas has become a very urgent problem for controlling air pollution.
The traditional method for treating the foul waste gas such as trimethylamine is a physical and chemical method, but the methods have the defects of high operation cost, easy generation of secondary pollution and the like. Compared with the traditional physical and chemical methods, the biological method has the advantages of low energy consumption, low cost, difficult generation of secondary pollution and the like, and is gradually the main method for treating the malodorous waste gas at home and abroad at present. At present, many research reports are reported on the biological method for treating inorganic malodors such as ammonia gas, hydrogen sulfide and the like, and few research reports are reported on the biological method for treating organic malodors such as trimethylamine and the like.
The adoption of the biological trickling filter is a popular biological method for treating the industrial malodorous waste gas at present. A solid filler bed is arranged in the biological trickling filter reactor to culture microorganisms, the microorganisms are attached to the surface of the solid filler to grow to form a firm biological film, waste gas flows through the biological reactor, and harmful substances are adsorbed on the biological film and are degraded by the microorganisms. In order to reduce the debugging time of the biological trickling filter, special microorganisms need to be inoculated into the bottom mud in the starting stage of the foul waste gas removal reactor. The efficient fungus capable of absorbing and degrading trimethylamine is screened from the bottom mud of the biological trickling filter, and the fungus is cultured in an artificial mode and then added into a biological trickling filter reactor, so that the effect of improving the biological trickling filter reactor is achieved. In addition, the screened high-efficiency bacteria lay an important foundation for researching the biodegradation mechanism of the trimethylamine-containing waste gas.
Disclosure of Invention
An object of the present invention is to provide a microorganism belonging to the genus Microbacterium sp ZB21, which is superior to the prior art.
The invention is realized by the following technical scheme:
the fungus ZB21 of Microbacterium (Exiguobacterium sp.) provided by the invention is preserved in China center for type culture Collection with the address: china, wuhan university, 430072, accession number: CCTCC NO, M2021370, preservation date: 21/4/2021.
The biological characteristics of the strain are as follows: the bacteria is gram-positive rod-shaped bacteria, the bacterial colony is circular, the diameter is 1.8-3.6mm, the bacterial colony is yellow white, and the surface is smooth.
The fungus ZB21 strain is obtained by screening active sludge of a biological trickling filter reactor of an industrial enterprise.
The second purpose of the invention is to provide the application of the fungus ZB21 in degrading the trimethylamine-containing waste gas.
Preferably, the application of the microorganism bacillus fungus ZB21 in the biological trickling filter reactor degradation and purification of trimethylamine waste gas is carried out.
Preferably, the filler in the biological trickling filter reactor adopts a matrix and fungus ZB21, wherein each gram of the matrix contains not less than 4.0X 107CFU fungus ZB21, the substrate is activated carbon, ceramsite and wood chip according to the weight ratio of 1: 1: 2 in mass ratio.
Preferably, the operating set-up parameters of the trickling bio-filter reactor are as follows: 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 3000m3H; the spraying density is 12m3/(m2H) residence time of 10 s.
The third purpose of the invention is to provide a fermentation product which is a fermentation liquid or liquid microbial inoculum of ZB21 which is a fungus of the genus Microbacterium.
Preferably, the method for preparing the fermentation liquid of the microorganism bacillus fungus ZB21 is as follows:
inoculating the purified Microbacterium fungus ZB21 cultured in PDA solid culture medium to the fermentation culture medium, and performing amplification culture according to the volume ratio of the seed solution to the fermentation culture medium of 1: 9; wherein the fermentation culture temperature is 32-37 deg.C, dissolved oxygen is greater than 1.8mg/L, pressure is 0.08MPa, and culture time is 60 h.
The fermentation medium comprises yeast powder 10.0g/L, peptone 10.0g/L, NaCl 8.0.0 g/L, and agar 15.0 g/L.
Preferably, the preparation method of the liquid microbial inoculum of the microorganism bacillus fungus ZB21 is as follows:
and (3) placing the fermentation liquor in a 4 ℃ ultra-low temperature centrifuge at 4000rpm, centrifuging for 15min, removing supernatant, collecting precipitated thalli, and re-suspending the thalli by using fresh sterile culture solution to obtain a liquid microbial inoculum with the final concentration of fungi of 0.5-0.8 g/L. The temperature of fermentation culture is 32-37 deg.C, dissolved oxygen is greater than 1.8mg/L, pressure is 0.08MPa, and culture time is 60 h.
The fermentation medium comprises yeast powder 10.0g/L, peptone 10.0g/L, NaCl 8.0.0 g/L, and agar 15.0 g/L.
Preservation description:
the strain of the microorganism bacillus (Exiguobacterium sp.) of the present invention, ZB21, was deposited in the chinese type culture collection with the following addresses: china, wuhan university, 430072, accession number: CCTCC NO, M2021370, preservation date: 21/4/2021.
The invention has the beneficial effects that:
(1) the screened microbacillus fungus strain ZB21 can obviously reduce the concentration of trimethylamine in the foul waste gas and has excellent treatment effect;
(2) the acclimation time of the active filler added with the microorganism bacillus fungus strain ZB21 is obviously shortened, and the biological trickling filter reactor can be quickly started;
(3) the application method of the microorganism bacillus fungus strain ZB21 in degrading the trimethylamine waste gas, provided by the invention, is simple to operate;
(4) the screened Microbacterium fungus ZB21 strain can be stored for a long time, can be continuously used after being activated after being stored in an ultra-low temperature refrigerator at minus 80 ℃ for one year, has no obvious reduction on the removal efficiency of trimethylamine-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 present patent and are not intended to limit the present invention.
Example 1: isolated culture of Microbacterium sp ZB21
The bio-trickling filter reactor involved in the isolation of the microorganism bacterium fungus ZB21 is shown in FIG. 1.
The separation method comprises the following steps: taking 15g of acclimatized bioreactor sludge, diluting the sludge to three different concentration gradients of 5 times, 50 times and 500 times 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 3 days in a constant temperature box at 32 ℃, picking mycelium from the edge of a colony, transferring to a fresh PDA plate, carrying out streaking separation culture, repeating until a pure culture is obtained, and transferring to a PDA inclined plane for storage.
Example 2: morphological and molecular biological identification of Microbacterium fungus ZB21
According to the manual of fungal identification, morphological characteristics of the Microbacterium fungi ZB21 were observed under a microscope, specifically by inoculating the selected strains on a PDA plate by a dibbling method, culturing at a constant temperature of 32 ℃, and visually observing morphological characteristics of the strains including colony morphology, color, size, edge characteristics, hyphal character, growth rate, and the like.
The morphological characteristics of the microorganism genus fungus ZB21 of the present invention are as follows:
as shown in FIG. 2, the strain of Microbacterium sp ZB21 was cultured on PDA medium, and the bacterium was a gram-positive rod-shaped bacterium, and the colony had a circular shape, a diameter of 1.8-3.6mm, a yellow-white color, and a smooth surface.
Example 3: molecular biological identification of Microbacterium fungus ZB21
1. Genomic DNA extraction
The method for extracting the genome DNA comprises the following steps: (1) selecting 2mL of bacterial liquid, centrifuging for 3 minutes at 4 ℃ and 12000rpm, and collecting to obtain thalli; (2) adding 600 μ L2 × CTAB (containing 2% β -mercaptoethanol), quickly 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; (3) 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; (4) adding isopropanol with the same volume into the supernatant, 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; (5) discarding the supernatant, washing the precipitate with 75% absolute ethanol, air-drying at room temperature to obtain fungal DNA, and adding ddH2And O, dissolving the DNA precipitate, and storing in a refrigerator for later use.
2. Micro bacillus fungus ZB21 ITS-PCR amplification and molecular identification
The rRNA gene Internal Transcribed Spacer (ITS) of the fungal genome is amplified by PCR technology by using the fungus universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', shown as SEQ ID NO.2) and 1492R (5'-TACGGCTACCTTGTTACGACTT-3', shown as SEQ ID NO. 3).
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 ddH2The volume of O was replenished to 20. mu.L.
Amplification conditions for PCR amplification on a PCR instrument: 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, purifying the PCR product and then sending the PCR product to a sample for sequencing; the nucleotide sequence obtained by sequencing was subjected to BLAST comparison in NCBI, and the comparison result showed that the homology with Microbacterium fungus (GenBank accession MH845736) was the highest and 99.86%, so that the strain was identified as Microbacterium fungus. The 16S rDNA sequence of the Microbacterium sp ZB21 fungus is shown as SEQ ID NO. 1.
Example 4: phylogenetic analysis of Microbacterium fungus ZB21
The obtained ZB21 sequence of the microorganism bacillus was subjected to BLAST search at NCBI, and ITS sequence of known species similar to the sequence was downloaded for use in construction of phylogenetic trees. Performing multi-sequence alignment by using clustalx1.83, setting a Bootstrap value as 1000 by using MEGA7.0 software and a Neighbor Joining statistical method according to the alignment 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 Microbacterium fungus ZB21 and preparation of filler
After being cultured and purified by PDA solid medium, the fungus ZB21 of the genus Microbacterium is inoculated to the fermentation medium and the seed solution and the fermentation medium are cultured in an enlarged scale at a volume ratio of 1: 9. The fermentation medium comprises yeast powder 10.0g/L, peptone 10.0g/L, NaCl 8.0.0 g/L, and agar 15.0 g/L. The temperature of fermentation culture is 32-37 deg.C, dissolved oxygen is greater than 1.8mg/L, pressure is 0.08MPa, and culture time is 60 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 ZB21 suspension was adsorbed onto the filler to complete the preparation of a filler enriched in ZB21 fungus.
Example 6: microbacterium fungus ZB21 for improving absorption and removal efficiency of trimethylamine-containing waste gas in biological trickling filter reactor
Two sets of parallel biotrickling filter reactors (control and treatment) were designed, each with a raw packing base without ZB21 fungusAnd mixing the activated sludge inoculated with the fungi obtained in the previous embodiment with a filler matrix by manual addition, and detecting the difference of absorption and removal efficiency of the two groups of equipment on the trimethylamine-containing waste gas in a working day period. After two sets of equipment are selected and started for 7 days, sampling is carried out to determine the required parameters. The trimethylamine concentration of the waste gas at the input port of the biological trickling filter reactor is respectively 200mg/m3、400mg/m3、600mg/m3、800mg/m3And 1000mg/m3And determining the degradation rate of trimethylamine. The measurement results are shown in table 1. As can be seen from the data results in Table 1, the removal efficiency of the treatment group bio-trickling filter reactor on the trimethylamine-containing waste gas is between 99.3% and 99.9%, which is significantly higher than that of the control group.
Example 7: method for shortening start-up time of biological trickling filter reactor by using microorganism bacillus fungus ZB21
Two sets of parallel biological trickling filter reactors of a control group and a treatment group are designed, original filler matrixes without ZB21 fungi and activated sludge inoculated by the fungi obtained in the previous examples are mixed in the filler matrixes by manual addition, and the trimethylamine-containing waste gas removal efficiency from the first day of starting to the 8 th day of starting is tested. Sampling at 8 am every day, sampling the waste gas at inlet and outlet, and measuring the content of trimethyl benzene gas in mg/m3. The measured data are shown in Table 2.
The results in table 2 show that the biotrickling reactor of the treatment group reached 95.4% efficiency from day 4 and 99.9% efficiency by day 7; whereas the control group of biotrickling reactors reached 87.6% efficiency from day 6 and then gradually stabilized. The statistical results of the data in table 2 show that the microorganism bacillus fungus ZB21 can greatly shorten the start-up time of the biotrickling reactor.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that are within the spirit and principle of the present invention are intended to be included in the scope of the present invention.
Sequence listing
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<213> fungus ZB21(Exiguobacterium)
<400> 1
atgcagtcga gcgcaggagc cgtctgaacc cttcgggggg acgacggtgg aatgagcggc 60
ggacgggtga gtaacacgta aagaacctgc ccataggtct gggataacca cgagaaatcg 120
gggctaatac cggatgtgtc atcggaccgc atggtccgct gatgaaaggc gctccggcgt 180
cgcccatgga tggctttgcg gtgcattagc tagttggtgg ggtaacggcc caccaaggcg 240
acgatgcata gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga 300
ctcctacggg aggcagcagt agggaatctt ccacaatgga cgaaagtctg atggagcaac 360
gccgcgtgaa cgatgaaggc tttcgggtcg taaagttctg ttgtaaggga agaacaagtg 420
ccgcaggcaa tggcggcacc ttgacggtac cttgcgagaa agccacggct aactacgtgc 480
cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagcgc 540
gcgcaggcgg cctcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggccattg 600
gaaactggga ggcttgagta taggagagaa gagtggaatt ccacgtgtag cggtgaaatg 660
cgtagagatg tggaggaaca ccagtggcga aggcgactct ttggcctata actgacgctg 720
aggcgcgaaa gcgtggggag caaacaggat tagataccct ggtagtccac gccgtaaacg 780
atgagtgcta ggtgttggag ggtttccgcc cttcagtgct gaagctaacg cattaagcac 840
tccgcctggg gagtacggtc gcaaggctga aactcaaagg aattgacggg gacccgcaca 900
agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccaa ctcttgacat 960
ccccctgacc ggtacagaga tgtatcttcc ccttcggggg caggggtgac aggtggtgca 1020
tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1080
tgtccttagt tgccagcatt tggttgggca ctctagggag actgccggtg acaaaccgga 1140
ggaaggtggg gatgacgtca aatcatcatg ccccttatga gttgggctac acacgtgcta 1200
caatggacgg tacaaagggc agcgaagccg cgaggtggag ccaatcccag aaagccgttc 1260
tcagttcgga ttgcaggctg caactcgcct gcatgaagtc ggaatcgcta gtaatcgcag 1320
gtcagcatac tgcggtgaat acgttcccgg gtcttgtaca caccgcccgt cacaccacga 1380
gagtttgcaa cacccgaagt cggtgaggta accgtaagag ccag 1424
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (Unknown)
<400> 2
agagtttgat cctggctcag 20
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence (Unknown)
<400> 3
tacggctacc ttgttacgac tt 22
Claims (10)
1. A fungal strain ZB21, characterized by being classified as a fungus of the genus microbacterium, classified under the name Exiguobacterium sp.zb21, deposited at the chinese type culture collection with the address: china, wuhan university, 430072, accession number: CCTCC NO, M2021370, preservation date: 21/4/2021;
the biological characteristics are as follows: the bacteria is gram-positive rod-shaped bacteria, the bacterial colony is circular, the diameter is 1.8-3.6mm, the bacterial colony is yellow white, and the surface is smooth.
2. The fungal strain ZB21, according to claim 1, wherein the rDNA ITS sequence is as set forth in SEQ ID NO: 1 is shown.
3. Use of a fungal strain for the degradation of trimethylamine-containing exhaust gas, wherein the fungal strain comprises the fungal strain ZB21 according to claim 1.
4. The use according to claim 3, wherein the trimethylamine-containing waste gas is degraded and purified in a bio-trickling filter reactor.
5. The use according to claim 4, wherein the bio-trickling filter reactor is packed with a matrix and the fungus ZB21, wherein the matrix comprises not less than 4.0 x 10/g7CFU fungus ZB 21.
6. The application of claim 5, wherein the substrate is a mixture of activated carbon, ceramsite and wood chips, and the mass ratio of the mixture is 1: 1: 2.
7. use according to any one of claims 3 to 6, 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 3000m3H; the spraying density is 12m3/(m2H) residence time of 10 s.
8. A fermented product characterized by being a fermentation broth of the fungal strain ZB21 of claim 1 or a liquid inoculum of the fungal strain ZB21 of claim 1.
9. A fermented product according to claim 8, wherein the fermentation broth of the fungal strain ZB21 is prepared by the process comprising:
inoculating a fungus strain ZB21 of claim 1 after PDA solid medium culture purification to a fermentation medium, and performing amplification culture according to the volume ratio of seed liquid to fermentation medium of 1: 9; wherein the fermentation culture temperature is 32-37 deg.C, dissolved oxygen is greater than 1.8mg/L, pressure is 0.08MPa, and culture time is 60 h; the fermentation medium comprises yeast powder 10.0g/L, peptone 10.0g/L, NaCl 8.0.0 g/L, and agar 15.0 g/L.
10. The fermented product according to claim 9, wherein the liquid microbial inoculum of the fungal strain ZB21 is prepared by the following method:
placing fermentation liquor of a fungus strain ZB21 in a 4 ℃ ultra-low temperature centrifuge at 4000rpm, centrifuging for 15min, removing supernatant, collecting precipitated thallus, and re-suspending thallus with fresh sterile culture solution to obtain liquid microbial inoculum with final concentration of fungus of 0.5-0.8 g/L; wherein the fermentation culture temperature is 32-37 deg.C, dissolved oxygen is greater than 1.8mg/L, pressure is 0.08MPa, and culture time is 60 h;
the fermentation medium comprises yeast powder 10.0g/L, peptone 10.0g/L, NaCl 8.0.0 g/L, and agar 15.0 g/L.
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