CN109735476B - Formaldehyde degrading strain and application thereof - Google Patents
Formaldehyde degrading strain and application thereof Download PDFInfo
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- CN109735476B CN109735476B CN201910215095.1A CN201910215095A CN109735476B CN 109735476 B CN109735476 B CN 109735476B CN 201910215095 A CN201910215095 A CN 201910215095A CN 109735476 B CN109735476 B CN 109735476B
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Abstract
The invention provides a strain of Methylobacterium (A), (B), (C)Methylobacterium podarium) The preservation number of the bacterium JQ-1 in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms is CGMCC No.17229. The strain has good tolerance to high-concentration formaldehyde, can grow in a culture medium with formaldehyde as a unique C source, and can completely degrade 54.8mg/L of formaldehyde within 120 h. The invention also provides a culture medium and a culture method of the strain. The invention provides a core excellent strain for effectively degrading formaldehyde, reduces the cost for degrading formaldehyde, saves resources, has no secondary pollution, is suitable for large-scale fermentation production and can be used for a long time.
Description
Technical Field
The invention relates to the field of applied microorganisms, in particular to a formaldehyde degrading strain and application thereof.
Background
The formaldehyde can denature protein, has carcinogenic, teratogenic and disabling effects, and can influence human health due to overproof formaldehyde in the air, and no effective treatment method for formaldehyde in indoor air exists at present.
Currently, there are 2 mainstream processing methods, the first one is: an adsorption method. The adsorption method needs corresponding adsorbents such as attapulgite, activated carbon and the like. The most common method is activated carbon adsorption, which is also a common method for removing formaldehyde in household rooms. The adsorption method is divided into natural adsorption and forced adsorption, wherein the natural adsorption method is to arrange an adsorption medium in a room and contact the adsorption medium through natural flow of air to realize limited-efficiency adsorption. The method has no effect on over-standard formaldehyde or the effect is small and can be ignored, the main reasons are that the air flow is limited, the contact amount of the active carbon (or other adsorption media, hereinafter referred to as active carbon) to the air is small, the contact area is small, and the contact probability of the formaldehyde and the active carbon is very small. The shallow contact may be repelled by a saturated layer on the surface of the activated carbon, and the adsorption reaction may not be sufficiently performed. Therefore, the formaldehyde removal effect of the natural adsorption method is very limited. The passive adsorption means that formaldehyde in the air enters an activated carbon chamber filled with activated carbon in a forced ventilation mode, an adsorption reaction occurs in the activated carbon chamber, the air can be in relatively complete contact with the activated carbon, and the activated carbon filters the air and adsorbs organic molecules such as formaldehyde in the air. The method has the defects that the adsorption effect of the activated carbon can be saturated, the saturated activated carbon can not continuously adsorb formaldehyde, and can release formaldehyde into the air to form a pollution source and cause secondary pollution, and the formaldehyde can be replaced periodically, so that the activated carbon becomes a consumable material, the demand is extremely high, the price of the activated carbon is high, and great resource waste is caused.
And the second method comprises the following steps: and (3) an oxidation removal method. The basic principle of the method is an oxidation method, and the method mainly comprises methods such as catalytic oxidation, plasma oxidation, high-temperature combustion oxidation, ozone oxidation and the like. High-temperature combustion oxidation: all gas components are heated to over 800 ℃ without difference by utilizing electric energy or combustion heat production, so that oxygen in the air and formaldehyde are subjected to oxidation-reduction reaction to generate H 2 O and CO 2 . However, this method is high in energy consumption and high in equipment cost, has an influence on the indoor air temperature, and is basically not suitable for practical application. Plasma oxidation method: by using the principle of high-voltage gas dischargeAir components are ionized indiscriminately, and formaldehyde ionized with oxygen undergoes oxidation-reduction reaction to generate H 2 O and CO 2 . The method also needs to consume a large amount of electric energy, has high-voltage potential safety hazard, and is not suitable for practical application because the method needs large floor area and heavy equipment volume for non-specific removal of formaldehyde; catalytic oxidation removal, such as photocatalytic oxidation, requires UV irradiation of TiO 2 The coating has limited reaction area, low oxidation efficiency, ultraviolet pollution and low practical value; the ozone oxidation method is characterized in that the ozone generated by an ozone generator is used for oxidizing formaldehyde gas molecules, the ozone generator has certain potential safety hazard due to the fact that extreme conditions such as high voltage electricity or ultraviolet rays are needed, and then redundant harmful gas O can be generated 3 Secondary pollution is generated, and the method has almost no practical value. The two methods have no ideal effect on removing formaldehyde in the air.
Disclosure of Invention
The invention aims to provide a technical scheme for overcoming the defects of the prior art, namely a formaldehyde degrading strain and application thereof.
The invention aims to provide a methylobacterium bacteriumMethylobacterium podariumJQ-1 (hereinafter referred to as JQ-1), the preservation number of JQ-1 strain in China general microbiological culture Collection center is CGMCC No.17229.
JQ-1 is characterized in that the strain can degrade formaldehyde. JQ-1 can completely degrade 54.8mg/L of formaldehyde within 120h under the condition that the OD value is 0.107 under the condition that the cell concentration is 30 ℃ in a known component inorganic culture medium taking the formaldehyde as the only C source. And the maximum tolerance concentration of JQ-1 to formaldehyde is: 120mg/L.
The invention also aims to provide a culture medium and culture conditions of JQ-1. Culture medium: formaldehyde 50mg/L, NH 4 Cl 0.5g/L、Na 2 HPO 4 3.0g/L、KH 2 PO 4 1.5g/L、MgSO 4 ·7H 2 O 0.1g/L、1M CaCl 2 0.1g/L; the culture conditions are as follows: inoculating 3-5%, culturing at 30 deg.C and 120rpm in dark.
The invention aims to provide a JQ-1 strain and application thereof in formaldehyde removal, and particularly provides a microorganism formaldehyde removal product taking JQ-1 as a core microbial inoculum.
Compared with the prior art, the invention has the following advantages and effects: the JQ-1 strain has good tolerance to high-concentration formaldehyde, and can be used for treating industrial wastewater containing high-concentration formaldehyde and industrial waste gas containing high-concentration formaldehyde; the JQ-1 strain degrades formaldehyde, and belongs to a biological metabolism method for removing formaldehyde, so that secondary pollution can not be generated; the determination of the special culture medium and the culture conditions ensures the excellent proliferation and subculture of the JQ-1 strain, can be fermented and produced in a large scale and used for a long time, and reduces the cost; the JQ-1 strain does not generate adsorption saturation effect, and is convenient to operate and maintain.
Preservation description:
the strain name is as follows: methylobacterium sp
Latin name:Methylobacterium podarium
the strain number is as follows:Methylobacterium podarium JQ-1
the preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 24 days in 2019, 1 month
Registration number of the preservation center: CGMCC No.17229
The sequencing result of JQ-1 shows that: the sequence is 16S ribosomal RNA gene, the sequence length is 1417bp, the sequence attachment sequence table shows, the NCBI database blast result: methylobacterium podarium strain LM-Pink 16S ribosomal RNA gene, partial sequence identity =99.9%.
Drawings
FIG. 1 is a photograph of a colony of the JQ-1 strain isolated in example 1.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The media used in the following examples are as follows:
formaldehyde culture medium: formaldehyde 50mg/L, NH 4 Cl 0.5g/L、Na 2 HPO 4 3.0g/L、KH 2 PO 4 1.5g/L、MgSO 4 ·7H 2 O 0.1g/L、1M CaCl 2 0.1g/L。
Example 1 isolation and characterization experiment of JQ-1
Preparing a formaldehyde culture medium, adding 20g/L of agarose into the culture medium, sterilizing at 120 ℃ for 30min, and cooling in a culture dish to form a flat plate. Diluting activated sludge containing formaldehyde organic industrial wastewater with sterile water, coating the diluted activated sludge on a culture plate, and culturing at 30 ℃ in a dark place. The culture is subjected to streak separation and purification to obtain JQ-1.
And (3) culturing the strain JQ-1 separated and purified in the steps by adopting a formaldehyde culture medium, extracting the total DNA of the strain to be used as a gene amplification template, and carrying out PCR reaction on a PCR amplification instrument by using a bacterial 16s rDNA universal primer. After the reaction, 2ul of PCR product was subjected to 1% agarose gel electrophoresis. Confirming the PCR amplified fragment. The PCR product was recovered using AxyPrep DNA gel recovery kit, and the specific procedures were performed according to the kit instructions. And taking the PCR product after each strain purification, and carrying out DNA sequencing by using a sequencer ABI 3730-XL. After homology comparison with known sequences in NCBI GenBank, the bacterial species were determined and the bacteria were classified into genus or species.
PCR gene amplification to obtain 16s rDNA gene fragment of strain JQ-1 of about 1.4kb, after sequence determination, on-line homology comparison with 16s rDNA sequence published in NCBI database shows that JQ-1 and JQ-1 have homology with each otherMethylobacterium podarium strainThe homology of the gene is the highest and reaches 99 percent.
Example 2 degradation experiment of JQ-1 on Formaldehyde
Sterilizing the formaldehyde culture medium at high temperature, standing to normal temperature, inoculating the strain JQ-1 with the inoculum size of 3-5%, culturing at 30 deg.C and 120rpm, and culturing in dark place. OD600 values and formaldehyde concentrations were measured at 0h,48h and 120h, respectively. The OD600 value can be obtained by a spectrophotometer method, which indirectly represents the cell concentration. A blank control group 1 was also set. The determination of the concentration of formaldehyde in the solution adopts the national environmental protection standard of the people's republic of China: measurement of Formaldehyde in Water by acetylacetone spectrophotometry HJ 601-2011. The results are as follows.
TABLE 1 growth status of cells
TABLE 2 Formaldehyde change in concentration
The experiment proves that: the JQ-1 strain has the degradation capability on formaldehyde in a solution; 2. at 30 ℃ and the OD value of the cell body concentration is 0.107, JQ-1 can completely degrade 54.8mg/L of formaldehyde within 120 h.
Example 3 resistance test of JQ-1 to Formaldehyde
In order to verify the tolerance degree of the strain of the invention to the concentration of formaldehyde, a series of formaldehyde-containing complete nutrient media with concentration gradients are prepared, and the formula of the media is as follows: glucose 10.0 g/L, NH 4 Cl 0.5g/L、Na 2 HPO 4 3.0g/L、KH 2 PO 4 1.5g/L、MgSO 4 ·7H 2 O 0.1g/L、1M CaCl 2 0.1g/L, agar g/L. Diluting the formaldehyde reagent by using an aseptic filter membrane to form 1000x formaldehyde standard solution. And (3) respectively packaging formaldehyde with different concentrations in different triangular flasks. The final concentration gradient formed was: 50. 75, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220mg/L of formaldehyde-containing medium. Sterilizing at 120 deg.C for 30min, cooling in culture dish to form plate, selecting the bacteria on the colony of pure culture, and coating to obtain colony capable of forming proliferation.
As a result: JQ-1 can positively proliferate and grow in a formaldehyde-containing culture medium of 50-120 mg/L. The highest formaldehyde tolerance concentration of the strain of the invention is judged as follows: 120mg/L.
Sequence listing
<110> institute of biological research of science institute of Gansu province
<120> formaldehyde degrading strain and application thereof
<130> 1
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1417
<212> DNA
<213> Methylobacterium podarium
<400> 1
ctcagagcga acgctggcgg caggcttaac acatgcaagt cgaacgggct tcttcggaag 60
tcagtggcag acgggtgagt aacacgtggg aacgtgccct tcggttcgga ataactcagg 120
gaaacttgag ctaataccgg atacgccctt acggggaaag gtttaccgcc gaaggatcgg 180
cccgcgtctg attagcttgt tggtggggta acggcctacc aaggcgacga tcagtagctg 240
gtctgagagg atgatcagcc acactgggac tgagacacgg cccagactcc tacgggaggc 300
agcagtgggg aatattggac aatgggcgca agcctgatcc agccatgccg cgtgagtgat 360
gaaggcctta gggttgtaaa gctcttttgt ccgggacgat aatgacggta ccggaagaat 420
aagccccggc taacttcgtg ccagcagccg cggtaatacg aagggggcta gcgttgctcg 480
gaatcactgg gcgtaaaggg cgcgtaggcg gccgattaag tcgggggtga aagcctgtgg 540
ctcaaccaca gaattgcctt cgatactggt tggcttgaga ccggaagagg acagcggaac 600
tgcgagtgta gaggtgaaat tcgtagatat tcgcaagaac accagtggcg aaggcggctg 660
tctggtccgg ttctgacgct gaggcgcgaa agcgtgggga gcaaacagga ttagataccc 720
tggtagtcca cgccgtaaac gatgaatgcc agccgttggt ctgcttgcag gtcagtggcg 780
ccgctaacgc attaagcatt ccgcctgggg agtacggtcg caagattaaa actcaaagga 840
attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgcagaa 900
ccttaccatc ccttgacatg gcatgttacc ctgggagacc ggggatcctc ttcggaggcg 960
tgcacacagg tgctgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1020
gcaacgagcg caacccacgt ccttagttgc catcattgag ttgggcactc tagggagact 1080
gccggtgata agccgcgagg aaggtgtgga tgacgtcaag tcctcatggc ccttacggga 1140
tgggctacac acgtgctaca atggcggtga cagagggacg cgaaggggcg acctggagca 1200
aatcccgaaa agccgtctca gttcggattg cactctgcaa ctcgggtgca tgaaggcgga 1260
atcgctagta atcgtggatc agcacgccac ggtgaatacg ttcccgggcc ttgtacacac 1320
cgcccgtcac accatgggag ttggtcttac ccgacggcgc tgcgccaacc gcaaggaggc 1380
aggcgaccac ggtagggtca gcgactgggg tgaagtc 1417
Claims (6)
1. Methylobacterium (A)Methylobacterium podarium) The preservation number of the bacterium JQ-1 in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms is CGMCC No.17229.
2. The Methylobacillus bacterium JQ-1 according to claim 1, wherein the bacterium is capable of degrading formaldehyde.
3. The Methylobacillus bacterium JQ-1 of claim 1, wherein the strain has a maximum tolerance concentration to formaldehyde of: 120mg/L.
4. The Methylobacillus bacterium JQ-1 of claim 1, wherein the strain completely degrades 54.8mg/L formaldehyde within 120h at 30 ℃ and the OD of the strain is 0.107.
5. The method for culturing Methylobacillus bacterium JQ-1 according to claim 1, wherein the culture is carried out at 30 ℃ and 120rpm in the absence of light at a ratio of 3 to 5%.
6. A microorganism formaldehyde removal product, characterized in that the product uses the Methylobacillus JQ-1 as the core microbial inoculum of claim 1 to remove formaldehyde.
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| CN102586149A (en) * | 2012-03-01 | 2012-07-18 | 黑龙江省科学院微生物研究所 | Methyl bacterium capable of degrading dichloromethane |
| CN102816714A (en) * | 2012-07-09 | 2012-12-12 | 浙江工业大学 | Formaldehyde degrading bacterium and its application |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101993839A (en) * | 2010-07-23 | 2011-03-30 | 浙江工业大学 | Methylobacterium rhodesianum H13 capable of efficiently degrading dichloromethane and application thereof |
| CN102533620A (en) * | 2012-03-01 | 2012-07-04 | 黑龙江省科学院微生物研究所 | Methyl bacterial strain for degrading methane gas |
| CN102586149A (en) * | 2012-03-01 | 2012-07-18 | 黑龙江省科学院微生物研究所 | Methyl bacterium capable of degrading dichloromethane |
| CN102816714A (en) * | 2012-07-09 | 2012-12-12 | 浙江工业大学 | Formaldehyde degrading bacterium and its application |
Non-Patent Citations (5)
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| Biodegradation of high concentrations of formaldehyde by lyophilized cells of Methylobacterium sp. FD1;Hiroshi Yonemitsu等;《Biosci Biotechnol Biochem》;20161130;2264-2270 * |
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| 甲基营养菌的研究进展;晁红军等;《微生物学通报》;20091231;1727-1737 * |
| 甲醛降解菌的分离、鉴定、降解条件优化及应用研究;牛成洁;《中国优秀硕士学位论文全文数据库》;20180515;B027-102 * |
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