CN111808770B - Air bacillus for degrading crude oil and application thereof - Google Patents

Air bacillus for degrading crude oil and application thereof Download PDF

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Publication number
CN111808770B
CN111808770B CN202010649671.6A CN202010649671A CN111808770B CN 111808770 B CN111808770 B CN 111808770B CN 202010649671 A CN202010649671 A CN 202010649671A CN 111808770 B CN111808770 B CN 111808770B
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bacillus
crude oil
oil
medium
culture medium
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CN111808770A (en
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孙立恒
吕福锦
杨帆
龙旭伟
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Nanjing University of Science and Technology
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Nanjing University of Science and Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Abstract

The invention relates to bacillus aerosidum for degrading crude oil and application thereof, belonging to the technical field of microorganisms. The strain is bacillus aerobica (A), (B), (C)Bacillus aerius) NUST-1, which has been registered and preserved in China general microbiological culture Collection center (CGMCC) at 12.12.2019, with the preservation number of CGMCC No. 19124. The bacillus aerosidus NUST-01 disclosed by the invention has high-efficiency crude oil degradation capability, can be used for bioremediation of crude oil contaminated soil, and has the characteristics of high efficiency, no pollution, difficulty in generation of drug resistance and the like.

Description

Air bacillus for degrading crude oil and application thereof
Technical Field
The invention belongs to the technical field of microorganisms and biology, and particularly relates to bacillus aerosus capable of degrading crude oil and application thereof.
Background
With the continuous development of social economy, people have more and more dependence on crude oil resources, a large amount of oil-containing pollutants are generated in the processes of crude oil exploitation, storage, transportation and processing, most of the pollutants contain toxic crude oil components such as long-chain alkane, aromatic hydrocarbon and the like, serious harm is caused to human health and ecological environment, and therefore the pollutants cannot be directly discharged and need to be properly treated.
Today, treatment methods for crude oil contaminants are largely classified into 3 types: physical, chemical and biological methods. The conventional physical method is mainly used for removing crude oil components from water by air flotation, flocculation, membrane separation, adsorption and other methods, but has the problems of low efficiency, high cost, incapability of meeting the actual production requirement and the like. Although the crude oil pollutants can be removed more efficiently by means of chemical methods of incineration and solvent extraction, a large amount of toxic and harmful gases are generated in the incineration process, so that serious secondary pollution is caused; the problems of operation danger, large use of toxic and harmful organic solvents and the like exist in the solvent extraction process, and serious secondary pollution is also brought. Biodegradation method using microorganism as antigenOil component is degraded and finally treated with CO2The method has the characteristics of green, environment-friendly, non-toxic, harmless, environment-friendly and the like, and is a technology with the most application prospect for treating the soil polluted by the crude oil. But the existing biodegradation treatment has the problems of low efficiency, poor universality, long period and the like, and cannot deal with the increasingly severe problem of crude oil pollutant treatment.
Disclosure of Invention
One of the purposes of the invention is to provide a bacillus aerobica for degrading crude oil, which can be applied to bioremediation of oil-containing soil to make up for the defects of the prior art and has good market prospect.
The purpose of the invention is realized by the following technical scheme:
the invention provides bacillus aerosidus which isBacillus aeriusNUST-1 with the preservation number of CGMCC number 19124 has been preserved in 12 months and 12 days in 2019 in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and the preservation address is No. 3 of Xilu No. 1 of Beijing, Chaoyang.
The invention also aims to provide the application of the bacillus aerosidus NUST-1 in bioremediation of oil-containing soil, which comprises the following specific steps:
(1) inoculating bacillus air NUST-1 into a sterilized seed culture medium, performing shake culture at a temperature of 30-40 ℃ in a shaking table to form a seed culture solution, and inoculating the seed culture solution into the sterilized fermentation culture medium according to an inoculation amount of 1% -10%;
(2) and (2) adding the culture medium in the step (1) into the soil polluted by the crude oil, namely the oil-containing soil, and culturing the mixed oil sludge aqueous solution in a shaking table at 37 ℃ for more than 5 days.
More preferably, the seed culture medium is LB culture medium.
More preferably, the fermentation medium consists of: NaNO3 2~8 g/L，MgSO4 0.1~0.3 g/L，NaCl 0.5~2 g/L，KCl 0.5~2 g/L，CaCl2 0.02~0.08g/L，H3PO4 3~6ml/L，pH=6.0~8.0。
More preferably, the ratio of the oil-containing soil to the culture medium in the step (1) is 1: 0.5-3 g/ml, and the crude oil content in the oil-containing soil is 0.5-5 wt%.
Compared with the prior art, the invention has the advantages that: has high degradation efficiency on crude oil and strong crude oil tolerance.
Detailed Description
The bacillus aerosidus NUST-1 of the invention is separated from the soil polluted by crude oil, and the strain is separated from the soil polluted by crude oil at 30 DEGoC grows on a flat plate on an LB solid culture medium, a bacterial colony is light yellow, and the surface of the bacterial colony is wet. 16sRNA sequencing is carried out on the pure strain obtained after screening and separation, and the 16sRNA gene sequence of the strain is shown in a sequence table. Determined to belong to the air bacillus by comparison with GenbankBacillus aerius) Named as air bacillusBacillus aerius。
Example 1 isolation, screening and identification of novel Bacillus aerosidus NUST-1:
1. enrichment of strains
2g of a soil contaminated with crude oil was placed in a shake flask containing 60mL of sterile distilled water, and 30 g of the soil was placedoAnd C, culturing for 15 minutes on a constant temperature shaking table, taking 2mL of supernatant, putting the supernatant into 60mL of LB liquid culture medium, and carrying out enrichment culture for 12 hours to obtain the strain capable of living in the crude oil soil. The composition of LB liquid medium (g/L) was: peptone 10, yeast extract 5, NaCl 10, pH = 7.0.
2. Screening of degradable crude oil strains
1) And (3) screening the strains by taking crude oil as a unique carbon source:
The strain is screened by using petroleum (also called as 'crude oil') as a unique carbon source. 2mL of the enriched liquid of the strain was added to 60mL of a liquid medium containing 1% petroleum, and the mixture was added to the medium at 37%oC, culturing by a constant temperature shaking table, wherein the liquid culture medium comprises the following components: NaNO3 4 g/L，MgSO4 0.2 g/L，NaCl 1 g/L，KCl 1 g/L，CaCl2 0.04 g/L，H3PO45 ml/L, petroleum 1%, pH = 7.0.
After 48 hours of culture, the culture broth was diluted in a gradient and spread on a screening mediumAt 37oC, culturing for 24 hours in an incubator to obtain 50 single colonies, wherein the screening culture medium is LB solid culture medium (g/L) and comprises the following components: peptone 10, yeast extract 5, NaCl 10, agar powder 15, pH = 7.0. Selecting single colony with vigorous growth, repeatedly screening and culturing under the same culture conditions to obtain pure strain named as Bacillus aereusBacillus aerius。
2) The strains were tested with crude oil as the sole carbon source:
carrying out enrichment culture on the obtained pure strain in 60mL LB liquid culture medium, taking 2mL of liquid after amplification culture, putting the liquid into 60mL of liquid culture medium with 1% crude oil as a unique carbon source, and carrying out enrichment culture on the liquid culture medium at 37 DEG CoC, culturing by a constant-temperature shaking table, wherein the culture medium comprises the following components: NaNO3 4 g/L，MgSO4 0.2 g/L，NaCl 1 g/L，KCl 1 g/L，CaCl2 0.04 g/L，H3PO41% of 5 ml/L crude oil, pH = 7.0;
after 48 hours of culture, the surface tension of the liquid in the culture medium is measured and compared with the surface tension of the liquid in the culture medium before inoculation, the surface tension of the liquid in the culture medium after inoculation is found to be reduced, which indicates that the bacteria can possibly generate the biosurfactant, and the content of the crude oil in the culture medium after inoculation is measured, so that the content of the crude oil in the culture medium is found to be reduced, and the bacteria have the capability of degrading the crude oil.
3. Taxonomic identification of strains
Identifying the screened high-yield enzyme strains by adopting a molecular biology method, measuring 16S rRNA sequences of the strains, comparing the strains in a GenBank nucleic acid database, and determining the strains to be bacillus aereus by sequence comparison, wherein the strains are named as bacillus aereusBacillus aeriusAnd the culture is registered and preserved in China general microbiological culture Collection center (CGMCC) at 12 months and 20 days in 2019, wherein the preservation number is CGMCC number 19124.
Example 2: determination of crude oil degradation capability of Bacillus aereus NUST-1
Inoculating separated Bacillus aeroponicus NUST-1 into culture medium with different crude oil (0.5, 1, 2, 3, 5 g/L) contents, and culturing in 37 deg.C shaking tableAfter 5 days of incubation, the residual crude oil content in the medium was analyzed and the degradation rate of the crude oil was calculated. The composition of the culture medium is: NaNO 34 g/L, MgSO40.2 g/L, NaCl 1 g/L, KCl 1 g/L, CaCl 20.04 g/L, H3PO 45 ml/L, crude oil 0.5-5 g/L, and pH =7.0.
Initial crude oil content [ g/L] Crude oil degradation Rate (%)
0.5 78.2
1 70.2
2 67.4
3 60.2
5 55.8
Example 3: treatment of oil-containing soil by using air bacillus NUST-1
Inoculating Bacillus aereus NUST-1 into sterilized LB seed culture medium, shake culturing at 37 deg.C with shaking table (200 rpm) to form seed culture solution, inoculating the seed culture solution into sterilized fermentation culture medium (NaNO) according to 3% inoculation amount 3 4 g/L，MgSO4 0.2 g/L，NaCl 1 g/L，KCl 1 g/L，CaCl2 0.04g/L，H3PO45 ml/L, pH = 7.0);
10 g of crude oil contaminated soil (hereinafter referred to as sludge) was taken and 20 ml of a culture medium was added thereto. Wherein the crude oil content in the oil sludge is 0.85% (wt), the mixed oil sludge aqueous solution is placed in a shaking table (220 rpm) at 37 ℃ for culture, and the residual oil content in the oil sludge is analyzed after 5 days. The results show that: the residual quantity of the oil sludge is 0.15 percent, and the degradation rate is 82.3 percent.
The above description is only a preferred example of the present invention, and is not intended to limit the scope of the present invention. In addition to the above embodiments, the present invention may have other embodiments. All technical solutions formed by adopting equivalent substitutions or equivalent changes fall into the protection scope of the claims of the present invention.
Sequence listing
<110> Nanjing university of science and technology
<120> bacillus aerosus for degrading crude oil and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 819
<212> DNA
<213> Bacillus aereus (Bacillus aerius)
<400> 1
atacatgcag tcgagcggac agaagggagc ttgctcccgg atgttagcgg cggacgggtg 60
agtaacacgt gggtaacctg cctgtaagac tgggataact ccgggaaacc ggagctaata 120
ccggatagtt ccttgaaccg catggttcaa ggatgaaaga cggtttcggc tgtcacttac 180
agatggaccc gcggcgcatt agctagttgg tgaggtaacg gctcaccaag gcgacgatgc 240
gtagccgacc tgagagggtg atcggccaca ctgggactga gacacggccc agactcctac 300
gggaggcagc agtagggaat cttccgcaat ggacgaaagt ctgacggagc aacgccgcgt 360
gagtgatgaa ggttttcgga tcgtaaagct ctgttgttag ggaagaacaa gtgcaagagt 420
aactgcttgc accttgacgg tacctaacca gaaagccacg gctaactacg tgccagcagc 480
cgcggtaata cgtaggtggc aagcgttgtc cggaattatt gggcgtaaag ggctcgcagg 540
cggtttctta agtctgatgt gaaagccccc ggctcaaccg gggagggtca ttggaaactg 600
ggaaacttga gtgcagaaga ggagagtgga attccacgtg tagcggtgaa aatgcgtaga 660
gatgtggagg aacaccagtg gcgaaggcga ctctctggtc tgtaactgac gctgaggagc 720
gaaagcgtgg ggagcgaaca ggattagata ccctgctact ccacgccgta aaacgaatga 780
gtgctaagtg ttagggggtt tccgcccctt aatgctgca 819

Claims (8)

1. A Bacillus aereus (Bacillus aereus) NUST-1 with preservation number of CGMCC number 19124 is provided.
2. Use of the bacillus aerosidum of claim 1 for degrading crude oil.
3. Use of the bacillus aerosidum of claim 1 for bioremediation of oil-containing soils.
4. Use according to claim 3, wherein oil-containing soil is soil contaminated with crude oil.
5. The use according to claim 3 or 4, characterized by the following specific steps:
(1) inoculating bacillus aerosidus NUST-1 into a sterilized seed culture medium, shaking and culturing in a shaking table at 30-40 ℃ to form a seed culture solution, and inoculating the seed culture solution into the sterilized fermentation culture medium according to 1% -10% of the inoculation amount;
(2) and (2) adding the culture medium in the step (1) into oil-containing soil, and culturing the mixed oil sludge aqueous solution in a shaking table at 37 ℃ for more than 5 days.
6. Use according to claim 5, wherein the seed medium is LB medium.
7. The use according to claim 5, wherein the fermentation medium consists of: NaNO3 2~8 g/L，MgSO40.1~0.3 g/L，NaCl 0.5~2 g/L，KCl 0.5~2 g/L，CaCl2 0.02~0.08g/L，H3PO4 3~6ml/L，pH=6.0~8.0。
8. The use according to claim 5, wherein the ratio of oil-containing soil to the medium of step (1) is 1: 0.5-3 g/ml, and the crude oil content in the oil-containing soil is 0.5-5 wt%.
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CN105670956A (en) * 2015-10-29 2016-06-15 常州大学 Medium thermophilic bacillus licheniformis strain and application thereof in treatment on high-temperature oil-bearing wastewater
CN105861388A (en) * 2016-05-26 2016-08-17 河南省科学院生物研究所有限责任公司 Oil field thermophilic degradation long chain hydrocarbon bacillus HNMC 11117 and application thereof

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
CN105670956A (en) * 2015-10-29 2016-06-15 常州大学 Medium thermophilic bacillus licheniformis strain and application thereof in treatment on high-temperature oil-bearing wastewater
CN105861388A (en) * 2016-05-26 2016-08-17 河南省科学院生物研究所有限责任公司 Oil field thermophilic degradation long chain hydrocarbon bacillus HNMC 11117 and application thereof

Non-Patent Citations (2)

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Petroleum Depletion Property and Microbial Community Shift After Bioremediation Using Bacillus halotolerans T-04 and Bacillus cereus 1-1;Zhenshan Deng et al.;《Front. Microbiol》;20200305;第1-9页 *
芽孢杆菌! " #$% 生物降解原油的特性及现场应用;姚建华等;《精细石油化工进展》;20061231;第7卷(第8期);第5-8和11页 *

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