CN110331113B - Mangrove forest bacillus and its application - Google Patents

Abstract

The invention relates to a mangrove bacillus and application thereof, the bacterial strain is mangrove bacillus (Mangroveibacter sp) 5092-2, the preservation number is CGMCC NO.17705, and the 16S rRNA sequence is shown in SEQ ID NO. 1. Is applied to the degradation treatment of kerosene pollution. The strain is used for fermenting and degrading kerosene, the reaction condition is mild, the required cost is low, and the strain is environment-friendly.

Description

Mangrove forest bacillus and its application

Technical Field

The invention belongs to the field of microorganisms and application thereof, and particularly relates to mangrove forest bacillus and application thereof.

Background

Petroleum and its products enter the marine environment and cause serious pollution in the course of exploitation, refining, storage and transportation and use; where oil spill pollution is the most harmful and oil leaks are known as super killers of marine pollution. Unlike conventional pollutants, petroleum pollutants are not easily destroyed after entering human bodies once polluting water areas or food chains, and inevitably endanger the human bodies when the persistence, the accumulation, the migration and the high toxicity of the petroleum pollutants are still maintained, so that the petroleum pollutants present carcinogenicity, denaturation and teratogenicity and seriously threaten the health of human beings. The treatment of oil spill pollution, the improvement and recovery of the ecological environment of polluted areas, the protection of marine environment and marine resources, and the promotion of sustainable development are the responsibility of the meaning of all countries in the world. Therefore, except taking effective measures to avoid the occurrence of the offshore oil spill accident, the marine oil spill treatment measures are also explored without the residual force, and the nature is protected by human intelligence.^[1-3]

At present, the treatment methods for oil pollution in the world mainly comprise 3 methods: the first is physical method, the second is chemical method, and the third is processing by biological technology. Because both physical and chemical methods have their own disadvantages that are difficult to overcome, e.g. by physical methodsThe oil film and dissolved oil on the surface of seawater are difficult to remove, and secondary pollution is likely to be caused by adopting a chemical method, so the research on the biological method is gradually paid attention. The biological method is to remove oil spill pollution by using oil as a nutrient substance for metabolism of microorganisms. The method has the advantages of high efficiency, economy, safety and no secondary pollution, and particularly shows the advantages in the case of dissolved oil which is permeated into seawater or thin oil layers which cannot be removed by mechanical devices.^[4-6]

Meanwhile, the problem of kerosene pollution still lacks an efficient and environment-friendly solution. The growth cycle of the microorganism is generally 1 to 3 days, the reaction condition is mild, the cost is low, the environment is friendly, and the problem of kerosene pollution can be well solved by utilizing the microorganism.

Plum, etc. takes soil microbes polluted by petroleum for a long time near a petrochemical plant as a bacteria source, takes kerosene as a unique carbon source, strains with good kerosene degradation effect are obtained through domestication, screening and separation, and the kerosene degradation efficiency is 42.6% through inspection.^[7]

Disclosure of Invention

The invention aims to solve the technical problem of providing the mangrove forest bacillus and the application thereof so as to overcome the defects that the problem of the coal oil pollution in the prior art can not be efficiently solved and the environment is friendly.

The invention provides a mangrove bacillus, which is mangrove bacillus (Mangroveibacter sp.)5092-2 with the preservation number of CGMCC NO.17705, and the 16S rRNA sequence of the mangrove bacillus is shown in SEQ ID NO. 1.

The characteristics of the mangrove bacillus are as follows:

the mangrove woods bacillus are arranged singly, doubly or quadruplely or are irregular in a three-dimensional wrapping shape; the length of the bacterial colony is 5.0-10.0 mu m, the bacterial colony is white, round, protruding, moist, flashing, full-edge and gram-negative.

The mangrove bacillus (Mangroovibacter sp.)5092-2 of the invention has been preserved in China general microbiological culture Collection center (CGMCC for short, institute of microbiology 3 of Zhongsco institute, No.1 of West Chen of the morning area of Beijing city) in 05.2019 at 05.month, with the preservation number of CGMCC NO. 17705.

According to BLAST cluster analysis and morphological identification results, the microorganism is determined to be Mangroovibacter sp (5092-2), and experiments prove that the kerosene can be degraded.

The invention determines the 16S rRNA partial sequence of the strain and carries out phylogenetic analysis.

The invention also provides an application of the mangrove bacillus in degradation treatment of kerosene pollution.

The method for degrading and treating the kerosene pollution comprises the following steps:

inoculating the mangrove forest bacillus into a kerosene liquid culture medium with the pH value of 7.0-7.4, culturing at the temperature of 30-35 ℃ and 180rpm for 120-168h, extracting the bacterial liquid (for example, extracting by using 30-50ml of dichloromethane), weighing the mass of the residual kerosene after the extraction solvent is volatilized, and ensuring that the degradation rate of the kerosene reaches more than fifty percent.

The kerosene liquid culture medium comprises the following components: NH (NH)₄Cl 5-10g/L、KH₂PO₄ 5-10g/L、KCl 0.1-0.2g/L、MgSO₄5-10g/L、CaCl₂ 0.02-0.05g/L、FeSO₄ 0.1-0.2g/L、NaCl 15-20g/L、K₂HPO₄1.0-2.0g/L and 30-50g/L of kerosene.

The method for degrading and treating the kerosene pollution comprises the following steps:

the mangrove forest bacillus is inoculated in kerosene liquid culture medium with pH 7.2 and cultured at 35 ℃ and 160rpm for 168 h.

The kerosene liquid culture medium comprises the following components: NH₄Cl 5g/L、KH₂PO₄ 5g/L、KCl 0.1g/L、MgSO₄ 5g/L、CaCl₂ 0.02g/L、FeSO₄ 0.1g/L、NaCl 15g/L、K₂HPO₄1.0g/L and 30g/L of kerosene.

The invention cultures and researches strains capable of degrading kerosene, gropes the optimal degradation conditions by changing the experimental conditions, and performs the experiment.

Advantageous effects

The invention utilizes the mangrove forest bacillus to ferment and degrade the kerosene, has mild reaction condition, low cost and environmental protection.

Drawings

FIG. 1 is a phylogenetic tree of the 16S rRNA sequence of the strain in example 1;

FIG. 2 is a morphological diagram of the strains of example 1 (the left panel shows colony morphology, and the right panel shows cell morphology);

FIG. 3 is a photograph of the DNA gel electrophoresis in example 1.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the teaching of the present invention, and these equivalents also fall within the scope of the claims appended to the present application.

Example 1

A new strain, namely mangroveibacterium (Mangroveobacter sp.)5092-2, is deposited in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms, and the deposit number is as follows: CGMCC NO. 17705.

The strain source is as follows: separating the Shanghai polluted soil, wherein the specific separation method comprises the following steps:

adding 10g of the oil-contaminated soil sample into a 250ml conical flask filled with 100ml of sterile water, and performing reaction at 28 ℃ and 160 r.min^-1Culturing for 24h in a constant-temperature shaking incubator. Standing after preparing soil suspension. And (3) configuring petroleum concentration gradients of 500, 1000, 1500 and 2000mg/L, and domesticating the strains by the method. Transferring 1ml soil suspension, inoculating into 100ml enrichment medium with petroleum concentration of 500mg/L, and culturing at 28 deg.C and 160r min^-1Culturing for 7 days in a constant-temperature shaking incubator. Then, 10ml of the suspension was added to a medium having a petroleum concentration of 1000mg/L, and the suspension was transferred 3 times. Thus obtaining the bacterial strain which grows in the environment polluted by high-concentration petroleum, and then selecting the bacterial strain with good degradation performance.

And (3) strain identification:

(1) and (3) observing colony morphology: colonies were white, round, protruding, wet, flashing, full-edged, gram-negative.

(2) And (3) observing the shape of the thallus: is rod-shaped and has a length of about 5.0 to 10.0 μm.

(3) The extraction method of the strain DNA comprises the following steps: a single colony is picked from the plate, inoculated into a 5ml LB liquid medium test tube, cultured for 12h in a constant temperature shaking table at 35 ℃ and 160rpm, 5 mul is absorbed, diluted by 100 times by sterile water and put into a water bath at 98 ℃ for 10min, cells are ruptured, and nucleic acid is released.

(4) Gene amplification of 16S rRNA

The PCR system was set up on ice: forward P2. mu.l, Reverse P2. mu.l, 10. mu.buffer 5. mu.l, Mg²⁺3μl，ddH₂O31.5. mu.l, dNTP 1. mu.l, template DNA 5. mu.l, Taq 0.5. mu.l. PCR procedure: 4min at 94 ℃ (pre-denaturation); 30s at 94 ℃ (denaturation), 30s at 53 ℃ (annealing), 90s at 72 ℃ (extension), and the cycle is 32 times; 72 ℃ 7min (extension). The PCR products were analyzed by agarose gel electrophoresis.

The primer sequences are respectively as follows:

SSU rRNA

BSF8/20 5’-AGA GTT TGA TCC TGG CTC AG-3’(SEQ ID NO.2)61.0℃ Position 8-27

BSR1541/20 5’-AAG GAG GTG ATC CAG CCG CA-3’(SEQ ID NO.3)61.0℃ Position1541-1522

and (3) performing 120V constant voltage electrophoresis on the PCR product, dyeing for 20min by using EB after the electrophoresis is finished, and observing and photographing in an ultraviolet imaging system.

(5) Analysis of the resulting sequences

DNA gel electrophoresis:

with the exception of well No.1, a clear band was evident, and the DNA molecular weight was about 15 kb.

The DNA gel electrophoresis is shown in FIG. 3.

And (3) carrying out gene sequencing on the PCR product with the band, wherein the sequencing result is shown as SEQ ID NO. 1.

And comparing the sequence with the sequence in a BLAST website to find the genus of the bacteria.

The bacterium is Proteobacteria (Proteobacteria), enterobacter, mangroovibacter sp.

The kerosene degradation process by the strain comprises the following steps: 1ml of seed liquid (NaCl 10g/L, yeast powder 5g/L, peptone 10g/L, pH 7.0, 2.0X 10/ml) was added⁸Individual bacteria) were inoculated into 100ml of kerosene liquid medium having a pH of 7.2 and cultured at 35 ℃ and 160rpm for 168 hours, the kerosene liquid medium consisting of: NH (NH)₄Cl 5g/L、KH₂PO₄ 5g/L、KCl 0.1g/L、MgSO₄ 5g/L、CaCl₂ 0.02g/L、FeSO₄ 0.1g/L、NaCl 15g/L、K₂HPO₄1.0g/L and 30g/L light oil. After the culture is finished, 50ml of dichloromethane is used for extraction, the mass of the residual kerosene is weighed after the dichloromethane is volatilized, and the efficiency of degrading the kerosene by the strains is calculated to be 52.3%.

The 16S rRNA sequence of the strain is shown in SEQ ID NO. 1.

Comparative example 1

Plum and the like take soil microorganisms which are polluted by petroleum for a long time near a petrochemical plant as a bacteria source and kerosene as a unique carbon source, and strains with good degradation effect on the kerosene are domesticated, screened and separated, wherein the temperature is 30 ℃, the pH is 7, the rotating speed of a constant-temperature shaking table is 190r/min, and the salinity is 2.5%. Under the optimal culture conditions: (NH)₄)₂SO₄ 1.0g/L、NaCl 30g/L、KH₂PO₄ 1.0g/L、MgSO₄·7H₂O 0.2g/L、CaC1₂ 0.02g/L、K₂HPO₄ 1.0g/L、FeCl₃Trace, 10g/L kerosene, and 42.6% kerosene degradation rate.

The effective kerosene degradation rate of the screened strains is 52.3 percent, which is higher than that of the comparative example, and the requirements on temperature, pH value and the like are lower. The present invention relates to the following references:

[1] wangxiwei, pure plum, Shennan, the influence of petroleum pollution on marine life [ J ]. southern Aquaculture, 2006,2(2):76-80.

[2] Wangweijie, Wu Changjiang river, the hazard of marine oil pollution to fishery and its control strategy [ J ]. Shandong environment, 1995, (2):20-21.

[3] Wangkingling, Li Shunpeng, environmental microbiology [ J ] microbiology, 1991,2:007.

[4] Bioremediation of microorganisms in marine petroleum pollution [ J ]. energy and environment, 2007, (1):42-43.

[5] Li Jie, processing and recovery of oil spills at sea [ J ] report on ocean lakes and marshes, 1996, (1):73-83.

[6] Yan Jihui. offshore oil spill and remediation [ J ] ocean technology, 1996,15(1):29-34.

[7] Liping et al, hydrocarbon pollutant degrading bacteria screening and growth condition research, Anhui agricultural science, 2012,40(21):11017-11019,11069.

SEQUENCE LISTING

<110> university of east China

<120> mangrove bacillus and application thereof

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atgcaagtcg agcggcagcg ggaagaagct tgcttctttg ccggcgagcg gcggacgggt 60

gagtaatgtc tgggaaactg cctgatggag ggggataact actggaaacg gtggctaata 120

ccgcataacg tcgcaagacc aaagaggggg accttcgggc ctcttgccat cagatgtgcc 180

cagatgggat tagcttgttg gtgaggtaac ggctcaccaa ggcgacgatc cctagctggt 240

ctgagaggat gaccagccac actggaactg agacacggtc cagactccta cgggaggcag 300

cagtggggaa tattgcacaa tgggcgcaag cctgatgcag ccatgccgcg tgtatgaaga 360

aggccttcgg gttgtaaagt actttcagtc aggaggaagg tggtgagctt aatacgctca 420

tcaattgacg ttactgacag aagaagcacc ggctaactcc gtgccagcag ccgcggtaat 480

acggagggtg caagcgttaa tcggaattac tgggcgtaaa gcgcacgcag gcggtctgtc 540

aagtcggatg tgaaatcccc gggctcaacc tgggaactgc attcgaaact ggcaggctgg 600

agtctcgtag agggaggtag aattccaggt gtagcggtga aatgcgtaga gatctggagg 660

aataccggtg gcgaaggcgg cctcctggac gaagactgac gctcaggtgc gaaagcgtgg 720

ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgtcg acttggaggc 780

tgtgcccttg aggcgtggct tccggagcta acgcgttaag tcgaccgcct ggggagtacg 840

gccgcaaggt taaaactcaa atgaattgac gggggcccgc acaagcggtg gagcatgtgg 900

tttaattcga tgcaacgcga agaaccttac ctggtcttga catccagaga atcctgcaga 960

gatgcgggag tgccttcggg agctctgaga caggtgctgc atggctgtcg tcagctcgtg 1020

ttgtgaaatg ttgggttaag tcccgcaacg agcgcaaccc ttatcctttg ttgccagcgg 1080

ttaggccggg aactcaaagg agactgccag tgataaactg gaggaaggtg gggatgacgt 1140

caagtcatca tggcccttac gaccagggct acacacgtgc tacaatggcg catacaaaga 1200

gaagcgaact tgcgagagta agcggacctc ataaagtgcg tcgtagtccg gattggagtc 1260

tgcaactcga ctccatgaag tcggaatcgc tagtaatcgc ggatcagaat gccgcggtga 1320

atacgttccc gggccttgta cacaccgccc gtcacaccat gggagtgggt tgcaaaagaa 1380

gtaggtagct taaccttcgg gagggcgctt accactttgt gattcatgac tggggtgaag 1440

tcg 1443

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Claims (2)

1. A mangrove forest bacillus, characterized in that: the mangrove bacillus is mangrove bacillus (A), (B), (C)Mangroveibacter sp.)5092-2 with preservation number of CGMCC NO.17705, and the 16S rRNA sequence is shown in SEQ ID NO. 1.

2. Use of the mangrove bacillus strain of claim 1 for the degradation of kerosene pollution.

Images