CN112011480B - Aromatic hydrocarbon degrading bacterium and application thereof - Google Patents
Aromatic hydrocarbon degrading bacterium and application thereof Download PDFInfo
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- 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
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- C02F2101/00—Nature of the contaminant
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Abstract
The invention discloses an aromatic hydrocarbon degrading bacterium and application thereof. The degrading bacteria are Rhodococcus sp SCSIO 21391, which are deposited in Guangdong province microorganism culture collection center at 7-13.2020, with the deposit numbers of GDMCC No: 61076. the strain can rapidly degrade various aromatic hydrocarbon compounds such as 2-chloro-4-nitrophenol, biphenyl and the like, and can be used for treating benzene organic pollutants. The application objects can comprise water or soil environment containing aromatic hydrocarbon organic pollutants and industrial and agricultural domestic wastewater and waste residues, and the harm of the aromatic hydrocarbon to the ecological environment safety is reduced. The aromatic hydrocarbon is degraded by the bacteria, so that the bacteria have the characteristics of energy conservation and environmental protection, and have wide application prospects.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to an aromatic hydrocarbon degrading bacterium and application thereof.
Background
Since the industrial revolution, the rapid development of science and technology brings unprecedented changes to human material life, and civilization is rapidly advanced, however, along with industrialization, global resource and environment problems are increasingly acute, environmental pollution, ecological destruction and resource environment become barriers for the healthy and sustainable development of human society, environmental problems become common problems for human beings all over the world, and the establishment of ecological civilization society for ensuring ecological safety is a new era national development majority proposed in China and is also an international scientific and technological research hotspot. The environmental pollutants are substances which have more than expected residual time after entering the environment and directly or indirectly harm human health or cause the decline of natural ecological environment. Pollutants in the environment can be divided into two categories according to their constituent elements: 1) inorganic contaminants: including heavy metals and their oxides, salts, sulfides, etc.; 2) organic contaminants: mainly comprises various artificially synthesized organic compounds such as pesticides, explosives, Persistent Organic Pollutants (POPs), rubber, plasticizers, various industrial raw materials (such as aniline, nitrobenzene and the like) and the like.
Among many organic pollutants, aromatic compounds are the most widely distributed substances in nature, and have the characteristics of stable six-membered ring structure, difficult decomposition, strong insulating property, low water solubility and the like, so that the aromatic compounds are often used as chemical raw materials and synthetic intermediates and widely applied to industry, agriculture, medical industry and daily life of people. Statistically, a large amount of these compounds are synthesized and utilized in millions of tons per year, but many aromatic compounds such as biphenyl, polychlorinated biphenyl, nitrophenol, Polycyclic Aromatic Hydrocarbons (PAHs), etc. have been proved to have strong toxicity, i.e., causing triproducity (mutagenicity, carcinogenicity, and teratogenicity), and are classified as the pollutants to be controlled by many countries. Therefore, how to repair the aromatic pollutants in the environment in a green and efficient manner becomes a hot point of research of scientists.
Compared with the traditional physical and chemical remediation, the microbial remediation has the advantages of low energy consumption, greenness, high efficiency and the like, and has a wide development prospect in the remediation direction of organic pollutant environments. It has been found that many microorganisms can utilize xenobiotics as energy sources to grow and reproduce in a nutrient-poor environment, and the conversion and decomposition of the xenobiotics are realized. The major research efforts for aromatic hydrocarbon organic-degrading bacteria are mainly focused on the members of the genera Pseudomonas, Rhodococcus, Bacillus, Arthrobacter, Ralstonia, and Mycobacterium. Although the above-mentioned degrading bacteria have high degradation efficiency for single aromatic hydrocarbon, there is only a few reports that one kind of bacteria can degrade multiple kinds of aromatic hydrocarbon organic matters simultaneously. Various pollutants often exist in environments such as water body soil and the like, whether the environment has a wide degradation substrate spectrum is an important consideration factor for reducing and applying environmental pollutant microorganisms, and the development of multifunctional degradation strains has practical significance in the aspect of guaranteeing the cleanness and safety of the environment.
Disclosure of Invention
In order to solve the problem of serious organic pollution of the aromatic hydrocarbon in the environment, the invention provides multifunctional aromatic hydrocarbon degrading rhodococcus and application thereof. Rhodococcus sp SCSIO 21391 of the present invention is capable of degrading 2-chloro-4-nitrophenol, biphenyl, diphenyl ether, dibromodiphenyl ether, beta-naphthol, pyrene and fluorene. Aims to promote the application of microbial technology in the treatment of the organic pollutants of the environmental aromatic hydrocarbons and solve the pollution problem of the organic pollutants of the environmental benzene series.
The invention is realized by the following technical scheme:
the invention finds that the rhodococcus has good degradation effect on aromatic compounds and can be used for degradation treatment of aromatic pollution.
The invention screens and obtains an aromatic hydrocarbon degrading bacterium from mangrove forest soil, which is named as Rhodococcus sp SCSIO 21391, which is preserved in Guangdong province microorganism culture collection center in 7-13 months in 2020, with the preservation number being GDMCC No: 61076.
therefore, the application of Rhodococcus sp SCSIO 21391 in degrading and converting aromatic compounds or improving the degradation and removal rate of aromatic hydrocarbons, and the application in treating water environment and soil polluted by aromatic hydrocarbons are all within the protection scope of the present invention.
Preferably, the aromatic hydrocarbon is benzene series organic pollutant.
Preferably, the benzene series organic pollutants are selected from at least one of 2-chloro-4-nitrophenol, biphenyl, diphenyl ether, dibromo diphenyl ether, beta-naphthol, pyrene and fluorene.
An aromatic hydrocarbon degrading bacterial agent, characterized by comprising the Rhodococcus sp SCSIO 21391.
Preferably, the degrading bacterial agent is a bacterial suspension of Rhodococcus sp (Rhodococcus sp.) SCSIO 21391 strain.
Preferably, the degrading microbial inoculum is prepared by activated culture of Rhodococcus sp SCSIO 21391 strain. Specifically, the Rhodococcus (Rhodococcus sp.) SCSIO 21391 strain is cultured and activated in a TSB culture medium or a bacterial culture medium, and precipitates are collected by centrifugation and are diluted after washing to prepare the Rhodococcus sp.
More preferably, the preparation method of the degrading microbial inoculum comprises the following steps: inoculating Rhodococcus (Rhodococcus sp.) SCSIO 21391 preserved by slant or frozen glycerol into a liquid TSB culture medium, and performing constant temperature shaking culture at 25-30 ℃ and 150-250 r/min for 24-48 h to prepare an activated seed solution; inoculating the seed solution into a fresh culture medium according to the inoculation amount of 1-10% v/v, and continuously carrying out constant-temperature oscillation amplification culture at 25-30 ℃ and 150-250 r/min for 36-48 h; and centrifuging at 25 ℃ for 10min at 5000r/min, removing the supernatant, collecting the bacteria, washing with sterile water for 2-3 times, and preparing into 1g/L bacteria suspension with sterile water to obtain the degrading bacteria agent.
The TSB culture medium comprises the following components: tryptone 15g/L, soybean peptone 5g/L, sodium chloride 5g/L, solvent water, pH 7.2, and sterilizing at high temperature and high pressure.
Compared with the prior art, the invention has the following beneficial effects:
the rhodococcus is obtained by screening, 2-chloro-4-nitrophenol, biphenyl, diphenyl ether, dibromo diphenyl ether and beta-naphthol can be degraded, and the removal rate is over 90 percent in 72 hours; pyrene and fluorene can be degraded, the removal rate is 10% after 72h, the method can be used for degradation treatment of aromatic hydrocarbon organic pollution, application objects can include water or soil containing aromatic hydrocarbon organic pollutants and the like, and the harm of aromatic hydrocarbon to the safety of ecological environment can be reduced. Compared with methods such as physical adsorption and chemical degradation, the method for treating aromatic hydrocarbon pollution by using the rhodococcus has the advantages of energy conservation, environmental protection and the like, and has wide application prospect.
Rhodococcus sp SCSIO 21391 of the present invention was deposited with the Guangdong province culture Collection of microorganisms (GDMCC) at 7/13/2020, address: the Guangzhou city first furious Zhonglu No. 100 large yard No. 59 building No. 5 building, the preservation number is: GDMCC NO: 61076.
drawings
FIG. 1 shows the plate growth pattern of Rhodococcus SCSIO 21391.
FIG. 2 is a transmission electron microscope image of Rhodococcus SCSIO 21391.
FIG. 3 is a phylogenetic tree of Rhodococcus SCSIO 21391.
FIG. 4 shows the degradation effect of Rhodococcus SCSIO 21391 on several aromatic hydrocarbon compounds, wherein the aromatic hydrocarbon compounds from left to right in the figure are pyrene, fluorene, beta-naphthol, biphenyl, 2-chloro-4-nitrophenol, dibromodiphenyl ether and diphenyl ether in sequence.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Example 1 isolation and identification of aromatic Hydrocarbon-degrading bacteria
1. Experimental Material
Inorganic salt culture medium: (NH)4)2SO4 2.0g/L，MgSO4 0.2g/L，CaCl2 0.01g/L，FeSO4 0.005g/L，Na2HPO4 1.5g/L，KH2PO41.5g/L, solvent is water, pH 7.0. When the solid culture medium is prepared, 18g/L agar powder is added on the basis of the components of the culture medium, and the components are mixed uniformly according to the content. The culture medium is placed in a high-pressure steam sterilization pot for sterilization treatment, and is sterilized for 15min at 121 ℃.
2. Isolation and characterization of strains
(1) Screening and purifying aromatic hydrocarbon degrading bacteria
Collecting a mangrove forest soil sample, adding the soil into 100mL of inorganic salt culture medium containing aromatic hydrocarbon compounds (20mg/L) in a super clean bench, and carrying out enrichment culture for 10d by constant temperature shaking culture at 30 ℃ and 150 r/min. 1mL of the enriched culture was added to 100mL of fresh inorganic salt medium containing aromatic compounds (50mg/L) for another 10 days. The second round of enrichment culture solution is added according to the proportion of 10-1-10-6Gradient dilution, spreading 200 μ L of the diluted solution on solid inorganic salt culture medium containing 20mg/L aromatic hydrocarbon compound, and culturing at 30 deg.C for 3 d. The grown colonies are numbered, and single colonies are picked out respectively and further streaked for separation and purification. And respectively inoculating the single colony into a liquid inorganic salt culture medium containing 50mg/L of aromatic hydrocarbon compounds to carry out degradation experiments, and investigating the degradation capability of each strain of bacteria on the aromatic hydrocarbon compounds. The aromatic hydrocarbon compound is 2-chloro-4-nitroPhenol.
(2) Identification of aromatic Hydrocarbon-degrading bacteria
Selecting the bacterial strain SCSIO 21391 with the highest degradation efficiency on the aromatic hydrocarbon compounds to a TSB solid plate, carrying out static culture in a constant temperature incubator at 30 ℃ for 3d, observing the morphological morphology of the bacterial strain as shown in figure 1, wherein the bacterial colony is orange red, and the surface is dry and uneven. The cultured bacteria were collected in an appropriate amount, washed with phosphate buffer (pH 7.2), fixed with 2.5% glutaraldehyde for 24 hours, dehydrated stepwise with ethanol, replaced with propylene oxide, impregnated and embedded with Spurr resin, and finally polymerized in an oven at 70 ℃ to give cells in the form of rods having a length of about 3 μm and a width of about 0.5. mu.m, without flagella, as shown in FIG. 2 by transmission electron microscopy of Hitachi JEM 1230.
The TSB culture medium comprises the following components in percentage by weight: tryptone 15g/L, soybean peptone 5g/L, sodium chloride 5g/L, solvent water, pH 7.2, sterilizing at 121 deg.C for 15min, and its preparation method comprises mixing the above components uniformly, and sterilizing. When preparing a solid medium, 18g/L agar powder was added to the above medium components.
Genomic DNA of the strain SCSIO 21391 was extracted and PCR-amplified using 16S rDNA amplification universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') using the genomic DNA as a template. The product is subjected to Sanger sequencing, the sequencing is completed by Shanghai biological engineering Co., Ltd, and the sequence of the 16S rDNA is shown as SEQ ID NO. 1. The obtained 16S rDNA was subjected to Blast alignment, and the relationship with Rhodococcus immethensis was found to be recent (99% identity). A phylogenetic tree was constructed as shown in figure 3.
Combining the above morphology and molecular identification results, the strain SCSIO 21391 obtained by screening in the invention is identified as Rhodococcus sp, which is named as: rhodococcus sp (Rhodococcus sp.) SCSIO 21391. And is preserved in Guangdong province microorganism culture collection center in 2020, 7 and 13 days, with the preservation number of GDMCC No: 61076; and (4) storage address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5.
Example 3 degradation experiment of Rhodococcus SCSIO 21391 on aromatic hydrocarbons
(1) Preparation of the bacterial suspension
Scraping 1-ring Rhodococcus erythropolis SCSIO 21391 from the slant or glycerol preservation solution, inoculating into sterilized TSB culture medium, and culturing in constant temperature shaking table (30 deg.C, 150r/min) for 48h to obtain activated seed solution; inoculating the seed solution into a fresh culture medium according to the inoculation amount of 1% v/v, and continuously carrying out constant-temperature oscillation and amplification culture at 30 ℃ and 150r/min for 48 h. Collecting Rhodococcus SCSIO 21391 culture solution, centrifuging at 25 deg.C and 5000r/min for 10min, discarding supernatant, collecting bacteria, washing with sterile water for 2 times, and resuspending with phosphate buffer (50mM, pH 7.2) to OD600About 2.0 to obtain bacterial suspension, and placing the bacterial suspension in a refrigerator at 4 ℃ for later use.
(2) Detection of aromatic compounds
The samples were subjected to GC-MS analysis using Shimadzu gas chromatography mass spectrometer (GCMS-QP2010 Ultra), the chromatographic column was Varian CP-Sil 8CB column (0.25 mm. times.30 m. times.0.25 μm), the temperatures of the inlet and detector were 300 ℃ and the column oven conditions were as follows: keeping the temperature at 40 ℃ for 1min, then increasing the temperature to 280 ℃ at 5 ℃/min, taking helium as a carrier gas, and setting the flow rate at 1mL/min and the sample injection amount at 1 mu L.
Detecting a sample by using an Agilent 1260 high-performance liquid phase, wherein the device is configured as follows: zorbax SB-C18ODS chromatography column (4.6X 150mm, 5 μm, Agilent Technologies, Palo Alto, Calif., USA), the detector was a two-stage array detector (DAD) with a detection wavelength of 320 nm. Mobile phase: a: deionized water containing 0.1% (v/v) glacial acetic acid; b: methanol containing 0.1% (v/v) glacial acetic acid. The gradient elution conditions were: 0-5min, 5% B; 5-25min, 5% B increased linearly to 100% B; and kept for 5 min. The flow rate was 1.0mL/min, and the column temperature was maintained at 30 ℃.
(3) Degradation effect of Rhodococcus SCSIO 21391 on aromatic Compounds
Inoculating the bacterial suspension obtained in the step (1) into an inorganic salt culture medium containing 50mg/L glucose, and respectively adding different aromatic hydrocarbon compounds including pyrene, fluorene, beta-naphthol, biphenyl, 2-chloro-4-nitrophenol, dibromodiphenyl ether and diphenyl ether. The final concentrations of the aromatic hydrocarbon compounds were 50mg/L, respectively. Controlling the final concentration of inoculated Rhodococcus SCSIO 21391 to OD6000.1, shaking-culturing at 30 deg.C and 150r/min for 72h, detecting aromatic hydrocarbon residue in the culture solution with gas phase/liquid phase, and mixing withComparison was made with a control group without added bacterial suspension. As shown in FIG. 4, under the above experimental conditions, the removal rate of 2-chloro-4-nitrophenol, biphenyl, diphenyl ether, dibromodiphenyl ether and β -naphthol was 90% or more, and the removal rate of pyrene and fluorene was 10%. The species Rhodococcus biotechensis RKJ300 most closely related to Rhodococcus erythropolis SCSIO 21391 was reported in the literature to degrade p-nitrophenol and 2-chloro-4-nitrophenol (Ghosh A, Khuran M, Chauhan A, et al. degradation of 4-nitrophenol,2-chloro-4-nitrophenol, and 2,4-dinitrophenol by Rhodococcus biotechensis RKJ300[ J].Environmental Science&Technology 2010,44:1069-].International Biodeterioration&Biodegrading, 2005,55: 103-. The Rhodococcus SCSIO 21391 has a wide spectrum of degrading aromatic hydrocarbon substrates, is superior to the species reported in the literature, and has application prospects in the degradation and repair of environmental organic pollutants due to the wide adaptability of the aromatic hydrocarbon substrates.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Sequence listing
<110> Nanhai ocean institute of Chinese academy of sciences
<120> aromatic hydrocarbon degrading bacterium and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1328
<212> DNA
<213> Rhodococcus erythropolis SCSIO 21391(Rhodococcus sp. SCSIO 21391)
<400> 1
ggtacacgag cggcgaacgg gtgagtaaca cgtgggtgat ctgccctgca cttcgggata 60
agcctgggaa actgggtcta ataccggata tgaccttcgg ctgcatggct gagggtggaa 120
aggtttactg gtgcaggatg ggcccgcggc ctatcagctt gttggtgggg taatggccta 180
ccaaggcgac gacgggtagc cgacctgaga gggtgaccgg ccacactggg actgagacac 240
ggcccagact cctacgggag gcagcagtgg ggaatattgc acaatgggcg aaagcctgat 300
gcagcgacgc cgcgtgaggg atgacggcct tcgggttgta aacctctttc agcagggacg 360
aagcgaaagt gacggtacct gcagaagaag caccggccaa ctacgtgcca gcagccgcgg 420
taatacgtag ggtgcaagcg ttgtccggaa ttactgggcg taaagagctc gtaggcggtt 480
tgtcgcgtcg tctgtgaaaa ctcgaggctc aacctcgagc ttgcaggcga tacgggcaga 540
cttgagtact gcaggggaga ctggaattcc tggtgtagcg gtgaaatgcg cagatatcag 600
gaggaacacc ggtggcgaag gcgggtctct gggcagtaac tgacgctgag gagcgaaagc 660
gtgggtagcg aacaggatta gataccctgg tagtccacgc cgtaaacggt gggcgctagg 720
tgtgggtttc cttccacggg atccgtgccg tagctaacgc attaagcgcc ccgcctgggg 780
agtacggccg caaggctaaa actcaaagga attgacgggg gcccgcacaa gcggcggagc 840
atgtggatta attcgatgca acgcgaagaa ccttacctgg gtttgacata taccggaaag 900
ccgtagagat acggcccccc ttgtggtcgg tatacaggtg gtgcatggct gtcgtcagct 960
cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgtct tatgttgcca 1020
gcacgtaatg gtggggactc gtaagagact gccggggtca actcggagga aggtggggac 1080
gacgtcaagt catcatgccc cttatgtcca gggcttcaca catgctacaa tggccagtac 1140
agagggctgc gagaccgtga ggtggagcga atcccttaaa gctggtctca gttcggatcg 1200
gggtctgcaa ctcgaccccg tgaagtcgga gtcgctagta atcgcagatc agcaacgctg 1260
cggtgaatac gttcccgggc cttgtacaca ccgcccgtca cgtcatgaaa gtcggtaaca 1320
cccgaagc 1328
Claims (7)
1. Rhodococcus capable of degrading aromatic hydrocarbonRhodococcussp.) SCSIO 21391 with accession number: GDMCC No: 61076, wherein the aromatic hydrocarbon is at least one of 2-chloro-4-nitrophenol, biphenyl, diphenyl ether, dibromodiphenyl ether, beta-naphthol, pyrene and fluorene.
2. Rhodococcus bacterium according to claim 1(Rhodococcussp.) use of SCSIO 21391 for degrading aromatic hydrocarbons selected from at least one of 2-chloro-4-nitrophenol, biphenyl ether, dibromobiphenyl ether, beta-naphthol, pyrene and fluorene.
3. Rhodococcus bacterium according to claim 1(Rhodococcussp.) application of SCSIO 21391 in bioremediation of aromatic hydrocarbon polluted soil and/or water, wherein the aromatic hydrocarbon is selected from at least one of 2-chloro-4-nitrophenol, biphenyl, diphenyl ether, dibromo diphenyl ether, beta-naphthol, pyrene and fluorene.
4. An aromatic hydrocarbon degrading bacterial agent comprising the Rhodococcus bacterium (R) according to claim 1Rhodococcussp.) SCSIO 21391, wherein the aromatic hydrocarbon is at least one selected from 2-chloro-4-nitrophenol, biphenyl ether, dibromobiphenyl ether, beta-naphthol, pyrene and fluorene.
5. The aromatic hydrocarbon degrading bacterial agent of claim 4, wherein the degrading bacterial agent is Rhodococcus (Rhodococcus)Rhodococcussp.) a bacterial suspension of the SCSIO 21391 strain.
6. The aromatic hydrocarbon degrading bacterial agent according to claim 4 or 5, wherein the degrading bacterial agent is Rhodococcus (Rhodococcus)Rhodococcussp.) SCSIO 21391 strain.
7. The aromatic hydrocarbon degradation bacterial agent of claim 6, wherein the preparation method of the degradation bacterial agent comprises the following steps: rhodococcus (A) to (B)Rhodococcussp.) the SCSIO 21391 is inoculated into a liquid TSB culture medium and is subjected to constant-temperature shaking culture to prepare an activated seed solution; inoculating the seed liquid into a fresh culture medium, continuously carrying out amplification culture, centrifuging, removing supernatant, collecting thalli, and carrying out resuspension to obtain bacterial suspension, namely the degrading microbial inoculum.
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