CN113637602B - Labrys portucalensis P49-1 and application thereof - Google Patents
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Abstract
The invention discloses a Labrys portucalensis P49-1 and application thereof. Labrys portucalensis P49-1 with a deposit number of: GDMCC No:61706.labrys portucalensis P49-1 can be degraded by using polychlorinated biphenyl as a carbon source, and the initial concentration of the polychlorinated biphenyl in PCB9 is 10 mg.L ‑1 After the inorganic salt culture solution is cultured for 10 days, the degradation rate can reach more than 60 percent. In conclusion, the Labrys portucalensis P49-1 is a bacterial strain which can degrade polychlorinated biphenyl and has strong tolerance to the polychlorinated biphenyl, has strong adaptability to the polychlorinated biphenyl and has good application potential in the aspect of bioremediation.
Description
The technical field is as follows:
the invention belongs to the field of organic pollutant degradation, and particularly relates to a polychlorinated biphenyl degrading strain Labrys portucalensis P49-1 and application thereof.
The background art comprises the following steps:
with the rapid development of modern industrial processes, industrial wastewater pollution is increasingly serious, so that various Persistent Organic Pollutants (POPs) are contained in the industrial wastewater and bottom sludge. Among them, polychlorinated biphenyls (PCBs) are a typical type of POPs, and are one of 12 POPs which are currently receiving wide attention internationally. PCBs are widely produced at home and abroad as main components of industrial raw materials, and a large amount of chlorinated biphenyls are leaked into the environment in the application process of early chlorinated biphenyls, so that the long-term pollution to the environment is caused. Because the substances have the characteristics of potential carcinogenicity, teratogenesis, mutagenicity, biological accumulation and the like, the substances can form great harm to the ecological environment and human health. Thus, as early as 2001, PCBs were listed in the stockholm convention and identified as "extremely toxic pollutants" that were prohibited from use, and were regulated to be eliminated from the environment by 2025.
The natural attenuation of toxic and harmful organic pollutants in the environment mainly depends on the metabolism of related microorganisms, and the bioremediation technology has the advantages of low cost, good effect, no secondary pollution and the like, so the method is the most potential remediation means for the polychlorinated biphenyl pollution remediation at present. At present, few polychlorinated biphenyl degrading strains have been reported, mainly including anaerobic degrading bacteria Desulfitobacterium, dehalosspirillum Multivorans, desfomonile tiedjei, dehalobacter restricus, deslformomonas chloroethyenica and aerobic degrading bacteria Burkholderia xenovorans, alcaligenes sp. However, most of the degrading bacteria belong to strict anaerobic bacteria, and high-chlorine PCBs (high-chlorine biphenyl with more than tetrachloro) are required to be dechlorinated under strict anaerobic conditions and degraded into low-chlorine biphenyl. The low-chlorine biphenyl is rarely subjected to an anaerobic dechlorination process, most of the low-chlorine biphenyl can be directly converted into the chlorobenzoic acid by aerobic degrading bacteria through a biphenyl degrading way, and the generated chlorobenzoic acid is further mineralized into carbon dioxide and water through other aerobic bacteria. Since most of the microorganisms in the environment are not culturable, many microorganisms, especially those with specific functions, cannot be isolated by pure culture. Therefore, the screening of the bacterial strain capable of effectively degrading the polychlorinated biphenyl has important application value and practical significance.
The invention content is as follows:
the first purpose of the invention is to provide Labrys portucalensis P49-1 with polychlorinated biphenyl degradation capability. The strain was deposited at the Guangdong province culture Collection (GDMCC) at 6/2/2021, address: building 5 of first furnance, large yard, 100, building 59, guangdong province, guangzhou, china, zip code: 510070, with a preservation number of: GDMCC No:61706.
the second purpose of the invention is to provide the application of Labrys portucalensis P49-1 in degrading polychlorinated biphenyl.
It is preferable to degrade polychlorinated biphenyl in an environment where the Labrys portucalensis P49-1 is contaminated with polychlorinated biphenyl.
The third purpose of the invention is to provide a polychlorinated biphenyl degrading bacterial agent which contains Labrys portucalensis P49-1 as an active ingredient.
Preferably, the polychlorinated biphenyl is PCB9.
The invention domesticates and separates 1 strain P49-1 which takes high-concentration polychlorinated biphenyl (PCB 9) as a carbon source from the sewage bottom mud of an electronic garbage recovery plant in Qingyuan, guangdong province, identifies the strain, researches the growth characteristic and the degradation characteristic of the strain on the PCB9, and provides a reference for the bioremediation of PCBs polluted environment.
Labrys portucalensis P49-1 was deposited at 2.6.2021 in Guangdong province culture Collection (GDMCC) at address: building 5 of first furnance, large yard, 100, building 59, guangdong province, guangzhou, china, zip code: 510070, the preservation number is: GDMCC No:61706.
description of the drawings:
FIG. 1 shows P49-1 grown on an inorganic salt solid medium using polychlorinated biphenyl as a carbon source.
FIG. 2 is a phylogenetic relationship of strain P49-1 and related bacteria based on 16S rRNA gene sequence, the construction method is a adjacency method, the setting of the self-expansion value is repeated 1000 times, only the result that the self-expansion value is more than 50% is shown in the figure, and the scale bar 0.1 represents the replacement rate of each nucleotide.
FIG. 3 shows the degradation efficiency of strain P49-1 in mineral salts medium with high concentration of PCB9 (initial concentration 10 mg. Multidot.L) -1 )。
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1: isolation and characterization of Labrys portucalensis P49-1
1. Materials and methods
1.1 sample sources
Collecting a sewage sample from sewage bottom mud of an electronic garbage recycling plant in Qing Yuan of Guangdong, taking high-concentration polychlorinated biphenyl (PCB 9) as a carbon source for long-term domestication, and obtaining the efficient polychlorinated biphenyl degrading bacteria through multiple screening, separation and purification.
1.2 culture Medium
1.2.1 inorganic salt Medium
The inorganic salt culture medium is used for enrichment culture of microorganisms in a sample and polychlorinated biphenyl degradation experiments under pure bacteria conditions. The formula of the culture medium is shown in table 1 (polychlorinated biphenyl solution), and the preparation method comprises adding the components into solvent water, mixing well, and sterilizing.
TABLE 1 inorganic salts culture medium formulation
1.2.2 nutrient Medium
The nutrient medium is used for culturing conventional microorganisms such as separation, purification, preservation, activation and the like of bacteria. The types and compositions of the liquid nutrient media used in this experiment are shown in Table 2. If the experiment needs to prepare a solid culture medium, only 1.5-2% of agar powder needs to be added on the basis of the original culture medium formula. If the culture conditions of the strains are not specified, the pH of the medium is adjusted to 7.0. The preparation method comprises adding the above materials into solvent water, mixing, adjusting pH, and sterilizing.
TABLE 2Luria-Bertani Medium (LB) composition
1.3 acclimatization, screening and isolation of strains
Adding the collected sewage into an enrichment medium (inorganic salt medium) with the concentration of 10 mg.L respectively -1 As a lower polychlorinated biphenylAnd placing the decomposed substrate in an incubator at 30 ℃ in a dark place for shake culture. And (3) carrying out strain domestication by using an inorganic salt culture medium taking polychlorinated biphenyl as a carbon source, wherein 7d is a domestication period. The inoculum size of 10% by volume was transferred to fresh enrichment medium with the same culture system and the enrichment process was repeated three times.
And (3) coating and separating the fourth generation enrichment culture sample obtained by the method of dilution plate, and separating the sample by using nutrient medium. Culturing the coated sample at the original culture temperature for about 48 hours to form obvious single colonies on the surface of the culture medium, selecting a plurality of different single colonies according to the characteristics of the colony such as morphology size, color, transparency and the like, and carrying out streak purification and culture on a nutrient medium flat plate. If single colonies of different characteristics are still observed on the streaked plates, they are streaked again until only single colonies of the same characteristics are observed on the same plate. 1 strain P49-1 with high-efficiency degradation performance on polychlorinated biphenyl is obtained by screening in the experiment. And (3) selecting the purified single bacterial colony to be cultured in a corresponding liquid nutrient medium to logarithmic phase, mixing the bacterial liquid and sterile glycerol, subpackaging the mixture into a sterile 2ml freezing tube (the concentration of the glycerol is 15 percent), and placing the tube at the temperature of minus 80 ℃ for long-term storage.
A. Morphological characteristics
The strain P49-1 is a bacterium separated from sewage bottom mud of an electronic garbage recycling plant far from Guangdong, and can form a white, round, smooth-surface, slightly-upward-convex, viscous liquid, spore-free, gram-negative and rod-shaped bacterial colony with the diameter of 1.0-2.2mm after being activated and growing on a flat plate made of the inorganic salt culture medium for 48 hours under the aerobic condition of 30 ℃. The bacterium is an obligate aerobic bacterium, and the community map of the bacterium growing on inorganic salt is shown in figure 1.
B. Molecular biological characteristics of Strain P49-1
The molecular biological characteristic identification mainly comprises sequencing and the construction of a phylogenetic tree. Before sequencing and construction of phylogenetic trees, bacterial DNA extraction is required (the bacterial genomic DNA rapid extraction kit used in the experiment is from Erdela biotechnology, inc. of Beijing). In order to study the taxonomy of bacteria, it is usually necessary to amplify the 16S rRNA gene, which is a DNA fragment of the coding rRNA component of prokaryotes, and construct phylogenetic trees, which are commonly used for detecting and identifying bacteria because of its high degree of conservation, specificity and appropriate sequence length.
Polymerase Chain Reaction (PCR) is mainly used to amplify different gene fragments, and different primers (27F and 1492R) are required for PCR, and the PCR amplification reaction system: 10 XBuffer 2.5. Mu.l, mg2+ (25 mmol/l) 1.5. Mu.l, dNTP (25 mmol/l) 0.3. Mu.l, forward primer (10 mmol/l) 0.5. Mu.l, reverse primer (10 mmol/l) 0.5. Mu.l, taq enzyme: 0.25. Mu.l, DNA set template 0.1. Mu.l, deionized water 19.35. Mu.l. Pcr amplification reaction conditions: denaturation at 95 deg.C, annealing at 55 deg.C, extension at 72 deg.C, circulating for 30 times, extension at 72 deg.C for 10min, and storing at 4 deg.C after pcr reaction. Amplifying the needed gene, adding 0.75-1% agarose and nucleic acid stain GelRed to prepare a gel block, adding PCR products and DNA markers (marker) containing fragments of various lengths into the gel block, placing the gel block into an electrophoresis apparatus, filling TBE (Tris boric acid) buffer solution into the electrophoresis apparatus, taking out the gel block after the electrophoresis apparatus works for 20min under a certain voltage, and placing the gel block under an ultraviolet lamp of 300nm for observation to confirm that the PCR product amplification reaction is successful. Then, the pcr product successfully amplified is sent to Huada Gene science and technology Limited for sequencing, and the sequencing primer is the same as the amplification primer.
The sequenced bacterial 16s rRNA gene sequence was uploaded to EzTaxon-e (http:// EzTaxon-e. Ezbiocloud. Net; (ii)Kim et al.,2012) In the field of genetic engineering, the website will compare the submitted sequence with the 16S rRNA gene sequence of a typical strain of a recognized species to obtain the similarity information between the sequences. According to the result analysis of sequence comparison, the corresponding typical strain can be selected as the model strain of the experimental isolated strain, and the 16S rRNA gene sequence of the model strain can be obtained, and phylogenetic analysis is constructed to prove that the model strain and the experimental isolated strain have difference, so that the isolated strain is identified. Construction of phylogenetic Tree Using MEGA 5.05 program (Tamura et al.,2011) Usually by using a neighborThe method of inoculation, minimum evolution and maximum reduction is used to construct the evolutionary tree, wherein the most common method is the adjacency method, and the self-expansion value is usually set to repeat 1000 times of calculation (Felsenstein, 1985).
A gene sequence of 1423bp 1696 rRNA (the nucleotide sequence is shown in SEQ ID NO. 1) is obtained through PCR and gene sequencing. The strain has 99.4 percent of gene similarity with Labrys portucalensis F11 (NR _ 042863.1) through 16S rRNA gene alignment.
A phylogenetic tree is made by using the 16S rRNA gene sequence of the strain P49-1 and the 16S rRNA gene sequence with higher similarity, so that the homology result between the 16S rRNA gene and the 16S rRNA gene with higher similarity is obtained. A phylogenetic tree constructed by the orthotopic grafting method is shown in FIG. 2.
From the above results, it can be seen that the bacterium P49-1 isolated in this experiment is of the Labrys portucalensis species. It was therefore designated Labrys portugalensis P49-1, deposited at 2.6.2021 in Guangdong province culture Collection (GDMCC) at address: building 5 of first furnance, large yard, 100, building 59, guangdong province, guangzhou, china, zip code: 510070, the preservation number is: GDMCC No:61706.
at present, there are few reports about the application of the strain in the environmental field. Therefore, the obtained efficient polychlorinated biphenyl degrading bacteria have important theoretical and practical significance for the treatment and deep remediation of the polluted water body, bottom mud and soil containing PCBs.
Example 2: growth conditions of Labrys portucalensis P49-1
Measurement of growth temperature: a liquid nutrient medium (LB nutrient medium in example 1) required for growth of the strain was prepared, and the prepared medium was sterilized in a sterilizer. The method comprises the steps of inoculating activated Labrys portucalensis P49-1 into 10ml of LB nutrient medium (added according to the proportion of 1% of the volume ratio) (an experimental group), taking the nutrient medium without inoculated bacteria as a control (a control group), putting the nutrient medium into different temperatures for culturing for 18h, repeating the control group and the experimental group corresponding to each temperature for three times, observing the growth condition of the bacteria every day, measuring the light absorption value of the culture medium at the position of lambda =600nm by using a visible-ultraviolet spectrophotometer when a result which is difficult to distinguish by naked eyes is met, and finally obtaining the growth temperature and the optimal growth temperature range of new bacteria. The test temperatures were as follows: 4 ℃,15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃ and 55 ℃.
In LB nutrient medium, P49-1 can grow at 15-40 deg.c, and the optimal growth temperature is 30 deg.c.
Example 3: determination of growth pH
Preparing a liquid nutrient medium (LB) required by the growth of the strain, and adjusting the pH of a culture solution by using a buffer system with the pH of 4.0-5.0,0.1mol/l of sodium citrate and 0.1mol/l of citric acid; pH 6.0-8.0,0.1mol/l NaOH and 0.1mol/l KH 2 PO 4 ；pH 9.0–10.0，0.1mol/l NaHCO 3 And 0.1mol/l Na 2 CO 3 (ii) a pH 11.0,0.1mol/l NaOH and 0.05mol/l Na 2 HPO 4 And then sterilized and disinfected. Inoculating activated Labrys portucalensis P49-1 into 10ml of culture medium (added according to the proportion of 1 percent by volume), repeating three times for each pH, taking the culture medium without inoculating bacteria as a control, putting the culture medium into the optimal growth temperature of new bacteria for culturing for 7d at 30 ℃, observing the growth condition of the bacteria every day, and when a result which is difficult to distinguish by naked eyes is met, measuring the light absorption value of the culture medium at the wavelength of lambda =600nm by using a visible-ultraviolet spectrophotometer to finally obtain the growth pH value and the optimal growth pH range of the new bacteria. The tested pH was as follows: 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0.
The strain can grow under the pH condition of 4.0-9.0, and the optimal growth pH is 7.0.
In summary, the optimal growth conditions for Labrys portucalensis P49-1 were determined to be 30 ℃ at pH 7.0. Degradation experiments of Labrys portucalensis P49-1 in high concentration polychlorinated biphenyl (PCB 9) were carried out under these conditions.
Example 4: polychlorinated biphenyl (PCB 9) degradation experiment
Labrys portucalensis P49-1 in logarithmic growth phase was inoculated in 10% inoculum size by volume respectively in mineral salt medium (example 1) containing PCB9 initially at 10mg/L, cultured for 10 days with shaking, and subjected to 3 parallel experiments.
Taking each processing sample for chemical analysis, and the specific steps are as follows: (1) sample pretreatment: each culture sample was extracted by adding methylene chloride, and 5. Mu.L of a recovery indicator having a concentration of 200mg/L was added thereto ( 13 C-PCB 9), fully shaking, transferring into a separating funnel and standing. And collecting organic phases after layering, putting the lower layer liquid back to a shake flask, repeatedly extracting with dichloromethane of the same volume, combining the extracts, transferring the combined extracts to a flat-bottomed flask containing a proper amount of activated copper sheets for rotary evaporation, concentrating to about 2mL, adding a small amount of n-hexane (about 5 mL), carrying out rotary evaporation to 2mL, repeatedly washing for three times, and replacing the organic solvent with the n-hexane. The concentrate after the substitution was purified by a glass packed column (diameter: about 9 mm). The column packing was from bottom to top 3cm 3% deactivated neutral alumina, 3cm 3% deactivated silica gel and 1cm anhydrous sodium sulfate. The column is activated by using a proper amount of n-hexane, the packed column is rinsed by 15mL of mixed reagent of n-hexane/dichloromethane (volume ratio is 1). (2) instrumental analysis: and (3) determining the content of PCBs in each processed sample by adopting an Agilent 7890 gas chromatograph-5975 mass spectrometer. The column used was an Agilent DB5-MS capillary column (column length 30m, inner diameter 0.25mm, film thickness 0.25 μm). The obtained data are processed by an Agilent chromatographic workstation, and the quantification of the polychlorinated biphenyl is carried out by a 6-point calibration curve and an internal standard method. The cell concentration of the microorganisms is measured by a photoelectric turbidimetric method and is expressed by OD, namely the optical density value of ultraviolet light which penetrates through a measured bacterial liquid sample when the wavelength is 600 nm.
According to GC-MS determination and analysis, it is obtained that Labrys portularensis P49-1 can degrade polychlorinated biphenyl, and the degradation rate can reach more than 60 percent after the polychlorinated biphenyl is cultured in an inorganic salt culture solution containing PCB9 with the concentration of 10mg/L for 10 days (figure 3). The bacterial strain P49-1 is a bacterial strain which can degrade polychlorinated biphenyl and has strong tolerance to polychlorinated biphenyl and strong adaptability to PCBs.
And (4) conclusion: 1 polychlorinated biphenyl degrading bacteria Labrys portucaensis P49-1 which can grow by taking polychlorinated biphenyl as a carbon source is obtained by enriching and separating sewage bottom mud of an electronic garbage recycling plant far from Guangdong. The strain is gram-negativeThe bacteria can form white, round, smooth, slightly upwards-convex, spore-free, flagellum-free and slime-like colonies with the diameter of 1.0-2.2 mm. According to the analysis of molecular biology means, the bacterium P49-1 separated in the experiment is obtained as a Labrys portucensis P49-1 strain, and the evolutionary tree thereof is drawn. At present, few reports are made on the application of the strain, and particularly, no report is found in research on the use of the norpolychlorinated biphenyl. The optimal growth conditions of the strain are that the optimal growth conditions of the strain are determined to be that the temperature is 30 ℃ and the pH is 7.0.Labrys portucalensis P49-1 can be degraded by using polychlorinated biphenyl as a carbon source, and the initial concentration of the polychlorinated biphenyl in PCB9 is 10 mg.L -1 After the inorganic salt culture solution is cultured for 10 days, the degradation rate can reach more than 60 percent. In conclusion, the Labrys portularensis P49-1 is a strain capable of degrading polychlorinated biphenyl and having strong tolerance to polychlorinated biphenyl, has strong adaptability to polychlorinated biphenyl and has good application potential in the aspect of bioremediation.
Sequence listing
<110> Guangzhou geochemistry institute of Chinese academy of sciences
<120> Labrys portucalensis P49-1 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1423
<212> DNA
<213> Labrys portucalensis
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tagagtttga tcctggctca gaacgaacgc tggcggcagg cttaacacat gcaagtcgaa 60
cgccccgcaa ggggagtggc agacgggtga gtaacgcgtg gggatgtgcc ttgaggtggg 120
gaataactgt gggaaactac agctaatacc gcatacgccc ttttggggaa agatttatcg 180
cctttagagc aacccgcgtc agattagcta gttggtaggg taatggccta ccaaggcgac 240
gatctgtagc tggtctgaga ggatgaccag ccacactggg actgagacac ggcccagact 300
cctacgggag gcagcagtgg ggaatattgg acaatgggcg caagcctgat ccagccatgc 360
cgcgtgagtg atgacggcct tagggttgta aagctctttt aacagggacg ataatgacgg 420
tacctgtaga ataagccccg gcaaacttcg tgccagcagc cgcggtaata cgaagggggc 480
tagcgttgtt cggaattact gggcgtaaag cgcacgtagg cggattgtta agtcgggggt 540
gaaatcctga ggctcaacct cagaactgcc ttcgatactg gcgatcttga gttcggaaga 600
ggttggtgga acagctagtg tagaggtgaa attcgtagat attagctaga acaccagtgg 660
cgaaggcggc caactggtcc gatactgacg ctgaggtgcg aaagcgtggg gagcaaacag 720
gattagatac cctggtagtc cacgccgtaa acgatgaatg ccagccgtcg gggagcttgc 780
tcttcggtgg cgcagctaac gctttaagca ttccgcctgg ggagtacggt cgcaagatta 840
aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgaag 900
caacgcgcag aaccttacca gcccttgaca tcccggtcgc ggatcacaga gatgagatcc 960
ttcagttcgg ctggaccgga gacaggtgct gcatggctgt cgtcagctcg tgtcgtgaga 1020
tgttgggtta agtcccgcaa cgagcgcaac cctcgcccct agttgccagc attcagttgg 1080
gcactctagg gggactgccg gtgataagcc gcgaggaagg tggggatgac gtcaagtcct 1140
catggccctt acgggctggg ctacacacgt gctacaatgg cggtgacagt gggaagcgaa 1200
ggggtgaccc ttagcaaatc tccaaaagcc gtctcagttc agattgcact ctgcaactcg 1260
agtgcatgaa ggtggaatcg ctagtaatcg cagatcagca tgctgcggtg aatacgttcc 1320
cgggccttgt acacaccgcc cgtcacatca tgggagttgg ttttactaga gtttgctgcg 1380
ccatcagcaa ggcgtcacaa ggtaactaga gtttaccgcg cta 1423
Claims (4)
1.Labrys portucalensis P49-1 with the preservation number: GDMCC No:61706.
2. the method of claim 1Labrys portucalensis The application of P49-1 in degrading polychlorinated biphenyl, wherein the polychlorinated biphenyl is PCB9.
3. Use according to claim 2, characterized in thatLabrys portucalensis The P49-1 is applied to the polychlorinated biphenyl polluted environment to degrade the polychlorinated biphenyl.
4. A polychlorinated biphenyl degrading bacterial agent comprising the compound according to claim 1Labrys portucalensis P49-1 is taken as an active ingredient, and the polychlorinated biphenyl is PCB9.
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