CN115197864A - Pseudomonas and method for degrading monocyclic aromatic hydrocarbon by using same - Google Patents
Pseudomonas and method for degrading monocyclic aromatic hydrocarbon by using same Download PDFInfo
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Abstract
The invention discloses pseudomonas and a method for degrading monocyclic aromatic hydrocarbon by applying the pseudomonas, belonging to the technical field of microbial treatment of environmental pollution. The aromatic hydrocarbon degrading bacteria CX20 belongs to pseudomonas (Pseudomonas sp), is deposited in Guangdong province institute of microbiology, and has the preservation number of GDMCC No. 61305. The bacillus subtilis is a gram-negative bacterium, has the biological characteristics of non-fermentation type and obligate aerobic property, has a bacillus-free thallus form, a round colony, light yellow opacity, smooth and moist surface, luster, smooth edge, a convex center and a diameter of 2-3 mm. The strain has good degradation capability on toluene, m-xylene and p-xylene, which shows that the strain has wide substrate utilization capability. The strain is suitable for degrading aromatic hydrocarbon substances in industrial wastewater and waste gas, and has wide application prospect.
Description
Technical Field
The invention relates to an aromatic hydrocarbon degrading bacterium, in particular to a pseudomonas and a method for degrading aromatic hydrocarbon by applying the pseudomonas, wherein the aromatic hydrocarbon is mainly a monocyclic aromatic hydrocarbon compound, and belongs to the technical field of biological treatment of environmental pollutants.
Background
With the continuous progress of social industrialization and urbanization, environmental problems caused by Volatile Organic Compounds (VOCs) are becoming more serious. How to control the emission of volatile organic compounds and the management of volatile organic compounds have become one of the hot spots of research in recent years. Aromatic hydrocarbon compounds in volatile organic compounds bring great threats to the safety and health of people, for example, benzene, toluene and xylene can produce paralysis on the central nervous system, cause acute toxicity and have stimulation on skin mucous membranes, wherein benzene is a clear carcinogen.
The physical and chemical method for treating the dimethylbenzene can achieve a good effect, but the cost is high, and secondary pollution is easily caused. Compared with the traditional treatment method, the biological treatment method is concerned at home and abroad due to the advantages of low operation cost, simple construction, no secondary pollution and the like, and gradually becomes the mainstream of the current volatile organic compound treatment. At present, biological treatment methods are widely applied to the field of treatment of waste gases such as malodors and VOCs. However, the technology still has many problems in practical application, one of which is that the biological method has a good effect of treating low-concentration organic waste gas, but has a very low effect of treating high-concentration waste gas, and one strain can only target one organic pollutant and has a very small effect on other pollutants. Therefore, there is an urgent need for a highly efficient and broad-spectrum degrading bacterium, which has a good effect on high-concentration aromatic hydrocarbon waste gas and a good degrading effect on various aromatic hydrocarbon substances, so as to improve the removal effect of a biological treatment system on target pollutants.
Disclosure of Invention
The invention mainly aims to solve the problems in the prior art, and provides pseudomonas capable of efficiently degrading various monocyclic aromatic hydrocarbons such as toluene, xylene and the like and a method for degrading aromatic hydrocarbons by using the pseudomonas, so that the pseudomonas is applied to treatment of chemical wastewater and waste gas.
The purpose of the invention is realized by the following technical scheme:
one pseudomonas is pseudomonas (pseudomonas sp), and is preserved with the code of gdmcc.no 61305.
The method for degrading monocyclic aromatic hydrocarbon by using pseudomonas comprises the following steps: the pseudomonas (pseudomonas sp) takes monocyclic aromatic hydrocarbon as a carbon source, and the monocyclic aromatic hydrocarbon is degraded by shake cultivation.
For further achieving the object of the present invention, preferably, the temperature of the shake culture is 25 to 30 ℃.
Preferably, the rotating speed of the shaking culture is controlled to be 120-160r/min.
Preferably, the time of the shake culture is 24-72h.
Preferably, the monocyclic aromatic hydrocarbon is one or more of benzene, toluene and xylene.
Preferably, the monocyclic aromatic hydrocarbon is xylene, such as m-xylene and p-xylene.
Preferably, the shake culture is performed by inoculating pseudomonas into an inorganic salt medium containing a carbon source substrate, and controlling the initial pH to be 7-8.
Preferably, the inorganic salt culture medium is a culture medium containing N, P, S, K, ca, mg and trace elements, wherein the trace elements comprise Fe, cu, zn and Co.
Preferably, the inoculation amount of the pseudomonas is 4-6wt%; the concentration of the carbon source is 365.6mg/L or less.
The pseudomonad (pseudomonas sp) of the present invention is named as pseudomonad CX20 or pseudomonad CX20 (pseudomonas sp CX 20).
The existing VOC biological treatment systems are generally micro-ecosystems that are gradually built up through environmental induction for a period of several tens of days, in which there are a large number of bacteria that can utilize volatile organic compounds, and some strains may have strong bioconversion ability. Benzene series (mainly monocyclic aromatic hydrocarbon) is used as a unique carbon source, and microorganisms in a VOC biological treatment system are enriched, domesticated, separated and purified, so that a strain with high conversion rate is expected to be obtained. The obtained high-conversion strain can be further subjected to pure culture in a fermentation tank and directly put into a VOC biological treatment system, so that the treatment efficiency is greatly improved.
The strain is obtained by screening solid filler in a biological trickling filter in a VOC treatment system of a certain company, domesticating, separating, purifying and screening.
The screening method of the strain comprises the following steps: taking an activated sludge sample by using a clean test tube, preserving at 4 ℃, acclimatizing and culturing by using an acclimatization culture medium with dimethylbenzene as a unique carbon source, taking 50 microliters of culture solution after acclimatization for one week, uniformly coating the culture solution on a plate screening culture medium, selecting a single colony with good growth vigor on the plate culture medium, carrying out streak separation, and repeating for three times to obtain a pure culture strain.
The domestication culture medium is a general inorganic salt culture medium and mainly contains N, P, S, K, ca, mg and trace elements, wherein the trace elements comprise Fe, cu, zn and Co. The following formulation is preferred: KH (Perkin Elmer) 2 PO 4 338.8mg,(NH 4 ) 2 SO 4 234.O mg,Na 2 CO 3 100.0mg,CaCl 2 ·2H 2 O 5.16mg,MgSO 4 ·7H 2 O 59.3mg,Na 2 HPO 4 ·12H 2 O 890.7mg, FeSO 4 ·7H 2 O0.37mg, 1mL of microelement mother liquor (FeCl) 2 ·4H 2 O 1500mg,CoCl 2 ·6H 2 O 190mg,ZnCl 2 70mg,MnSO 4 ·7H 2 O 100mg,NiCl 2 ·6H 2 O 24mg,Na 2 MoO4·2H 2 O 24mg,MnCl 2 ·4H 2 O 6mg,CuCl 2 ·2H 2 O2 mg, distilled water 1000mL, solid medium plus 1.5% agar.
Culture conditions of the strain: the temperature is 25-30 ℃, the initial pH is 7-8, and the rotating speed of the shaking table is 150rpm.
The Pseudomonas sp CX20 degrading bacteria can take 100-210mg/L of volatile organic pollutants such as toluene, xylene and the like as a carbon source, and can be cultured for 24-72h at the temperature of 25-30 ℃ and at the speed of 120-160r/min by a shaking table, and the quantity of the bacteria reaches10 8 More than cfu/mL.
Pseudomonas sp CX20 (pseudomonas sp CX 20) has a strong tolerance to toluene and xylene. When the concentration of the toluene is lower than 130.5mg/L, CX20 can completely degrade the toluene within 48h, and then the degradation rate gradually decreases with the increasing of the concentration of the toluene, but when the concentration of the toluene reaches 261mg/L, the degradation rate of the toluene is still 52.55%, and when the concentration of the toluene reaches 365.6mg/L, the degradation rate of the toluene is 20%. From the growth state of the cells, when the toluene concentration is lower than 208.8mg/L, the cell concentration increases with the increase of the substrate concentration, and the peak value is 2.13X 10 8 cfu/mL。
Strain CX20 is also very tolerant to meta-xylene and para-xylene. When the concentration of the m-xylene is lower than 129mg/L, the strain can completely degrade the m-xylene in the culture medium within 48 hours, when the concentration of the m-xylene is 129-180.6 mg/L, the degradation rate of the strain to the m-xylene is 80% -90%, and only when the concentration of the m-xylene exceeds 193.5mg/L, the degradation rate of the m-xylene is rapidly reduced. When the concentration of the paraxylene is lower than 180.6mg/L, the strain can completely degrade the paraxylene in the culture medium within 48 hours, and when the concentration of the paraxylene exceeds 193.5mg/L, the degradation rate of the paraxylene is rapidly reduced. From the growth condition of the strain, the maximum growth concentration of the strain is 2.53 multiplied by 10 when the substrate is meta-xylene 8 cfu/mL; the maximum growth concentration of the strain is 1.07 x 10 when the substrate is p-xylene 8 cfu/mL。
When pseudomonas CX20 (Pseudomonas sp CX 20) is used for degrading monocyclic aromatic hydrocarbon, the carbon source is at an initial concentration of 100-210mg/L, the degradation rate of CX20 to toluene, m-xylene and p-xylene is 60-100% within 24-72h, and the degradation effect to o-xylene, benzene and butyl acetate is also certain.
Compared with the monocyclic aromatic hydrocarbon degradation strain in the prior art, the pseudomonas CX20 (Pseudomonas sp CX 20) provided by the invention has the following advantages:
1) The pseudomonas CX20 (pseudomonas sp CX 20) can degrade various aromatic hydrocarbon compounds such as toluene, p-xylene and m-xylene in waste water, and is particularly suitable for application in paint shops.
2) The invention has higher degradation efficiency on a plurality of aromatic hydrocarbon compounds such as toluene, m-xylene, p-xylene and the like, when the concentration of a substrate is about 100mg/L, the degradation rate of the toluene and the p-xylene is more than 99.9 percent, the degradation rate of the m-xylene is more than 88 percent, and when the concentration of the toluene is 206mg/L, the degradation rate of the toluene in 24 hours of pseudomonas CX20 reaches 70 percent.
3) The invention has fast degradation rate and good growth of various aromatic hydrocarbon compounds such as p-toluene, m-xylene, p-xylene and the like, and the thallus concentration reaches 1.80 multiplied by 10 when being used for 48 hours 8 cfu/mL。
Drawings
FIG. 1 is a macroscopic colony map of Pseudomonas sp CX20 (Pseudomonas sp CX 20) as a degrading bacterium according to the present invention.
Fig. 2 is an oil microscope micrograph of pseudomonas degrading CX20 (pseudomonas sp CX 20) of the present invention.
FIG. 3 is a phylogenetic analysis tree of Pseudomonas sp CX20 (Pseudomonas sp CX 20) as a degradative bacterium according to the present invention.
FIG. 4 is a graph showing the degradation of Pseudomonas sp CX20 (Pseudomonas sp CX 20) in a medium containing six single-substrate inorganic salts of benzene, toluene, (o-, m-, p-) xylene and n-butyl acetate.
Fig. 5 shows the utilization of different concentrations of toluene by pseudomonas degradans CX20 (pseudomonas sp CX 20) according to the present invention.
FIG. 6 shows the utilization of different concentrations of meta-xylene by Pseudomonas bacteria according to the present invention.
FIG. 7 shows the utilization of different concentrations of paraxylene by Pseudomonas degradans according to the present invention.
FIG. 8 is a graph showing the degradation curve of Pseudomonas sp CX20 (Pseudomonas sp CX 20) in a medium containing toluene single substrate inorganic salt at a concentration of 206mg/L for 72 hours along with the change of the cell concentration.
Detailed Description
The present invention will be described in more detail below with reference to specific embodiments and drawings.
Example 1: acclimatization and screening of Pseudomonas CX20 (Pseudomonas sp CX 20)
Inoculating 5mL of an activated sludge sample in a biological filter of Guangdong Shaoguan place into a 250mL conical flask containing 100mL of domestication culture medium, then adding a certain amount of dimethylbenzene as a unique carbon source, sealing the bottle mouth to prevent the dimethylbenzene from volatilizing, culturing for 2d at 30 ℃ and the rotating speed of a shaking table of 150rpm, then transferring by 5 percent of inoculation amount and continuing domestication with a fresh culture medium under the same culture condition, sequentially increasing the content of the dimethylbenzene added in the culture medium, and sequentially controlling the concentration of the dimethylbenzene in the culture medium to be 10mg/L, 30mg/L, 50mg/L, 80mg/L and 120mg/L. After the acclimatization is finished, taking 100 mu L of the final acclimatization culture, coating the final acclimatization culture on a flat plate screening culture medium, sealing the culture medium by using a sealing film, putting the culture medium into a constant temperature incubator at 30 ℃ for inverted culture for 1d, and then putting the culture dish for upright culture for 1d. And selecting single colonies with good growth on a plate culture medium, streaking and separating the colonies for three times to obtain pure culture strains. The pure culture bacterial colony is circular, light yellow and opaque, the surface is smooth, moist and glossy, the edge is smooth, the center is convex, and the diameter is 2-3 mm, as shown in figure 1.
Example 2: pseudomonas sp CX20 (Pseudomonas sp CX 20) physiological and biochemical test
The form of the Pseudomonas CX20 was observed with a 10X 100 oil scope, and it was found from the figure that the bacterium was a short rod-shaped bacterium without spores.
The bacteria is determined to be gram negative, positive line of catalase test, positive line of citrate test, negative of starch hydrolysis test, negative of esterase test, negative of methyl red test, negative of V-P test, negative of indole test and negative of nitrate reduction test by performing physiological and biochemical tests such as gram staining, catalase test, starch hydrolysis test, esterase test, citrate test and the like on the bacteria.
Example 3: identification of Pseudomonas CX20 (Pseudomonas sp CX 20)
Extracting and separating strain DNA by using a silica gel membrane centrifugal column method according to the steps detailed in the operation manual of the bacterial genome extraction kit, and extracting and separating the strain DNA by using a 16S rDNA universal primer 27F: -AGA GTT TGA TCCTGC TCA G-3 'and 1492r 5' seq.id.no3: GGT TAC CTT GTT ACG ACT T-3' and PCR amplification system is as follows: a50. Mu.L PCR reaction contained 1. Mu.L of DNA, 2. Mu.L of the forward primer, 2. Mu.L of the reverse primer, 20. Mu.L of 2 XPCR Mix, and 25. Mu.L of ultrapure water. Then, a 1% (w/v) agarose gel prepared by using 0.5 XTBE buffer solution is added with Gold view nucleic acid dye, and an appropriate amount of PCR products are spotted and subjected to agarose gel electrophoresis, and then observed by gel electrophoresis. After confirming that the target band was clear and bright and in the correct molecular weight range, the DNA fragment was recovered from the PCR reaction solution using the PCR product purification kit. The recovered DNA fragments were sent to Biotechnology engineering (Guangzhou) for DNA sequencing, and the determined DNA sequences were copied to NCBI (national center of biotechnology information) for BLAST (basic local alignment search tool) alignment. The cell was identified as Pseudomonas plecoglossicida (Pseudomonas plecoglossicida) by alignment. The strains were then further identified as P.plecoglossus, using MEGA-6 software to construct a phylogenetic tree of strains, see FIG. 3. The nucleotide sequence of 16S rRNA of the pseudomonas (pseudomonas sp) is shown in SEQ ID No. 1.
The pseudomonas (pseudomonas.sp) is preserved in a microbial strain preservation center in Guangdong province in 11 months and 20 days in 2020, the preservation address is No. 59 floor 5 of Michelia Tokyo No. 100 of Guangzhou city in Guangzhou province, the preservation number is GDMCC No. 61305, and the preservation date is 11 months and 20 days in 2020.
Example 4 application of the growing cell strains in the degradation of six single substrates, benzene, toluene, three xylenes and n-butyl acetate
Inoculating pseudomonas degrading strain CX20 in logarithmic growth phase into 100mg/L inorganic salt culture medium containing single substrate (benzene, toluene, three kinds of xylene and butyl acetate respectively) according to the inoculation amount of 5wt%, controlling the initial pH to be 7.0, performing shake cultivation at 30 ℃ and 140r/min for 48h, measuring the content of benzene, toluene, xylene and butyl acetate in the culture solution by using headspace gas chromatography, and calculating the environmental loss by taking a blank with the addition of equal amount of sterile water as a reference. The inorganic salt culture medium may be a common inorganic salt culture medium of the present invention, and is a culture medium containing N, P, S, K, ca, mg and trace elementsThe elements include Fe, cu, zn, and Co. Preferably, the inorganic salt culture medium comprises: KH (Perkin Elmer) 2 PO 4 1g,K 2 HPO 4 ·3H 2 O 1g,(NH 4 ) 2 SO 4 0.5g,MgSO 4 ·7H 2 0 0.36g,KNO 3 0.5g,CaCl 2 0.001g,1mL microelement mother liquor (FeCl) 2 ·4H 2 O 1500mg,CoCl 2 ·6H 2 O 190mg,ZnCl 2 70mg,MnSO 4 ·7H 2 O 100mg,NiCl 2 ·6H 2 O 24mg,Na 2 MoO4·2H 2 O 24mg,MnCl 2 ·4H 2 O 6mg,CuCl 2 ·2H 2 O2 mg, distilled water 1000 mL) agar powder 17g, distilled water 1000mL, autoclaved at 121 ℃ for 15min.
The degradation effect of the pseudomonas CX20 on a single substrate is shown in figure 4, the degradation effect of the CX20 on toluene is best, the toluene is completely degraded in 48 hours under the initial concentration of 100mg/L, the m-xylene and the p-xylene have stronger degradation effects, the degradation rates under the initial concentration of 100mg/L are respectively 88.34% and 99.89%, the degradation rates on o-xylene, benzene and butyl acetate also have certain degradation effects, and the degradation rates are 3.76%,7.25% and 18.07%.
EXAMPLE 5 tolerance of the strains to toluene and xylene
To investigate the tolerance of strain CX20 to toluene and xylene, a series of concentration gradients were set up for degradation experiments, with toluene concentration (mg/L): 104.4 130.5, 156.6, 208.8, 261, 313.2, 365.4, 417.6; xylene concentration (mg/L): 103.2, 129, 154.8, 167.7, 180.6, 193.5, 206.4, 258. The experimental conditions were the same as in example 4.
The tolerance of strain CX20 to different concentrations of toluene is shown in fig. 5. When the concentration of the toluene is lower than 130.5mg/L, CX20 can completely degrade the toluene within 48h, and then the degradation rate of the toluene is continuously reduced along with the continuous increase of the concentration of the toluene; when the concentration of toluene reaches 261mg/L, the degradation rate is reduced to 52.55%; when the concentration of the toluene exceeds 365.6mg/L, the degradation rate of the toluene is less than 20 percent. From the aspect of the growth condition of the thallus, when the concentration of the toluene is lower than 208.8mg/L, the concentration of the thallus increases along with the increase of the concentration of the substrate, and the peak valueIs 2.13X 10 8 cfu/mL, which shows that toluene has promotion effect on the growth of strains in the concentration range and is the main nutrient limiting condition for the growth of strains. And when the concentration of the substrate is between 208.8 and 313.2mg/L, the concentration of the thalli begins to continuously decrease, which shows that the toluene in the concentration range has a certain inhibition effect on the growth of the strains, probably because the toluene has a certain cytotoxicity effect on the strains, so that the growth of the strains is inhibited, and the degradation rate of the strains on the substrate is reduced.
The strain CX20 has similar utilization to meta-xylene (FIG. 6) and para-xylene (FIG. 7). When the substrate is metaxylene, the strain can completely degrade the metaxylene in the culture medium when the concentration is lower than 129mg/L, the degradation rate of the strain is between 80 and 90 percent when the concentration of the metaxylene is between 129 and 180.6mg/L, and the degradation rate of the metaxylene is rapidly reduced to below 5 percent when the concentration of the metaxylene is higher than 193.5 mg/L. When the substrate is p-xylene, the strain can completely degrade the p-xylene with the concentration of less than 180.6mg/L within 48 hours, and when the p-xylene concentration exceeds 193.5mg/L, the degradation rate of p-xylene is also rapidly reduced. From the growth condition of the strain, the change rule is similar. When the concentration of the target substrate is lower than 180.6mg/L, the concentration of the bacteria increases with the increase of the substrate concentration and reaches a peak value. The maximum growth concentration of the strain is 2.53X 10 when the substrate is m-xylene 8 cfu/mL; the maximum growth concentration of the strain is 1.07 x 10 when the substrate is p-xylene 8 cfu/mL. The growth rule of the strain is consistent overall, but the growth condition of the strain is obviously better than that of p-xylene when the substrate is m-xylene. When the concentration of the substrate exceeds 193.5mg/L, the strain basically does not grow in the presence of the two substrates and is consistent with the change rule of the degradation rate of the dimethylbenzene.
Example 6: time-dependent degradation of a growing cell strain in a toluene single substrate mineral salt medium at a concentration of 206mg/L
Inoculating pseudomonas degrading strain CX20 in logarithmic growth phase into 206mg/L inorganic salt culture medium containing single substrate toluene in an inoculation amount of 5%, controlling the initial pH to be 7.0, and performing shake culture at 30 ℃ and 140r/minMeasuring the toluene content in the culture solution every 12h by using headspace gas chromatography, and measuring OD 600 And calculating the bacterial concentration, drawing a curve of the toluene concentration and the bacterial concentration changing along with time, and adding a blank of the same amount of sterile water as a reference to calculate the environmental loss.
As shown in FIG. 8, CX20 was able to grow stably in the medium containing 206mg/L of toluene as the sole carbon source, and the concentration of bacteria reached the maximum at 48h, which was 1.80X 10 8 cfu/mL, the toluene content is sharply reduced in the first 24 hours and then tends to be stable, about 50mg/L, and the degradation efficiency can reach 75.06 percent.
Sequence listing
The applicant: guangzhou Rigge Biotechnology Ltd
Application name: pseudomonas and method for degrading monocyclic aromatic hydrocarbon by using same
1. Pseudomonas sp 16S rRNA nucleotide sequence
GCAGTCGAGCGGATGACGGGAGCTTGCTCCTTGATTCAGCGGCGGACGGGTGAGTAA TGCCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCG CATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCT AGGTCGGATTAGCTAGTTGGTGGGGTAATGGCTCACCAAGGCGACGATCCGTAACTGG TCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAG GCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGT GTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTT AATACCTTGCTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGC AGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCG CGTAGGTGGTTTGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATC CAAAACTGGCAAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGA AATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACT GACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCAC GCCGTAAACGATGTCAACTAGCCGTTGGAATCCTTGAGATTTTAGTGGCGCAGCTAAC GCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGA CGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACC TTACCAGGCCTTGACATGCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACT CTGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTC CCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAAG GAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCC CTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCG CGAGGTGGAGCTAATCTCACAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCG ACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTT CCCGGGCCTTGTACACACCGC
2. PCR amplification with 26s rDNA sequence universal primer 27f: -AGA GTT TGA TCCTGC TCA G-3'
3. PCR amplification with 26s rDNA sequence universal primer 1492r: -GGT TAC CTT GTT ACG ACT T-3'
Sequence listing
<110> Guangzhou Rigger Biotechnology Ltd
<120> pseudomonas and method for degrading monocyclic aromatic hydrocarbon by using same
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1340
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> nucleotide sequence of 16S rRNA of Pseudomonas sp
<400> 1
gcagtcgagc ggatgacggg agcttgctcc ttgattcagc ggcggacggg tgagtaatgc 60
ctaggaatct gcctggtagt gggggacaac gtttcgaaag gaacgctaat accgcatacg 120
tcctacggga gaaagcaggg gaccttcggg ccttgcgcta tcagatgagc ctaggtcgga 180
ttagctagtt ggtggggtaa tggctcacca aggcgacgat ccgtaactgg tctgagagga 240
tgatcagtca cactggaact gagacacggt ccagactcct acgggaggca gcagtgggga 300
atattggaca atgggcgaaa gcctgatcca gccatgccgc gtgtgtgaag aaggtcttcg 360
gattgtaaag cactttaagt tgggaggaag ggcagtaagt taataccttg ctgttttgac 420
gttaccgaca gaataagcac cggctaactc tgtgccagca gccgcggtaa tacagagggt 480
gcaagcgtta atcggaatta ctgggcgtaa agcgcgcgta ggtggtttgt taagttggat 540
gtgaaagccc cgggctcaac ctgggaactg catccaaaac tggcaagcta gagtacggta 600
gagggtggtg gaatttcctg tgtagcggtg aaatgcgtag atataggaag gaacaccagt 660
ggcgaaggcg accacctgga ctgatactga cactgaggtg cgaaagcgtg gggagcaaac 720
aggattagat accctggtag tccacgccgt aaacgatgtc aactagccgt tggaatcctt 780
gagattttag tggcgcagct aacgcattaa gttgaccgcc tggggagtac ggccgcaagg 840
ttaaaactca aatgaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg 900
aagcaacgcg aagaacctta ccaggccttg acatgcagag aactttccag agatggattg 960
gtgccttcgg gaactctgac acaggtgctg catggctgtc gtcagctcgt gtcgtgagat 1020
gttgggttaa gtcccgtaac gagcgcaacc cttgtcctta gttaccagca cgttatggtg 1080
ggcactctaa ggagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaagtcat 1140
catggccctt acggcctggg ctacacacgt gctacaatgg tcggtacaga gggttgccaa 1200
gccgcgaggt ggagctaatc tcacaaaacc gatcgtagtc cggatcgcag tctgcaactc 1260
gactgcgtga agtcggaatc gctagtaatc gcgaatcaga atgtcgcggt gaatacgttc 1320
ccgggccttg tacacaccgc 1340
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 27F
<400> 2
agagtttgat cctgctcag 19
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 1492R
<400> 3
ggttaccttg ttacgactt 19
Claims (10)
1. Pseudomonas, characterized in that the strain is Pseudomonas sp, with the preservation number GDMCC No. 61305.
2. A method for degrading monocyclic aromatic hydrocarbon by using Pseudomonas sp, which comprises subjecting Pseudomonas sp (Pseudomonas sp) as defined in claim 1 to shake culture using monocyclic aromatic hydrocarbon as a carbon source to degrade monocyclic aromatic hydrocarbon.
3. The method for degrading monocyclic aromatic hydrocarbon using Pseudomonas as claimed in claim 2, wherein said shake cultivation temperature is 25-30 ℃.
4. The method of claim 2, wherein the rotation speed of the shaking culture is controlled to be 120-160r/min.
5. The method of claim 2, wherein the shaking culture is carried out for 24-72 hours.
6. The method for degrading monocyclic aromatic hydrocarbons using Pseudomonas sp as in any one of claims 2 to 4, wherein said monocyclic aromatic hydrocarbons are one or more of benzene, toluene and xylene.
7. The method for degrading monocyclic aromatic hydrocarbon using Pseudomonas sp as claimed in claim 6, wherein said monocyclic aromatic hydrocarbon is xylene selected from m-xylene and p-xylene.
8. The method for degrading monocyclic aromatic hydrocarbons using Pseudomonas bacteria as claimed in any of claims 2 to 4, wherein said shake culture is a culture in which Pseudomonas bacteria are inoculated into an inorganic salt medium containing a carbon substrate and the initial pH is controlled to 7 to 8.
9. The method for degrading monocyclic aromatic hydrocarbons using Pseudomonas bacteria as claimed in claim 6, wherein said inorganic salt medium is a medium containing N, P, S, K, ca, mg and trace elements including Fe, cu, zn, co.
10. The method for degrading monocyclic aromatic hydrocarbons using Pseudomonas sp as claimed in claim 6, wherein the inoculum size of said Pseudomonas sp is 4-6wt%; the concentration of the carbon source is 365.6mg/L or less.
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CN101955896A (en) * | 2010-06-18 | 2011-01-26 | 上海交通大学 | Organic solvent tolerance Pseudomonas and application thereof to degrading polycyclic aromatic hydrocarbons (PAHs) |
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CN105670962A (en) * | 2016-01-22 | 2016-06-15 | 江南大学 | Efficient aromatic hydrocarbon degradation bacterium and application thereof |
CN113462622A (en) * | 2021-09-03 | 2021-10-01 | 佛山市玉凰生态环境科技有限公司 | Pseudomonas for efficiently degrading various aromatic pollutants and application thereof |
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CN101955896A (en) * | 2010-06-18 | 2011-01-26 | 上海交通大学 | Organic solvent tolerance Pseudomonas and application thereof to degrading polycyclic aromatic hydrocarbons (PAHs) |
CN105087429A (en) * | 2015-07-17 | 2015-11-25 | 武汉科技大学 | O-xylene degradation bacterium and application thereof |
CN105670962A (en) * | 2016-01-22 | 2016-06-15 | 江南大学 | Efficient aromatic hydrocarbon degradation bacterium and application thereof |
CN113462622A (en) * | 2021-09-03 | 2021-10-01 | 佛山市玉凰生态环境科技有限公司 | Pseudomonas for efficiently degrading various aromatic pollutants and application thereof |
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