CN104830737A - Pseudomonas aeruginosa strain and application thereof - Google Patents

Pseudomonas aeruginosa strain and application thereof Download PDF

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Publication number
CN104830737A
CN104830737A CN201510260525.3A CN201510260525A CN104830737A CN 104830737 A CN104830737 A CN 104830737A CN 201510260525 A CN201510260525 A CN 201510260525A CN 104830737 A CN104830737 A CN 104830737A
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grams
gram
bacterial strain
aeruginosa bacterial
pseudomonas aeruginosa
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CN104830737B (en
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吴涓
左珊珊
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Anhui University
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Anhui University
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Abstract

The invention discloses a pseudomonas aeruginosa strain, of which the collection number is CCTCC M 2015272. The invention further provides an application of the pseudomonas aeruginosa strain CCTCC M 2015272 in synthesis of a glycolipid biosurfactant, and a method for synthesizing the glycolipid biosurfactant by employing the pseudomonas aeruginosa strain CCTCC M 2015272. The pseudomonas aeruginosa strain has the beneficial effects that 1 the pseudomonas aeruginosa strain CCTCC M 2015272 disclosed by the invention can be applied to synthesis of the glycolipid biosurfactant, and the yield of the glycolipid biosurfactant of the pseudomonas aeruginosa strain CCTCC M 2015272 is much higher than that of the glycolipid biosurfactant of an existing pseudomonas aeruginosa strain; and 2 compared with the glycolipid biosurfactant synthesized by the existing pseudomonas aeruginosa strain, the glycolipid biosurfactant synthesized by the pseudomonas aeruginosa strain CCTCC M 2015272 has higher temperature tolerance and wider pH adaptability.

Description

P. aeruginosa bacterial strain and application thereof
Technical field
The invention belongs to the development and application field of microorganism, relate in particular to a kind of P. aeruginosa bacterial strain and application thereof.
Background technology
Bio-surfactant (Biosurfactants) be microorganism secrete in metabolic process there is surfactivity, contain the amphoteric substance of hydrophilic group and hydrophobic group simultaneously, comprise glycolipid, lipopeptid, lipoprotein, phosphatide and neutral lipid derivative etc.Compared with the tensio-active agent of chemosynthesis, bio-surfactant has many obvious advantages: (1) low micelle-forming concentration and high surface and interface activity; (2) to the stability of ionic strength; (3) higher biological degradability and hypotoxicity; (4) progressively absorption and lasting activity; (5) excellent demulsification performance.At present, petroleum pollution is the outstanding environment difficulties of of facing in countries in the world, and how each state all uses bioremediation technology to carry out the reparation of oil-polluted soils in research, and the application of bio-surfactant is a key factor of biological restoration.
But the subject matter of synthesising biological tensio-active agent is that output is too low at present, and extraction purification expense is too high, and the cost of bio-surfactant is higher than the cost of chemical surfactant 3-10 times.Therefore reduce production cost, seed selection bio-surfactant superior strain, optimize fermentation culture conditions and the effective recovery process of exploiting economy, be the emphasis of research at present.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind ofly can synthesize the P. aeruginosa bacterial strain obtaining high yield, high-quality Glycolipids Biosurfactants via.
In order to solve the problems of the technologies described above, the invention provides a kind of P. aeruginosa bacterial strain filtered out from the polluted soil of oil field, the Panjin City Liaohe River, Liaoning Province, the preserving number of this bacterial strain is: CCTCC M 2015272, preservation date is on May 4th, 2015, Chinese name (Latin name) is P. aeruginosa bacterial strain (Pseudomonas aeruginosa), depositary institution is China typical culture collection center, in the Wuhan, China institution of higher education of address.
The present invention also provides the application of P. aeruginosa bacterial strain CCTCCM2015272 in synthesis Glycolipids Biosurfactants via.
The present invention also provides the method utilizing P. aeruginosa bacterial strain CCTCCM2015272 to synthesize Glycolipids Biosurfactants via, comprise the following steps: be that the P. aeruginosa bacterial strain of CCTCC M 2015272 is seeded in seed culture medium by preserving number, in 30-35 DEG C, constant-temperature shaking culture 1-2 days under the fermentation condition of 120-150r/min, then be inoculated in fermention medium with the inoculum size of 5-10%, constant-temperature shaking culture 5-7 days under identical fermentation condition, from fermented liquid, Glycolipids Biosurfactants via is extracted finally by the precipitator method or solvent extration,
Described seed culture medium is extractum carnis liquid nutrient medium, and described fermention medium is dextrose broth.
Further, the consisting of of described extractum carnis liquid nutrient medium: containing 3.0 grams of extractum carniss, 1.0 grams of NaNO in every 1000 ml waters 3, 1.0 grams of (NH 4) 2sO 4, 1.2 grams of Na 2hPO 412H 2o, 1.0 grams of KH 2pO 4, 0.01 gram of MgSO 47H 2o and 0.002 gram CaCl 2;
Consisting of of described dextrose broth: containing 35.0 grams of glucose, 4.0 grams of NaNO in every 5 milliliters of trace element solutions 3, 1.1 grams of KCl, 4.0 grams of NaCl, 0.028 gram of FeSO 47H 2o, 1.5 grams of KH 2pO 4, 1.5 grams of K 2hPO 4, 0.5 gram of MgSO 47H 2o and 0.5 gram yeast powder;
Wherein the consisting of of trace element solution: containing 0.029 gram of ZnSO in every 1000 ml waters 4, 0.024 gram of CaCl 2, 0.025 gram of CuSO 4with 0.017 gram of MgSO 4.
The substratum of above-mentioned formula can provide good growing nutrient and environment for P. aeruginosa bacterial strain CCTCCM2015272, is conducive to reducing synthesis cycle, and improves the output of Glycolipids Biosurfactants via.
Beneficial effect of the present invention is:
1. P. aeruginosa bacterial strain CCTCCM2015272 of the present invention can be used in synthesizing Glycolipids Biosurfactants via, and the output of the Glycolipids Biosurfactants via of P. aeruginosa bacterial strain CCTCCM2015272 of the present invention is far away higher than the output of the Glycolipids Biosurfactants via of existing verdigris pseudomonas strains;
2. P. aeruginosa bacterial strain CCTCCM2015272 of the present invention synthesizes the Glycolipids Biosurfactants via of Glycolipids Biosurfactants via compared to existing verdigris pseudomonas strains synthesis gained of gained, it is stronger to the tolerance of temperature, and has adaptability widely to pH.
3. provided by the invention utilize P. aeruginosa bacterial strain CCTCCM2015272 to synthesize the method technique of Glycolipids Biosurfactants via is simple, the cycle is short, easy to implement, be applicable to industrialization scale operation.
Accompanying drawing explanation
Fig. 1 is the phylogeny tree graph that the embodiment of the present invention 1 screens obtained strains.
Fig. 2 is the infrared absorpting light spectra that P. aeruginosa bacterial strain CCTCCM2015272 synthesizes the bio-surfactant of gained.
Embodiment
Below in conjunction with accompanying drawing, embodiment, the invention will be further described:
Embodiment 1
The screening of P. aeruginosa bacterial strain and qualification, comprise the following steps:
Experiment material:
Enrichment medium: NaCl 3.0g, NH 4cl 0.1g, MgSO 47H 2o 0.02g, NaNO 30.2g, KH 2pO 40.1g, K 2hPO 40.2g, crude oil 3.0g, distilled water 1000mL.
Oil plate culture medium: peptone 5.0g, NaCl 3.0g, NH 4cl 0.1g, MgSO 47H 2o 0.02g, NaNO 30.2g, KH 2pO 40.1g, K 2hPO 40.2g, agar 20.0g.Plate is down flat, at the crude oil that its surface coated one deck dilutes through sherwood oil after sterilizing.
Seed culture medium: extractum carnis 3.0g, NaNO 31.0g, (NH 4) 2sO 41.0g, Na 2hPO 412H 2o 1.2g, KH 2pO 41.0g, MgSO 47H 2o 0.01g, CaCl 20.002g, distilled water 1000mL.
Fermention medium: glucose 35.0g, NaNO 34.0g, KCl 1.1g, NaCl 4.0g, FeSO 47H 2o 0.028g, KH 2pO 41.5g, K 2hPO 41.5g, MgSO 47H 2o 0.5g, yeast powder 0.5g, trace element solution 5mL.
Trace element solution: ZnSO 40.029g, CaCl 20.024g, CuSO 40.025g, MgSO 40.017g, distilled water 1000mL.
1.1 primary dcreening operation
Take soil sample (being collected in oil field, the Panjin City Liaohe River, Liaoning Province polluted soil) 10g, add 90mL sterilized water, 150r/min shaking table vibration 2h, getting supernatant liquor 10mL after leaving standstill 30min is inoculated in the shaking flask that 90mL enrichment medium is housed, in 30 DEG C, shaking culture in the constant-temperature table of 120r/min, getting nutrient solution after 3d is forwarded in enrichment medium, under similarity condition, carry out second incubation.Get the nutrient solution 1mL of second incubation, carry out gradient dilution with sterilized water, the enrichment culture liquid getting different concns gradient coats oily plate culture medium, and cultivate 24h in 30 DEG C of incubators after, picking has single bacterium colony of larger oil extraction circle, further separation and purification, obtains primary dcreening operation bacterial strain.
1.2 multiple sieves
By primary dcreening operation inoculation in seed culture medium, in 30 DEG C, shaking culture 1d in 120r/min constant-temperature table, be then inoculated in fermention medium with the inoculum size of 5%, under similarity condition, cultivate 3d.Get the culture dish that is added with 20mL water, the water surface adds 0.1mL paraffin oil (adding the dyeing of appropriate Sudan red) and forms red oil film, at oil film center instillation shake flask fermentation liquid, measure oil extraction loop diameter size, the bacterial strain reservation that diameter is greater than 3cm makes further research.Meanwhile, measure the surface tension of fermented liquid, filter out the dominant bacteria of biosurfactant production.
1.3 measurement of surface tension
By filtering fermentation liquor oil removing, the centrifugal 20min of 12000r/min, gets supernatant liquor, adopts Auto-tensiometer QBZY-1 surface and interface tensiometer platinum plate method chart surface tension.
1.4 identification of strains
By Shanghai biotechnology Services Co., Ltd, 16S rRNA order-checking is carried out to the bacterial strain that above-mentioned screening obtains, by check order to be listed in GenBank GenBank and carry out similarity analysis with other 16S rRNA sequence datas existing.
DNA rapid extraction test kit is adopted to extract the genome of degradation bacteria as template, carry out the amplification of 16S rRNA gene, after pcr amplification product being carried out the agarose gel electrophoresis of 1%, carry out 16S rRNA order-checking, by check order to be listed in GenBank GenBank and carry out similarity analysis with other 16S rRNA sequence datas existing.See Fig. 1, pcr amplification is carried out with 16s rRNA gene universal primer, obtain the fragment of a treaty 1440bp, after BLAST comparison, find that the similarity of this bacterial strain and Pseudomonas aeruginosa Tsaydam-5-ASA (KC137277.1) is 100%.Therefore, the identification of strains that above-mentioned screening obtains is P. aeruginosa bacterial strain.
Embodiment 2
Utilize the method for P. aeruginosa bacterial strain CCTCCM2015272 synthesising biological tensio-active agent, comprise the following steps:
P. aeruginosa bacterial strain CCTCCM2015272 is seeded in seed culture medium, in 30 DEG C, constant-temperature shaking culture 1 day under the fermentation condition of 120r/min, then be inoculated in fermention medium with the inoculum size of 5%, under identical fermentation condition, constant-temperature shaking culture 5 days, extracts bio-surfactant finally by solvent extration from fermented liquid.
Embodiment 3
Utilize the method for P. aeruginosa bacterial strain CCTCCM2015272 synthesising biological tensio-active agent, comprise the following steps:
P. aeruginosa bacterial strain CCTCCM2015272 is seeded in seed culture medium, in 32 DEG C, constant-temperature shaking culture 2 days under the fermentation condition of 150r/min, then be inoculated in fermention medium with the inoculum size of 8%, under identical fermentation condition, constant-temperature shaking culture 7 days, extracts bio-surfactant finally by solvent extration from fermented liquid.
Embodiment 4
Utilize the method for P. aeruginosa bacterial strain CCTCCM2015272 synthesising biological tensio-active agent, comprise the following steps:
P. aeruginosa bacterial strain CCTCCM2015272 is seeded in seed culture medium, in 35 DEG C, constant-temperature shaking culture 1.5 days under the fermentation condition of 135r/min, then be inoculated in fermention medium with the inoculum size of 10%, under identical fermentation condition, constant-temperature shaking culture 6 days, extracts bio-surfactant finally by the precipitator method from fermented liquid.
In above-described embodiment 2 to embodiment 3, described seed culture medium is extractum carnis liquid nutrient medium, consisting of of extractum carnis liquid nutrient medium: containing 3.0 grams of extractum carniss, 1.0 grams of NaNO in every 1000 ml waters 3, 1.0 grams of (NH 4) 2sO 4, 1.2 grams of Na 2hPO 412H 2o, 1.0 grams of KH 2pO 4, 0.01 gram of MgSO 47H 2o and 0.002 gram CaCl 2;
Described fermention medium is dextrose broth, consisting of of dextrose broth: containing 35.0 grams of glucose, 4.0 grams of NaNO in every 5 milliliters of trace element solutions 3, 1.1 grams of KCl, 4.0 grams of NaCl, 0.028 gram of FeSO 47H 2o, 1.5 grams of KH 2pO 4, 1.5 grams of K 2hPO 4, 0.5 gram of MgSO 47H 2o and 0.5 gram yeast powder, wherein the consisting of of trace element solution: containing 0.029 gram of ZnSO in every 1000 ml waters 4, 0.024 gram of CaCl 2, 0.025 gram of CuSO 4with 0.017 gram of MgSO 4.
The process that the described precipitator method extract bio-surfactant is: by centrifugal for fermented liquid (12000r/min, 30min, 4 DEG C), get supernatant liquor 6mol/L hydrochloric acid and be adjusted to pH2.0, occur flocks, 4 DEG C of hold over night.The centrifugal 20min of 12000r/min, collecting precipitation.With the hydrochloric acid soln repetitive scrubbing 2 times of pH2.0, centrifugal collecting precipitation.Subsequently precipitation is dissolved in the NaOH solution of 1mol/L, regulates pH to 7.0, obtain light brown sample solution (crude product).With chloroform extraction 3 times, merge organic phase.Carry out rotary evaporation again, obtain viscous brown thing, by dope lyophilize, obtain sterling.
The process that described solvent extration extracts bio-surfactant is: by centrifugal for fermented liquid (12000r/min, 30min, 4 DEG C), get supernatant liquor 100mL equal-volume extraction agent and repeatedly extract 3 times, merges organic phase.Carry out rotary evaporation again, obtain viscous brown thing, by dope lyophilize, obtain sterling.Extraction agent can select CH 2cl 2, CHCl 3, CHCl 3/ CH 3oH (V:V=1:1) or CHCl 3/ CH 3oH (V:V=2:1).
Embodiment 5
Performance analysis is carried out to the bio-surfactant of embodiment 2 to embodiment 4 gained
Analytical results is as follows:
The bio-surfactant extracted from the fermented liquid of P. aeruginosa bacterial strain CCTCCM2015272 is thick in brown color after freeze drier drying.
By the bio-surfactant sample dissolve with methanol after lyophilize, carry out thin layer chromatography analysis.Respectively with phenolsulfuric acid reagent, phospho-molybdic acid-ethanol reagent and triketohydrindene hydrate for developer is tested, observe colour developing spot color.The analytical results display dot of thin-layer chromatography is brown color, can belong to glycolipid class by preliminary judgement institute biosurfactant production.
As seen from Figure 2, at 3500 ~ 3000cm -1there is stronger absorption band at place, and this explanation has a large amount of-OH to exist; 1400 ~ 1200cm -1there are some charateristic avsorption bands of carbohydrate at wave band place.As 1400cm -1place is the angle vibration of carbohydrate C-H, 1100cm -1place is the stretching vibration of C-O-C key.C=O stretching vibration appears at 1900 ~ 1630cm -1, 1750cm -1the absorption peak at place is the stretching vibration of C=O.Analytical results in conjunction with thin-layer chromatography is known, and this bio-surfactant belongs to Glycolipids Biosurfactants via.
Embodiment 6
P. aeruginosa bacterial strain CCTCCM2015272 and existing verdigris pseudomonas strains (purchased from Bian Zhen bio tech ltd, Nanjing) synthesize the simultaneous test of Glycolipids Biosurfactants via under the same conditions
Synthetic method is with embodiment 2, and the extraction of the Glycolipids Biosurfactants via of P. aeruginosa bacterial strain CCTCCM2015272 and existing verdigris pseudomonas strains the results are shown in Table 1:
The extraction result of table 1 Glycolipids Biosurfactants via
*in table, the unit of data is g/L
The Glycolipids Biosurfactants via output of P. aeruginosa bacterial strain CCTCCM2015272 is up to 14.97g/L as can be seen from Table 1, and the Glycolipids Biosurfactants via output of existing verdigris pseudomonas strains only has 5.742g/L, therefore, it is possible to draw, the output of the Glycolipids Biosurfactants via of P. aeruginosa bacterial strain CCTCCM2015272 of the present invention is far away higher than the output of the Glycolipids Biosurfactants via of existing verdigris pseudomonas strains;
Glycolipids Biosurfactants via P. aeruginosa bacterial strain CCTCCM2015272 and existing verdigris pseudomonas strains being synthesized to gained carries out stability test respectively, stability experiment shows, the Glycolipids Biosurfactants via that P. aeruginosa bacterial strain CCTCCM2015272 and existing verdigris pseudomonas strains synthesize gained is all very strong to the tolerance of NaCl, substantially without impact; The Glycolipids Biosurfactants via of existing verdigris pseudomonas strains synthesis gained can tolerate the high temperature of 90 DEG C, and the Glycolipids Biosurfactants via of P. aeruginosa bacterial strain CCTCCM2015272 synthesis gained can tolerate the high temperature of 150 DEG C, stronger to the tolerance of temperature; The Glycolipids Biosurfactants via of existing verdigris pseudomonas strains synthesis gained has adaptability (pH 6.5 ~ 11.0) more widely to pH, and the Glycolipids Biosurfactants via that P. aeruginosa bacterial strain CCTCCM2015272 synthesizes gained has adaptability (pH 1 ~ 13) widely to pH.In sum, the quality that P. aeruginosa bacterial strain CCTCCM2015272 synthesizes the Glycolipids Biosurfactants via of gained is higher, and the scope of application is wider.
Be to be understood that example as herein described and embodiment only in order to illustrate, those skilled in the art can make various amendment or change according to it, when not departing from spirit of the present invention, all belong to protection scope of the present invention.

Claims (4)

1. P. aeruginosa bacterial strain, its preserving number is CCTCC M 2015272.
2. the application of P. aeruginosa bacterial strain as claimed in claim 1 in synthesis Glycolipids Biosurfactants via.
3. utilize the method for the P. aeruginosa bacterial strain synthesis Glycolipids Biosurfactants via described in claim 1, comprise the following steps: be that the P. aeruginosa bacterial strain of CCTCC M 2015272 is seeded in seed culture medium by preserving number, in 30-35 DEG C, constant-temperature shaking culture 1-2 days under the fermentation condition of 120-150r/min, then be inoculated in fermention medium with the inoculum size of 5-10%, under identical fermentation condition, constant-temperature shaking culture 5-7 days, extracts Glycolipids Biosurfactants via finally by the precipitator method or solvent extration from fermented liquid;
Described seed culture medium is extractum carnis liquid nutrient medium, and described fermention medium is dextrose broth.
4. utilize P. aeruginosa bacterial strain to synthesize the method for Glycolipids Biosurfactants via as claimed in claim 3, it is characterized in that:
Consisting of of described extractum carnis liquid nutrient medium: containing 3.0 grams of extractum carniss, 1.0 grams of NaNO in every 1000 ml waters 3, 1.0 grams of (NH 4) 2sO 4, 1.2 grams of Na 2hPO 412H 2o, 1.0 grams of KH 2pO 4, 0.01 gram of MgSO 47H 2o and 0.002 gram CaCl 2;
Consisting of of described dextrose broth: containing 35.0 grams of glucose, 4.0 grams of NaNO in every 5 milliliters of trace element solutions 3, 1.1 grams of KCl, 4.0 grams of NaCl, 0.028 gram of FeSO 47H 2o, 1.5 grams of KH 2pO 4, 1.5 grams of K 2hPO 4, 0.5 gram of MgSO 47H 2o and 0.5 gram yeast powder;
Wherein the consisting of of trace element solution: containing 0.029 gram of ZnSO in every 1000 ml waters 4, 0.024 gram of CaCl 2, 0.025 gram of CuSO 4with 0.017 gram of MgSO 4.
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CN113897407A (en) * 2021-10-27 2022-01-07 长春工业大学 Pseudomonas aeruginosa and application thereof

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