CN102978135A - Crude oil degrading bacterium for producing lipid biosurfactant and application - Google Patents
Crude oil degrading bacterium for producing lipid biosurfactant and application Download PDFInfo
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Abstract
The invention discloses a crude oil degrading bacterium for producing a lipid biosurfactant and application. The crude oil degrading bacterium has a name of pseudomonas aeruginosa MZ01 and is collected in the CGMCC (China General Microbiological Culture Collection Center) of Institute of Microbiology, Chinese Academy of Sciences, Datun road, Chaoyang, Beijing, China on July 12, 2012, and the collection number is CGMCC No.6354. The strain is mainly used for producing lipid, the CMC value is 0.1g/l, the surface tension of water can be reduced from 72.0mN/m to 29.9mN/m, an obvious emulsifying and solubilizing effect is realized on hydrophobic organisms, and the effect is lasting; and under the conditions that the temperature is 25 DEG C, the pH value is 7 and the rotation speed is 150rpm, the crude oil can be reduced from 200mg/l to about 92mg/l in 5 days, the total removal rate of petroleum exceeds 52%, and the degradation rate of the strain against crude oil is over 30%.
Description
Technical field
The present invention relates to a kind of bacterial strain, particularly a kind of oil degradation bacterium and application of producing the lipid bio-surfactant.
Background technology
Tensio-active agent is the material that a kind of existing hydrophilic radical has again lipophilic group, it has wetting or anti-stick, emulsification or breakdown of emulsion, foaming or froth breaking and solubilising, dispersion, washing, the effect such as anticorrosion, antistatic, is very widely fine chemical product of a kind of purposes.As washing composition, other application almost can cover all field of fine chemical to tensio-active agent except in daily life.
Yet a large amount of uses of chemical surfactant may cause the secondary pollution of soil or water body, and most of chemical surfactant all is difficult to again be utilized thereby can be detained in environment.Therefore, environment amenable bio-surfactant more and more receives people's concern.
Bio-surfactant is a kind of of natural surface active agent, and some that refer to mainly that microorganism produces under certain culture condition have the capillary meta-bolites of obvious reduction.Compare with the tensio-active agent of chemosynthesis, bio-surfactant is except having the identical characteristics such as reduction surface tension, stable emulsion and foaming, also have the not available environmental friendliness characteristic of general chemical surfactant, but such as advantages such as nontoxic natural biology degraded, ecological safeties.Bio-surfactant also often has higher surfactivity than chemical surfactant in addition, and emulsifying capacity is also stronger, has potential using value in industrial aspect such as medicine, makeup, washing composition and food.Particularly in the reparation of difficult degradation hydrophobic organic compound such as polycyclic aromatic hydrocarbons, polychlorobiphenyl, petroleum hydrocarbon, bio-surfactant plays an important role, and it makes hydrophobic organic compound emulsification and obvious solubilising, so that these materials of the easier degraded of degradation bacteria.
In addition, bio-surfactant still can play a role in extreme environment, and it is not subjected to the impact of the external environments such as temperature, and general chemical surfactant is strict to requirement for environmental conditions, can not get due effect at extreme environment.And bio-surfactant can be utilized by microorganism, can be not residual in the environment midium or long term, can not produce harmful effect to environment.Yet bio-surfactant still is difficult to the commercialization utilization at present, mainly is that the purge process investment is large because its productive rate is low and the substrate cost is high.Obtaining in a large number, the bacterial classification of the bio-surfactant of metabolism is to solve the high a kind of approach of present bio-surfactant cost.
Summary of the invention
The shortcoming that primary and foremost purpose of the present invention is to overcome prior art provides a kind of oil degradation bacterium that produces the lipid bio-surfactant with not enough.
Another object of the present invention is to provide the application of the oil degradation bacterium of described product lipid bio-surfactant.
Purpose of the present invention is achieved through the following technical solutions: a kind of oil degradation bacterium that produces the lipid bio-surfactant, name is called Pseudomonas aeruginosa (Pseudomonas aeruginosa) MZ01, be preserved in the China Committee for Culture Collection of Microorganisms common micro-organisms center that is positioned at Chaoyang District, BeiJing, China city Da Tun road Institute of Microorganism, Academia Sinica on July 12nd, 2012, deposit number is CGMCC No.6354;
The form of described Pseudomonas aeruginosa MZ01 is: smooth, the ball-type of bacterium colony, and the edge is smooth, is light green, belongs to Gram-negative bacteria, and the form of this thalline of observed under electron microscope is coccus;
The 16S rDNA of described Pseudomonas aeruginosa MZ01 is shown in SEQ ID NO.1;
The application of the oil degradation bacterium of described product lipid bio-surfactant in preparation bio-surfactant and oil degradation.
The present invention has following advantage and effect with respect to prior art:
(1) bacterial strain provided by the invention mainly produces lipid, and its CMC value is 0.1g/l, and the surface tension of water can be down to 29.9mN/m from 72.0mN/m, and it has obvious emulsification and solublization to hydrophobic organic compound, and effect is lasting.
(2) 25 ℃ of bacterial strains provided by the invention, pH value are 7, and rotating speed is that 5d can be down to crude oil about about 92mg/l from 200mg/l under the condition of 150rpm, and the total clearance of oil is for reaching more than 52%, wherein this bacterial strain to the degradation rate of crude oil more than 30%.
Description of drawings
Fig. 1 is the morphologic observation figure of bacterial strain MZ01, and wherein, A is flat-plate bacterial colony photo figure, and B is Electronic Speculum figure.
Fig. 2 is the infrared spectrogram of the tensio-active agent that produces of MZ01 bacterial strain.
Fig. 3 is the tensio-active agent that MZ01 produces
1The H spectrum analysis is figure as a result.
Fig. 4 is the tensio-active agent that MZ01 produces
13The C spectrum analysis is figure as a result.
Fig. 5 is the CMC pH-value determination pH figure as a result of bio-surfactant.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited to this.
The screening and separating of MZ01.
Gather near the oil-polluted soils of Guangzhou refinery, get 2g soil and add in the culture medium A, place 30 ℃, shaken overnight in the 150rpm shaking table.Crude oil is added into culture medium A as sole carbon source repeatedly tames cultivation to the bacterial strain in the soil and (places 30 ℃, 5d vibrates in the 150rpm shaking table, again be seeded in the culture medium A of the fresh 2g of containing crude oil after the 5d, this step of triplicate is tamed the bacterial strain in the soil), the addition of crude oil (deriving from Guangzhou China Petrochemical Industry Company) is to add 2g crude oil in every liter of culture medium A.
The 1ml trace element is added in consisting of of culture medium A (composition of the culture medium A of following use is with herein) in every liter of enrichment medium, the pH value is 7.0;
Contain in every liter of enrichment medium: 0.5 gram MgSO
4.7H
2O, 0.02 gram CaCl
2.2H
2O, 1 gram NH
4NO
3, 1 the gram NaCl, 3 the gram yeast extracts, 1.5 the gram KH
2PO
4, 2 the gram K
2HPO
4, water is settled to 1L;
Contain in every liter of trace element: 0.1 gram ZnSO
4.7H
2O, 0.3 gram MnSO
4.H
2O, 0.1 gram FeSO
4.7H
2O, 0.1 gram CaCl
2.2H
2O, 0.1 gram CoCl
2.6H
2O, 0.01 gram CuSO
4.5H
2O, 0.01 gram AlK (SO
4)
2.12H
2O, 0.01 gram H
3BO
3, 0.0137 the gram (NH
4)
2Mo
2O
7, 0.0107 the gram MgSO
4.7H
2O, water is settled to 1L.
Through domestication and separation and purification obtain the bacterial strain that tensio-active agent and degrading crude oil are produced in some strains repeatedly, utilize the cave in test of experiment, emulsification index and fermented liquid measurement of surface tension of oil droplet to filter out the bacterial strain that can better produce tensio-active agent, the selection result is as shown in table 1.Utilize simultaneously the redox color reaction of DCPIP reagent to filter out the effectively bacterial strain of degrading crude oil, obtain bacterial strain MZ01 and be not only producing tensio-active agent but also bacterial strain that can fine degrading crude oil.Dull and stereotyped form (as shown in Figure 1) of observing this bacterium colony, it is light green, irregular shape, the edge is smooth, and the bacterium colony smooth surface is moistening.The form of this thalline of observed under electron microscope is coccus, and diameter is 707～870nm.It is delivered to order-checking company (Guangdong Province's microbiological analysis inspection center) survey its 16SrDNA, the sequence that obtains is as follows, by NCBI BLAST, itself and Pseudomonas aeruginosa symbolic animal of the birth year reach 100% like homology, therefore identify that this bacterium is Pseudomonas aeruginosa (Pseudomonas aeruginosa).With its called after Pseudomonas aeruginosa (Pseudomonas aeruginosa) MZ01, be preserved in the China Committee for Culture Collection of Microorganisms common micro-organisms center that is positioned at Chaoyang District, BeiJing, China city Da Tun road Institute of Microorganism, Academia Sinica on July 12nd, 2012, deposit number is CGMCC No.6354.
Table 1
The oil droplet experiment of caving in
1. with the bacterial strain of purifying with initial fermention medium (add 5g glucose every liter of culture medium A and obtain, lower with) (150rpm is about 30 ℃) fermentation culture 2d, the centrifugal 10min of 4000rpm obtains supernatant liquor.
2. add crude oil in the heart in 96 orifice plate plates lid, every hole 2 μ l left standstill 24 hours, and the 96 orifice plate plates lid that obtains handling well is then toward wherein adding the supernatant liquor that 1. step obtains, every hole 2 μ l.Choose the bacterial strain that can produce the oil extraction circle in a minute and carry out subsequent experimental.Every strain bacterial strain is done three groups of Duplicate Samples.
The emulsification assessment of indices
The bacterium liquid of cultivating through initial fermention medium (150rpm, about 30 ℃ cultivate 2d) is in the centrifugal 10min of 4000rpm, get respectively 2ml supernatant liquor and 2ml n-hexadecane in the 10ml centrifuge tube, then ultrasonication 15min leaves standstill 2h, observes emulsion layer height and emulsification layered effect thereof.Every strain bacterial strain is done three groups of Duplicate Samples.
Measurement of surface tension
, get supernatant liquor and measure its capillary size in the centrifugal 10min of 4000rpm through the bacterium liquid of initial fermention medium (150rpm, about 30 ℃ cultivate 2d).Required instrument is the QBZY-1 type surface tension instrument of being produced by the auspicious instrument company in Shanghai side.Every strain bacterial strain is done three groups of Duplicate Samples.
DCPIP reagent colour development reaction screening oil degradation bacterium
1. in the centrifugal 15min of 5000rpm, get precipitation through the bacterium liquid of initial fermention medium (150rpm, about 30 ℃ cultivate 2d); Then use the NaCl solution washing of mass percent 0.85% to use its suspension twice, adjusting its OD580 is 0.4～0.6 again.
2. in 96 orifice plates, add 200 μ l culture medium A by every hole, 30 μ l oil, 20mg DCPIP(2,6-Dichlorophenol indophenol) as redox agent.Add 25 μ l and place incubated at room temperature behind the thallus suspension liquid of above-mentioned processing, oily degradation bacteria can become colorless culture from blueness, with the capability standard of the remarkable situation of colour-change behind the 24h as measurement degraded oil.Every strain bacterial strain is done three groups of Duplicate Samples.
The sequence of 16S rDNA is as follows:
1catgcaagtc?gagcggatga?agggagcttg?ctcctggatt?cagcggcgga?cgggtgagta
61atgcctagga?atctgcctgg?tagtggggga?taacgtccgg?aaacgggcgc?taataccgca
121tacgtcctga?gggagaaagt?gggggatctt?cggacctcac?gctatcagat?gagcctaggt
181cggattagct?agttggtggg?gtaaaggcct?accaaggcga?cgatccgtaa?ctggtctgag
241aggatgatca?gtcacactgg?aactgagaca?cggtccagac?tcctacggga?ggcagcagtg
301gggaatattg?gacaatgggc?gaaagcctga?tccagccatg?ccgcgtgtgt?gaagaaggtc
361ttcggattgt?aaagcacttt?aagttgggag?gaagggcagt?aagttaatac?cttgctgttt
421tgacgttacc?aacagaataa?gcaccggcta?acttcgtgcc?agcagccgcg?gtaatacgaa
481gggtgcaagc?gttaatcgga?attactgggc?gtaaagcgcg?cgtaggtggt?tcagcaagtt
541ggatgtgaaa?tccccgggct?caacctggga?actgcatcca?aaactactga?gctagagtac
601ggtagagggt?ggtggaattt?cctgtgtagc?ggtgaaatgc?gtagatatag?gaaggaacac
661cagtggcgaa?ggcgaccacc?tggactgata?ctgacactga?ggtgcgaaag?cgtggggagc
721aaacaggatt?agataccctg?gtagtccacg?ccgtaaacga?tgtcgactag?ccgttgggat
781ccttgagatc?ttagtggcgc?agctaacgcg?ataagtcgac?cgcctgggga?gtacggccgc
841aaggttaaaa?ctcaaatgaa?ttgacggggg?cccgcacaag?cggtggagca?tgtggtttaa
901ttcgaagcaa?cgcgaagaac?cttacctggc?cttgacatgc?tgagaacttt?ccagagatgg
961attggtgcct?tcgggaactc?agacacaggt?gctgcatggc?tgtcgtcagc?tcgtgtcgtg
1021agatgttggg?ttaagtcccg?taacgagcgc?aacccttgtc?cttagttacc?agcacctcgg
1081gtgggcactc?taaggagact?gccggtgaca?aaccggagga?aggtggggat?gacgtcaagt
1141catcatggcc?cttacggcca?gggctacaca?cgtgctacaa?tggtcggtac?aaagggttgc
1201caagccgcga?ggtggagcta?atcccataaa?accgatcgta?gtccggatcg?cagtctgcaa
1261ctcgactgcg?tgaagtcgga?atcgctagta?atcgtgaatc?agaatgtcac?ggtgaatacg
1321ttcccgggcc?ttgtacacac?cgcccgtcac?accatgggag?tgggttgctc?cagaagtagc
1381tagtctaacc?gcaaggggga?cggt
Embodiment 2: the evaluation of tensio-active agent bacterial strain
The purification of bio-surfactant
Add Semen Maydis oil and to culture medium A (addition of Semen Maydis oil is to add 2g Semen Maydis oil in every liter of culture medium A), bacterial strain MZ01 is carried out fermentation culture, behind the 7d, the bacterium liquid that obtains is regulated pH value to 8.0 with 2N NaOH, then in the centrifugal 15min of 10000rpm, collect supernatant liquor, regulate again the pH value to 2.0 of supernatant liquor with 12N HCl, and put into 4 ℃ of refrigerator hold over night.Then with this solution in the centrifugal 15min of 10000rpm, collecting precipitation.The by volume chloroform of 2:1 proportioning of precipitation: methanol solution carries out three extractions, takes off layer, with 0.22 μ m membrane filtration, gets filtrate, is rotated evaporation (45 ℃) again, namely obtains required product.Through measuring, 25 ℃, initial pH value is 7, carry out fermentation culture with the culture medium A that contains Semen Maydis oil 2g/L, extracted afterwards and its tunning of purifying in 3 days, the bio-surfactant that can get its generation is 2.27g/l, and under 25 ℃, this tensio-active agent can drop to 26.7mN/m from 56.4mN/m with fermented liquid.
The evaluation of bio-surfactant
(1) thin-layer chromatography
In chromatography cylinder, pour the developping agent (chloroform: methyl alcohol: the ammoniacal liquor of mass percent 28% by volume 65:35:5 mixes) of about 20ml into, and be allowed to condition at that volatilization makes it to reach vapor liquid equilibrium in the chromatography cylinder.Silica-gel plate with pencil mark lower end and upper end, is got the tensio-active agent point of extraction on the lower end line of silica-gel plate with the pencil mark with kapillary, silica-gel plate is put into chromatography cylinder.Notice that developping agent can not surpass the lower end of pencil mark.When treating developping agent closely to upper end line, take out thin layer plate, spray respectively three kinds of developers, put into 105 ℃ of baking oven calcination 5min, observe colour-change.The thin-layer chromatography developer has: I) phenolsulfuric acid: 3g phenol and the 5mL vitriol oil are dissolved in the 95mL ethanol, and glycolipid shows brown spot; II) ammonium molybdate-potassium permanganate: phosphatide developer; III) triketohydrindene hydrate: 0.5% anhydrous propanone solution, lipopeptid is aobvious red.
Test result shows that therefore aobvious redness behind this tensio-active agent spray triketohydrindene hydrate developer judges tentatively that this material belongs to the lipopeptid class.
(2) the infrared conversion spectrum of Fourier (FTIR)
Tensio-active agent through purifying is yellow body of paste, picks a small amount of this body of paste applying materials with cotton swab and carries out the FTIR analysis at compressing tablet.
As shown in Figure 2, broad peak 3359cm
-1It is the stretching vibration peak (C-H that the molecule interchain hydrogen bond causes) that is caused by O-H; Stretching vibration 3008cm
-1The flexible bands of a spectrum of C-H that the intramolecular hydrogen bond of C-H group causes; 2926cm
-1And 2855cm
-1The connection CH of fatty acid group
2And CH
3The C-H stretching vibration of group; 1745cm
-1The flexible vibration of the C=O bands of a spectrum of acetyl ester bond; 1166cm
-1The flexible bands of a spectrum of C-H mutation in the alkene; 1097 and 1062cm
-1Peak value shows is the C-OH slip key; In this collection of illustrative plates, 916 and 810cm
-1Peak value shows is the component relevant with anomers; 983 and 732cm
-1Flexible vibration shows is the O-H group that firmly is connected on the carbon head of end position, the i.e. water-wet side of this tensio-active agent; Characteristic differentiation zone 1238cm in this collection of illustrative plates
-1And 1462cm
-1Between the zone refer to the flexible vibration of mutation of the C-H stretching vibration of typical fatty acid group and O-H group, what show is the hydrophobic part of this bio-surfactant herein.
Figure can verify thus, and this tensio-active agent is a kind of lipid material.(3) nucleus magnetic resonance
The bio-surfactant that the MZ01 that purifies is produced carries out H
1Spectrum and C
13Spectrum analysis is dissolved in the chloroform with kapillary this material that takes a morsel, and analyzes under 400MHZ and 297K and obtains respectively
1H spectrum and
13C composes spectrogram.
Such as Fig. 3 and 4,
1H and
13Collection of illustrative plates shown in the C nuclear magnetic resonance spectroscopy, this material mainly is comprised of lipid as can be known.In 1H spectrum, the zone shown between the 0.88-2.78ppm shows a kind of long chain hydrocarbon of this material, and in this long-chain compound, the chemical bond that 0.88ppm shows is-CH
3, 1.28,1.30ppm is (CH
2)-CH
2-, 2.31ppm is-CH
2-COO-, what 4.12-4.31ppm showed is carbon-carbon double bond, 5.31-5.38ppm is expressed as carbon carbon triple bond.
13In the C spectrum, 13ppm is depicted as-CH
3-, what 70ppm represented is carbon carbon triple bond, what represent about 170ppm is-COO-that the zone between the 20ppm to 30ppm shows is (CH
2)-CH
2-, the 130ppm near zone is depicted as carbon-carbon double bond.The bio-surfactant that further specifies bacterial strain MZ01 generation is a kind of lipid material.
Embodiment 3: the CMC of surfactant pH-value determination pH that bacterial strain MZ01 produces
To be mixed with that concentration is 0.025,0.04,0.05,0.1,0.2,0.25, a series of aqueous solution of 0.3g/l by the embodiment 2 dried tensio-active agent of purifying, the QBZY-1 type surface tension instrument of using the auspicious instrument company in Shanghai side to produce is measured its surface tension under 25 ℃, the result as shown in Figure 5.According to Tu Kede, its CMC value of tensio-active agent that bacterial strain MZ01 produces is 0.1g/l, and can be down to 30mN/m by 72mN/m this moment with the surface tension of water.The result shows that this tensio-active agent has preferably emulsification solubilizing effect to hydrophobic organic compound.
Embodiment 4: the experiment of strains for degrading crude oil
With bacterial strain MZ01 centrifugal 5min under the 6000rpm condition of nutrient broth incubated overnight, then with sterilized water with the thalline washing of precipitation three times, centrifugal 5min under the same terms, and make bacteria suspension and regulate OD with sterilized water
600To 0.5.Get this bacteria suspension of 1ml to the culture medium A that contains crude oil 200mg/l, under 25 ℃, the condition of 150rpm, cultivated five days.Blank is set, for not containing the MZ01 bacterium, two parallel laboratory test groups.After five days, utilize n-hexadecane extraction oil component, and use the n-hexadecane constant volume.Then use ultraviolet-visible spectrophotometer (UV-2550) to measure the residual content of crude oil in 224nm.The result is as shown in table 2, and the clearance of five days crude oil reaches more than 50%, and bacterial strain to the degradation rate of crude oil more than 30%, show that bacterial strain MZ01 has crude oil to utilize preferably effect.
The effect of table 2 bacterial strain MZ01 degrading crude oil
Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (5)
1. oil degradation bacterium that produces the lipid bio-surfactant, it is characterized in that name is called Pseudomonas aeruginosa (Pseudomonas aeruginosa) MZ01, be preserved in the China Committee for Culture Collection of Microorganisms common micro-organisms center that is positioned at Chaoyang District, BeiJing, China city Da Tun road Institute of Microorganism, Academia Sinica on July 12nd, 2012, deposit number is CGMCC No.6354.
2. the oil degradation bacterium of product lipid bio-surfactant according to claim 1, it is characterized in that: the form of described Pseudomonas aeruginosa MZ01 is: smooth, the ball-type of bacterium colony, the edge is smooth, be light green, belong to Gram-negative bacteria, the form of this thalline of observed under electron microscope is coccus.
3. the oil degradation bacterium of product lipid bio-surfactant according to claim 1, it is characterized in that: the 16S rDNA of described Pseudomonas aeruginosa MZ01 is shown in SEQ ID NO.1.
4. the oil degradation bacterium of each described product lipid bio-surfactant of claim 1～3 is in the application of preparation bio-surfactant.
5. the application of oil degradation bacterium in oil degradation of each described product lipid bio-surfactant of claim 1～3.
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| CN105886426A (en) * | 2016-03-18 | 2016-08-24 | 天津大学 | Spilled oil repairing bactericide and applications thereof in environment pollution |
| CN106497818A (en) * | 2015-09-07 | 2017-03-15 | 粮华生物科技（北京）有限公司 | Pseudomonas aeruginosa and microbial inoculum and their applications in degraded oil and/or oil product |
| CN111394419A (en) * | 2019-01-03 | 2020-07-10 | 中国石油天然气集团有限公司 | Method for detecting petroleum hydrocarbon degrading ability of petroleum hydrocarbon degrading bacteria in micro-scale mode |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104342392A (en) * | 2014-10-30 | 2015-02-11 | 中国石油天然气股份有限公司 | Microbacterium oxydans for degrading polycyclic aromatic hydrocarbon and application thereof |
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| CN106497818B (en) * | 2015-09-07 | 2019-06-25 | 粮华生物科技（北京）有限公司 | Pseudomonas aeruginosa and microbial inoculum and their applications in degraded oil and/or oil product |
| CN105886426A (en) * | 2016-03-18 | 2016-08-24 | 天津大学 | Spilled oil repairing bactericide and applications thereof in environment pollution |
| CN105886426B (en) * | 2016-03-18 | 2019-11-15 | 天津大学 | Oil spilling remediation microbial inoculum and its application in pollution environment |
| CN111394419A (en) * | 2019-01-03 | 2020-07-10 | 中国石油天然气集团有限公司 | Method for detecting petroleum hydrocarbon degrading ability of petroleum hydrocarbon degrading bacteria in micro-scale mode |
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