CN105316269B - The pseudomonas aeruginosa and its application in degraded oil of the micro- oxygen of one plant of tolerance and hypersaline environment - Google Patents
The pseudomonas aeruginosa and its application in degraded oil of the micro- oxygen of one plant of tolerance and hypersaline environment Download PDFInfo
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Abstract
The invention discloses the pseudomonas aeruginosas of one plant of tolerance micro- oxygen and hypersaline environment, the Strain Designation is pseudomonas aeruginosa (Pseudomonas aeruginosa) Ya1, the bacterial strain is preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms center " in September in 2015 on the 18th, and preservation registration number is CGMCC NO.11426.The application in degraded oil that the invention also discloses the bacterial strains is being degraded by the application of the oil in the soil or water body of oil pollution.Experiment display：The bacterial strain of the present invention can be using oil as the inorganic salts cultured on solid medium of sole carbon source, generate apparent degradation circle, and the bacterial strain can be with normal growth under 2%~6% micro-oxygen conditions, the well-grown under the condition of culture of 5% or so total salinity, and maintain under micro- oxygen and high salt conditions the ability of degrading crude oil.Indicate that Ya1 bacterial strains are applied to the fields such as remedying oil-polluted soils and processing oil pollution waste water under micro- oxygen and hypersaline environment and have good development prospect.
Description
Technical field
The present invention relates to a Pseudomonas aeruginosa strain and its application more particularly to the copper of one plant of tolerance micro- oxygen and hypersaline environment
Green pseudomonad Ya1 and its application in degraded oil or in degradation answering by the oil in the soil or water body of oil pollution
With belonging to biotechnology.
Background technology
With the development of China Petroleum, oil exploitation dynamics rises year by year with total demand.However oil is to people
While bring tremendous economic interests, huge threat also is caused to periphery ecological environment.Prospecting, exploitation, transport with
And during storage, the soil contamination problem caused by oil is a long-standing environmental problem.It is petroleum-polluted at present
Land area constantly expanding, pollution level is more serious, is brought to the ecological environment in oil field and periphery serious broken
Bad (2002, Rahman etc.).After petroleum hydrocarbon enters soil, soil texture can be destroyed, disperses grogs, makes the water penetration of soil
It reduces；Its reactive group being rich in can be combined with inorganic nitrogen, phosphorus and be limited nitrification and dephosphorylation, to keep soil effective
The content reduction of phosphorus, nitrogen；Polycyclic aromatic hydrocarbon especially therein, because having carcinogenic, mutagens, teratogenesis isoreactivity and capable of being existed by food chain
It is enriched with step by step in animal and plant body, thus more endanger (1985, Bossert and Bartha；1980, Liu).Meanwhile soil
Petroleum component meeting polluted surface water body in earth, or even simultaneously polluted underground water, also, oil is leaked as time goes by and gradually
Recovery process also will produce a large amount of oily waste waters.Reference carries out agriculture filling by the water source of oil pollution, can influence crops and normally give birth to
Long development, reduces the resistance of crops.And with the growth of crops, petroleum pollution can be progressively enriched with, final to influence
To the health (2002, Abed etc.) of the mankind.Therefore the soil and water pollution administered caused by oil have very heavy
The realistic meaning wanted, and the research hotspot in field of environment protection at present.
The processing method of oil pollution at present can be divided into three kinds, i.e. physical method, chemical method and biology side by its property
Method.In contrast, biological method has physical method and the unrivaled advantage of chemical method：It is that processing cost is low first,
The one third of processing cost only about physical chemistry method；It followed by repairs easy to operate, can be repaired in situ；It is secondary again
The reparation of object method is smaller to the secondary pollution of environment (2012, Zhen-Yu W etc.).But it using microbial degradation oil and repaiies
The soil of multiple oil pollution faces the challenge of natural environment with water body in practical applications, is mainly shown as with high salt and micro- oxygen etc. no
Sharp factor limits the growth and breeding that part has the microorganism of efficient degradation petroleum capability, to significantly limit reality
Application effect (2011,Deng).Currently, degrading under the conditions ofs with high salt and micro- oxygen etc. stone for pseudomonas aeruginosa
The relevant report of oily ability is less.One plant, which is isolated, from Daqing oil field Produced Liquid and crude oil pollution object has oil degradation ability
Pseudomonas aeruginosa, 2% salinity condition of culture to the bacterium growth have certain facilitation, drop oil cut rate can reach
17.4%, but the bacterial strain is only capable of maintaining slowly growing in 5% salinity or more, drops oil cut rate and viscosity break ratio significantly declines
(2007, Hao Donghui etc.) cannot be satisfied and adapt to the large-scale application of industrial practical application or different geographical.
Bibliography：
1. Rahman K S M,Thahira-Rahman J,Lakshmanaperumalsamy P,et
al.J.Bioresource technology,2002,85(3):257-261.
2. Bossert I,and Bartha R.J.Soil Science,1985,140(1):75-77.
3. Liu D.J.Bulletin of environmental contamination and toxicology,
1980,25(1):616-622.
4. Abed R M M,Safi N M D,J,et al.J.Applied and Environmental
Microbiology,2002,68(4):1674-1683.
5. Zhen-Yu W,Ying X U,Hao-Yun W,et al.J.Pedosphere,2012,22(5):717-
725.
6.M,ZborowskaE,Karwowska E,et al.J.Ecological Engineering,
2011,37(11):1895-1900.
7. Hao east brightness, Song Jianrong, Wang Zihao wait .J. Chinese science and technology papers online, 2007,1-8.
Invention content
In view of the deficiencies of the prior art, the problem to be solved in the present invention is to provide the copper of one plant of tolerance micro- oxygen and hypersaline environment
Green pseudomonad Ya1 and its application in degraded oil or in degradation answering by the oil in the soil or water body of oil pollution
With.
The pseudomonas aeruginosa of the tolerance micro- oxygen and hypersaline environment of the present invention, it is characterised in that：The Strain Designation is copper
Green pseudomonad (Pseudomonas aeruginosa) Ya1, the bacterial strain are preserved in " the micro- life of China on the 18th in September in 2015
Object culture presevation administration committee common micro-organisms center ", deposit number are CGMCC NO.11426.
The pseudomonas aeruginosa strains of the degradable oil of the micro- oxygen of above-mentioned tolerance and hypersaline environment, biological property are：
Rod-short (see Fig. 1) is presented in cell, and individually, size is (0.6 μm~0.8 μm) × (1.0 μm~1.3 μm), movable；Bacterium colony is flat
It is flat unformed, it spreads or slightly spreads, surface wettability to periphery；Bacterium colony is in canescence, and surrounding media often has water colo(u)r
Diffusion；Can when being cultivated on the inorganic salts solid medium using oil as sole carbon source can degraded oil, while generating degradation
It encloses (see Fig. 2).Physiological and biochemical property is：Gram-negative, micro- aerobic, chemoheterotrophic bacteria, the sour aerogenesis of glucose fermentation production,
Catalase test, methyl red test and gelatin hydrolysis experiment present it is positive, acid-fast stain experiment, indole test, VP experiments,
Starch Hydrolysis experiment presents negative.Optimum growth temperature is 28~37 DEG C, and it is 6.5~7.5 to be most suitable for growth pH value.Physiology and biochemistry
Experimental result refers to table 1, completely the same compared with pseudomonas aeruginosa type strain.
Table 1：Ya1 bacterial strains (CGMCC NO.11426) are compared with the physiological and biochemical property of pseudomonas aeruginosa reference culture
Note："+" indicates that bacterial strain is positive；"-" indicates that bacterial strain is negative.
*：Eastern show pearl, Cai Miaoying etc., common bacteria system identification handbook, Science Press, 2001, the first edition, p364-
398。
The above-mentioned fluid nutrient medium composition for thalli morphology observation：Contain tryptone in per 1000ml deionized waters
17g, soy peptone 3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, and adjust pH 7.5.
The above-mentioned solid medium composition for thalli morphology observation：Contain tryptone in per 1000ml deionized waters
17g, soy peptone 3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, agar powder 15g, and adjust pH 7.5.
The experimental method of above-mentioned observation of morphological is write with reference to the elegant pearl in east, Cai Miaoying etc.《Common bacteria system identification
Handbook》, Science Press, 2001, the first edition, p353-363.
What above-mentioned physiological and biochemical test culture medium and experimental method were write with reference to the elegant pearl in east, Cai Miaoying etc.《Common bacteria system
System identification handbook》, Science Press, 2001, the first edition, p364-398.
Pseudomonas aeruginosa of the present invention (Pseudomonas aeruginosa) Ya1CGMCC NO.11426 are measured
The gene order of 16S rRNA the results show that its gene order length be 1396bp, nucleotide sequence such as SEQ ID NO.1 institutes
Show.
By using U.S. Biotechnology Information center (National Center for Biotechnology
Information, NCBI) comparison of BLASTN programs, it is found that the gene order of the Ya1 bacterial strain 16S rRNA of the present invention is noted with NCBI
The gene order of more Pseudomonas aeruginosa strains (Pseudomonas aeruginosa) 16S rRNA of volume has high homology
(>97%), the similitude of individual bacterial strains is even higher, it may be determined that Ya1 bacterial strains are a Pseudomonas aeruginosa strains
(Pseudomonas aeruginosa).Choose the high typical pseudomonas aeruginosa (Pseudomonas of tetraploid rice
Aeruginosa 16S rRNA gene orders) are as reference subject；It is used using neighbouring method (Neighbour-Joining)
Systematic evolution tree between 5 software building Ya1 bacterial strains of Mega and reference strains selects Bacillus licheniformis to make
For Wai Qun branches, attached drawing 3 is as a result seen.
The selection of the pseudomonas aeruginosa Ya1 strains of the micro- oxygen of tolerance of the present invention and hypersaline environment is：
The pedotheque of collected oil pollution is dissolved with sterile saline, soil supension is made in oscillation mixing,
It is applied to after gradient dilution using oil as in the inorganic salts solid medium tablets of sole carbon source, stationary culture under the conditions of 30 DEG C,
Existing to there is apparent oil degradation to iris out, the larger bacterium colony of picking oil degradation circle, scribing line is transferred on solid medium, 30 DEG C
Under the conditions of stationary culture, until there is apparent single bacterium colony to generate, the verdigris through after purification, obtaining being resistant to micro- oxygen and hypersaline environment twice
Bacterium is protected in pseudomonad strain, number Ya1, glycerine cold storage.The bacterial strain is preserved in " China Microbiological bacterium on 18th in September in 2015
Kind preservation administration committee common micro-organisms center ", deposit number is CGMCC NO.11426.
Application of the pseudomonas aeruginosa of the micro- oxygen of tolerance of the present invention and hypersaline environment in degraded oil is being degraded
By the application of the oil in the soil or water body of oil pollution.
Wherein：The micro- oxygen and the pseudomonas aeruginosa of hypersaline environment of being resistant to can realize the fast of cell on nutrient medium
Speed breeding and amplification, can realize the fully degraded to oil on containing oil medium；Its cultivation temperature is 28~37 DEG C, shaking table
Culture rotating speed is 180~220rpm.
The pseudomonas aeruginosa degradation of the present invention for being resistant to micro- oxygen and hypersaline environment is by the soil or water body of oil pollution
In oil micro-oxygen conditions be oxygen concentration be 2%~6%；Degradation temperature is preferably 30~35 DEG C.
The pseudomonas aeruginosa of the present invention for being resistant to micro- oxygen and hypersaline environment is in degradation by the soil or water of oil pollution
During oil in body, it is 0.5%~5% to be resistant to total salt amount.
The pseudomonas aeruginosa Ya1 of the micro- oxygen of tolerance of the present invention and hypersaline environment is under 2%~6% micro-oxygen conditions
It can be with normal growth.
Detect the bacterial strain is to the method and step of micro- oxygen tolerance：
(1) strain selects：Select pseudomonas aeruginosa Ya1 of the present invention (CGMCC NO.11426)；
(2) inclined-plane culture activates：Strain is inoculated in slant medium, under the conditions of 28~37 DEG C, stationary culture 20~40
Hour, it is spare；
(3) seed culture：The bacterial strain that step (2) is cultivated, aseptically with oese connect 1~2 ring in 40~
In 50mL (250mL triangular flasks) liquid seed culture medium, set rotary rpm be 180~220 revs/min, radius of turn 40mm
Shaking table on, 28~37 DEG C cultivate 18~24 hours, obtain seed liquor；
(4) in using oil as the inorganic salts solid medium of sole carbon source P. aeruginosa is accessed according to 1% inoculum concentration
Bacterium Ya1 suspensions are placed in the micro- aerobic cell culture systems of anaerobism.Oxygen concentration is respectively set to 0%, 2%, 6%,
21%；Stationary culture and timing sampling under the conditions of 28~37 DEG C.3 parallel laboratory tests are set.It is united using visible spectrophotometer
It counts bacterium solution and absorbance value and draws growth curve at 600nm.
Above-mentioned slant medium group becomes：Contain tryptone 17g, soy peptone 3g in per 1000ml deionized waters,
Sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, agar powder 15g, and adjust pH 7.5.
Aforesaid liquid seed culture medium group becomes：Contain tryptone 17g, soy peptone in per 1000ml deionized waters
3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, and adjust pH 7.5.
The above-mentioned group of minimal medium containing oil becomes：NaNO31.5g/L, (NH4)2SO41.5g/L, K2HPO41g/L,
MgSO40.5g/L, KCl 0.5g/L, FeSO40.01g/L, CaCl20.002g/L, 3~8g/L of oil (preferably 5g/L),
And adjust pH 7.0.
In the above method, step (2), (3), preferably 30 DEG C of (4) described cultivation temperature.
In the above method, step (2), (3) described incubation time preferably 24 hours.
The result shows that pseudomonas aeruginosa CGMCC NO.11426 of the present invention can under 2%~6% micro-oxygen conditions
With normal growth, under complete anaerobic condition, growth is inhibited (attached drawing 4) by apparent.
Pseudomonas aeruginosa CGMCC NO.11426 of the present invention can be with normal growth in high salinity medium.
Detect the bacterial strain is to the method and step of condition of culture tolerance with high salt：
(1) strain selects：Select pseudomonas aeruginosa CGMCC NO.11426 of the present invention；
(2) inclined-plane culture activates：Strain is inoculated in slant medium, under the conditions of 28~37 DEG C, stationary culture 20~40
Hour, it is spare；
(3) seed culture：The bacterial strain that step (2) is cultivated, aseptically with oese connect 1~2 ring in 40~
In 50mL (250mL triangular flasks) liquid seed culture medium, it is 180~220 revs/min to set rotary rpm, radius of turn 40mm
Shaking table on, 28~37 DEG C cultivate 18~24 hours, obtain seed liquor；
(4) it adds NaCl respectively in using oil as the inorganic salts solid medium of sole carbon source and obtains different total salinity
Culture medium.Ya1 strain suspensions are accessed according to 1% inoculum concentration, shaken cultivation and are periodically taken under the conditions of 28~37 DEG C, 180rpm
Sample.3 parallel laboratory tests are set.The quantity (cfu/ml) of viable bacteria is counted on Solid media for plates using colony counting method and is painted
Growth curve processed.
Above-mentioned inclined-plane and Solid media for plates group become：Contain tryptone 17g, soybean in per 1000ml deionized waters
Peptone 3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, agar powder 15g, and adjust pH 7.5.
Aforesaid liquid seed culture medium group becomes：Contain tryptone 17g, soy peptone in per 1000ml deionized waters
3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, and adjust pH 7.5.
The above-mentioned group of minimal medium containing oil becomes：NaNO31.5g/L, (NH4)2SO41.5g/L, K2HPO41g/L,
MgSO40.5g/L, KCl 0.5g/L, FeSO40.01g/L, CaCl20.002g/L, 3~8g/L of oil (preferably 5g/L),
And adjust pH 7.0.By adding the NaCl of 15g/L, 45g/L and 95g/L respectively into the culture medium, the total of culture medium is adjusted
Salinity is 2%, 5% and 10%.
In the above method, step (2), (3), preferably 30 DEG C of (4) described cultivation temperature.
In the above method, step (2), (3) described incubation time preferably 24 hours.
The result shows that pseudomonas aeruginosa CGMCC NO.11426 of the present invention are under 2% total salt content condition of culture
It still can be grown under 5% total salt content condition of culture with normal growth, but after total salt content is increased to 10%,
Growth is inhibited (attached drawing 5) by apparent.
The step of pseudomonas aeruginosa CGMCC NO.11426 strains for degrading oil pollution abilities of the present invention measure
It is：
(1) strain selects：Select pseudomonas aeruginosa Ya1 of the present invention (CGMCC NO.11426)；
(2) inclined-plane culture activates：Strain is inoculated in slant medium, under the conditions of 28~37 DEG C, stationary culture 20~40
Hour, it is spare；
(3) seed culture：The bacterial strain that step (2) is cultivated, aseptically with oese connect 1~2 ring in 40~
In 50mL (250mL triangular flasks) liquid seed culture medium, set rotary rpm be 180~220 revs/min, radius of turn 40mm
Shaking table on, 28~37 DEG C cultivate 18~24 hours, obtain seed liquor；
(4) oil resolution ratio detects：It is with the inoculum concentration of 1~5% volume ratio, seed liquor access is former containing 250mg
In the 50mL minimal mediums of oil (250mL triangular flasks), set that rotary rpm is 160~220rpm, radius of turn is 40mm's
On shaking table, 28~37 DEG C are cultivated 8~10 days, realize the fully degraded to oil.
Above-mentioned slant medium group becomes：Contain tryptone 17g, soy peptone 3g in per 1000ml deionized waters,
Sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, agar powder 15g, and adjust pH 7.5.
Aforesaid liquid seed culture medium group becomes：Contain tryptone 17g, soy peptone in per 1000ml deionized waters
3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, and adjust pH 7.5.
Above-mentioned minimal medium group becomes：NaNO31.5g/L, (NH4)2SO41.5g/L, K2HPO41g/L,
MgSO40.5g/L, KCl 0.5g/L, FeSO40.01g/L, CaCl20.002g/L, and pH 7.0 is adjusted, distilled water is prepared.Training
The total salinity for supporting base is about 0.5% (being calculated according to soluble solid).
In above-mentioned application, step (2), (3), preferably 30 DEG C of (4) described cultivation temperature.
In above-mentioned application, step (2), (3) described incubation time preferably 24 hours.
In above-mentioned application, step (4) the inoculum concentration volume ratio preferably 1%.
In above-mentioned application, step (4) the culture rotating speed is preferably 180rpm.
In above-mentioned application, step (4) described incubation time preferably 9 days.
Culture medium after taking step (4) to cultivate, is extracted with carbon tetrachloride, goes moisture removal to remain with machine phase, with tetrachloro
Change carbon and make reference solution, using infrared spectrophotometer in 2930cm-1、2960cm-1、3030cm-1Place measures its absorbance value.
The culture medium for not accessing Ya1 bacterial strains is handled under the same conditions, as blank control, according to state of the People's Republic of China (PRC)
It is listed in family's environment protectment protection (the measurement infrared spectrophotometer of HJ 637-2012 water-quality petroleums and animals and plants oils)
Formula calculates the content of total petroleum hydrocarbon in sample, and calculates crude oil removal.
The results show that under above-mentioned preferred condition of culture (oxygen content 21%, total salinity 0.5%), Ya1 bacterial strains are accessed
Afterwards, the content of total petroleum hydrocarbon drops to 91 ± 7mg from initial 250mg in culture medium, and the removal rate of oil is 63.6% or so
(being shown in Table 2).
Pseudomonas aeruginosa CGMCC NO.11426 bacterial strains of the present invention are under low oxygen content and condition of culture with high salt
Can be with degraded oil, determination step：
(1) strain selects：Select pseudomonas aeruginosa Ya1 of the present invention (CGMCC NO.11426)；
(2) inclined-plane culture activates：Strain is inoculated in slant medium, under the conditions of 28~37 DEG C, stationary culture 20~40
Hour, it is spare；
(3) seed culture：The bacterial strain that step (2) is cultivated, aseptically with oese connect 1~2 ring in 40~
In 50mL (250mL triangular flasks) liquid seed culture medium, set rotary rpm be 180~220 revs/min, radius of turn 40mm
Shaking table on, 28~37 DEG C cultivate 18~24 hours, obtain seed liquor；
(4) with the inoculum concentration of 1~5% volume ratio, 50mL difference salt of the seed liquor access containing 250mg crude oil is dense
In the minimal medium of degree (250mL triangular flasks), it is placed in the micro- aerobic cell culture systems of anaerobism, oxygen concentration is distinguished
It is set as 10%, 6%, 2%, 28~37 DEG C are cultivated 8~10 days, realize the fully degraded to oil.
Above-mentioned slant medium group becomes：Contain tryptone 17g, soy peptone 3g in per 1000ml deionized waters,
Sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, agar powder 15g, and adjust pH 7.5.
Aforesaid liquid seed culture medium group becomes：Contain tryptone 17g, soy peptone in per 1000ml deionized waters
3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, and adjust pH 7.5.
Above-mentioned minimal medium group becomes：NaNO31.5g/L, (NH4)2SO41.5g/L, K2HPO41g/L,
MgSO40.5g/L, KCl 0.5g/L, FeSO40.01g/L, CaCl20.002g/L, and pH 7.0 is adjusted, distilled water is prepared.It is logical
The NaCl for adding 15g/L, 45g/L and 70g/L respectively into the culture medium is crossed, the total salinity for adjusting culture medium is 2%, 5% and
7.5%.
In above-mentioned application, step (2), (3), preferably 30 DEG C of (4) described cultivation temperature.
In above-mentioned application, step (2), (3) described incubation time preferably 24 hours.
In above-mentioned application, step (4) the inoculum concentration volume ratio preferably 1%.
In above-mentioned application, step (4) described incubation time preferably 9 days.
Culture medium after taking step (4) to cultivate, is extracted with carbon tetrachloride, goes moisture removal to remain with machine phase, with tetrachloro
Change carbon and make reference solution, using infrared spectrophotometer in 2930cm-1、2960cm-1、3030cm-1Place measures its absorbance value.
The culture medium for not accessing Ya1 bacterial strains is handled under the same conditions, as blank control, according to state of the People's Republic of China (PRC)
It is listed in family's environment protectment protection (the measurement infrared spectrophotometer of HJ 637-2012 water-quality petroleums and animals and plants oils)
Formula calculates the content of total petroleum hydrocarbon in sample, and calculates crude oil removal.
Under above-mentioned preferred condition of culture, after accessing Ya1 bacterial strains, the removal rate of total oil the results are shown in Table 2 in culture medium.
Table 2：The removal rate of Ya1 bacterial strains crude oil under different oxygen and salt content condition of culture
The results show that Ya1 bacterial strains (CGMCC NO.11426) still maintain higher stone in micro- oxygen and hypersaline environment
Oil degradation capabilities.
Above-mentioned experiment confirms：Pseudomonas aeruginosa (Pseudomonas aeruginosa) Ya1 disclosed by the invention has
Preferable oil degradation ability, and higher crude oil removal is still maintained in micro- oxygen and hypersaline environment, repairing stone
The fields such as soil contaminated by crude oil and processing oil pollution waste water have preferable application prospect and commercial value.
To sum up, advantageous effect possessed by the present invention is：
(1) the advantage of the invention is that the pseudomonas aeruginosa Ya1 bacterial strains (CGMCC NO.11426) are in oxygen content
21% and total salinity 0.5% condition of culture under, can be with efficient degradation oil, the removal rate of crude oil is 63.6% or so.
(2) micro-oxygen conditions of the pseudomonas aeruginosa Ya1 bacterial strains (CGMCC NO.11426) of the present invention 2%~6%
Under can be with normal growth.
(3) pseudomonas aeruginosa Ya1 bacterial strains (CGMCC NO.11426) of the present invention are in 2% total salt content condition of culture
Under can be grown under 5% total salt content condition of culture with normal growth.
(4) pseudomonas aeruginosa Ya1 bacterial strains (CGMCC NO.11426) of the present invention in micro- oxygen and hypersaline environment still
Higher oil degradation ability is so maintained, there is preferable application prospect and commercial value.
Description of the drawings
Bacterial strain pseudomonas aeruginosa (Pseudomonas aeruginosa) Ya1 bacterial strains provided by the invention are in 2015
September is deposited in " China Committee for Culture Collection of Microorganisms's common micro-organisms center " (address on the 18th：Chaoyang District, Beijing City north
The institute 3 of occasion West Road 1), preserving number is CGMCC NO.11426.
Fig. 1 is the cellular morphology of pseudomonas aeruginosa Ya1 bacterial strains under the microscope.
Fig. 2 is bacterium colony of the pseudomonas aeruginosa Ya1 bacterial strains on the inorganic salts solid medium using oil as sole carbon source
Form.
Fig. 3 is pseudomonas aeruginosa Ya1 bacterial strain phylogenetic analysis.
Fig. 4 is tolerance of the pseudomonas aeruginosa Ya1 bacterial strains to oxygen.
Fig. 5 is tolerance of the pseudomonas aeruginosa Ya1 bacterial strains to salinity.
Specific implementation mode
The present invention provides one plant can with the pseudomonas aeruginosa Ya1 bacterial strains (CGMCC NO.11426) of degraded oil, under
It elaborates in face of the content of present invention, but the content is explanation of the invention rather than limits.
Embodiment 1：The screening of degradable oil bacterial strain
The pedotheque for taking the collected oil pollutions of 1g is put into 250ml triangular flasks, and the sterile lifes of 9ml are added thereto
Brine is managed, soil supension is made in oscillation mixing, and doubling dilution is carried out by 10 times of concentration gradients.The 100 μ l dilutions are taken to be respectively
10-3、10-4、10-5With 10-6Soil supension, be added drop-wise to using oil as on the inorganic salts solid medium of sole carbon source, with coating
Stick uniformly smears uniform, the stationary culture 7 days under the conditions of 37 DEG C.
It has waited for that apparent oil degradation is irised out now, with the bacterium colony that oese picking oil degradation circle is larger, has been trained in TSA solids
It supports and crosses on base, stationary culture under the conditions of 37 DEG C, until there is apparent single bacterium colony to generate.Switching scribing line is twice.
It with oese picking single bacterium colony, is transferred in the test tube equipped with 8ml fluid nutrient mediums, in 37 DEG C, 180rpm conditions
Lower shaken cultivation is stayed overnight, that is, obtains pseudomonas aeruginosa Ya1 bacterium solutions.200 μ l glycerine and 800 μ l are separately added into cold storage pipe
Bacterium solution places preserved in -80 DEG C of ultra low temperature freezers after mixing, strain number Ya1.The bacterial strain is in September, 2015
It is deposited in " China Committee for Culture Collection of Microorganisms's common micro-organisms center " within 18th, preserving number CGMCC
NO.11426。
Above-mentioned sterile saline group becomes：0.85%NaCl solution.
Above-mentioned inorganic salts solid medium group becomes：NaNO31.5g/L, (NH4)2SO41.5g/L, K2HPO41g/L,
MgSO40.5g/L, KCl 0.5g/L, FeSO40.01g/L, CaCl20.002g/L, agar powder 15g/L, crude oil 5g/L, pH
7.0, distilled water configuration.
Above-mentioned TSA solid mediums group becomes：Tryptone 17g/L, soy peptone 3g/L, sodium chloride 5g/L, phosphoric acid
Hydrogen dipotassium 2.5g/L, glucose 2.5g/L, pH7.3 ± 0.2, agar powder 15g/L, distilled water are prepared.
Embodiment 2：The morphologic observation of Ya1 bacterial strains and physiological and biochemical property identification
The form of Ya1 bacterial strains is observed using the oil mirror of Nikon inverted microscope.Ya1 bacterial strain physiological and biochemical properties reflect
Determine what method was write with reference to the elegant pearl in east, Cai Miaoying etc.《Common bacteria system identification handbook》(Science Press, 2001, first
Version), qualification test cultivation temperature is set as 37 DEG C.Observation analysis is gone back simultaneously measures Ya1 bacterial strains using oil as sole carbon source
Optimum temperature when inorganic salts cultured on solid medium and most suitable growth pH value.
The biological property of Ya1 bacterial strains is：Rod-short (see Fig. 1) is presented in cell, and individually, size is (0.6 μm~0.8 μm)
× (1.0 μm~1.3 μm), it is movable；Bacterium colony is flat unformed, spreads or slightly spreads, surface wettability to periphery；Bacterium colony is in ash
White, surrounding media often have water colo(u)r diffusion；It can be trained on the inorganic salts solid medium using oil as sole carbon source
When foster can degraded oil, while generate degradation circle (see Fig. 2).Physiological and biochemical property is：Gram-negative, it is micro- aerobic,
Sun is presented in chemoheterotrophic bacteria, the sour aerogenesis of glucose fermentation production, catalase test, methyl red test and gelatin hydrolysis experiment
Property, feminine gender is presented in acid-fast stain experiment, indole test, VP experiments, Starch Hydrolysis experiment.Optimum growth temperature is 28~37 DEG C,
It is 6.5~7.5 to be most suitable for growth pH value.Bio-chemical characteristics are the results detailed in Table 1.Show bacterial strain Ya1 and Pseudomonas
The physiological and biochemical property of aeruginosa is identical.
Embodiment 3：The 16S rRNA identified for genes of Ya1 bacterial strains
According to the operating instruction of TIANGEN bacterial genomes extracts kit (TianGen companies, article No. DP302-02),
The total genomic dna of the Ya1 bacterial strains of extraction separation and purification.The total genomic dna of extraction uses 0.8% agarose gel electrophoresis
It is detected, injects loading wells after taking 3.0 μ l total genomic dnas samples and 0.6 μ 6 × sample-loading buffers of l to be sufficiently mixed uniformly
In, molecular weight marker selects λ-Hind III DNA Marker, electrophoretic voltage 120V, electrophoresis time 35 minutes.Upstream is selected to draw
Object 27F (5 ' -3 '：) and downstream primer 1492R (5 ' -3 ' AGAGTTTGATCCTGGCTCAG：GGTTACCTTGTTACGACTT) expand
Increase the 16S rRNA genes of isolated strains, PCR reaction systems and condition are as shown in the table：
PCR product is detected using 1.2% agarose gel electrophoresis, take 3.0 μ l PCR products and 0.6 μ l 6 ×
After Loading Buffer are sufficiently mixed uniformly in injection loading wells, Marker selects 2000 DNA Marker of DL, electrophoresis electricity
Press 120V, electrophoresis time 30 minutes.According to TIANGEN Ago-Gels QIAquick Gel Extraction Kit (TianGen companies, article No. DP209-
02) operating instruction carries out purifying recycling to PCR product, the PCR product after recycling with 1.2% agarose gel electrophoresis into
Row detection.Electrophoresis method is same as above.PCR product after purifying recycling is sent to sequencing company and is sequenced.The results show that its gene sequence
Row length is 1396bp, and nucleotide sequence is as shown in SEQ ID NO.1.
The 16S rRNA gene orders of sequencing gained are delivered to ncbi database (http://www.ncbi.nlm.nih.
Gov BLASTN comparisons are carried out in).As a result show that 16S rRNA gene orders and other verdigris in ncbi database of the bacterial strain are false
The 16S rRNA gene orders of monad have high homology (>97%), the similitude of individual bacterial strains is even higher, can be true
It is a Pseudomonas aeruginosa strain (Pseudomonas aeruginosa) to determine Ya1 bacterial strains.
The high typical Pseudomonas aeruginosa16S rRNA gene orders of tetraploid rice are chosen as ginseng
Compare as；It is used between 5 software building Ya1 bacterial strains of Mega and reference strains using neighbouring method (Neighbour-Joining)
Systematic evolution tree selects Bacillus licheniformis as Wai Qun branches (see Fig. 3).
Embodiment 4：Ya1 bacterial strains are resistant to the detection of micro- oxygen environment ability
The step of detection method, is sequentially as follows：
(1) strain selects：Select pseudomonas aeruginosa Ya1 of the present invention；
(2) inclined-plane culture activates：By strain be inoculated in slant medium (tryptone 17g/L, soy peptone 3g/L,
Sodium chloride 5g/L, dipotassium hydrogen phosphate 2.5g/L, glucose 2.5g/L, pH7.3, agar powder 15g/L, distilled water are prepared), 30 DEG C of items
Under part, stationary culture 24 hours is spare；
(3) seed culture：The bacterial strain that step (2) is cultivated, aseptically connects 1 ring in 50mL (250mL with oese
Triangular flask) (tryptone 17g/L, soy peptone 3g/L, sodium chloride 5g/L, dipotassium hydrogen phosphate in liquid seed culture medium
2.5g/L, glucose 2.5g/L, pH7.3, distilled water are prepared), set that rotary rpm is 180 revs/min, radius of turn is 40mm's
On shaking table, 30 DEG C are cultivated 24 hours, and seed liquor is obtained；
(4) bacterial strain Ya1 detects micro- oxygen concentration tolerance：Using oil as the inorganic salts solid medium of sole carbon source
(NaNO31.5g/L, (NH4)2SO41.5g/L, K2HPO41g/L, MgSO40.5g/L, KCl 0.5g/L, FeSO40.01g/L,
CaCl20.002g/L, oil 5g/L, pH 7.0, deionized water prepare) according to 1% inoculum concentration access pseudomonas aeruginosa
Ya1 suspensions are placed in the micro- aerobic cell culture systems of anaerobism.Oxygen concentration is set as 6%；Training is stood under the conditions of 30 DEG C
Support simultaneously timing sampling.3 parallel laboratory tests are set.Bacterium solution absorbance value at 600nm is counted using visible spectrophotometer.
The result shows that using oil as in the inorganic salts solid medium of sole carbon source, when oxygen concentration is 6%, verdigris
Pseudomonad Ya1 bacterial strains can still be grown, and when culture was to the 9th day, absorbance value is 0.69 ± 0.05 under 600nm.This shows
Ya1 bacterial strains have certain tolerance to micro- oxygen environment.
Embodiment 5：Detection of the Ya1 bacterial strains to salinity tolerance
The step of detection method, is sequentially as follows：
(1) strain selects：Select pseudomonas aeruginosa Ya1 of the present invention；
(2) inclined-plane culture activates：By strain be inoculated in slant medium (tryptone 17g/L, soy peptone 3g/L,
Sodium chloride 5g/L, dipotassium hydrogen phosphate 2.5g/L, glucose 2.5g/L, pH7.3, agar powder 15g/L, distilled water are prepared), 30 DEG C of items
Under part, stationary culture 24 hours is spare；
(3) seed culture：The bacterial strain that step (2) is cultivated, aseptically connects 1 ring in 50mL (250mL with oese
Triangular flask) (tryptone 17g/L, soy peptone 3g/L, sodium chloride 5g/L, dipotassium hydrogen phosphate in liquid seed culture medium
2.5g/L, glucose 2.5g/L, pH7.3, distilled water are prepared), set that rotary rpm is 180 revs/min, radius of turn is 40mm's
On shaking table, 30 DEG C are cultivated 24 hours, and seed liquor is obtained；
(4) bacterial strain Ya1 detects salinity tolerance：Consolidate in the inorganic salts for being 5% as total salinity of sole carbon source using oil
Body culture medium (NaNO31.5g/L, (NH4)2SO41.5g/L, K2HPO41g/L, MgSO40.5g/L, KCl 0.5g/L, FeSO4
0.01g/L, CaCl20.002g/L, NaCl 45g/L, oil 5g/L, pH 7.0, deionized water prepare) according to 1% inoculation
Amount access pseudomonas aeruginosa Ya1 suspensions, shaken cultivation and timing sampling under the conditions of 30 DEG C, 180rpm.3 parallel realities are set
It tests.Using colony counting method in TSA culture mediums (tryptone 17g/L, soy peptone 3g/L, sodium chloride 5g/L, phosphoric acid hydrogen two
Potassium 2.5g/L, glucose 2.5g/L, pH 7.3 ± 0.2, agar powder 15g/L, distilled water prepare) on count viable bacteria quantity
(cfu/ml) and growth curve is drawn.
The result shows that using oil as in the inorganic salts solid medium of sole carbon source, when total salinity reaches 5%, copper
Green pseudomonad Ya1 bacterial strains can still be grown, and when culture was to the 8th day, the quantity of viable bacteria is 0.92 ± 0.16cfu/ml.This table
Bright Ya1 bacterial strains have certain tolerance to hypersaline environment.
Embodiment 6：Application of the Ya1 bacterial strains in biodegradable oil pollution：
The step of application process is related to sequence is as follows：
(1) strain selects：Select pseudomonas aeruginosa Ya1 of the present invention；
(2) inclined-plane culture activates：By strain be inoculated in slant medium (tryptone 17g/L, soy peptone 3g/L,
Sodium chloride 5g/L, dipotassium hydrogen phosphate 2.5g/L, glucose 2.5g/L, pH7.3, agar powder 15g/L, distilled water are prepared), 30 DEG C of items
Under part, stationary culture 24 hours is spare；
(3) seed culture：The bacterial strain that step (2) is cultivated, aseptically connects 1 ring in 50mL (250mL with oese
Triangular flask) (tryptone 17g/L, soy peptone 3g/L, sodium chloride 5g/L, dipotassium hydrogen phosphate in liquid seed culture medium
2.5g/L, glucose 2.5g/L, pH7.3, deionized water are prepared), set rotary rpm be 180 revs/min, radius of turn 40mm
Shaking table on, 30 DEG C cultivate 24 hours, obtain seed liquor；
(4) crude oil resolution ratio detects：With the inoculum concentration of 1% volume ratio, seed liquor access is contained and is derived from Shengli Oil Field
Oily waste water in (50mL), the shaking table culture 9 days under the conditions of 30 DEG C, 180rpm.The waste water of Ya1 bacterial strains will not accessed identical
Under the conditions of handled, as a contrast.
(5) oily waste water of culture is transferred in separatory funnel, 10ml carbon tetrachloride is added and vibrates 3min, it is during which frequent
Open cock exhaust.After stratification, lower layer's organic phase is transferred to and has been added in the conical flask of 3g anhydrous sodium sulfates in advance, is shaken
It is dynamic to remove remaining moisture for several times.If anhydrous sodium sulfate crystallization is blocking, need to add anhydrous sodium sulfate, stand.With four chlorinations
Carbon makees reference solution, using infrared spectrophotometer in 2930cm-1、2960cm-1、3030cm-1Place measures its absorbance value.It will
The waste water for not accessing Ya1 bacterial strains is handled under the same conditions, as a contrast.It is protected according to People's Republic of China's National Environmental
The formula listed in shield standard (the measurement infrared spectrophotometer of HJ 637-2012 water-quality petroleums and animals and plants oils) calculates
The content of total petroleum hydrocarbon in sample, and calculate crude oil removal.
The results show that after access Ya1 bacterial strains, the content of total petroleum hydrocarbon is dropped to from initial 7.91g/L in waste water
4.37g/L, the removal rate of crude oil is close to 44.8%.
Embodiment 7：Ya1 bacterial strains are degraded the application of petroleum capability under micro- oxygen environment
The step of application process is related to sequence is as follows：
(1) strain selects：Select pseudomonas aeruginosa Ya1 of the present invention；
(2) inclined-plane culture activates：By strain be inoculated in slant medium (tryptone 17g/L, soy peptone 3g/L,
Sodium chloride 5g/L, dipotassium hydrogen phosphate 2.5g/L, glucose 2.5g/L, pH7.3, agar powder 15g/L, distilled water are prepared), 30 DEG C of items
Under part, stationary culture 24 hours is spare；
(3) seed culture：The bacterial strain that step (2) is cultivated, aseptically connects 1 ring in 50mL (250mL with oese
Triangular flask) (tryptone 17g/L, soy peptone 3g/L, sodium chloride 5g/L, dipotassium hydrogen phosphate in liquid seed culture medium
2.5g/L, glucose 2.5g/L, pH7.3, distilled water are prepared), set that rotary rpm is 180 revs/min, radius of turn is 40mm's
On shaking table, 30 DEG C are cultivated 24 hours, and seed liquor is obtained；
(4) crude oil resolution ratio detects：With the inoculum concentration of 1% volume ratio, seed liquor access is contained and is derived from Shengli Oil Field
Oily waste water in (50mL), be placed in the micro- aerobic cell culture systems of anaerobism and oxygen concentration be respectively set to 6%；
Stationary culture 9 days under the conditions of 30 DEG C.The waste water for not accessing Ya1 bacterial strains is handled under the same conditions, as a contrast.
(5) according to the content of total petroleum hydrocarbon in the method determination sample of 6 step of embodiment (5).
The results show that after access Ya1 bacterial strains, the content of total petroleum hydrocarbon is dropped to from initial 7.91g/L in waste water
5.15g/L, the removal rate of crude oil is close to 35.0%.
Embodiment 8：The application of Ya1 bacterial strains degraded oil ability in sample with high salt
The step of application process is related to sequence is as follows：
(1) strain selects：Select pseudomonas aeruginosa Ya1 of the present invention；
(2) inclined-plane culture activates：By strain be inoculated in slant medium (tryptone 17g/L, soy peptone 3g/L,
Sodium chloride 5g/L, dipotassium hydrogen phosphate 2.5g/L, glucose 2.5g/L, pH7.3, agar powder 15g/L, distilled water are prepared), 30 DEG C of items
Under part, stationary culture 24 hours is spare；
(3) seed culture：The bacterial strain that step (2) is cultivated, aseptically connects 1 ring in 50mL (250mL with oese
Triangular flask) (tryptone 17g/L, soy peptone 3g/L, sodium chloride 5g/L, dipotassium hydrogen phosphate in liquid seed culture medium
2.5g/L, glucose 2.5g/L, pH7.3, distilled water are prepared), set that rotary rpm is 180 revs/min, radius of turn is 40mm's
On shaking table, 30 DEG C are cultivated 24 hours, and seed liquor is obtained；
(4) crude oil resolution ratio detects：With the inoculum concentration of 1% volume ratio, seed liquor access is contained and is derived from Shengli Oil Field
High-salt oil-containing wastewater in (50mL, total salinity be 3.7%), the shaking table culture 9 days under the conditions of 30 DEG C, 180rpm.It will not access
The waste water of Ya1 bacterial strains is handled under the same conditions, as a contrast.
(5) according to the content of total petroleum hydrocarbon in the method determination sample of 6 step of embodiment (5).
The results show that after access Ya1 bacterial strains, the content of total petroleum hydrocarbon is from initial in the waste water that total salinity is 3.7%
6.53g/L drops to 4.98g/L, and the removal rate of crude oil is close to 23.7%.
Sequence table
<110>Dongying City Zheng Ze Environmental Protection Technology Co., Ltd of Shandong University
<120>The pseudomonas aeruginosa and its application in degraded oil of the micro- oxygen of one plant of tolerance and hypersaline environment
<141> 2015-12-2
<160> 1
<170> PatentIn Version 3.5
<210> 1
<211> 1396
<212> DNA
<213>Pseudomonas aeruginosa（Pseudomonas aeruginosa）
<221>Pseudomonas aeruginosa（Pseudomonas aeruginosa）Ya1 CGMCC NO.11426 16S rDNA
<222>（1）…（1396）
<400> 1
cgtccccctt gcggttagac tagctacttc tggagcaacc cactcccatg gtgtgacggg 60
cggtgtgtac aaggcccggg aacgtattca ccgtgacatt ctgattcacg attactagcg 120
attccgactt cacgcagtcg agttgcagac tgcgatccgg actacgatcg gttttatggg 180
attagctcca cctcgcggct tggcaaccct ttgtaccgac cattgtagca cgtgtgtagc 240
cctggccgta agggccatga tgacttgacg tcatccccac cttcctccgg tttgtcaccg 300
gcagtctcct tagagtgccc acccgaggtg ctggtaacta aggacaaggg ttgcgctcgt 360
tacgggactt aacccaacat ctcacgacac gagctgacga cagccatgca gcacctgtgt 420
ctgagttccc gaaggcacca atccatctct ggaaagttct cagcatgtca aggccaggta 480
aggttcttcg cgttgcttcg aattaaacca catgctccac cgcttgtgcg ggcccccgtc 540
aattcatttg agttttaacc ttgcggccgt actccccagg cggtcgactt atcgcgttag 600
ctgcgccact aagatctcaa ggatcccaac ggctagtcga catcgtttac ggcgtggact 660
accagggtat ctaatcctgt ttgctcccca cgctttcgca cctcagtgtc agtatcagtc 720
caggtggtcg ccttcgccac tggtgttcct tcctatatct acgcatttca ccgctacaca 780
ggaaattcca ccaccctcta ccgtactcta gctcagtagt tttggatgca gttcccaggt 840
tgagcccggg gatttcacat ccaacttgct gaaccaccta cgcgcgcttt acgcccagta 900
attccgatta acgcttgcac ccttcgtatt accgcggctg ctggcacgaa gttagccggt 960
gcttattctg ttggtaacgt caaaacagca aggtattaac ttactgccct tcctcccaac 1020
ttaaagtgct ttacaatccg aagaccttct tcacacacgc ggcatggctg gatcaggctt 1080
tcgcccattg tccaatattc cccactgctg cctcccgtag gagtctggac cgtgtctcag 1140
ttccagtgtg actgatcatc ctctcagacc agttacggat cgtcgccttg gtaggccttt 1200
accccaccaa ctagctaatc cgacctaggc tcatctgata gcgtgaggtc cgaagatccc 1260
ccactttctc cctcaggacg tatgcggtat tagcgcccgt ttccggacgt tatcccccac 1320
taccaggcag attcctaggc attactcacc cgtccgccgc tgaatccagg agcaagctcc 1380
cttcatccgc tcgact 1396
Claims (1)
1. the pseudomonas aeruginosa of one plant of tolerance micro- oxygen and hypersaline environment, it is characterised in that：The Strain Designation is that verdigris is false single
Born of the same parents bacterium (Pseudomonas aeruginosa) Ya1, the bacterial strain are preserved in " Chinese microorganism strain on 18th in September in 2015
Preservation administration committee common micro-organisms center ", deposit number are CGMCC NO.11426.
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| "氧浓度变化对铜绿假单胞菌生物被膜生成的影响";崔东清;《中国博士学位论文全文数据库（电子期刊）医药卫生科技辑》;20091031(第10期);摘要 * |
| "铜绿假单胞菌在高盐度下用于原油降解的可行性研究";姚斌;《中国优秀硕士论文全文数据库（电子期刊）工程科技Ⅰ辑》;20070228(第02期);摘要 * |
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