CN106754473B - Bacillus safensis and application thereof in remediation of open sea pollutants - Google Patents
Bacillus safensis and application thereof in remediation of open sea pollutants Download PDFInfo
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Abstract
The invention relates to the field of water environment treatment by using microorganisms, and particularly relates to bacillus safensis and application thereof. The bacillus safensis has been deposited in the general microbiological center of the China Committee for culture Collection of microorganisms (CCTCC) in 2016, 9 and 28, and the preservation numbers are as follows: CGMCC No. 13059. The method obtains the indigenous degraded strains by screening, and the obtained strains are loaded, flocculated, embedded and then repaired by applying the sea area; the microbial inoculum does not need complex instruments and equipment when being put in, and the putting effect and the repairing effect can be ensured.
Description
Technical Field
The invention relates to the field of water environment treatment by using microorganisms, in particular to bacillus safensis and application thereof in repairing open sea pollutants.
Background
The problems of marine oil spill and oil pollution are always important problems influencing marine environment and marine ecology, and at present, emergency treatment of marine oil pollution, particularly sudden oil spill accidents is mainly carried out by adopting physical interception, recovery, chemical dispersing agents and other modes for emergency treatment of marine surface seawater at the initial stage of oil spill. However, no good physical and chemical treatment method exists for the petroleum pollution caused by the seabed spilled oil or the petroleum pollution settled to the seabed sediments along with the movement of water, and the environmental self-purification and artificial introduction of the petroleum degrading microorganisms in the sediments are the main ways for solving the petroleum pollution of the marine sediments at present.
At present, the treatment of organic pollution of river sediments has a more successful engineering implementation case, and mainly adopts physical, chemical and biological modes and adopts a mode of sediment plowing and putting related repair reagents. However, at present, the microbial remediation of the petroleum pollution of the offshore sediments is mostly in the aspects of theoretical research and small-scale laboratory simulation marine environment experiment, and the demonstration and the engineering of the microbial remediation of the petroleum pollution of the offshore sediments in a large scale are still few. On the one hand, the marine environment is special, the requirements of offshore construction on weather, sea conditions, construction ships and offshore operation experience are strict, most of the existing engineering demonstration is mainly focused on a beach area aiming at the remediation of marine organic pollution, particularly petroleum pollution, and a microbial remediation method and application for open sea area sediment pollution are lacked.
Disclosure of Invention
The invention aims to provide bacillus safensis and application thereof in remediation of open sea pollutants.
In order to achieve the purpose, the invention adopts the technical scheme that:
the bacillus safensis is preserved in the China general microbiological culture Collection center in 2016, 9 and 28 days, and has the preservation numbers as follows: CGMCC No. 13059.
The bacillus safensis is a gram-positive stain bacterium, the cell is rod-shaped, the diameter is less than 1 micron, spores are formed, cysts do not expand, and polar flagellum movement is achieved. Strictly aerobic bacteria, positive for VP test, do not reduce nitrate and normally produce acid from glucose, arabinose, xylose and mannitol. Has wide physiological characteristics, can degrade macromolecular substances of animal and plant sources, such as protein, and other various compounds, but cannot produce amylase. The bacillus safensis and the bacillus pumilus cannot be effectively identified according to the 16S rRNA gene sequence, the similarity value of the 16S rRNA gene sequences is 99.9 percent, and the two species can be distinguished by utilizing the gyrB gene sequence.
1. Results of cell morphology and physicochemical experiments
2.16 results of determination of SrRNA Gene sequence
CTCACCGACTTCGGGTGTTGCAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTATGGGATTGGCTAAACCTTGCGGTCTTGCAGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTtGTCACCGgCAGTCACcTtAGAGTGCCCAACTGAaTGCTGGCAaCTAaGATCAaGGGTtGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACcTGTCACTCTGTCCCCGAAGGGAAAGCCCTATCTCTAGGGtTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTC TACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGgCTTTCTGGTTAGGTACCGTCAAGGTGCGAGCAGTTACTCTCGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCATTACCCCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGACAGCCGAAACCGTCTTTCATCCTTGAACCATGCGGTTCAAGGAACTATCCGGTATTAGCTCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCCGGGAGCAAG
3. Results of antibiotic susceptibility experiments
Application of bacillus safensis in repairing deposited petroleum pollutants in open sea areas.
The method specifically comprises the steps of loading a strain by zeolite, coating the strain by a flocculating agent, and then putting the coated microbial inoculum into a water area to be treated for application in remediation of petroleum pollutants in the water area.
A microbial inoculum for repairing deposited pollutants in open sea areas comprises the active component of the bacillus safensis;
or, Bacillus safensis with Acinetobacter calcoaceticus and Pseudomonas putida.
Acinetobacter calcoaceticus has been deposited in the general microbiological culture collection center of China Committee for culture Collection of microorganisms at 11/6 2010 with the following collection numbers: CGMCC No. 4311; the pseudomonas putida has been deposited in the common microorganism center of the China Committee for culture Collection of microorganisms at 6 months and 1 day 2010, and the deposit numbers are as follows: CGMCC No. 3887; the bacillus safensis has been deposited in the general microbiological center of the China Committee for culture Collection of microorganisms (CCTCC) in 2016, 9 and 28, and the preservation numbers are as follows: CGMCC No. 13059.
The general microbiological center address of the above-mentioned China Committee for culture Collection of microorganisms is No.3 Xilu No.1 on North Chen of the Korean-Yang district in Beijing.
The strains adopted when the petroleum polluted sediments in the open sea area are Acinetobacter calcoaceticus, Pseudomonas putida and Bacillus sabdarius which are mixed according to the ratio of the number of bacteria to be 3:2: 4.
The invention has the advantages that:
the method obtains the indigenous degraded strains by screening, and the obtained strains are loaded, flocculated, embedded and then repaired by applying the sea area; the microbial inoculum does not need complex instruments and equipment when being put in, and the putting effect and the repairing effect can be ensured. In conclusion, the method is scientific, reasonable, efficient, safe, simple to operate and suitable for restoring the sediment pollution of the open water area with the water depth of 2-50 meters, and specifically comprises the following steps:
the screening of the indigenous degradation microorganisms is the screening and the use of pollution remediation microorganisms, foreign species are avoided, indigenous strains in the sediments in the remediation sea area are used as strain sources, pollutants to be remediated (petroleum or other organic matters) are used as a unique carbon source, and microbial strains capable of efficiently degrading the pollutants to be remediated are screened through enrichment culture and separation culture;
the bacterial strain screens sediment samples collected in an area to be repaired, and screens indigenous degradation bacterial strains from the sediment samples, so that introduction of foreign species can be effectively avoided, large-scale biological pollution is avoided, and marine ecology is prevented from being damaged; for a single strain, a biological acute toxicity test is required to evaluate the safety, and for strains with obvious lethal effect, the strains should be rejected in the product. The combination of different strains can adopt a 96-well plate method, and the workload and the working time during strain combination can be effectively reduced. Meanwhile, the composite bacterial strain is obtained through the synergistic effect among the bacterial strains, the petroleum pollution in the environment is degraded and repaired, and in addition, the composite putting of various bacterial strains is more favorable for forming a biological film, so that the survival probability of the bacterial strains and the pollutant degradation effect are increased.
Drawings
FIG. 1 is a gram-stained microscopic image of Bacillus safensis.
FIG. 2 is a graph comparing the petroleum degradation efficiency of different strains in a mixed ratio.
FIG. 3 is a diagram showing the result of the evaluation of the oil pollution remediation effect of the marine sediments according to the embodiment of the present invention (where A is C in oil)12-C27Repairing effect; b is the reduction of the ratio of light hydrocarbon to heavy hydrocarbon; c is the polycyclic aromatic hydrocarbon repairing effect in petroleum).
FIG. 4 is a graph showing the degradation rate of Bacillus safensis on petroleum.
Detailed Description
The examples serve to further illustrate the invention, but the invention is not limited to the examples.
The strain adopted by the invention obtains the indigenous degradation strain from the sediment sample of the area to be repaired, which effectively avoids the introduction of foreign species, avoids the large-scale biological pollution and damages to marine ecology; the selection of the microbial inoculum adsorption material (such as zeolite and the like) can be selected according to the water depth, the zeolite is suitable for the sediment repair of a deeper water area, and the particle size of the zeolite is more than 1 mm, so that the microbial inoculum can be ensured to be rapidly settled to the seabed, and the loss of the microbial inoculum in a water body in the falling process is reduced; and the loss of the microbial inoculum during putting can be reduced by adopting the agglomeration and embedding of the flocculating agent.
Wherein, the selection of the flocculating agent needs to consider the pH and the biodegradability, and the pH of the flocculating agent during working is maintained between 5 and 8 due to the limitation of the growth conditions of microorganisms; the green degradable flocculant can not only enable the microbial inoculum to agglomerate, but also provide a certain carbon source for the microorganisms, and guarantee the growth of the repaired microorganisms.
And when the microbial inoculum is obtained, the microbial inoculum is packaged by adopting a pure natural degradable material, so that difficultly degradable pollutants cannot be introduced into the sea area to be repaired, and the release effect of pollutant degrading bacteria can be ensured by quickly degrading the material. The specific subpackage weight needs to be determined according to the water depth and the water flow condition, the specific subpackage standard is that smaller packages are selected as much as possible within the range (offset range) of guaranteeing the putting of the fungus bags, but the offset range of the fungus bags when the fungus bags are settled to the seabed sediments is directly related to the size of the fungus bags and the water flow speed, and the judgment can be carried out through numerical simulation before the size of the fungus bags is determined.
The above-mentioned security that obtains operation construction should be guaranteed to the microbial inoculum fungus package process of puting in, but security is guaranteed to the at utmost that small package fungus package sea is shed, the transport of conveyer belt, reducible manpower expenditure for put in speed and the degree of consistency of puting in. In addition, in the process of putting the microbial inoculum, the throwing effect can be determined by matching with an underwater monitoring system; the underwater monitoring system needs to ensure sufficient illumination, and the disturbance of the sediment in the throwing process can influence the observation effect.
The tracking and monitoring after the restoration mainly evaluates the effects of the organic pollution content of the sea area sediment and the overlying water, the microbial and bacterial density of the sea area sediment and the overlying water and the zeolite particles in the sea area sediment.
Example 1 screening and combination of highly effective Petroleum-degrading bacteria
The method comprises the steps of taking microorganisms in sediments in the sea area of the cigarette platform as a source of efficient petroleum degrading bacteria, screening indigenous strains with good degrading effect on petroleum pollution, mixing the strains according to the proportion of the maximum degrading effect, adsorbing the strains on the surface of zeolite, wrapping the strains by a film coating agent, and finally subpackaging and putting the strains to finish bioremediation of the sediments in the designated sea area of the Bohai sea.
(1) Strain screening: weighing 2g of petroleum-polluted seawater sediment sample, respectively adding the sample into a triangular flask containing a seawater culture medium and a fresh water culture medium, carrying out shaking culture on a constant-temperature shaking table at 30 ℃ and 180r/min for 3d, transferring 1mL of culture solution into a screening culture medium, carrying out shaking culture on a constant-temperature shaking table at 30 ℃ and 180r/min for 5d, transferring 1mL of culture solution into a new screening culture medium, carrying out 5d culture under the same condition, and repeating for 3 times. Taking culture solution after 3 cycles of culture, respectively coating the culture solution on a seawater solid culture medium and a fresh water solid culture medium after gradient dilution, culturing for 12-24h at 30 ℃, selecting single bacterial colonies with different forms, and carrying out streaking, purification and culture until obtaining single bacterial colonies without mixed bacteria.
(2) And (3) microbial strains: acinetobacter calcoaceticus (T32), Pseudomonas putida (SP1), and Bacillus safensis (Bb). And finally, screening three strains with the degradation efficiency of more than 50 percent for 7 days of petroleum, and identifying the three strains to be respectively acinetobacter calcoaceticus, pseudomonas putida and bacillus safensis.
(3) The maximum degradation effect ratio is as follows: combining strains according to the proportion in the horizontal orthogonal experiment, comparing the petroleum degradation effects of different combination proportions by adopting a 96-pore plate mode, and selecting the optimal degradation proportion. Through tests, the optimal combination ratio of the acinetobacter calcoaceticus, the pseudomonas putida and the bacillus safensis is 3:2: 4. (FIG. 2)
The formula of the culture medium in the step (1) is as follows:
seawater culture medium: trypton 5g, Yeast Extract 1g, Fe2(PO4)30.01g, adding aged seawater to1L, and adjusting pH to 7.0-7.2. Seawater solid culture medium: 20g of agar was added to the seawater culture medium.
Fresh water culture medium: 10g of Trypton, 5g of Yeast Extract and 10g of NaCl, adding distilled water to1L, and adjusting the pH to 7.0-7.2. Fresh water solid medium: 20g of agar was added on the basis of fresh water medium.
Screening a culture medium: 2g of crude oil (oil from the Burley oil spill) were added to1L of MM medium.
MM medium: NH (NH)4NO31g，K2HPO41.5g，KH2PO40.5g，NaCl 0.5g，MgSO40.2g, 1mL of chelated iron solution (FeSO)4·7H20.246g of O, 0.331g of EDTA disodium, 0.331g of dH2O up to 100mL), 1mL of trace element solution (Na)2MoO4·2H2O 0.01g，MnCl20.02g，CoCl2·6H2O 0.0002g，ZnSO4·7H2O 0.01g，CuSO4·5H2O 0.02g，ddH2O up to 100mL，2％wt/vol K2CO3Adjusting pH to 7.0-7.2), ddH2O up to1L, and adjusting the pH to 7.0-7.2.
In the step (3), the specific experimental method comprises the following steps:
A. respectively dissolving a certain amount of crude oil in a certain amount of mixed solution of petroleum ether, n-hexane and 1:1 petroleum ether-n-hexane to prepare 0.5% petroleum extract solution, centrifugally filtering, injecting the uniformly mixed petroleum solution into a 96-well plate, adding 300ul of the petroleum solution into each well, and standing overnight in a ventilated place to volatilize the organic solvent, wherein the petroleum is remained on the side wall and the bottom of the well plate.
B.3 taking out the strains, respectively activating, carrying out amplification culture (the culture medium for amplification culture is the seawater culture medium in the step (1)), carrying out normal-temperature centrifugation for 1min until OD is 1, 8000r, collecting the strains, carrying out heavy suspension washing by using an MM culture medium for three times, carrying out heavy suspension by using MM, mixing the heavy-suspended strains according to different proportions of 1:1:1, 1:2:2, 1:3:3, 1:4:4, 2:1:2, 2:2:1, 2:3:4, 2:4:3, 3:1:3, 3:2:4, 3:3:1, 3:4:2, 4:1:4, 4:2:3, 4:3:2 and 4:4:1 of bacterial colonies, supplementing 300ul by using MM as a control, repeating the proportions for three times, placing the mixture into an incubator, and keeping the temperature of the incubator at 28 ℃.
And C.7 days later, taking out the pore plate, drying at low temperature, re-dissolving the petroleum in the pore plate by respectively using petroleum ether, n-hexane and a petroleum ether-n-hexane mixed solution in a ratio of 1:1, measuring the ultraviolet absorbance of each pore plate at a wavelength of 225nm, and determining the degradation efficiency of the petroleum (see figure 2).
The method comprises the following steps of (1) carrying out strain mixing on different types of petroleum pollutants according to the research on the most suitable degradation ratio of three different petroleum hydrocarbons to achieve the best effect, namely, aiming at the pollutants which are short-chain petroleum hydrocarbon components, the colony number ratio of the strains is 1:2: 2; aiming at the pollutants of long-chain petroleum hydrocarbon components, the colony number ratio of the strain is 4:3: 2; the colony number ratio of the strain is 3:2:4 aiming at the pollutants of mixed petroleum hydrocarbon components. Most of the petroleum pollution is mixed petroleum hydrocarbon pollution.
Example 2 microbial remediation of marine sediment oil contamination
And repairing the petroleum pollution of the deposits in the Bohai sea area.
(1) Repairing the strain: acinetobacter calcoaceticus, pseudomonas putida, and bacillus safensis.
(2) Fermenting the three strains respectively in large scale, mixing the fermented bacteria solutions with the concentration of 2-3 OD. respectively, and mixing the bacteria solutions according to the ratio of the colony count of 3:2:4, wherein the total bacteria amount in the mixed bacteria solution is about 1 × 109CFU/ml, and refrigerating and storing in a refrigerator at 4 ℃ after mixing.
(3) Adsorption and wrapping of bacterial liquid: and (3) mixing and adsorbing the mixed bacteria liquid according to the mass ratio of the bacteria liquid to the zeolite of 2:1, wherein the adsorption time is 2 hours, after mixing, sieving the air water in water, spraying 7 wt% of polyglutamic acid solution after the water content of the zeolite of the adsorbed bacteria liquid is 60% -70%, continuously mixing and stirring until the zeolite is agglomerated and no flowing liquid exists, preparing the petroleum remediation microbial inoculum, and storing the petroleum remediation microbial inoculum in a ton bag internally attached with a waterproof and air-impermeable film at 4 ℃. The mass ratio of the 7% polyglutamic acid to the mixed bacterial liquid is 1: 10.
(4) Subpackaging and putting of microbial inoculum: numerical simulation is carried out according to the conditions of water depth (25-30 meters) and water flow (0.1-0.6m/s), the size of a fungus bag is determined to be 5kg, a natural easily-degradable jute bag is used as a packaging material, and after subpackaging is carried out rapidly, every 20 bags are packaged uniformly in a ton bag with a waterproof and air-impermeable film inside, so that the transportation is facilitated. And (5) after subpackaging, immediately carrying out marine throwing.
(5) Tracking and monitoring after repairing: and (3) carrying out primary restoration effect monitoring and evaluation one month after the microbial inoculum is put, wherein the main monitoring items comprise petroleum hydrocarbon content of sea sediments and overlying water, microbial bacterial density of the sea sediments and the overlying water, zeolite particles in the sea sediments and the like, and through the restoration effect evaluation, the petroleum degradation efficiency of most restoration stations is over 50 percent, and the station degradation efficiency of the part with higher petroleum content can be as high as 70-80 percent. (see FIG. 2)
And (4) adopting a conveyor belt throwing method in the marine throwing method in the step (4), and setting conveyor belt intervals according to the width of the ship body and the microbial inoculum throwing amount:
conveying a belt: the length is 9.6m, and the marking is carried out every 1.6 m;
conveying belt interval: 3 m;
number of conveyor belts: 3, cutting;
belt speed of the conveyor: 0.8 m/s;
the ship speed is as follows: section 3 (adjustable according to actual conditions) (about 1.5 m/s);
throwing speed: one bag every 2 seconds;
the ship sails back and forth at intervals of 9 m.
Displaying a tracking monitoring result after repairing in the step (5): for the first-time collected samples after restoration, the station positions 5 and 6 with higher background petroleum content have the best restoration effect, and the normal alkane content is respectively degraded by 65.0 percent and 39.6 percent. Is affected by microbial degradation, wherein the ratio of light to heavy hydrocarbons LMW/HMW is reduced relative to the total before repair. The combined bacterium has better utilization of alkane before C15-C27 and higher degradation rate which reaches 18.05-89.50%; the utilization effect on the alkane of the high-carbon alkane n-C28-n-C36 is poor, and the degradation efficiency is low. Polycyclic aromatic hydrocarbons are similar to normal alkanes, wherein the station 5 and 6 with high background petroleum content have the best repairing effect, the polycyclic aromatic hydrocarbon content is respectively degraded by 48.54 percent and 78.62 percent, and the degradation rate of other stations is 25 to 52 percent. (FIG. 3)
Example 3 method of degrading Petroleum hydrocarbons with Bacillus saffron
(1) And (3) microbial strains: bacillus safensis (Bb);
(2) seed culture: selecting a ring of colonies from a flat plate, inoculating the colonies into an LB culture medium, shaking the liquid to fill 30%, culturing at 28-30 ℃, at a culture rotation speed of 180-200 rpm for 10-16h, centrifuging at 13,000 rpm for 5min, and collecting thalli;
(3) and (3) degrading petroleum hydrocarbon by Bb, namely inoculating the Bb thallus collected in the step (2) into an inorganic salt peptone liquid culture medium containing 2g/L of petroleum hydrocarbon according to the initial concentration of 1 × 108 for culture, taking the inorganic salt peptone liquid culture medium containing the petroleum hydrocarbon which is not inoculated with the Bb as a control, taking the experimental group and the control after 7 days, extracting the residual petroleum hydrocarbon by using petroleum ether, and taking the experimental group and the control after 14 days, and extracting the residual petroleum hydrocarbon by using the petroleum ether.
(4) Detection of petroleum hydrocarbons: diluting the petroleum ether containing the petroleum hydrocarbon in the step (3), detecting an absorption peak under 225nm wavelength by a spectrophotometry to obtain the concentration of the petroleum hydrocarbon, and calculating the degradation rate of the petroleum hydrocarbon (figure 4).
The LB culture medium used in the step (2) has the formula as follows: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride, and sterilizing for 30 minutes at 121 ℃.
The formula of the inorganic salt peptone medium in the step (3) is as follows: na (Na)2HPO4·12H2O 3g/L,KH2PO41g/L,NaCl 3g/L,MgSO40.3g/L, 2.5g/L of Tryptone; adjusting pH to 6.0-8.0, and sterilizing at 121 deg.C for 30 min.
Through the examples, the feasibility, effectiveness and convenience of the method are fully proved, the best marine sediment petroleum remediation effect can be achieved with the least cost, the method is also suitable for the remediation of other marine organic pollution, and only efficient degradation microorganisms aiming at different pollutants need to be screened. Most of the functional sea areas/water areas in China are seriously polluted, but the cases of the real marine/fresh water sediment remediation are not many, and a corresponding guidance method is lacked. The method well compensates the defects in the aspect.
SEQUENCE LISTING
<110> institute of tobacco pipe coastal zone of Chinese academy of sciences
<120> Bacillus safensis and application thereof in remediation of open sea pollutants
<130>
<160>1
<170>PatentIn version 3.1
<210>1
<211>1369
<212>DNA
<213> Bacillus saffron
<220>
<221>gene
<222>(1)..(1369)<223>
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ctcaccgact tcgggtgttg caaactctcg tggtgtgacg ggcggtgtgt acaaggcccg 60
ggaacgtatt caccgcggca tgctgatccg cgattactag cgattccagc ttcacgcagt 120
cgagttgcag actgcgatcc gaactgagaa cagatttatg ggattggcta aaccttgcgg 180
tcttgcagcc ctttgttctg tccattgtag cacgtgtgta gcccaggtca taaggggcat 240
gatgatttga cgtcatcccc accttcctcc ggtttgtcac cggcagtcac cttagagtgc 300
ccaactgaat gctggcaact aagatcaagg gttgcgctcg ttgcgggact taacccaaca 360
tctcacgaca cgagctgacg acaaccatgc accacctgtc actctgtccc cgaagggaaa 420
gccctatctc tagggttgtc agaggatgtc aagacctggt aaggttcttc gcgttgcttc 480
gaattaaacc acatgctcca ccgcttgtgc gggcccccgt caattccttt gagtttcagt 540
cttgcgaccg tactccccag gcggagtgct taatgcgtta gctgcagcac taaggggcgg 600
aaacccccta acacttagca ctcatcgttt acggcgtgga ctaccagggt atctaatcct 660
gttcgctccc cacgctttcg ctcctcagcg tcagttacag accagagagt cgccttcgcc 720
actggtgttc ctccacatct ctacgcattt caccgctaca cgtggaattc cactctcctc 780
ttctgcactc aagtttccca gtttccaatg accctccccg gttgagccgg gggctttcac 840
atcagactta agaaaccgcc tgcgagccct ttacgcccaa taattccgga caacgcttgc 900
cacctacgta ttaccgcggc tgctggcacg tagttagccg tggctttctg gttaggtacc 960
gtcaaggtgc gagcagttac tctcgcactt gttcttccct aacaacagag ctttacgatc 1020
cgaaaacctt catcactcac gcggcgttgc tccgtcagac tttcgtccat tgcggaagat 1080
tccctactgc tgcctcccgt aggagtctgg gccgtgtctc agtcccagtg tggccgatca 1140
ccctctcagg tcggctacgc atcgtcgcct tggtgagcca ttaccccacc aactagctaa 1200
tgcgccgcgg gtccatctgt aagtgacagc cgaaaccgtc tttcatcctt gaaccatgcg 1260
gttcaaggaa ctatccggta ttagctccgg tttcccggag ttatcccagt cttacaggca 1320
ggttacccac gtgttactca cccgtccgcc gctaacatcc gggagcaag 1369
Claims (5)
1. A Bacillus safensis characterized in that: the bacillus safensis has been deposited in the general microbiological center of the China Committee for culture Collection of microorganisms (CCTCC) in 2016, 9 and 28, and the preservation numbers are as follows: CGMCC No. 13059.
2. Use of bacillus safensis according to claim 1 wherein: the application of the bacillus safensis in repairing deposited pollutants in open sea areas is provided.
3. Use of bacillus safensis according to claim 2 wherein: the bacterial strain is loaded by zeolite and coated by a flocculating agent, and then the coated microbial inoculum is put into a water area to be treated and applied to the restoration of petroleum in the water area.
4. A microbial inoculum for repairing deposited pollutants in open sea areas is characterized in that: the active ingredient of the microbial inoculum is the bacillus safensis of claim 1;
or, the Bacillus safensis strain of claim 1 in combination with Acinetobacter calcoaceticus and Pseudomonas putida.
5. The microbial inoculum for remediating open sea sediment contaminants as set forth in claim 4, wherein: the strains adopted when the petroleum polluted sediments in the open sea area are Acinetobacter calcoaceticus, Pseudomonas putida and Bacillus sabdarius which are mixed according to the ratio of the number of bacteria to be 3:2: 4.
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| CN102181384B (en) * | 2011-02-25 | 2012-11-28 | 中国科学院烟台海岸带研究所 | Acinetobacter calcoaceticus T32 capable of metabolizing furazolidone and application thereof |
| CN102676418A (en) * | 2011-10-28 | 2012-09-19 | 中国科学院烟台海岸带研究所 | Application of acinetobacter calcoaceticus capable of degrading petroleum |
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Non-Patent Citations (2)
| Title |
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| Classification and Identification of Petroleum Microorganisms by MALDI-TOF Mass Spectrometry;Célio F. F. Angolini等;《J. Braz. Chem. Soc.》;20150120;第26卷(第3期);第513-520页 * |
| Genome Sequence of Bacillus safensis CFA06, Isolated from Biodegraded Petroleum in Brazil;Prianda R. Laborda等;《Genome Announcements》;20140724;第2卷(第4期);第e00642-14页 * |
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