CN104357350B - One plant of deep-sea actinomyces and its application - Google Patents
One plant of deep-sea actinomyces and its application Download PDFInfo
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- CN104357350B CN104357350B CN201410534724.4A CN201410534724A CN104357350B CN 104357350 B CN104357350 B CN 104357350B CN 201410534724 A CN201410534724 A CN 201410534724A CN 104357350 B CN104357350 B CN 104357350B
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Abstract
The invention discloses one plant of deep-sea actinomyces and its application.The present invention discloses one plant of cock Salmonella (Kocuria sp.), and its deposit number is CGMCC No.9648.Deep-sea actinomyces FXJ8.057 disclosed by the invention can reduce Fe (III).Iron reduction can promote the degraded and purification of polluter in water body under earth's surface, therefore, bacterial strain disclosed by the invention has important application prospect in the disposal of the environmental problems such as residues of pesticides, heavy metal toxicity and body eutrophication.
Description
Technical field
The present invention relates to one plant of deep-sea actinomyces and its application, belong to biological technical field.
Background technology
Be richly stored with microbial resources in ocean, deep-sea due to its distinctive high pressure, low temperature (caldera exception),
The special ecological environment such as oligotrophic, dark, creates the highly various of seabed microbial species, Physiology and biochemistry and ecological functions
Property.The imagination of as many as the microbe groups out of the mankind in deep-sea.Acidophilus, basophilic, thermophilic is widely present in deep-sea extreme environment
Micro- life out of limit of life such as salt, thermophilic cold (up to less than 0 DEG C), thermophilic (more than 120 DEG C), piezophilic (more than 500 atmospheric pressure)
Thing.Comprising many microorganisms being not yet separately cultured in deep sea water and deposit, Deep-Sea Microorganisms research is not only to micro- life
Thing development of resources has important practical significance, and also has important scientific meaning with the rule of environmental interaction to understanding life.
Actinomyces be a type genomic dna G+C contents are high, form of diverse gram-positive bacterium, because its bacterium colony is normal
It is radial and gain the name.It is an important monoid in marine microorganism, is widely distributed in the various environment in ocean.People's morning
Phase just isolates 3 monoids of Micromonospora, Rhodococcus and Streptomyces from marine environment, is ocean
The dominant groups of actinomyces.With the further investigation to marine actinomycete, more actinomyces monoids are being found and are describing.
The content of the invention
It is an object of the invention to provide one plant of deep-sea actinomyces and its application.
The present invention provides one plant of cock Salmonella (Kocuria sp.), and its deposit number is CGMCC No.9648.
The zymotic fluid or tunning of above-mentioned cock Salmonella fall within protection scope of the present invention.
The method that one kind goes back Fe in raw sample (III) falls within protection scope of the present invention, be by above-mentioned cock Salmonella or on
State zymotic fluid or tunning to mix with the sample containing Fe (III), obtain mixed liquor, then mixed liquor is cultivated；
The Fe (III) is ferric iron.
In the above method, the sample is to contain FeCl3, ferrihydrite and/or goethite sample.
In any of the above-described described method, the condition of the culture is that aerobic, pH is the condition of 6-8；
The pH is specially 7 or 8.
In any of the above-described described method, the condition of the culture also includes normal pressure or condition of high voltage.
In any of the above-described described method, the high pressure is 1MPa-2.5MPa, specially 1MPa or 2.5MPa.
The application of above-mentioned cock Salmonella or above-mentioned zymotic fluid or tunning in reduction Fe (III) falls within of the invention
Protection domain；
The Fe (III) is ferric iron.
The application of above-mentioned cock Salmonella or above-mentioned zymotic fluid or tunning in heavy metal toxicity is reduced falls within this hair
Bright protection domain；
Or,
The application of above-mentioned cock Salmonella or above-mentioned zymotic fluid or tunning in residues of pesticides are eliminated falls within the present invention
Protection domain.
Above-mentioned cock Salmonella or above-mentioned zymotic fluid or tunning answering in purifying water or eliminating body eutrophication
With falling within protection scope of the present invention.
The bacterium with Fe (III) reducing power reported in existing document is all to play it under anaerobism, pH acid conditions
Fe (III) reducing power, the bacterium that the present invention is provided is aerobic bacteria, and Fe (III) is played under conditions of the neutral left and right of aerobic, pH also
Proper energy power.
The deep-sea actinomyces FXJ8.057 that the present invention is provided can reduce Fe (III).Iron reduction can promote under earth's surface in water body
The degraded and purification of polluter, therefore, the bacterial strain that the present invention is provided is in residues of pesticides, heavy metal toxicity and body eutrophication
There is important application prospect in disposal etc. environmental problem.
Brief description of the drawings
Fig. 1 is positions of the bacterial strain FXJ8.057 in 16S rRNA Phylogenetic Trees.
Specific embodiment
Experimental technique used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.
The separation and identification of embodiment 1, bacterial strain FXJ8.057
First, the separation of bacterial strain FXJ8.057
Collection south west Indian Ocean seawater sample, 100mL is concentrated into by the seawater of about 100L, obtains concentrated seawater sample.
Glycerine and trehalose is added to be stored in -20 DEG C in sample.To after laboratory, 5-10mL concentrated seawater samples, 55 DEG C of heat treatments are taken
5min, takes 1mL gradient dilutions 10-1, 10-2, obtain dilution.Take 0.2mL dilutions and coat DNB culture mediums (peptone 1g/
100ml, beef extract 0.5g/100ml, NaCl 0.5g/100ml, balance of water) in, 16 DEG C culture, picking single bacterium colony, by its in
GYM culture mediums (glucose 0.4g/100ml, oat extract 1g/100ml, yeast extract 0.4g/100ml, balance of water)
Middle purifying culture, obtains bacterial strain FXJ8.057.
2nd, the identification of bacterial strain FXJ8.057
(1) bacterial strain FXJ8.057 is cultivated 48 hours for 28 DEG C in GYM culture mediums, bacterium colony light red, moistening, circular, edge
It is smooth, it is opaque；Cell is spherical (diameter is about 0.8-1.5 μm).
(2) physiological and biochemical property
Bacterial strain FXJ8.057 is identified as gram-positive bacteria, aerobic, middle temperature.Urase is positive, catalase positive, nitre
Hydrochlorate is also Antigen positive hybridomas, and gelatin liquefaction is positive, and milk solidifies and peptonizes feminine gender, and M-R, V-P experiment are negative, H2S produces negative, starch
Hydrolysis is positive.
Bacterial strain FXJ8.057 utilization of carbon source and other physiological and biochemical properties are as shown in table 1.
The bacterial strain FXJ8.057 utilization of carbon source of table 1 and other physiological and biochemical properties
| Test event | As a result | Test event | As a result | Test event | As a result | Test event | As a result |
| The pool of fiber two | + | Synanthrin | - | Glycerine | + | Polysorbas20 | - |
| Fructose | + | Galactolipin | + | Maltose | + | Polysorbate40 | - |
| Lactose | + | Rhamnose | + | Mannitol | + | Tween 80 | + |
| Arabinose | + | Xylose | + | Guanine | - | Temperature (DEG C) | 4-40 |
| Ribose | - | Raffinose | + | Xanthine | - | Nacl is tolerated | 0-7% |
| Mannose | + | Erythrite | - | Casein | - | pH | 6-10 |
| Sucrose | + | Trehalose | + | Adenine | - | Cellulase | - |
"+" represents that test result is the positive；"-" represents that test result is feminine gender.
(3) 16S rRNA genetic analysis
The genomic DNA of bacterial strain FXJ8.057 is extracted, 16S rRNA genes is expanded using bacterial universal primers and is purified survey
Sequence, the 16S rRNA gene orders of bacterial strain FXJ8.057 are as shown in SEQ ID No.1.By sequencing results by NCBI's
Blast program carries out similarity system design.Result shows that most like with bacterial strain 16S rRNA gene orders is Kocuria
Flava JCM15621, Kocuria turfanensis JCM15622 and Kocuria rosea JCM11614, similitude is equal
Less than 98.68%.
Positions of the bacterial strain FXJ8.057 in 16S rRNA Phylogenetic Trees is as shown in Figure 1.
(4) chemical classification feature
Bacterial strain FXJ8.057 main polar lipid component is phosphatide DPG；Breathing quinone is methylnaphthoquinone MK-7 (H2), content is about
It is 60.5%；Its topmost aliphatic acid is 15:0anteiso, content is about 66%.
The Classification And Nomenclature of bacterial strain FXJ8.057 is cock Salmonella (Kocuria sp.), and the bacterial strain is in 09 month 2014 11
Day is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address：Beijing is exposed to the sun
The institute 3 of area North Star West Road 1, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.9648.
The iron reducing power analysis of the bacterial strain FXJ8.057 under the conditions of embodiment 2, pH7
First, the synthesis of mineral
The synthesis (alpha-feooh) of goethite powder:180mL concentration is stirred for the KOH solution of 5mol/L is rapidly joined
The 100ml concentration mixed is the Fe (NO of 1mol/L3)3·9H2In O solution, distilled water is added liquor capacity is reached 2L immediately.
60 hours are stood at 70 DEG C, is centrifuged (3000 × g, 15min), be precipitated thing, distilled water washing precipitate 3 times, by sediment
Ground after 40 DEG C of drying, obtained final product.
The synthesis of ferrihydrite powder：By 180mL concentration for the KOH solution of 5mol/L rapidly joins the 100ml for stirring
Concentration is the Fe (NO of 1mol/L3)3·9H2In O solution, distilled water is added liquor capacity is reached 2L immediately.Quick spin
(3000 × g, 15min), is precipitated thing, and distilled water washing precipitate 3 times grinds after sediment normal temperature is dried, obtains final product.
2nd, the preparation of culture medium
FeCl is separately added into the GYM culture mediums (it is 7 that phosphate buffer adjusts pH) with one times of distilled water diluting3It is molten
Liquid, the ferrihydrite or goethite powder (containing Fe (III)) of step one synthesis so that FeCl3, ferrihydrite and goethite end it is dense
Degree is 1mM, is made containing FeCl3, ferrihydrite or goethite 1/2 dilution GYM culture mediums.
Wherein FeCl3The prior filtration sterilization of solution, the ferrihydrite and goethite powder of step one synthesis are in advance using interval
Sterilization sterilizes, and condition is：After 100 DEG C of sterilizing 30min, 12h is placed, sterilized again, repeat sterilizing 3 times.
3rd, the preparation of bacteria suspension
Bacterial strain FXJ8.057 is collected by centrifugation thalline in the GYM fluid nutrient mediums after culture three days, aseptic water washing three times,
And it is made bacteria suspension with sterilized water.
4th, strain culturing
By step 3 prepare bacteria suspension be seeded in respectively step 2 preparation containing FeCl3, ferrihydrite or goethite 1/2
In the GYM culture mediums of dilution, inoculum concentration is 1 × 105Cells/mL, under aerobic conditions, 28 DEG C of shaking table cultures obtain each after 7 days
Nutrient solution, detects iron reducing power.
5th, iron reducing power detection
Fe is detected using spectrophotometer method (luxuriant and rich with fragrance Lip river piperazine development process)2+Concentration.The development step of wherein sample is specific as follows：
200 each nutrient solutions of μ l are drawn, the HCl/water solution that 200 μ l concentration are 1M is separately added into, piping and druming is mixed, and obtains mixed liquor, is put
After putting 12h, (HEPES that Ferrozine is dissolved in 50mM is molten to 1mL phenanthrene Lip river piperazine (Ferrozine) solution to take 100 μ l mixed liquors
In liquid, the concentration of Ferrozine is 1g/L) in, after standing 5min, spectrophotometer reads light absorption value under wavelength 562nm.With
Corresponding each culture medium of bacterium is not connect respectively as blank.
Result shows, bacterial strain FXJ8.057 can pH7, it is aerobic under conditions of reduction contain FeCl3, ferrihydrite or goethite
1/2 dilution GYM culture mediums FeCl3, ferrihydrite or goethite, the Fe for ultimately forming2+Content be respectively 0.2,0.06
And 0.121mM.Show that bacterial strain FXJ8.057 has Fe (III) reducing power.
The iron reducing power analysis of the bacterial strain FXJ8.057 under the conditions of embodiment 3, pH8
Because mineral structure is relatively stablized, and also proper energies of the bacterial strain FXJ8.057 to goethite under the conditions of pH7 in example 2
Power is preferable, so the present embodiment only have detected reduction of the bacterial strain FXJ8.057 to goethite under conditions of pH8.
First, the synthesis of goethite
The step of with embodiment 2 one.
2nd, the preparation of culture medium
Step one is added to synthesize in the GYM culture mediums (it is 8 that phosphate buffer adjusts pH) with one times of distilled water diluting
Goethite powder so that the final concentration of 1mM of goethite, be made containing goethite 1/2 dilution GYM culture mediums.
The step of wherein sterilizing of goethite powder is with embodiment 2 two.
3rd, the preparation of bacteria suspension
The step of with embodiment 2 three.
4th, strain culturing
By the bacterial suspension inoculation of step 3 preparation in the GYM culture mediums of containing goethite 1/2 dilution prepared by step 2,
Inoculum concentration is 1 × 105Cells/mL, under aerobic conditions, 28 DEG C of shaking table cultures obtain nutrient solution, detection iron also proper energy after 7 days
Power.
5th, iron reducing power detection
The step of method is with embodiment 2 five.
Result shows, bacterial strain FXJ8.057 can pH8, it is aerobic under conditions of 1/2 dilution of the reduction containing goethite GYM
The goethite of culture medium, the Fe for ultimately forming2+Content be 0.122mM.Show that bacterial strain FXJ8.057 has Fe (III) also proper energies
Power.
Embodiment 4, bacterial strain FXJ8.057 iron reducing power analysis under high pressure
First, the preparation of culture medium
The step of embodiment 2 is added in the GYM culture mediums (it is 7 that phosphate buffer adjusts pH) with one times of distilled water diluting
The goethite powder of a rapid synthesis so that the final concentration of 1mM of goethite, is made the GYM cultures of 1/2 dilution containing goethite
Base.
The step of wherein sterilizing of goethite powder is with embodiment 2 two.
2nd, the preparation of bacteria suspension
With the step three in embodiment 2.
3rd, the culture of bacterial strain
Step 2 prepare bacterial suspension inoculation step one prepare containing goethite 1/2 dilution GYM culture mediums in,
Inoculum concentration is 1 × 105Cells/mL, obtains mixed liquor, and it is drawn into 2ml with syringe, and (culture medium is not removed under aerobic conditions
Oxygen, so being aerobic conditions), it is put into autoclave and is cultivated in 1MPa and 2.5MPa respectively, each nutrient solution is obtained after 14 days,
Detection iron reducing power.
4th, iron reducing power detection
Nutrient solution is only replaced with each nutrient solution that step 3 is obtained respectively with the step five in embodiment 2 for method.
Result shows that bacterial strain FXJ8.057 can reduce goethite under the high pressure of 1MPa and 2.5MPa, ultimately form
Fe2+Content difference 0.098,0.15mM, show bacterial strain FXJ8.057 under high pressure also have Fe (III) reducing power.
Claims (12)
1. one plant of cock Salmonella (Kocuria sp.), its deposit number is CGMCC No.9648.
2. the zymotic fluid of the cock Salmonella described in claim 1；The zymotic fluid is bacteria suspension.
3. the method that one kind goes back Fe in raw sample (III), is by described in the cock Salmonella described in claim 1 or claim 2
Zymotic fluid mixes with the sample containing Fe (III), obtains mixed liquor, then mixed liquor is cultivated.
4. method according to claim 3, it is characterised in that：The sample is to contain FeCl3, ferrihydrite and/or goethite
Sample.
5. the method according to claim 3 or 4, it is characterised in that：The condition of the culture is that aerobic, pH is the bar of 6-8
Part.
6. method according to claim 5, it is characterised in that：The condition of the culture also includes normal pressure or condition of high voltage；
The high pressure is 1MPa-2.5MPa.
7. method according to claim 6, it is characterised in that：The high pressure is 1MPa or 2.5MPa.
8. the application of the cock Salmonella described in claim 1 or the zymotic fluid described in claim 2 in reduction Fe (III).
9. the application of the cock Salmonella described in claim 1 or the zymotic fluid described in claim 2 in heavy metal toxicity is reduced；
Heavy metal is Fe (III).
10. the application of the cock Salmonella described in claim 1 or the zymotic fluid described in claim 2 in residues of pesticides are eliminated；
The residues of pesticides are participated in or caused by Fe (III).
The application of the zymotic fluid described in cock Salmonella or claim 2 described in 11. claims 1 in purified water；It is described net
It is based on purifying water that iron reduction is produced to change water quality.
The answering in body eutrophication is eliminated of the zymotic fluid described in cock Salmonella or claim 2 described in 12. claims 1
With；The body eutrophication is participated in or caused by Fe (III).
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