CN104357350A - Deep-sea actinomycete and applications thereof - Google Patents
Deep-sea actinomycete and applications thereof Download PDFInfo
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- CN104357350A CN104357350A CN201410534724.4A CN201410534724A CN104357350A CN 104357350 A CN104357350 A CN 104357350A CN 201410534724 A CN201410534724 A CN 201410534724A CN 104357350 A CN104357350 A CN 104357350A
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- C—CHEMISTRY; METALLURGY
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- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
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Abstract
The invention discloses a deep-sea actinomycete and applications thereof, and discloses Kocuria sp. with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.9648. The deep-sea actinomycete FXJ8.057 can reduce Fe (III). Iron reduction can promote degradation and purification of pollutants in subsurface water bodies, so that the strain has important application prospects in solving environmental problems of pesticide residues, heavy-metal toxicity, water body eutrophication and the like.
Description
Technical field
The present invention relates to strain deep-sea actinomycetes and an application thereof, belong to biological technical field.
Background technology
Be richly stored with in ocean Microbial resources, deep-sea, due to the special ecotope of its distinctive high pressure, low temperature (caldera exception), oligotrophic, dark etc., creates the great diversity of seabed microbial species, Physiology and biochemistry and ecological functions.The imagination out of the mankind more than microbe groups in deep-sea.Extensively also exist in deep-sea extreme environment addicted to acid, basophilic, addicted to salt, addicted to the microorganism out of limit of life such as cold (less than 0 DEG C can be reached), thermophilic (more than 120 DEG C), piezophilic (more than 500 normal atmosphere).Comprise the microorganism of many not yet separated cultivations in deep sea water and settling, Deep-Sea Microorganisms research not only has important practical significance to Microbial resources exploitation, also has important scientific meaning to the rule of understanding life and environmental interaction.
Actinomycetes are that the G+C content of a type genomic dna is high, the gram positive bacterium of form of diverse, because its bacterium colony is often radially gained the name.It is an important monoid in marine microorganism, is extensively distributed in the various environment in ocean.People just isolate Micromonospora, Rhodococcus and Streptomyces 3 monoids in early days from ocean environment, are the dominant groups of marine actinomycete.Along with the further investigation to marine actinomycete, more actinomycetes monoid is being found and is describing.
Summary of the invention
The object of this invention is to provide strain deep-sea actinomycetes and an application thereof.
The invention provides a strain cock Salmonella (Kocuria sp.), its deposit number is CGMCC No.9648.
Fermented liquid or the tunning of above-mentioned cock Salmonella also belong to protection scope of the present invention.
The method that one goes back Fe in raw sample (III) also belongs to protection scope of the present invention, is by above-mentioned cock Salmonella or above-mentioned fermented liquid or tunning and the sample mix containing Fe (III), obtains mixed solution, then cultivated by mixed solution;
Described Fe (III) is ferric iron.
In aforesaid method, described sample is for containing FeCl
3, ferrihydrite and/or pyrrhosiderite sample.
In above-mentioned arbitrary described method, the condition of described cultivation is aerobic, pH is the condition of 6-8;
Described pH is specially 7 or 8.
In above-mentioned arbitrary described method, the condition of described cultivation also comprises normal pressure or condition of high voltage.
In above-mentioned arbitrary described method, described high pressure is 1MPa-2.5MPa, is specially 1MPa or 2.5MPa.
Above-mentioned cock Salmonella or above-mentioned fermented liquid or the application of tunning in reduction Fe (III) also belong to protection scope of the present invention;
Described Fe (III) is ferric iron.
Above-mentioned cock Salmonella or above-mentioned fermented liquid or tunning also belong to protection scope of the present invention reducing the application in heavy metal toxicity;
Or,
Above-mentioned cock Salmonella or above-mentioned fermented liquid or tunning also belong to protection scope of the present invention eliminating the application in pesticide residue.
Above-mentioned cock Salmonella or above-mentioned fermented liquid or tunning are purifying water or the application eliminated in body eutrophication also belongs to protection scope of the present invention.
The bacterium with Fe (III) reducing power reported in existing document all plays its Fe (III) reducing power under anaerobism, pH acidic conditions, bacterium provided by the invention is aerobic bacteria, under the condition of aerobic, the neutral left and right of pH, play Fe (III) reducing power.
Deep-sea actinomycetes FXJ8.057 provided by the invention can reduce Fe (III).Fe3+ reduction can promote degraded and the purification of pollution substance in water body under earth's surface, and therefore, bacterial strain provided by the invention has important application prospect in the disposal of the environmental problems such as pesticide residue, heavy metal toxicity and body eutrophication.
Accompanying drawing explanation
Fig. 1 is the position of bacterial strain FXJ8.057 in 16S rRNA Phylogenetic Tree.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The Isolation and Identification of embodiment 1, bacterial strain FXJ8.057
One, the separation of bacterial strain FXJ8.057
Gather south west Indian Ocean seawater sample, the seawater of about 100L is concentrated into 100mL, obtains concentrated seawater sample.Add glycerine in the sample to which and trehalose is kept at-20 DEG C.Behind laboratory, get 5-10mL concentrated seawater sample, 55 DEG C of thermal treatment 5min, get 1mL gradient dilution 10
-1, 10
-2, obtain diluent.Get 0.2mL diluent and coat DNB substratum (peptone 1g/100ml, extractum carnis 0.5g/100ml, NaCl 0.5g/100ml, surplus is water) in, 16 DEG C of cultivations, picking list bacterium colony, by it in GYM substratum (glucose 0.4g/100ml, oat extract 1g/100ml, yeast extract 0.4g/100ml, surplus is water) middle purifying cultivation, obtain bacterial strain FXJ8.057.
Two, the qualification of bacterial strain FXJ8.057
(1) bacterial strain FXJ8.057 in GYM substratum 28 DEG C cultivate 48 hours, bacterium colony light red, moistening, circular, the smooth of the edge, opaque; Cell spherical (diameter is about 0.8-1.5 μm).
(2) physiological and biochemical property
Bacterial strain FXJ8.057 is through being accredited as gram-positive microorganism, aerobic, middle temperature.Urase is positive, catalase positive, and nitrate reduction is positive, and gelatine liquefication is positive, and milk solidifies and peptonizes feminine gender, and M-R, V-P test feminine gender, H
2s produces feminine gender, and Starch Hydrolysis is positive.
Bacterial strain FXJ8.057 utilization of carbon source and other physiological and biochemical property as shown in table 1.
Table 1 bacterial strain FXJ8.057 utilization of carbon source and other physiological and biochemical property
| Test event | Result | Test event | Result | Test event | Result | Test event | Result |
| Fiber two pool | + | Synanthrin | - | Glycerol | + | Polysorbas20 | - |
| Fructose | + | Semi-lactosi | + | Maltose | + | Polysorbate40 | - |
| Lactose | + | Rhamnosyl | + | N.F,USP MANNITOL | + | Tween 80 | + |
| Pectinose | + | Wood sugar | + | Guanine | - | Temperature (DEG C) | 4-40 |
| Ribose | - | Raffinose | + | Xanthine | - | Nacl tolerates | 0-7％ |
| Seminose | + | Tetrahydroxybutane | - | Casein | - | pH | 6-10 |
| Sucrose | + | Trehalose | + | VITAMIN B4 | - | Cellulase | - |
"+" represents that test result is positive; "-" represents that test result is negative.
(3) 16S rRNA genetic analysis
Extract the genomic dna of bacterial strain FXJ8.057, adopt bacterial universal primers amplification 16S rRNA gene and purifying order-checking, the 16S rRNA gene order of bacterial strain FXJ8.057 is as shown in SEQ ID No.1.Sequencing results is carried out similarity system design by the blast program of NCBI.Result shows, that the most similar to this bacterial strain 16S rRNA gene order is Kocuria flava JCM15621, Kocuria turfanensis JCM15622 and Kocuria roseaJCM11614, and similarity is all less than 98.68%.
The position of bacterial strain FXJ8.057 in 16S rRNA Phylogenetic Tree as shown in Figure 1.
(4) chemical classification feature
The main polar lipid component of bacterial strain FXJ8.057 is phosphatide DPG; Breathing quinone is methyl naphthoquinone MK-7 (H
2), content is about 60.5%; Its topmost lipid acid is 15:0anteiso, and content is about 66%.
The Classification And Nomenclature of bacterial strain FXJ8.057 is cock Salmonella (Kocuria sp.), this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (has been called for short CGMCC on 09 11st, 2014, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.9648.
The Fe3+ reduction capability analysis of the bacterial strain FXJ8.057 under embodiment 2, pH7 condition
One, the synthesis of mineral
The synthesis (alpha-feooh) of pyrrhosiderite powder: be that the KOH solution of 5mol/L adds the Fe (NO that the 100ml concentration stirred is 1mol/L fast by 180mL concentration
3)
39H
2in O solution, add distilled water immediately and make liquor capacity reach 2L.60 hours are left standstill at 70 DEG C, centrifugal (3000 × g, 15min), be precipitated thing, distilled water washing precipitate 3 times, grinding after being dried by throw out 40 DEG C, to obtain final product.
The synthesis of ferrihydrite powder: be that the KOH solution of 5mol/L adds the Fe (NO that the 100ml concentration stirred is 1mol/L fast by 180mL concentration
3)
39H
2in O solution, add distilled water immediately and make liquor capacity reach 2L.Quick spin (3000 × g, 15min), be precipitated thing, distilled water washing precipitate 3 times, dries rear grinding by throw out normal temperature, to obtain final product.
Two, the preparation of substratum
FeCl is added respectively in the GYM substratum (phosphate buffered saline buffer regulates pH to be 7) with distilled water diluting one times
3the ferrihydrite of solution, step one synthesis or pyrrhosiderite powder (all containing Fe (III)), make FeCl
3, ferrihydrite and pyrrhosiderite final concentration be 1mM, make containing FeCl
3, ferrihydrite or pyrrhosiderite the GYM substratum of 1/2 dilution.
Wherein FeCl
3the prior filtration sterilization of solution, the ferrihydrite that step one is synthesized and pyrrhosiderite powder adopt tyndallization sterilizing in advance, and condition is: after 100 DEG C of sterilizing 30min, place 12h, again sterilizing, repeat sterilizing 3 times.
Three, the preparation of bacteria suspension
Bacterial strain FXJ8.057 cultivated after three days in GYM liquid nutrient medium, and collected by centrifugation thalline, sterilized water washs three times, and makes bacteria suspension with sterilized water.
Four, strain culturing
Bacteria suspension prepared by step 3 be seeded in prepared by step 2 respectively containing FeCl
3, ferrihydrite or pyrrhosiderite 1/2 dilution GYM substratum in, inoculum size is 1 × 10
5cells/mL, under aerobic conditions, after 28 DEG C of shaking tables cultivate 7 days, obtains each nutrient solution, detects Fe3+ reduction ability.
Five, Fe3+ reduction ability detects
Spectrophotometer method (luxuriant and rich with fragrance Lip river piperazine development process) is adopted to detect Fe
2+concentration.Wherein the development step of sample is specific as follows: draw each nutrient solution of 200 μ l, add the HCl aqueous solution that 200 μ l concentration are 1M respectively, piping and druming mixing, obtain mixed solution, after being placed 12h, get 100 μ l mixed solutions in luxuriant and rich with fragrance Lip river piperazine (Ferrozine) solution (Ferrozine is dissolved in the HEPES solution of 50mM, and the concentration of Ferrozine is 1g/L) of 1mL, after leaving standstill 5min, spectrophotometer reads light absorption value under wavelength 562nm.Not connect each substratum of the correspondence of bacterium as blank.
Result shows, and bacterial strain FXJ8.057 can reduce containing FeCl under the condition of pH7, aerobic
3, ferrihydrite or pyrrhosiderite the FeCl of GYM substratum of 1/2 dilution
3, ferrihydrite or pyrrhosiderite, the final Fe formed
2+content be respectively 0.2,0.06 and 0.121mM.Show that bacterial strain FXJ8.057 has Fe (III) reducing power.
The Fe3+ reduction capability analysis of the bacterial strain FXJ8.057 under embodiment 3, pH8 condition
Because mineral structure is more stable, and in example 2 under pH7 condition bacterial strain FXJ8.057 better to the reducing power of pyrrhosiderite, so the present embodiment only have detected the reduction of bacterial strain FXJ8.057 to pyrrhosiderite under the condition of pH8.
One, the synthesis of pyrrhosiderite
With the step one of embodiment 2.
Two, the preparation of substratum
In the GYM substratum (phosphate buffered saline buffer regulates pH to be 8) with distilled water diluting one times, add the pyrrhosiderite powder that step one is synthesized, the final concentration making pyrrhosiderite is 1mM, makes the GYM substratum of 1/2 dilution containing pyrrhosiderite.
Wherein the sterilizing of pyrrhosiderite powder is with the step 2 of embodiment 2.
Three, the preparation of bacteria suspension
With the step 3 of embodiment 2.
Four, strain culturing
What bacterial suspension inoculation step 3 prepared was prepared in step 2 contains in the GYM substratum of 1/2 dilution of pyrrhosiderite, and inoculum size is 1 × 10
5cells/mL, under aerobic conditions, after 28 DEG C of shaking tables cultivate 7 days, obtains nutrient solution, detects Fe3+ reduction ability.
Five, Fe3+ reduction ability detects
Method is with the step 5 of embodiment 2.
Result shows, and bacterial strain FXJ8.057 can reduce the pyrrhosiderite of GYM substratum containing 1/2 dilution of pyrrhosiderite under the condition of pH8, aerobic, the Fe of final formation
2+content be 0.122mM.Show that bacterial strain FXJ8.057 has Fe (III) reducing power.
Embodiment 4, bacterial strain FXJ8.057 Fe3+ reduction capability analysis under high pressure
One, the preparation of substratum
The pyrrhosiderite powder that the step one adding embodiment 2 in the GYM substratum (phosphate buffered saline buffer regulates pH to be 7) with distilled water diluting one times is synthesized, the final concentration making pyrrhosiderite is 1mM, makes the GYM substratum of 1/2 dilution containing pyrrhosiderite.
Wherein the sterilizing of pyrrhosiderite powder is with the step 2 of embodiment 2.
Two, the preparation of bacteria suspension
With the step 3 in embodiment 2.
Three, the cultivation of bacterial strain
What bacterial suspension inoculation prepared by step 2 was prepared in step one contains in the GYM substratum of 1/2 dilution of pyrrhosiderite, and inoculum size is 1 × 10
5cells/mL, obtains mixed solution, and it is drawn 2ml with syringe, under aerobic conditions (substratum does not have deoxygenation, so be aerobic conditions), put into autoclave and cultivate at 1MPa and 2.5MPa respectively, obtain each nutrient solution after 14 days, detect Fe3+ reduction ability.
Four, Fe3+ reduction ability detects
Nutrient solution, with the step 5 in embodiment 2, is only replaced with each nutrient solution that step 3 obtains by method respectively.
Result shows, bacterial strain FXJ8.057 all can reduce pyrrhosiderite under the high pressure of 1MPa and 2.5MPa, the final Fe formed
2+content respectively 0.098,0.15mM, show that bacterial strain FXJ8.057 under high pressure also has Fe (III) reducing power.
Claims (10)
1. a strain cock Salmonella (Kocuria sp.), its deposit number is CGMCC No.9648.
2. the fermented liquid of cock Salmonella according to claim 1 or tunning.
3. go back the method for Fe in raw sample (III) for one kind, by cock Salmonella according to claim 1 or fermented liquid according to claim 2 or tunning and the sample mix containing Fe (III), obtain mixed solution, then mixed solution is cultivated.
4. method according to claim 3, is characterized in that: described sample is for containing FeCl
3, ferrihydrite and/or pyrrhosiderite sample.
5. the method according to claim 3 or 4, is characterized in that: the condition of described cultivation is aerobic, pH is the condition of 6-8.
6., according to the arbitrary described method of claim 3-5, it is characterized in that: the condition of described cultivation also comprises normal pressure or condition of high voltage.
7., according to the arbitrary described method of claim 3-6, it is characterized in that: described high pressure is 1MPa-2.5MPa, is specially 1MPa or 2.5MPa.
8. cock Salmonella according to claim 1 or fermented liquid according to claim 2 or the application of tunning in reduction Fe (III).
9. cock Salmonella according to claim 1 or fermented liquid according to claim 2 or tunning are reducing the application in heavy metal toxicity;
Or,
Cock Salmonella according to claim 1 or fermented liquid according to claim 2 or tunning are eliminating the application in pesticide residue.
10. cock Salmonella according to claim 1 or fermented liquid according to claim 2 or tunning are purifying water or are eliminating the application in body eutrophication.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2012133823A1 (en) * | 2011-03-30 | 2012-10-04 | 独立行政法人海洋研究開発機構 | Novel useful deep-sea bacteria |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012133823A1 (en) * | 2011-03-30 | 2012-10-04 | 独立行政法人海洋研究開発機構 | Novel useful deep-sea bacteria |
Non-Patent Citations (2)
| Title |
|---|
| CHUN-YUAN WU等: "Kocuria rosea HN01, a newly alkaliphilic humus-reducing bacterium isolated from cassava dreg compost", 《J SOILS SEDIMENTS》 * |
| DEREK R. LOVLEY: "Dissimilatory Fe(III) and Mn(IV) Reduction", 《MICROBIOLOGICAL REVIEWS》 * |
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Effective date of registration: 20160606 Address after: 100101 Beichen West Road, Beijing, No. 1 hospital, No. 3, No. Applicant after: Institute of Microbiology, Chinese Academy of Sciences Applicant after: China Ocean Mineral Resources Research and Development Association Address before: 100101 Beichen West Road, Beijing, No. 1 hospital, No. 3, No. Applicant before: Institute of Microbiology, Chinese Academy of Sciences |
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