CN102168039B - Method for screening extracellular algae-killing protein marine bacteria - Google Patents
Method for screening extracellular algae-killing protein marine bacteria Download PDFInfo
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Abstract
The invention provides a method for screening extracellular algae-killing protein marine bacteria and relates to a method for screening bacteria. The extracellular algae-killing protein marine bacteria are pseudoalteromonas DHQ25 which has been collected in China Center for Type Culture Collection. The method comprises the following steps of: separating 13 strains of bacteria capable of killing harmful red tide alga (alexandrium tamarense) from a Yangtze River estuary, culturing the bacteria until the bacteria are in the stabilized phase, and centrifuging to obtain culture supernatant; incubating partial culture supernatant, and testing the algicidal activity of the incubated culture supernatant to obtain 6 strains of bacteria with algicidal activity, namely HH2, DHY3, DHY11, DHY20, DHY36 and DHQ28; performing ultra-filtration of another part of culture supernatant, and testing the algicidal activity of the supernatant which is subjected to ultra-filtration to obtain 6 strains of bacteria with algicidal activity, namely HH5, DHY3, DHY28, DHQ5, DHQ19 and DHQ25; and selecting the pseudoalteromonas DHQ25 with algicidal activity.
Description
Technical field
The present invention relates to the screening method of a kind of bacterium, especially relate to a kind of changing conditions that high temperature deactivation is handled and uf processing is observed active reservation of using, in order to obtain the having active method of killing the algae bacterium of extracellular protein.
Background technology
In the aquatic ecosystem, the relation of microalgae and bacterium more and more causes people's attention.Bacterium is mainly reflected in the influence of algae: bacterium absorbs the organic substance that algae produces on the one hand, and for algae grows provides nutritive salt and necessary growth factor, thereby regulate algae grows; On the other hand, bacterium also can suppress algae grows through direct or indirect effect, even the cracking frustule, thereby shows as algae effect extremely.
Middelboe etc. find that along with the development of wawter bloom, the amount of the cell exocrine thing of bacterium increases more than 1 times in the water body when the wawter bloom of the lake water generation of research eutrophication, and the activity of bacterium extracellular enzyme also has remarkable enhancing.This shows that bacterium possibly be to kill algae through cell exocrine thing and special extracellular enzyme.Loveioy etc. are separated to 1 strain Pseudoalteromonas in Australia and belong to bacterium; This bacterium can cause red tide algae Gymnodinium catenatum, Chattonella marina and Heterosigma akashiwo lysis in 3h; Further research shows that this bacterium is through working to the sea water medium release of active agent.Thereby Doucette etc. are separated to 1 strain bacterium Cytophaga 41-DBG2 and can produce a kind of solvability and kill the algae mixture and kill G.breve effectively.
Pseudomonas aeruginosa can produce a large amount of antibiotics materials such as diffustivity azophenlyene coloring matter, and other bacterium and algae grows are all had restraining effect.Imai etc. find that in Japanese Hiroshima bay the bacterium that 4 strain Alteromonas belong to also can kill Chattonella antiqua.From Pseudomonas stutzer, extract " the dinoflagellate growth inhibitor (Dinoflagellate Growth Inhibitor; DGI) " active higher; And toxicity stable (4 ℃ time 3 months remain unchanged), and harmless to fish is the more satisfactory algae material that kills.About killing algae mechanism, one thinks it through acting on physiological process like the blocking-up respiratory chain, and it is synthetic to suppress cell walls, suppresses the aspects such as formation of spore, to reach the result who suppresses the frustule growth or kill frustule.
([1] Lee, S.O., Kato, J. such as Lee; Takiguchi, N., Kuroda, A.; Ikeda, T., Mitsutani; A., Ohtake, H.Involvement of an extracell μ Lar protease in algicidal activity of the marine bacterium Pseudoalteromonas sp.strain A28 [J] .Applied and Environmental Microbiology; 2000,66 (10): 4334-4339) from 1 strain can be killed the nutrient solution of Pseudoalteromonas sp.A28 of diatom (Skeleonema costatum), extract and be purified to a kind of proteolytic enzyme, this enzyme has algicdal activity; Further research shows, the about 50kDa of this enzyme molecular weight, and its ph optimum and temperature are respectively 8.8 and 30 ℃.([2] Yuan Junfeng such as Yuan Junfeng; Meng Zhifang; Chen Dehui; Ge Dongling; Chapter the ancestor relate to. and neutral citric acid fungus is to the allelopathy [J] of several frequently seen algal grown. fresh water fishery; 1999,29 (4): the growth that 12-15.) secretory product of one of isolating dominant bacteria neutral citric acid fungus (Citrobacter intermedius) is examined chlorella (Chlorella pyrenoidosa), goat's horn crescent moon algae (Selenastrum capricormutum), wawter bloom anabena (Anabaena flos-aquae) and variable anabena (Anabaena vqriabilis) to scenedesmus obliquus (Scenedesmus obliquus), powder from green Euglena (Euglena viridis) wawter bloom has promoter action; Growth to silver grey plane fracture algae (Merismopedia glauca), Chlamydomonas reinhardtii (Chlamydononas reinhardii) and microcystic aeruginosa (Microcystis aerugiuosa) is inhibited.The exocytosis thing of neutral citric acid fungus can promote the individual growth of powder nuclear chlorella cells, mainly is the size that influences cell to other algae.([3] Kodani, S., Imoto such as Kodani; A., Mitsutani, A.; Murakami, M.Isolation and identification of the antialgal compound, harmane (1-methyl-beta-carboline); Produced by the algicidal bacterium, Pseudomonas sp.K44-1 [J] .Journal of Applied Phycology, 2002; 14 (2): 109-114) from Shinobazu pond, Tokyo, be separated to the bacterium that 12 strains have algicdal activity, 9 strains belong to Pseudoalteromonas sp., and wherein the methanol extract of K44-1 has tangible algicdal activity; Further therefrom extract 1-methyl-β carboline, when concentration at 30 μ g disc
-1The time can suppress the growths of several kinds of blue-green algaes.
Because these complicated relations between bacterium and the algae; Make people when the generation of research plant plankton wawter bloom and red tide, development, decline and extinction mechanism; Must consider the importance of bacterium, wherein bacterium kills the algae phenomenon and more utilizes the microbial control red tide that possible approach is provided.In a word, the facility of bacterium aboundresources and indoor cultivation make People more and more pay attention to research its with the relation of little algae, attempt to explore the new way that a control red tide breaks out.
Summary of the invention
The object of the present invention is to provide the product born of the same parents to kill algae albumen marine bacteria outward.
Another object of the present invention is to provide the product born of the same parents to kill the screening method of algae albumen marine bacteria outward.
It is that false monospore replaces bacterium DHQ25 (Pseudoalteromonas sp.DHQ25) that said product born of the same parents kill algae albumen marine bacteria outward; False monospore replaces bacterium DHQ25 (Pseudoalteromonas sp.DHQ25) and has been preserved in Chinese typical culture collection center on August 30th, 2010; Deposit number is CCTCC NO:M2010210, and the address at Chinese typical culture collection center is Chinese Wuhan Wuhan University.
The screening method that said born of the same parents kill algae albumen marine bacteria outward may further comprise the steps:
1) will separate bacterium that 13 strains that obtain can kill poisonous red tide algae Alexandrium tamarense from the entrance of Changjiang River as starting strain, cultivate stationary phase, centrifugal acquisition culture supernatant liquid;
2) get and carry out algicdal activity after the part culture supernatant liquid hatching of step 1) gained and detect, obtain the bacterium that 6 strains have algicdal activity, be designated as HH2, DHY3, DHY11, DHY20, DHY36, DHQ28 respectively;
In step 2) in, the condition of said hatching can be at 100～110 ℃ hatches 30～40min down.
Another part culture supernatant liquid of 3) getting the step 1) gained is through ultrafiltration, gets supernatant after the ultrafiltration and carries out algicdal activity and detect, and obtains the bacterium that 6 strains have algicdal activity, is designated as HH5, DHY3, DHY28, DHQ5, DHQ19, DHQ25 respectively;
In step 3), said ultrafiltration can be adopted the ultra-filtration centrifuge tube ultrafiltration, and said ultra-filtration centrifuge tube can be selected the 3kD ultra-filtration centrifuge tube for use.
4) pick out bacterium DHQ25, be false monospore and replace bacterium DHQ25 (Pseudoalteromonas sp.DHQ25) with algicdal activity.
Because DHQ25 has lost algicdal activity after pyroprocessing; And after uf processing, kept algicdal activity; The ultrafiltrated characteristic that in protein characteristic reaction, is positive; Show that DHQ25 is that a strain can be produced the outer proteic marine bacteria of algicdal activity of born of the same parents, the false monospore of called after replaces bacterium DHQ25 (Pseudoalteromonas sp.DHQ25).
The present invention is so that the poisonous aseptic dinoflagellate of a strain---Alexandrium tamarense (ATGD98-006) is an experimental subjects; To be separated to the 13 strain marine bacterias that can kill Alexandrium tamarense from MATTER IN CHANGJIANG ESTUARY is starting strain; The two screening systems of integrated use, i.e. (mwco membrane specification: 3kD) to observe the algicdal activity changing conditions of each bacterium, wherein HH5 is handled in the high temperature of culture supernatant liquid (100 ℃) inactivation treatment and ultra-filtration; DHY28, DHQ5 and DHQ19 have kept algicdal activity after two kinds of processing; DHY31, DHQ4 and DHQ17 have lost algicdal activity after two kinds of processing; HH2, DHY11, DHY20, DHY36 and DHQ28 have kept algicdal activity after pyroprocessing, and after uf processing, have lost algicdal activity; DHQ25 has lost algicdal activity after 100 ℃ of pyroprocessing, and its algicdal activity still just keeps after uf processing; The uf processing liquid of DHQ25 culture supernatant is in protein characteristic reaction (ninhydrin reaction) characteristic that is positive; Above experiment conclusion shows that DHQ25 is that a strain can be produced the outer proteic bacterial isolates of algicdal activity of born of the same parents.
Description of drawings
Fig. 1 is the algicdal activity microscope figure of the supernatant after the ultrafiltration.
Fig. 2 is the algicdal activity microscope figure of supernatant behind the high-temperature inactivation.
Fig. 3 replaces the different growth phases of bacterium DHQ25 and kills algae rate graph of a relation for false monospore.In Fig. 3, X-coordinate is time T ime (h), and left ordinate zou is light absorption value OD (640nm), and right ordinate zou is for killing algae rate Algicidal actiyvity (%); Histogram is the OD value that false monospore replaces the different growth phases of bacterium DHQ25; ■ representes algae rate extremely.
Fig. 4 is different supernatant add-ons and the graph of a relation that kills the algae rate.In Fig. 4, X-coordinate is time T ime (h), and ordinate zou is for killing algae rate Algicidal actiyvity (%); ▲ the algae rate of killing when adding the 0.2mL supernatant, the kill algae rate of ■ when adding 20 μ L supernatants, ● the algae rate of killing when adding 2 μ L supernatants.
Fig. 5 is preclinical Alexandrium tamarense and the graph of a relation that kills the algae rate.In Fig. 5, X-coordinate is time T ime (h), and ordinate zou is for killing algae rate Algicidal actiyvity (%); The kill algae rate of ■ when adding the 0.3mL supernatant, ● the algae rate of killing when adding 30 μ L supernatants.
Fig. 6 is the Alexandrium tamarense and the graph of a relation that kills the algae rate of logarithmic phase.In Fig. 6, X-coordinate is time T ime (h), and ordinate zou is for killing algae rate Algicidal actiyvity (%); The kill algae rate of ■ when adding the 0.3mL supernatant, ● the algae rate of killing when adding 30 μ L supernatants.
Fig. 7 is the Alexandrium tamarense and the graph of a relation that kills the algae rate of stationary phase.In Fig. 7, X-coordinate is time T ime (h), and ordinate zou is for killing algae rate Algicidal actiyvity (%); The kill algae rate of ■ when adding the 0.3mL supernatant, ● the algae rate of killing when adding 30 μ L supernatants.
Embodiment
1) will from the MATTER IN CHANGJIANG ESTUARY seawater sample, (comprise top layer, middle level and bottom) and separate that bacterium that 13 strains that obtain can kill poisonous red tide algae Alexandrium tamarense is as starting strain; Cultivate 14～18h to stationary phase, centrifugal acquisition culture supernatant liquid; The prescription of the substratum (2216E) that adopts is: peptone (Peptone) 5g, and yeast extract (Yeast Extract) 1g, high ferric phosphate 0.1g, agar powder 10g (solid medium), pH7.6-7.8, the Chen Haishui constant volume is to 1L.
2) get and carry out algicdal activity after the part culture supernatant liquid hatching of step 1) gained and detect, obtain the bacterium that 6 strains have algicdal activity, be designated as HH2, DHY3, DHY11, DHY20, DHY36, DHQ28 respectively; The condition of said hatching is to hatch 30～40min down at 100～110 ℃.
The algae strain Alexandrium tamarense (AT) that carries out the algicdal activity detection is available from hydrobiont institute of Ji'nan University, and the Alexandrium tamarense that obtains through the degerming of the aseptic algae technology of the applicant does not have bacterial strain (referring to Chinese patent CN1887353).The used nutrient solution of algae is the f/2 nutrient solution.Algae places indoor triangular flask to cultivate, and temperature is 20 ± 1 ℃, and illumination condition is 12h illumination, and 12h is dark.
Another part culture supernatant liquid of 3) getting the step 1) gained is through ultrafiltration, gets supernatant after the ultrafiltration and carries out algicdal activity and detect, and obtains the bacterium that 6 strains have algicdal activity, is designated as HH5, DHY3, DHY28, DHQ5, DHQ19, DHQ25 respectively; The centrifugal 1.5h of ultrafiltration under the ultra-filtration centrifuge tube 5000g is adopted in said ultrafiltration, and said ultra-filtration centrifuge tube is selected 3kD ultra-filtration centrifuge tube (referring to table 1) for use.
4) pick out bacterium DHQ25, be false monospore and replace bacterium DHQ25 (Pseudoalteromonas sp.DHQ25) with algicdal activity.
DHQ25 has kept algicdal activity (referring to Fig. 1) after uf processing; After 100 ℃ of pyroprocessing, lost algicdal activity (referring to Fig. 2); And ultrafiltrated reacts the characteristic that is positive in (ninhydrin reagent reaction) at protein characteristic; Demonstration is albumen/polypeptide/amino acids material, shows that DHQ25 is that a strain can be produced the outer proteic marine bacteria of algicdal activity of born of the same parents.
Table 1
The detection of DHQ25 nutrient solution algicdal activity is following:
Fluorescein diacetate (FDA) is made into the solution that concentration is 5mg/mL, places 4 ℃ of refrigerators to keep in Dark Place.During dyeing, get 200 μ L samples (the even frustule nutrient solution of different treatment) in the 1.5mL centrifuge tube; Add fluorescein diacetate FDA 4 μ L, making its final concentration is 100 μ g/mL, and room temperature is put into the ice chest lucifuge and treated microscopy after leaving standstill dyeing 5min; Behind the stained specimens mixing, get 20 μ L, under (495nm) and visible light under the blue-light excited light, observe counting respectively with fluorescent microscope in the frustule tally; The cell that sends bright green fluorescence is a viable cell; The cell that sends red fluorescence is a dead cell, and living cell counting 3 times is averaged.Calculate algae efficient extremely according to following formula:
Kill algae rate (%)=(Nc-Nt)/Nc * 100
Wherein, Nc representes to add the frustule number alive of 2216E control group, and Nt representes to add the frustule number alive of bacteria-free filtrate control group or different treatment mode bacteria-free filtrate experimental group.
The different cultivation periods of embodiment 2 bacterium are to the influence of algae killing effect
In the microbial culture process, whenever get final proof (0.4mL joins 40mL algae liquid) at a distance from 3h, serve as that the algicdal activity detection is carried out in contrast respectively with 0.4mL sterilization back 2216E substratum.
Begin to grow into from bacterium and stablize the later stage (36h), the every detection at a distance from the 3h sampling killed algae rate, the volume ratio of supernatant and algae culturing liquid (1: 100); Find out that from Fig. 3 (0～6h) bacterium supernatant does not almost have algae killing effect, to 9～15h, kills algae rate fast rise in latent period; 18～21h algae killing effect is the most obvious; Kill the algae rate behind the 24h and decrease, change not quite, maintain basically on the level but after this kill the algae rate.
Embodiment 3 supernatant add-ons are to the influence of algae killing effect
Get 0.2mL respectively, 20 μ L, the supernatant of 2 μ L join 30mL and are cultured in the algae liquid of stationary phase, serve as that the algae detection is killed in contrast with 0.2mL sterilization back 2216E substratum.
0.2ml, 20 μ L, 2 μ L microbial culture supernatants join 30mL algae liquid, are that blank kills the algae detection with the 0.2mL aseptic culture medium.When algae is cultivated stationary phase, from Fig. 4, draw DHQ25 and in the experimental group of 0.2ml, show algae killing effect, and in 20 μ L and two experimental group of 2 μ L, algae killing effect is not remarkable, kill the algae rate and obviously reduce compared with the algae rate of killing under the 0.2ml processing.
The vegetative period of embodiment 4 algaes is to the influence of algae killing effect
Getting 0.3mL joins the different during cultivations of 45mL respectively with 30 μ L bacterium supernatants (lag phase is cultured to 6d, and frustule concentration is 3.0 * 10
3～3.3 * 10
3Cells mL
-1The index later stage is cultured to 15d, and frustule concentration is 1.6 * 10
4～1.8 * 10
4Cells mL
-1Be cultured to 22d stationary phase, and frustule concentration is 1.8 * 10
4～2.0 * 10
4Cells mL
-1) the algae liquid handled of not asepticize and the algae liquid after the aseptically process in, serve as that the algae detection is killed in contrast with 0.3mL sterilization back 2216E substratum.
The best moment of in the governance process of algae, introducing algicide is also unknown; Algae produces the poison amount and reaches the highest when stationary phase; The physiology of algae also is in vigorous state, and one thinks that algicide at this time can embody reasonable algae killing effect, and then the algae to any one period all has the algae ability of killing preferably; In fact kill the algae effect if can select the fragile relatively growth phase of algae to drop into algicide, then often can improve algae efficient extremely greatly.So the present invention has provided the influence of the vegetative period of different algaes to algae killing effect.0.3mL join the algae culturing liquid of 45mL different growing stages respectively with the supernatant of 30 μ L, kill the algae detection.Learn that from Fig. 5～7 when algae during in latent period and stationary phase, the 0.3mL supernatant shows good algae killing effect.And when algae during in logarithmic phase, because the sharp increase of frustule quantity, the algae ability of killing of DHQ25 supernatant has hysteresis quality to a certain degree, and this utilization for reality has good directive function.
Claims (1)
1. produce born of the same parents and kill algae albumen marine bacteria outward; It is characterized in that replacing bacterium DHQ25 (Pseudoalteromonas sp.DHQ25) for false unit cell; False unit cell replaces bacterium DHQ25 (Pseudoalteromonas sp.DHQ25) and is preserved in Chinese typical culture collection center on August 30th, 2010, and deposit number is CCTCC NO:M2010210.
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| CN102167732B (en) * | 2010-12-10 | 2013-08-21 | 厦门大学 | Protein type algicidal compound and preparation method thereof |
| CN103146619B (en) * | 2013-03-21 | 2014-05-14 | 青岛蔚蓝天成生物科技有限公司 | Halomonas sulfidaeris and application thereof |
| CN106967647B (en) * | 2017-05-03 | 2020-06-30 | 大连市现代农业生产发展服务中心 | Pseudoalteromonas foli and application method thereof |
| CN112481339B (en) * | 2019-09-12 | 2022-04-19 | 台湾海洋大学 | Preparation method for improving phycoerythrin yield |
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| CN101037661A (en) * | 2007-02-12 | 2007-09-19 | 浙江大学 | Pseudoalteromonas and its usage |
| CN101200699A (en) * | 2007-11-21 | 2008-06-18 | 淮海工学院 | Ocean microorganism low-temperature amylase and producing strain pseudoalteromonas GS230 thereof |
| CN101843261A (en) * | 2010-05-05 | 2010-09-29 | 秦旭坤 | Method and application for extracting alexandrium tamarense inhibitor from enteromorpha |
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| CN101037661A (en) * | 2007-02-12 | 2007-09-19 | 浙江大学 | Pseudoalteromonas and its usage |
| CN101200699A (en) * | 2007-11-21 | 2008-06-18 | 淮海工学院 | Ocean microorganism low-temperature amylase and producing strain pseudoalteromonas GS230 thereof |
| CN101843261A (en) * | 2010-05-05 | 2010-09-29 | 秦旭坤 | Method and application for extracting alexandrium tamarense inhibitor from enteromorpha |
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