CN103952359A - Brevundimonas sp. and application thereof - Google Patents
Brevundimonas sp. and application thereof Download PDFInfo
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- CN103952359A CN103952359A CN201410186617.7A CN201410186617A CN103952359A CN 103952359 A CN103952359 A CN 103952359A CN 201410186617 A CN201410186617 A CN 201410186617A CN 103952359 A CN103952359 A CN 103952359A
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Abstract
The invention provides brevundimonas sp. Brevundimonas sp. is named brevundimonas sp.J4, with collection number of CGMCC (China General Microbiological Culture Collection Center) NO.8976. The strain screened from the water body of the Taihu Lake has strong lytic effects on the dominant strain-microcystis aeruginosa in the cyanobacterial bloom, can be used for developing and producing novel algicides, and is finally applied to control of the fresh water cyanobacterial bloom.
Description
Technical field
The present invention relates to microorganism, be specifically related to strain shortwave Zymomonas mobilis and an application thereof.
Background technology
A large amount of be rich in nutritive substance industrial or agricultural and sanitary sewage discharge enters natural water, cause Eutrophication Status to be on the rise.As one of important sign of body eutrophication, outburst frequency and the area of algal bloom worldwide present cumulative year after year trend, cause very serious environmental influence and financial loss to many countries, caused national governments and the people's extensive concern.In China, many freshwater lakes (as Taihu Lake, Dian Chi, Chaohu etc.) are all subject to having a strong impact on of blue-green alga bloom.Therefore, effectively control blue-green alga bloom and seem very necessary.
At present, the method for control blue-green alga bloom mainly contains physical method, chemical process and biological method.Physical method mainly contains manually and fishes for, machinery removes algae etc.Chemical process mainly comprises adding kills algae chemical substance copper sulfate, weedicide etc.But, thereby physics and chemistry method is due to expensive, secondary pollution and the potential hazard of Freshwater ecosystems has been limited to it and effectively used.Therefore, biological method receives increasing concern because of its high efficiency, specificity and advantages of environment protection.
Molten algae bacterium is the general name that a class can be killed the bacterium of alga cells, because it can kill the blue-green algae of causing in blue-green alga bloom effectively, therefore extensively thought and can be applicable to the control of blue-green alga bloom.Therefore, from natural water, filter out and there is the molten algae bacterium that advantage algae strain in blue-green alga bloom is even killed in inhibition, for development of new algicide, there is important more practical value, finally can be applicable to control blue-green alga bloom.
Summary of the invention
Object of the present invention, the shortwave Zymomonas mobilis in order to provide a strain to have molten algae activity, for the development and production of novel algicide, is finally applied to control blue-green alga bloom exactly.
The shortwave Zymomonas mobilis the present invention relates to, Latin is by name: Brevundimonas sp., this bacterial strain is Gram-negative bacteria, shaft-like thalline, size is 0.4-0.5 * 1-2 μ m.This bacterial strain obligate is aerobic, chemoheterotrophy.On beef-protein medium flat board, bacterium colony is smooth, circular and white, median size.The most suitable growth pH is 6.0-8.0, and optimum growth temperature is 25-30 ℃, does not grow at 4 ℃.Through 16S rRNA gene sequencing and homology comparison, itself and certain shortwave aeromonas strain have 99% homology, therefore be accredited as shortwave zygosaccharomyces bacterium, called after shortwave Zymomonas mobilis J4, the accession number of the 16S rRNA gene order of this bacterium in GenBank is KC429672.
Shortwave Zymomonas mobilis J4 provided by the invention is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number CGMCC NO.8976, preservation date on March 28th, 2014.Preservation mechanism address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, postcode: 100101 phones: 86-10-64807596.
Technical scheme of the present invention is: a strain shortwave Zymomonas mobilis (Brevundimonas sp.), and called after shortwave Zymomonas mobilis J4, preserving number is CGMCC NO.8976.
Above-mentioned shortwave Zymomonas mobilis J4 is used for controlling blue-green alga bloom.
Above-mentioned application is by shortwave Zymomonas mobilis J4 being inoculated in to sterilizing beef-protein medium (the 3g/L extractum carnis of pH7.0; 10g/L peptone; 5g/L sodium-chlor), under 30 ℃, 200rpm condition, cultivate 24h and obtain its fermented liquid, be applied to molten algae.
Above-mentioned application, be by the fermented liquid of shortwave Zymomonas mobilis J4 is first obtained after the filtering with microporous membrane with 0.22 μ m aperture after the centrifugal 20min of 12000g again J4 without fermented liquid, be applied to molten algae.
Above-mentioned application, is dry again with obtaining its extract after methanol extraction through Rotary Evaporators without fermented liquid by shortwave Zymomonas mobilis J4, is applied to molten algae.
Main effect of the present invention and advantage:
1, bacterial screening method is simple, repeatable high.The shortwave Zymomonas mobilis J4 that screening obtains has very strong algicidal effect to the advantage algae strain microcystic aeruginosa in the blue-green alga blooms such as Taihu Lake, Dian Chi.
2, shortwave Zymomonas mobilis without without fermented liquid, microcystic aeruginosa also being had to very strong algicidal effect after fermented liquid, pyroprocessing.In addition, shortwave Zymomonas mobilis J4 has very strong algicidal effect to microcystic aeruginosa 9110 without the methanol extraction thing of fermented liquid under 0.05mg/mL concentration.
3, shortwave Zymomonas mobilis J4 and methanol extraction thing thereof all have very strong algicidal effect, environmentally friendly, can not cause secondary pollution, can be used for the development and production of novel algicide, are finally applied to control blue-green alga bloom.
Accompanying drawing explanation
Fig. 1 is the action effect schematic diagram of the separated different strains obtaining to microcystic aeruginosa 9110;
Fig. 2 is the algicidal effect schematic diagram of shortwave Zymomonas mobilis J4 to microcystic aeruginosa 9110;
Fig. 3 is the algicidal effect schematic diagram of the shortwave Zymomonas mobilis J4 fermented liquid after different modes is processed, in figure A, B, C, D be respectively shortwave Zymomonas mobilis J4 fermented liquid, without after fermented liquid, pyroprocessing without fermented liquid, without the mycetocyte liquid of supernatant;
Fig. 4 is that shortwave Zymomonas mobilis J4 is without the dull and stereotyped result schematic diagram of algae of the methanol extraction thing of fermented liquid;
Fig. 5 is the dull and stereotyped result schematic diagram of algae of the methanol extraction thing of beef-protein medium.
Embodiment
With example in detail the present invention is described in detail below, but protection scope of the present invention is not limited to this.
1. the screening of molten algae bacterium
The natural water samples 1mL that Mei Liangwan waters, Taihu Lake is gathered is inoculated in enrichment culture 24h in 100mL beef-protein medium (30 ℃, 200rpm condition under cultivate), 10mL nutrient solution is joined to the algae liquid (algae cell density approximately 1 * 10 of the microcystic aeruginosa 9110 (separated from Taihu Lake) that 100mL is cultured to logarithmic phase
7individual/mL) in.After one week, get yellow algae liquid and coat on beef-protein medium flat board by gradient dilution method, 30 ℃ of overnight incubation, get the flat board that bacterium colony density is moderate, according to the difference of colonial morphology, select a series of bacterial strains from flat board.
Select bacterial strain is inoculated in 100mL beef-protein medium according to 1% ratio, under 30 ℃, 200rpm condition, cultivate 24h, subsequently 10mL bacterium liquid is added respectively to (algae cell density approximately 1 * 10 in the microcystic aeruginosa 9110 algae liquid of 100mL logarithmic phase
7individual/mL).In addition the aseptic beef-protein medium of 10mL is added in 100mL algae liquid in contrast.The algae liquid of all experimental group and control group is all positioned in illumination box and cultivates after 6 days and measure microcystic aeruginosa 9110 cell densities, calculates its molten algae rate.Choose a strain bacterial strain wherein with efficient algicidal effect and carried out later stage research, this bacterial strain called after J4.
Algae liquid culture condition: microcystic aeruginosa 9110 use BG11 liquid nutrient mediums are cultivated, is positioned in illumination box, and 25 ℃, intensity of illumination 40 μ mol photons m-2s-1, light dark period ratio is 12: 12.
Molten algae rate method of calculation: molten algae rate=(control group algae cell density-experimental group algae cell density)/control group algae cell density * 100%.Wherein use blood cell plate counting process to measure the algae cell density of microcystic aeruginosa.
Fig. 1 is the action effect figure of the separated part bacterial strain obtaining to microcystic aeruginosa 9110.After adding the bacterium liquid of different strains, after 6 days, in each experimental group, the algae cell density of microcystic aeruginosa 9110 is different, calculates different molten algae rates, has reflected the algicidal effect of these bacterial strains to microcystic aeruginosa 9110 significant differences.Wherein molten algae bacterium J4 has the strongest algicidal effect to microcystic aeruginosa 9110, and bacterial strain H10 can promote the growth of microcystic aeruginosa 9110.
Fig. 2 is the algicidal effect of shortwave Zymomonas mobilis J4 to microcystic aeruginosa 9110.After adding shortwave Zymomonas mobilis J4, the cell density of microcystic aeruginosa 9110 constantly declines in time, and microcystic aeruginosa 9110 cell densities in contrast constantly rise in time, and the molten algae rate after 6 days is 93.0%.
2. the evaluation of molten algae bacterium J4
According to methods such as morphologic observation, dyeing, 16S rRNA gene sequencings, molten algae bacterium J4 is identified.This bacterial strain is Gram-negative bacteria, shaft-like thalline, and size is 0.4-0.5 * 1-2 μ m.This bacterial strain obligate is aerobic, chemoheterotrophy.On beef-protein medium flat board, bacterium colony is smooth, circular and white, median size.The most suitable growth pH is 6.0-8.0, and optimum growth temperature is 25-30 ℃, does not grow for 4 ℃.Through 16S rRNA gene sequencing and homology comparison, learn (the National Center of Biotechnology Information of the Qi Yu U.S. state-run biotechnology information center, NCBI) in GenBank word bank, certain shortwave aeromonas strain has 99% homology, therefore be accredited as shortwave zygosaccharomyces bacterium, called after shortwave Zymomonas mobilis J4.This bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is CGMCC NO.8976, and preservation date is on March 28th, 2014.The 16S rRNA gene order of this bacterial strain is KC429672 in the accession number of GenBank.
3. the molten algae ability of molten algae bacterium J4 to different algal species
Shortwave Zymomonas mobilis J4 is inoculated in the beef-protein medium after sterilizing according to 1% inoculum size, and under 30 ℃, 200rpm condition, shaking table is cultivated 24h and is obtained its fermented liquid.9 portions of cultured 10mL shortwave Zymomonas mobilis J4 fermented liquids are joined respectively in the 100mL algae liquid of different algal species (table 1) (all algae have all been cultured to logarithmic phase), the aseptic beef-protein medium of equivalent is added in 9 kinds of algae liquid (100mL) in contrast simultaneously.All experimental group and control group are all placed in illumination box and cultivate after 6 days, measure respectively the chlorophyll concentration (for microcystic aeruginosa, measuring its algae cell density) of all experimental group and control group algae liquid, calculate its molten algae rate.Each sample all has three Duplicate Samples, with mean+SD form, represents.
In the algae strain relating in this experiment, have 6 strain separation from Mei Liangwan waters, Taihu Lake, all the other 4 strains are purchased from the aquatic institute in Wuhan algae kind storehouse.Experimental result shows, shortwave Zymomonas mobilis J4 has promotion growth to two strain algae strains wherein, and the strain of overwhelming majority test algae is had to algicidal effect, especially the advantage algae strain microcystic aeruginosa in TAIHU LAKE Central China is had to very strong algicidal effect (table 1).This result shows that molten algae bacterium J4 can kill the advantage algae strain microcystic aeruginosa in blue-green alga bloom specifically, is the molten algae bacterium that a strain has application prospect.
The algicidal effect of table 1 shortwave Zymomonas mobilis J4 to different algae strains
Note: the algae strain separation marking with asterisk (*) in table is from Mei Liangwan waters, Taihu Lake, and all the other are from the aquatic institute in Wuhan algae kind storehouse.
4. the research of shortwave Zymomonas mobilis J4 algicidal mode
Shortwave Zymomonas mobilis J4 is cultivated and obtains its fermented liquid according to step 3.Shortwave Zymomonas mobilis J4 fermented liquid obtains it without fermented liquid after the filtering with microporous membrane with 0.22 μ m aperture again after the centrifugal 20min of 12000g.This is placed in 121 ℃ of high temperature high pressure process 21min of steam sterilizer without fermented liquid, obtain after pyroprocessing without fermented liquid.Shortwave Zymomonas mobilis J4 fermented liquid is abandoned supernatant after the centrifugal 20min of 5000g, then rinses sedimentation cell also centrifugal 3 times with aseptic high purity water, and the cell of the last resuspended precipitation of high purity water with equivalent obtains the mycetocyte liquid without supernatant.By 10mL fermented liquid, 10mL without in the microcystic aeruginosa 9110 algae liquid that join respectively 100mL logarithmic phase without fermented liquid, 10mL without the mycetocyte liquid of supernatant of fermented liquid, 10mL pyroprocessing (algae cell density is at 1 * 107 mL-1).In addition the aseptic beef-protein medium of equivalent is added in 100mL microcystic aeruginosa 9110 algae liquid in contrast.All experimental group and control group microcystic aeruginosa 9110 algae liquid are all placed in illumination box and cultivate, and measure all algae liquid algae cell densities after 6 days, calculate its molten algae rate.
Fig. 3 is the algicidal effect schematic diagram of the shortwave Zymomonas mobilis J4 fermented liquid after different modes is processed, in figure A, B, C, D be respectively shortwave Zymomonas mobilis J4 fermented liquid, without after fermented liquid, pyroprocessing without fermented liquid, without the molten algae rate (6 days) of the mycetocyte liquid of supernatant.The molten algae rate (6 days) of shortwave Zymomonas mobilis J4 fermented liquid is 93.0%, in addition the molten algae rate (6 days) without fermented liquid is 84.3%, and be only 28.8% without the molten algae rate (6 days) of the mycetocyte liquid of supernatant, this shows that shortwave Zymomonas mobilis J4 carrys out molten algae by the outer molten algae active substance of secretion born of the same parents.Shortwave Zymomonas mobilis J4 after pyroprocessing is 84.2% without the molten algae rate (6 days) of fermented liquid in addition, and this molten algae active substance that shows shortwave Zymomonas mobilis J4 secretion is non-protein matter.
5. the preparation of the molten algae active substance of shortwave Zymomonas mobilis J4 crude extract
Shortwave Zymomonas mobilis J4 is cultivated and obtains it without fermented liquid according to step 4.Use Rotary Evaporators at 50 ℃ dry this without fermented liquid after, then add methanol extraction to spend the night according to the ratio of 1: 10 (methyl alcohol: dry without fermented liquid), use subsequently separating funnel to obtain extraction supernatant liquor.This extraction supernatant liquor is removed the methyl alcohol of extraction in supernatant liquor through Rotary Evaporators, then adds and in high purity water, dissolve the mixture that is configured to 0.05mg/mL.
In order to check whether contain molten algae active substance in this mixture, the methanol extraction thing of getting the 200 μ L shortwave Zymomonas mobilis J4 fermented liquids of handling well splashes into the white circular scraps of paper on microcystic aeruginosa agar plate, in addition by beef-protein medium according to above step same treatment after in contrast.Agar plate is put into illumination box and is cultivated, by observe the white circular scraps of paper around molten algae circle have or not judge whether extract contains molten algae active substance.
The making method of microcystic aeruginosa agar plate: in flat board, pour appropriate BG11 solid medium (agar content 1.5%, about 30mL/ piece is dull and stereotyped) into, standby after it solidifies.To after the centrifugal 20min of cultured microcystic aeruginosa algae liquid 5000g, abandon supernatant, collect frustule and join (1% agar in the BG11 soft agar solid medium after sterilizing, being placed in 53 ℃ of water-baths prevents from solidifying), after shaking up, pour prefabricated BG11 solid plate (about 20mL/ piece dull and stereotyped) into, treat that it solidifies to be placed in illumination box, to cultivate and standby.
Fig. 4 is that shortwave Zymomonas mobilis J4 is without the dull and stereotyped result schematic diagram of algae of the methanol extraction thing of fermented liquid; Fig. 5 is the dull and stereotyped result schematic diagram of algae of the methanol extraction thing of beef-protein medium.In the methanol extraction thing of the molten algae circle explanation shortwave Zymomonas mobilis J4 occurring on algae flat board in Fig. 4 without fermented liquid, contain molten algae active substance, and without molten algae circle, illustrate that molten algae active substance secreted by shortwave Zymomonas mobilis J4 really on algae flat board in Fig. 5.Therefore the molten algae active substance that, the present invention can secrete with methanol extraction shortwave Zymomonas mobilis J4.
Claims (5)
1. a strain shortwave Zymomonas mobilis (Brevundimonas sp.), called after shortwave Zymomonas mobilis J4, protecting minus sign is CGMCC NO.8976.
2. the application of shortwave Zymomonas mobilis J4 in controlling blue-green alga bloom described in claim 1.
3. application according to claim 2, is characterized in that, by shortwave Zymomonas mobilis J4 being inoculated in the aseptic beef-protein medium of pH7.0, cultivates 24h and obtain its fermented liquid under 30 ℃, 200rpm condition, is applied to molten algae.
4. application according to claim 3, is characterized in that, by the fermented liquid of shortwave Zymomonas mobilis J4 is obtained to it without fermented liquid after the filtering with microporous membrane with 0.22 μ m aperture again after the centrifugal 20min of 12000g, is applied to molten algae.
5. application according to claim 4, is characterized in that, by after shortwave Zymomonas mobilis J4 dry through Rotary Evaporators without fermented liquid, then with the thick extract obtaining after methanol extraction, is applied to molten algae.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105385642A (en) * | 2015-12-29 | 2016-03-09 | 深圳市铁汉生态环境股份有限公司 | Brevundimonas and application thereof |
| CN105567589A (en) * | 2015-12-29 | 2016-05-11 | 深圳市铁汉生态环境股份有限公司 | Brevundimonas diminuta and application thereof |
| CN110438033A (en) * | 2019-07-02 | 2019-11-12 | 浙江工业大学 | A kind of grease degrading strain, application and oils degradation method |
| CN112175887A (en) * | 2020-11-04 | 2021-01-05 | 塔里木大学 | Brevundimonas bacterium endophytic bacterium brevundimonas bacterium and application thereof |
Citations (1)
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| CN103667135A (en) * | 2013-12-09 | 2014-03-26 | 上海交通大学 | Stenotrophomonas and applications thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103667135A (en) * | 2013-12-09 | 2014-03-26 | 上海交通大学 | Stenotrophomonas and applications thereof |
Non-Patent Citations (4)
| Title |
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| LIN S.ET AL: "Brevundimonas sp.J4 16S ribosomal RNA gene，partial sequence", 《GENBANK 登录号为 KC429672.1》 * |
| SHENGQIN LIN: "Characterization of an algicidal bacterium Brevundimonas J4 and chemical defense of Synechococcus sp. BN60 against bacterium J4", 《HARMFUL ALGAE》 * |
| 吴鑫: "太湖梅梁湾冬季浮游细菌的多样性", 《生态学杂志》 * |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105385642A (en) * | 2015-12-29 | 2016-03-09 | 深圳市铁汉生态环境股份有限公司 | Brevundimonas and application thereof |
| CN105567589A (en) * | 2015-12-29 | 2016-05-11 | 深圳市铁汉生态环境股份有限公司 | Brevundimonas diminuta and application thereof |
| CN105385642B (en) * | 2015-12-29 | 2019-02-05 | 深圳市铁汉生态环境股份有限公司 | Shortwave monad and its application |
| CN105567589B (en) * | 2015-12-29 | 2019-06-25 | 深圳市铁汉生态环境股份有限公司 | Defect shortwave monad and its application |
| CN110438033A (en) * | 2019-07-02 | 2019-11-12 | 浙江工业大学 | A kind of grease degrading strain, application and oils degradation method |
| CN110438033B (en) * | 2019-07-02 | 2021-04-27 | 浙江工业大学 | Grease degrading bacterium, application and grease degrading method |
| CN112175887A (en) * | 2020-11-04 | 2021-01-05 | 塔里木大学 | Brevundimonas bacterium endophytic bacterium brevundimonas bacterium and application thereof |
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Application publication date: 20140730 |