CN101264944A - Blue algae water bloom controlling method - Google Patents
Blue algae water bloom controlling method Download PDFInfo
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- CN101264944A CN101264944A CNA2008100250020A CN200810025002A CN101264944A CN 101264944 A CN101264944 A CN 101264944A CN A2008100250020 A CNA2008100250020 A CN A2008100250020A CN 200810025002 A CN200810025002 A CN 200810025002A CN 101264944 A CN101264944 A CN 101264944A
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- methionin
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- microcystis aeruginosa
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Abstract
The invention discloses a method for controlling blue algae bloom, which is characterized in that the method is implemented by adding lysine and conducting artificial lighting. The inventive method is simple and feasible, can promote the algae-inhibiting effect of lysine via lighting, and has distinct blue algae bloom controlling effect.
Description
Technical field
The present invention relates to environmental protection technical field, be specifically related to a kind of method of controlling blue-green alga bloom.
Background technology
Wawter bloom is a kind of natural, ecological phenomenon in the fresh water, is only to be caused by algae, as blue-green algae, green alga, diatom etc., the just eutrophication of water.When wawter bloom took place, one was blue or green water.This wawter bloom phenomenon that just has at nature, just on the books on Ancient Times in China history.In their very fast disappearances of occurring in nature, do not bring harm to aquatic products animal and human class.Wawter bloom can be described as lakelands " red tide " phenomenon.
After the eutrophication, wawter bloom frequently occurs in the fresh water, and area spreads year by year, and the time length prolongs year by year.All there is wawter bloom in Taihu Lake, Dian Chi, Chaohu, Hongchehu Lake, even the mobile river, as maximum tributary, the Changjiang river---wawter bloom also appears in the section of river, Hankow, downstream, Han River.The maximum harm that wawter bloom causes in the fresh water is: the drinking water source is on the hazard, and the algae toxin influences human beings'health by food chain, and the secondary metabolite MCRST of blue-green alga bloom can damage liver, has short cancer effect, directly threatens human beings'health and existence.In addition, the filtration unit of waterworks is clogged by algal bloom, and the wawter bloom that swims on the water surface influences view, and unpleasant stink is arranged.
When the algae raised growth, these algae can discharge toxin---water bloom, and fish are had toxic action.After the algae mass mortality, in corrupt, the process that is decomposed, also want dissolved oxygens a large amount of in the consume water, make the serious stench of water body.And cause the appearance of wawter bloom phenomenon, major cause still is that the waters is used chemical fertilizer, resident living sewage and trade effluent along the line in a large number and entered rivers and lakes in a large number, causes nitrogen in the rivers and lakes, phosphorus, potassium equal size to rise.
Lake eutrophication still is that China quite grows the great water environmental problems in one period at present and from now on.The method of seeking a kind of efficient control blue-green alga bloom is extremely urgent.At present, the technology broken out of control lake eutrophication algae comprises physics method, chemical method, biological process and restoration of the ecosystem engineering method etc.The physics method have bed mud dredging, dilution or water transfer, algae filter with fish for, clay method etc., chemical method adds algicide, algae-inhibiting agent etc., biological process has the input of microorganism control algae, protozoon control algae, aquatic macrophyte control algae and fish etc., and the restoration of the ecosystem engineering method has technology such as careless type ecosystem structure, ecological regulation and control.The whole bag of tricks respectively has the limitation of relative merits and use, and what have is not suitable for large-size lake, and what have has a secondary pollution, the economy that has a little less than, therefore, in today that China's lake eutrophication is on the rise, development environment close friend's algal tufa control techniques has necessity very much.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method of controlling blue-green alga bloom.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of method of controlling blue-green alga bloom adopts the mode of adding Methionin and implementing illumination.
Wherein, the add-on of described Methionin is 3-5g/m
3
Wherein, described illumination comprises natural light and artificial light filling on daytime.Described artificial light filling be on the water surface incandescent light light filling or/and underwater incandescent light light filling.Adopt artificial light filling in night (white lamp) can improve the effect of algal control; Adopt light filling under water, can improve the light utilising efficiency, reach effect of algae restraint efficiently.
Beneficial effect: the method for control blue-green alga bloom of the present invention is simple, can promote the effect of algae restraint of Methionin greatly by illumination, and the effect of control wawter bloom is obvious.
Description of drawings
Fig. 1 suppresses the toxic limit medium dose (EC of microcystic aeruginosa for Methionin
50).
Fig. 2 is the influence of Methionin to the microcystic aeruginosa growth.
Fig. 3 is the influence of Methionin to the microcystic aeruginosa content of soluble protein, wherein
*Expression and 0mg/L control group significant difference, P＜0.05;
*The expression difference extremely remarkable, P＜0.01, below identical.
Fig. 4 is the influence of Methionin to microcystic aeruginosa SOD enzymic activity.
Fig. 5 is the influence of Methionin to microcystic aeruginosa MDA content.
Fig. 6 is that Methionin is to microcystic aeruginosa Ca
2+Mg
2+The active influence of-ATPase.
Fig. 7 be under the illumination condition Methionin to the influence of microcystic aeruginosa chlorophyll a content.
Fig. 8 be under the dark condition Methionin to the influence of microcystic aeruginosa chlorophyll a content.
Fig. 9 for the low light level according to the influence of Methionin under the condition to microcystic aeruginosa chlorophyll a content.
Embodiment:
Embodiment 1: Methionin and the test of illumination combined action control wawter bloom
At diameter be in the plastic tank of 50cm respectively on the limit, Taihu Lake in June, 2007, adds 90L lake water and microcystic aeruginosa, and initial algae density is 1.3 * 10
6Cell/L, adding the Methionin scope is 0-5mg/L, simultaneously in each bucket under water mid-mounting 40w waterproof incandescent light carry out light filling.Comparing not add Methionin, can effectively suppress the growth of microcystic aeruginosa behind the 96h, is 5mg/L at lysine concentration, and microcystic aeruginosa density is half of blank, embodies the ability of its control algae, the results are shown in Table 1.
Table 1 Methionin and illumination combined action control blue-green algae break out test-results (algae liquid OD value)
Simultaneously, revision test repeatedly, find: the Methionin of (1) fresh configuration, after handling 48 hours, the level of 5mg/L can be killed Microcystis aeruginosa; (2) Methionin after placement for some time, action effect time lag, effect dosage rising-may decompose relevant with Methionin.By to Methionin toxic limit medium dose (EC
50) research, show Methionin toxic limit medium dose (EC
50) be 2.6mg/L, the results are shown in Figure 1.
Embodiment 2: Methionin and the research of illumination combined action control wawter bloom mechanism
1 material and method
1.1. algae kind and substratum: microcystic aeruginosa (Microcystis aeruginosa FACHB469) is provided by Wuhan hydrobiont institute of the Chinese Academy of Sciences.The BG11 substratum is adopted in test, dress 150ml algae liquid in the 250ml Erlenmeyer flask.The algae liquid inoculation of taking the logarithm vegetative period, inoculating at the beginning, density is 1.5 * 10
6About cell/ml.Culture condition: 25 ℃ of temperature, Light To Dark Ratio 12h: 12h, illuminance 2200Lx.Methionin is made into mother liquor after the sterilization of 0.22 μ m membrane filtration is deposited for 4 ℃; After the algae kind inserts substratum, in proportion Methionin is added in the algae liquid.
1.2. the mensuration of microcystic aeruginosa growth curve: the optical density(OD) OD that surveys the microcystic aeruginosa nutrient solution
460nmWith the concentration with blood bead tally counting microcystic aeruginosa, the line style equation that obtains optical density(OD) (x) and cell quantity (y) is y=8 * 10
-8X+0.0166, (r
2=0.997).
1.3. the mensuration of physiological and biochemical index:
1.3.1. measuring, content of soluble protein adopts Xylene Brilliant Cyanine G G-250 staining.
1.3.2. the mensuration of enzymic activity:
The preparation of crude enzyme liquid: get each treatment group algae liquid 20ml, 4 ℃, 10000rpm frozen centrifugation 10min remove supernatant liquor, add 3ml buffered soln and (survey SOD and MDA PBS damping fluid, 0.05M, 7.0,4 ℃ of precoolings of pH; Survey ATPase STN damping fluid, pH 7.8, contain 0.05mol/L Tris-HCl, 0.4mol/L sucrose, 0.01mol/L NaCl, 4 ℃ of precoolings), ultrasonication under the ice bath (3min, 5s, 5s), at 4 ℃, 7000rpm frozen centrifugation 10min, supernatant liquid is crude enzyme liquid, is used for enzyme assay.
Nitroblue tetrazolium(NBT) (NBT) method is adopted in the SOD enzyme assay; The MDA assay adopts thiobarbituricacid (TBA) method; Ca
2+Mg
2+-ATPase determination of activity adopts Nanjing to build up the ATP enzyme testing cassete that bio-engineering research provides.
1.3.3. the chlorophyll a assay adopts 95% ethanol low temperature dark place to extract back spectrophotometry.
1.3.4. phycobiliprotein assay: get each treatment group algae sample 20ml, the centrifugal 5min of 10000rpm, remove supernatant liquor after, add 3ml PBS (0.05M, pH 6.8), put into the freezing 8h of low temperature refrigerator (86 ℃), the dark place thaws then, so multigelation is 3 times, the centrifugal 10min of 7000rpm again, supernatant liquor is measured 620nm with microplate reader, 650nm, absorbance value under the 565nm is calculated as follows each component concentration of phycobiliprotein:
Phycocyanins, C-(PC, mg/L)=(OD620-0.7 * OD650)/7.38;
Allophycocyanin (APC, mg/L)=(OD650-0.19 * OD620)/5.65;
Phycoerythrin (PE, mg/L)=[OD565-2.8 (PC)-1.34 (AP)]/1.27.
1.4. experimental data adopts SPSS 13.0 software packages to carry out the analysis of independent sample T inspection statistics, is significant difference with P＜0.05, P＜0.01 is a utmost point significant difference.
2 results and discussion
2.1 Methionin is to the influence of microcystic aeruginosa growth
As shown in Figure 2, the microcystic aeruginosa of handling with different concns Methionin can continued growth in 24h.After 24h, 0.6mg/L Methionin has no significant effect Microcystis aeruginosa; 2.0mg/L Methionin has faint restraining effect to Microcystis aeruginosa; And 5.0mg/L and 10.0mg/L Methionin begin obviously to suppress the growth of Microcystis aeruginosa, and most of frustule is suppressed when 96h.As shown in Figure 3, the frustule content of soluble protein is suppressed process and frustule is similar, and microscopy can be seen most of frustule generation cracking when 96h.
When utilizing Methionin to handle Microcystis aeruginosa, the algae liquid color that each concentration is handled in the 24h does not have considerable change; And after the 48h, the algae liquid that 5.0mg/L and 10.0mg/L handle begins to bleach, flavescence, and nutrient solution becomes oyster white gradually behind the 96h, even frond caking, wall sticking phenomenon occur, the frustule death of breaking in a large number.Analogy state basic policy etc. is mentioned these the four kinds of organic existence of lactic acid, citric acid, L-glutamic acid and glycine has restraining effect to the growth of anabena 7120 (Anabaena sp.7120), after cultivating 6d, gradually colourless [the analogy state basic policy of nutrient solution, Cong Wei, Cai Zhaoling etc. the organic carbon compound is to the influence [J] of anabena 7120 growths. the hydrobiont journal, 2003,27 (3): 238-242.].This is very consistent with this test-results, therefore can infer that may there be similar inhibition mechanism in some acid organism to the blue-green algae cell.
2.2 Methionin is to the influence of microcystic aeruginosa antioxidant system
As shown in Figure 4, different concns Methionin SOD enzymic activity to the Microcystis aeruginosa cell in 48h does not influence, and when 96h, 5.0mg/L and 10.0mg/L Methionin significantly suppress its activity.As shown in Figure 5, different concns Methionin does not influence Microcystis aeruginosa MDA content when 24h, and 5.0mg/L and 10.0mg/L Methionin make Microcystis aeruginosa cell MDA content significantly descend when 48h, then extremely significantly descend during 96h.Analysis according to Fig. 2 and Fig. 3,5.0mg/L and 10.0mg/L Methionin begins to suppress the growth of Microcystis aeruginosa cell during from 24h, and the SOD enzymic activity of frustule does not have noticeable change, MDA content not to raise when 48h in 48h and descends on the contrary, illustrate that Methionin does not suppress the SOD enzymic activity of frustule, also do not bring out frustule and produce MDA.During 48h during as the decline of the MDA content of ester matter peroxidation product, 96h the decline of SOD enzymic activity should frustule itself be tending towards dead, cracking and cause.SOD is one of enzyme of eliminate activity oxyradical most critical in the cell paste; MDA is the snperoxiaized key product of ester matter, and it has represented the snperoxiaized degree of cell ester matter basically.By above analysis as can be known, thus Methionin does not further damage this approach of frustule and does not suppress Microcystis aeruginosa by inducing frustule to produce free radical.
2.3 Methionin is to microcystic aeruginosa Ca
2+Mg
2+The active influence of-ATPase
As shown in Figure 6, when 24h different concns Methionin to the Ca of Microcystis aeruginosa cell
2+Mg
2+The active not influence of-ATPase, 5.0mg/L and 10.0mg/L Methionin then extremely significantly suppress the atpase activity of frustule when 48h and 96h.Ca
2+Mg
2+There are a plurality of positions of biomass cells in-ATPase, and as on the cytoplasmic membrane and on the interior utricule, the transhipment and the photosynthesis of pair cell nutritive substance play an important role.Hehmann (2002) thinks that Methionin may circulate by atpase activity or the phosphoric acid that suppresses the Microcystis aeruginosa cell, thereby has suppressed the absorption of Microcystis aeruginosa to calcium, magnesium or other mineral element, and then causes the cracking of Microcystis aeruginosa cell.By the analysis of Fig. 6 as can be known, Methionin suppresses very remarkable to the atpase activity of Microcystis aeruginosa cell, and therefore, the ATP enzyme may be that Methionin plays an inhibiting target site to the Microcystis aeruginosa cell.
2.4 Methionin is to the influence of microcystic aeruginosa photosynthesizer
As shown in Figure 7, under illumination condition, begin 5.0mg/L and 10.0mg/L Methionin promptly has utmost point significant inhibitory effect to Microcystis aeruginosa chlorophyll a content from 24h.As shown in Table 2, when 48h, Phycocyanins, C-(PC) relative content descends in each component of phycobiliprotein, and phycoerythrin (PE) relative content increases, and allophycocyanin (APC) relative content is constant.Illustrate under the illumination condition that 5.0mg/L and 10.0mg/L Methionin can influence frustule phycobiliprotein component and constitute, its point of application is Phycocyanins, C-(PC) and phycoerythrin (PE).Phycobiliprotein is the major function group that blue-green algae photosynthesis is caught optical antenna, be positioned at and catch optical antenna---phycobilisome, it comprises PC and APC and three kinds of albumen of PE, has to catch luminous energy and energy efficient is passed to photosystem II (PS II) reactive center---the function of chlorophyll a.Therefore, the variation of Microcystis aeruginosa phycobiliprotein component formation can directly have influence on its photoresponse efficient.
Under table 2 illumination condition, each component concentration of microcystic aeruginosa phycobiliprotein during 48h
Methionin (mg/L) PC (%) APC (%) PE (%)
0 20.78±1.02 17.90±0.40 61.32±1.42
2.0 19.20±0.54 17.74±0.35 63.06±0.85
5.0 5.39±0.49
** 19.83±3.26 74.78±3.48
**
10.0 6.41±0.55
** 20.39±3.41 73.19±3.52
**
(
*Expression and 0mg/L control group significant difference, P＜0.05;
*Expression difference is extremely remarkable, P＜0.01; As follows)
Find in the test, (Erlenmeyer flask that will cultivate Microcystis aeruginosa tightly wraps up with aluminium foil under dark condition, place in the illumination box and cultivate), different concns Methionin does not all have the obvious suppression effect to Microcystis aeruginosa, just flavescence death gradually of algae liquid after a week in the 96h.It is reported that microcystic aeruginosa has the ability of utilizing organic substrates to carry out the chemoheterotrophy growth under dark condition.As shown in Figure 8, within 96h, the Methionin of different concns does not have restraining effect to Microcystis aeruginosa chlorophyll a content under the dark condition, and wherein significantly increasing appears in the chlorophyll a content of 2.0mg/L Methionin processing.As shown in Table 3, Methionin constitutes not influence to Microcystis aeruginosa phycobiliprotein component.Explanation is under dark condition, and Microcystis aeruginosa has the ability of utilizing Methionin to carry out the chemoheterotrophy growth, but this ability is very faint, is not enough to keep the lasting growth of frustule.
Under table 3 dark condition, each component concentration of microcystic aeruginosa phycobiliprotein during 96h
Methionin (mg/L) PC (%) APC (%) PE (%)
0 19.85±1.08 20.43±0.16 59.72±0.93
2.0 19.48±1.29 20.29±0.71 60.23±1.99
5.0 20.02±0.84 19.87±0.34 60.11±1.13
10.0 17.88±0.84 19.70±1.35 62.41±0.69
Also find in the test, under faint illumination condition (illuminance 600Lx～1100Lx), different concns Methionin does not have restraining effect to Microcystis aeruginosa yet, thus the author to Microcystis aeruginosa at the low light level according to the ability of utilizing Methionin under the condition, promptly its light heterotrophic growth ability is studied.The light heterotrophic growth can by in containing the substratum of organic carbon source, add suitable concn inhibitor of photosynthesis---dichlorophenyl dimethyl urea (DCMU) is realized.The activity inhibited of cell photosynthesizer II this moment (PSII), non-ring type electron transport is prevented from, and NADPH no longer produces, CO
2No longer be reduced assimilation, but photosystem I (PSI) still in action, cell still can carry out the synthetic of ATP by cyclic phosphorylation.Experiment shows that 10 μ mol/L DCMU can suppress the Microcystis aeruginosa growth fully.After the inoculation, add 10 μ mol/L DCMU when adding Methionin, and cultivate according under the condition, and handle as blank with 10 μ mol/L DCMU under the normal illumination condition at the low light level.By Fig. 9 and table 4 as can be known, each treatment group and DCMU blank are basically identicals to the situation that influences that Microcystis aeruginosa chlorophyll a content and phycobiliprotein component constitute.Therefore, play the inhibiting DCMU of being at the low light level according under the condition, promptly Microcystis aeruginosa can not be kept growth at the low light level by the PSI system according under the condition, and it does not utilize Methionin to carry out the ability of light heterotrophic growth.
By above analysis as can be known, Methionin constitutes and Ca the chlorophyll a content in the Microcystis aeruginosa photosynthesizer, phycobiliprotein component
2+Mg
2+-ATPase activity all has remarkably influenced; Illumination condition is that Methionin plays an inhibiting responsive condition to Microcystis aeruginosa, illustrates that also photosynthesizer is the action site that Methionin suppresses Microcystis aeruginosa.
Table 4 low light level shines under the condition, each component concentration of microcystic aeruginosa phycobiliprotein during 96h
Lysine concentration (mg/L) PC (%) APC (%) PE (%)
0 24.66±0.75 15.93±0.15 59.41±0.60
2.0 17.45±0.27
**?18.88±0.47
** 63.67±0.64
**
5.0 17.83±1.24
**?19.08±0.34
** 63.09±0.98
**
10.0 16.91±0.44
**?18.13±0.45
** 64.96±0.42
**
DCMU (contrast) 19.62 ± 0.11 20.89 ± 0.10
*59.49 ± 0.03
3 discuss
Blue-green algae is except utilizing NO
3 -Grow etc. inorganic nitrogen-sourced, also can utilize some organonitrogens to grow, as Neilson ﹠amp; Larsson has studied 7 kinds of blue-green algaes to the growing state of organonitrogen as only nitrogen source, found that these 7 kinds of blue-green algaes can only utilize urea, urate and a handful of amino acid (L-glutamic acid, Tianmen propylhomoserin, arginine and ornithine) to grow; Other unicellular blue green algae (as Synechococcus PCC 7002) utilizes the scope of organic nitrogen source wider, can utilize most amino acid.But some amino acid also can play restraining effect to the growth of blue-green algae, can suppress the growth of Synechocystis sp.29108 as phenylalanine, but it suppresses mechanism at present and unclear.In general, the algal control mechanism of chemical substance mainly is to suppress synthetic, the enzymic activity or the photosynthesis of cell walls.The L-Methionin that this test discovery adds mainly shows its photosynthesis of inhibition to the restraining effect of microcystic aeruginosa.
Blue-green algae is prokaryotic organism, no chloroplast(id) in the cell, but there is non-buttress to fold thylakoid, thylakoid surface bonding phycobilisome, 4 kinds of albumen such as PSI, PSII, Cytb6/f complex body and ATP synthetic enzyme are all on thylakoid.Photosynthetic photoresponse, promptly photochemical reaction, electron transport and photophosphorylation carry out on 4 species complexs.PSI and PSII are multimeric proteins, mainly comprise antenna system and reactive center part, and the former main effect is to catch luminous energy, carry out photochemical reaction originally and the latter mainly is the luminous energy that utilizes the former to catch.Under illumination condition, Methionin light reflex center chlorophyll a has significant inhibitory effect; Under dark condition, Methionin does not play restraining effect to microcystic aeruginosa, and PSI can not keep the lasting growth of frustule separately, and the damage that PSII is described is the major reason that Methionin suppresses the Microcystis aeruginosa growth.Generally speaking, there are PSII oxidation side, PSII reactive center or PSII primary electron acceptor in the position of PSII damaged.Methionin may suppress the PSII reactive center of Microcystis aeruginosa, upset the turnover of D1 albumen, reduce the photoresponse efficient of reactive center, the most of luminous energy that makes antenna system catch can only dissipate with the thermal radiation form, be that Microcystis aeruginosa utilizes the usefulness of light to reduce, its semi-saturation light intensity may descend, so the intensity of illumination of normal illumination may surpass the boundary of photoresponse at this moment and produce the light inhibition.
Add L-Methionin the decline of PC relative content, the PE relative content of Microcystis aeruginosa are raise, and dark condition constitutes not influence to the phycobiliprotein component, illustrates that illumination condition is the factor of influence that Methionin influences phycobiliprotein component formation.Because different light quality and light intensity can cause the type of blue-green algae phycobiliprotein and the variation of content, blue-green algae can catch luminous energy effectively by the component and the content that change phycobiliprotein, therefore when PSII sustains damage, during the reduction of photoresponse efficient, the antenna system-phycobiliprotein that may bring out photoresponse constitutes and adapts to this variation by regulating its component.Can infer by above analysis, add L-Methionin may be mainly by suppressing the PSII reactive center of Microcystis aeruginosa cell, and suppress photoresponse center chlorophyll a content, upset phycobiliprotein component formation etc., the photosynthesizer of Microcystis aeruginosa is damaged.
Can set up electrochemical proton gradient in the photosynthetic membrane both sides in the electron transfer process between PSI and the PSII, it can drive ATP enzymic synthesis ATP.Therefore, when PSII sustains damage, can make the synthetic driving energy that lacks of this ATP, cause atpase activity to reduce.In addition on the one hand, since in live body no matter be oxidative phosphorylation or photophosphorylation, ATP is synthetic all to be joined with the electron transport coupling, therefore synthetic being suppressed of ATP also can cause the sub-transfer chain of endocellular electricity to be restricted conversely, may further cause the cell function disorder thus, as suppress cell to the absorption of mineral elements such as calcium magnesium etc., and then the cell growth is suppressed.
Claims (4)
1, a kind of method of controlling blue-green alga bloom is characterized in that adopting the mode of adding Methionin and implementing illumination.
2, the method for control blue-green alga bloom according to claim 1, the add-on that it is characterized in that described Methionin is 3-5g/m
3
3, the method for control blue-green alga bloom according to claim 1 is characterized in that described illumination comprises natural light and artificial light filling on daytime.
4, the method for control blue-green alga bloom according to claim 1, it is characterized in that described artificial light filling be on the water surface incandescent light light filling or/and underwater incandescent light light filling.
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