CN102943046B - Scenedesmus obliquus producing self-flocculation substance, and application of same in microalgae harvesting - Google Patents
Scenedesmus obliquus producing self-flocculation substance, and application of same in microalgae harvesting Download PDFInfo
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- CN102943046B CN102943046B CN 201210420662 CN201210420662A CN102943046B CN 102943046 B CN102943046 B CN 102943046B CN 201210420662 CN201210420662 CN 201210420662 CN 201210420662 A CN201210420662 A CN 201210420662A CN 102943046 B CN102943046 B CN 102943046B
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- 238000005189 flocculation Methods 0.000 title abstract description 84
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- 238000003306 harvesting Methods 0.000 title abstract 4
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Abstract
The invention relates to a strain of Scenedesmus obliquus producing a self-flocculation substance, and an application of the Scenedesmus obliquus in microalgae harvesting, and belongs to the technical field of microbial applications. The strain has a classification name of Scenedesmus obliquus and a preservation number of CGMCC No.6567, and is preserved in China General Microbiological Culture Collection Center (CGMCC) on September 14, 2012, wherein the address is Institute of Microbiology, Chinese Academy of Sciences, 3# Court No.1, Beichen West Road, Chaoyang District, Beijing. With a flocculation substance produced by the Scenedesmus obliquus AS-6-1, non-flocculation strains such as freshwater scenedesmus FSP-3, freshwater chlorella CNW-11 and seawater chlorella DUT01 can be subjected to rapid flocculation harvesting so as to provide broad application prospects in microalgae harvesting. The flocculation substance is separated and purified from the self-flocculation Scenedesmus obliquus AS-6-1 cell wall, and is a water-soluble polysaccharide, wherein the non-flocculation scenedesmus FSP-3 is treated with 0.6 mg/L of the flocculation substance, and the flocculation efficiency can reach 80%; and the most suitable pH value is 7.0, the most suitable temperature is 30 DEG C, the flocculating activity does not require metal ions, and characteristics of time saving, efficiency, good pH value stability and good thermal stability are provided.
Description
Technical field
The present invention relates to a strain and produce scenedesmus obliquus and the application in micro-algae is gathered thereof of autoflocculation material, belong to technical field of microbe application.
Background technology
In recent years, along with the rapid growth of global economy, the consumption of the fossil energies such as oil and coal is significantly risen, and fossil energy shortage crisis is extremely urgent, and the concern of the renewable energy sources such as biomass energy is gradually become to focus.In numerous non-grain biomass, that algae has is widely distributed, adaptive capacity to environment is strong, photosynthetic efficiency high (utilization ratio of sun power can reach 3-8%), growth cycle short (general Dai Shiwei 1-10 days), fat content high (fat content can reach the 40-86% of dry weight), high (but the annual per hectare produce oil 58 of output, 700L, be the 1-2 of other produce oil crops doubly) etc. characteristics, utilize algae to produce biofuel and there is vast potential for future development.In addition, micro-algae can also produce the high value added products such as pigment, lipid acid and medicine, therefore has important using value.
Due to microalgae cell individuality little (being about 5 to 50 μ m), culture density low water content large (being 15 to 108g/L), cell surface is with characteristics such as negative charges, and gathering of micro-algae is more difficult, and the collecting method of existing micro-algae mainly contains centrifuging, filtration method, By Bubble-floating Method and chemical flocculation.But centrifuging consumes energy, large cost is high; Filtration method depends on the frustule size, and inapplicable extensive micro-algae is collected; And the high complicated operation of By Bubble-floating Method cost; Though chemical flocculation can reach micro-algae collecting effect preferably by contrast, but the interpolation of a large amount of metal ion and difficult degradation high polymers has increased the work difficulty of downstream processing undoubtedly, and environment is had to certain toxicity.Therefore, develop micro-algae cheaply efficient collecting method be the important guarantee that carries out micro-algae biorefinery and the micro-algae energy of large-scale industrial production.
Summary of the invention
Technical problem to be solved by this invention is exactly for prior art recited above, provide a kind of flocculating effect obviously, the collecting method of environmental friendliness, Biosafety-by the frustule autoflocculation, carry out gathering of micro-algae.
The scenedesmus obliquus growing state and the cell that the object of the present invention is to provide a strain to have natural autoflocculation ability form, the evaluation of autoflocculation material is provided, the method of the above-mentioned autoflocculation material of a kind of separation and purification is provided, flocculating property and the application of autoflocculation material are provided.
The technical solution used in the present invention is: the registering on the books of scenedesmus obliquus (Scenedesmus obliquus) that the autoflocculation material is produced in a strain is numbered CGMCC No.6567, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, the depositary institution address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preservation date is on September 14th, 2012.
Described scenedesmus obliquus comprises Chlorophyta, Chlorococcale, really assembles suborder, grid algae section and Scenedesmus.
In described scenedesmus obliquus, separation and purification obtains agglutinating matter and is applied in during micro-algae gathers.Described agglutinating matter is a kind of macromolecule polysaccharide, and its molecular weight is 1.279 * 105Da; 0.6mg/L agglutinating matter is processed non-flocculation grid algae FSP-3 algae strain 20min, flocculation efficiency can reach 80%; During pH4-9, flocculation activity is good, optimum pH value 7.0; Temperature flocculation activity in the time of 30 ℃ is good, and after 121 ℃ of pyroprocessing 20min, flocculation activity still remains on more than 50%, has good thermostability.
Above-mentioned scenedesmus obliquus is the algae strain that the strain that screens from South Taiwan's fresh water has natural autoflocculation ability.Through 23S rDNA sequence alignment, itself and Scenedesmus obliquus have higher similarity (96%), in conjunction with physiology and appearance, identify, determine that this algae strain is scenedesmus obliquus.This algae strain is in the DM liquid nutrient medium of optimizing, and 28 ℃ of cultivations 4-5 days are that visible frustule flocks together, and the granular size naked eyes are visible; By the static standing over night of the nutrient solutions of 13 days, frustule flocks together and is deposited to the Erlenmeyer flask bottom, and supernatant liquor is limpid visible.
The further isolation identification of the present invention cause scenedesmus obliquus to produce the material of autoflocculation phenomenon.The agglutinating matter that separation and purification obtains is a kind of macromolecule polysaccharide, has and saves time simultaneously, efficiently and preferably the characteristic such as pH stability and thermostability.0.6mg/L agglutinating matter is processed non-flocculation grid algae FSP-320min, flocculation efficiency can reach 80%; In the pH5-9 scope, flocculation activity is good, all more than 40%, and optimum pH value 7.0; In 20-60 ℃ of temperature range, flocculation activity is good, and all more than 66%, after 121 ℃ of pyroprocessing 20min, flocculation activity still remains on more than 50%; In addition, flocculation activity does not rely on metal ion, although the Al of high price
3+, Fe
3+, Ca
2+and Mg
2+the agglutinating matter flocculation activity is had to certain promoter action, but facilitation effect is not obvious.At present, the agglutinating matter of the micro-algae of this natural autoflocculation had not also had report.
In the present invention, autoflocculation agglutinating matter that scenedesmus obliquus is produced can comprise fresh water grid algae FSP-3 to multiple non-flocculation microalgae, the micro-plan ball of limnetic chlorella CNW-11 and seawater algae DUT-01 has good throwing out, wherein to flocculating effect the best of fresh water grid algae FSP-3.
The invention has the beneficial effects as follows: the non-flocculation algae strain but the agglutinating matter rapid flocculation of this scenedesmus obliquus production is gathered: fresh water grid algae FSP-3, limnetic chlorella CNW-11 and marine chlorella DUT01 have broad application prospects aspect gathering in micro-algae.The separation and purification from autoflocculation scenedesmus obliquus cell walls of above-mentioned agglutinating matter obtains, and is a kind of water-soluble polysaccharide, with this agglutinating matter of 0.6mg/L, processes non-flocculation grid algae FSP-3, and flocculation efficiency can reach 80%; Optimum pH value 7.0,30 ℃ of optimal temperatures, flocculation activity does not need metal ion, have save time, efficient characteristics, and there is good pH stability and thermostability.
The accompanying drawing explanation
The growth that Fig. 1 is the autoflocculation scenedesmus obliquus and flocculation situation.
Fig. 2 is autoflocculation scenedesmus obliquus growth conditions.
The distribution situation that Fig. 3 is flocculation activity in autoflocculation scenedesmus obliquus nutrient solution.
Fig. 4 is autoflocculation scenedesmus obliquus agglutinating matter original position color reaction result.
In figure: A. aniline-O-phthalic amine reaction; B: ninhydrin reaction; C: phosphomolybdic acid reaction.1. reference material: A1: glucose, B1: Histidine, C1: Yelkin TTS; 2. contrast; 3, the 4. micro-algae agglutinating matter of autoflocculation.
The infared spectrum that Fig. 5 is autoflocculation scenedesmus obliquus agglutinating matter.
Fig. 6 is autoflocculation scenedesmus obliquus agglutinating matter high efficiency result.
Fig. 7 is autoflocculation scenedesmus obliquus agglutinating matter treatment time result.
Fig. 8 is autoflocculation scenedesmus obliquus agglutinating matter pH stability result.
Fig. 9 is autoflocculation scenedesmus obliquus agglutinating matter thermostability result.
Embodiment
Experiment material and reagent
1. bacterial strain
Scenedesmus obliquus separates acquisition from South Taiwan's fresh water.
2. biochemical reagents
D-MANNOSE, D-semi-lactosi, D-wood sugar, glucuronic acid AR, Sigma company; L-fucose, L-rhamnosyl, AR, Treechem company; Other reagent is domestic analytical pure.
3. substratum
The DM substratum of optimizing: ddH
2o, 1L; Ca (NO
3)
24H
2o, 1.00g; KH
2pO
4, 0.26g; MgSO
47H
2o, 0.55g; KCl, 0.25g; FeSO
47H
2o, 0.02g; EDTA2Na, 0.2g; H
3bO
3, 0.0029g; ZnCl
2, 0.00011g; MnCl
24H
2o, 0.00181g; (NH
4)
6mo
7o
244H
2o, 0.000018g; CuSO
45H
2o, 0.00008g, pH6.0-6.2.
Illustrate: do not make the experimental methods of molecular biology illustrated in following examples, with reference to the listed concrete grammar of " molecular cloning experiment guide " (third edition) J. Pehanorm Brooker one book, carry out, the biochemical test method, carry out or carry out according to test kit and product description with reference to listed concrete grammars of a book such as " biochemical theory and method " (first version) Li Jianwu.
Embodiment 1: the cultivation of autoflocculation scenedesmus obliquus of the present invention.
Autoflocculation scenedesmus obliquus of the present invention derives from the poisons in freshwater of South Taiwan, and the optimum culture condition is as follows: the pH 6.0-6.2 of initial medium; Initial concentration is about O.D.685nm 0.05-0.1; Culture temperature is 28 ℃; Light intensity 60 μ mol m
-2s
-1; Periodicity of illumination: 16h illumination cultivation, 8h dark culturing.While in the DM liquid nutrient medium of optimizing, being cultured to 4-5 days, this frustule is state of aggregation more, and aggregate size naked eyes are visible, and by the static standing over night of the nutrient solutions of 13 days, frustule flocks together and is deposited to the Erlenmeyer flask bottom, and supernatant liquor is limpid visible.Observe under Electronic Speculum and find that frustule has membranaceous material that cell is wrapped up to adhere to each other (Fig. 1) outward.Embodiment 2: extract autoflocculation scenedesmus obliquus genome, with and the acquisition of 23S rDNA.
1. extract autoflocculation scenedesmus obliquus genomic dna
The autoflocculation scenedesmus obliquus is inoculated in the DM liquid nutrient medium of 500mL, cultivates 11 days under 28 ℃, now be in logarithmic phase.The centrifugal 5min of 8000r/min, collect frond, and washings cleans frond, repeats once to clean after centrifugal.Adopt test kit Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA) to extract and obtain autoflocculation scenedesmus obliquus genomic dna.
2.23SrDNA the acquisition of sequence
23S rDNA PCR primer is as follows:
P1：5’-GGA?CAG?AAA?AAC?CCT?ATG-3’
P2：5’-CCT?TTT?CC?CTA?GAG?TAAC-3’
The PCR reaction conditions is as shown in table 1:
Table 1 PCR reaction conditions
The PCR reaction system is as shown in table 2:
Table 2 PCR reaction system
The PCR product send the order-checking of precious biotech firm, and its 23S rDNA sequence is shown in sequence table.
Embodiment 3: the growing state of autoflocculation scenedesmus obliquus and the mensuration of biomass content
The autoflocculation scenedesmus obliquus is inoculated in the DM liquid nutrient medium of optimization, while being cultured to 5-13 days, and the algae Fast Growth, the frustule number increases sharply, and nitrogen content obviously reduces, and in whole culturing process, pH changes little, maximum is no more than 8, and has no inorganic sediment (Fig. 2).Cultivate after 16 days protein content in the autoflocculation algal biomass and can reach 28.4 ± 1.3% of dry weight, carbohydrate accounts for 25.3 ± 1.0%, and lipid accounts for 22.9 ± 1.1%, and chlorophyll, up to 13.0 ± 0.6%, shows that algae strain of the present invention is with a wide range of applications.
Embodiment 4: the separation and purification of autoflocculation agglutinating matter that scenedesmus obliquus produces
1. the mensuration of flocculation efficiency
Agglutinating matter crude extract or purified are dissolved in to distilled water, make 1mg/mL solution, be added in the non-flocculation microalgae of 10mL, concussion mixes 30s, surveys immediately each algae liquid and now under 680nm, surveys absorbancy OD680 (t
0); After standing 20min, the algae liquid of getting same liquid level is surveyed it and survey absorbancy OD680 (t under 680nm
1); Calculated flocculation activity according to following calculation formula [1].
OD680 (t in formula
0): algae and the absorbancy of the 680nm of mensuration immediately after flocculation crude extract solution mixes; OD680 (t
1): algae is with after throw out solution mixes, the absorbancy of standing 20min.
2. the separation of autoflocculation agglutinating matter that scenedesmus obliquus produces of the present invention
Measure 500mL autoflocculation scenedesmus obliquus nutrient solution and carry out centrifugally, separate to obtain frond and supernatant.Supernatant liquor 40-50 ℃ rotary evaporation is concentrated, the 37 ℃ of E of the Pronase with final concentration 5mg/mL enzymolysis 1h of algae liquid after concentrated, supernatant liquor adds the long-pending 4 ℃ of alcohol precipitations of 95% ethanol of triploid to spend the night, and next day is centrifugal, throw out carries out vacuum lyophilization, and gained is the outer agglutinating matter crude extract of white born of the same parents; Frond, after disrupt red cell, adopts method as described as supernatant to be processed, and can obtain the crude extract of cell wall agglutinating matter.The material that separation is obtained carries out the flocculation activity test, and result mainly is present in cell walls as flocculation activity as indicated in Fig. 3.
By cell walls agglutinating matter runic thing, after ultrafiltration and concentration, ethanol precipitation, washing with acetone, lyophilize, obtain white powder, bulk, odorless tasteless, soluble in water, slightly be dissolved in low-concentration ethanol, be insoluble to the agglutinating matter runic thing of high concentration ethanol, acetone and other organic solvent.
3. the purifying of autoflocculation agglutinating matter that scenedesmus obliquus produces
(1) dialysis
The flocculation crude extract is redissolved in equal volume algae liquid in long-pending distilled water, after the dialysis tubing of packing into (seeing through molecular weight 8000) tap water flowing water dialysis 48h, distill water dialysis 24h.
(2) ion-exchange chromatography
By the agglutinating matter of dialysing, the DE52 cellulose ion exchange column that upper Tris damping fluid pre-equilibration is good.First use the distilled water wash-out, then use the NaCl solution gradient wash-out of 0-2.0mol/L, flow velocity 1.0mL/min, every pipe 5mL fraction collection, take foregoing flocculation activity measuring method, will have to non-flocculation microalgae the component merging phase of good throwing out.
(3) gel filtration chromatography of flocculation crude extract
The component with flocculation activity that ion-exchange is collected is further purified through Sephacryl S-400 column chromatography, 0.1mol/L the NaCl eluant solution, flow velocity 0.2mL/min, amount with the 5mL/ pipe is collected automatically, and detection method is the same, collects, merges the elutriant of flocculation activity component, vacuum concentration, flowing water dialysis 48h, distill water dialysis 48h, obtain white agglutinating matter sterling after lyophilize.
Embodiment 5: autoflocculation agglutinating matter that scenedesmus obliquus the produces of the present invention physico-chemical property of being correlated with
By the agglutinating matter that in embodiment 4, purifying obtains, utilize several different methods to carry out analysis of physical and chemical property.
1. the qualitative analysis of autoflocculation agglutinating matter that scenedesmus obliquus produces of the present invention
(1) the original position color reaction of autoflocculation agglutinating matter that scenedesmus obliquus produces of the present invention
The agglutinating matter that separation and purification is obtained is identified its character by the In Situ Thin Layer color reaction.Test result, as Fig. 4, finds that agglutinating matter only becomes coloring matter with phenol-O-phthalic amine reagent react, and all negative with ninhydrin reaction, phosphomolybdic acid reaction, in this agglutinating matter of the results show, contains polysaccharide.
(2) ultraviolet spectral analysis of autoflocculation agglutinating matter that scenedesmus obliquus produces of the present invention
It is 1mg/mL solution that the autoflocculation agglutinating matter that scenedesmus obliquus produces of the present invention of separation and purification is made to concentration, do blank with distilled water, carry out continuous sweep with ultraviolet spectrophotometer UV5500 in wavelength 220-400nm scope, take absorbancy as ordinate zou, wavelength is the X-coordinate mapping.Result shows on a declining curve to the 400nm light absorption value from 220nm, the charateristic avsorption band of nucleic acid (260nm) and protein (280nm) do not occur, and this illustrates in this agglutinating matter and does not contain nucleic acid and protein component.
(3) autoflocculation scenedesmus obliquus of the present invention produces agglutinating matter infared spectrum structure elucidation
The autoflocculation scenedesmus obliquus of the present invention of getting separation and purification produces agglutinating matter 1mg, add the 20mg Potassium Bromide in mortar, grind and be the fine flour shape under infrared(ray)lamp, be pressed into thin slice in pelleting press, use NICOLET560 (U.S.) type infrared spectrometer at 4000-500cm
-1the infrared spectra that the wavelength region interscan obtains.Autoflocculation scenedesmus obliquus of the present invention produces the general feature absorption peak that agglutinating matter Ci district all has polysaccharose substance as shown in Figure 5.At 3200cm
-1-3600cm
-1near a kind of strong absorption peak appears, in glycan molecule-stretching vibration peak of H, show in polysaccharide to exist in molecule and intermolecular hydrogen bond; At 2800cm
-1-3000cm
-1(2920cm
-1near) absorption peak be the stretching vibration peak of C-H, this regional absorption peak is the charateristic avsorption band of carbohydrate; 1730cm
-1-1600cm
-1between (1630cm
-1place) in strong absorption peak be-the C=O stretching vibration of CHO causes; 1455cm
-1near absorption peak may be the characteristic peak of COOH stretching vibration in uronic acid; 1775cm
-1-1735cm
-1between without absorption peak, illustrate without acetonyl ester; 1000cm
-1-1200cm
-1between larger absorption peak be caused by two kinds of C-O stretching vibrations, a kind of C-O-H that belongs to wherein, another kind is the C-O-C of sugar ring; 1047cm
-1near strong absorption peak proof monose wherein exists with the form of pyranoside; 890cm
-1neighbouring without absorption peak, illustrate that polysaccharide is containing α-glycosidic link, containing β-glycosidic link; 600cm
-1near without absorption peak, the stretching vibration peak (table 1) that does not contain C-X (halogen) is described.
Table 1. autoflocculation scenedesmus obliquus agglutinating matter infared spectrum analysis result
(4) ultimate analysis
Accurately take autoflocculation scenedesmus obliquus of the present invention and produce agglutinating matter 10mg, after 105 ℃ of drying and processing 30min, with PE-2400 type elemental analyser, carry out ultimate analysis, the results are shown in Table 2.According to C in table, H, the atom number ratio of O, can calculate C:H:O=1:2:1, basically meets sugared general formula Cn (H
2o) n, consistent with analytical results before, further determine that the agglutinating matter of purifying is saccharide compound.Contain in addition a small amount of nitrogen and element sulphur in this sugar.INFRARED SPECTRUM is analyzed at 1623cm
-1absorption peak, can determine it is a kind of mucopolysaccharide; Contain amino and sulfate radical in this sugar.
Table 2. autoflocculation scenedesmus obliquus agglutinating matter results of elemental analyses
2. the qualitative analysis of autoflocculation agglutinating matter that scenedesmus obliquus produces of the present invention
(1) sugar analysis of autoflocculation agglutinating matter that scenedesmus obliquus produces of the present invention
Measure neutral sugar, uronic acid and aminosugar composition in agglutinating matter by anthrone development process and carbazole sulfuric acid process Elson-Morgan method.By Anthrone Sulphuric acid reagent is joined in this agglutinating matter sample solution, and after reaction, solution is blue-greenish colour, proves that this agglutinating matter contains neutral sugar; Carbazole sulfuric acid process reagent joins in sample solution, and after reaction, solution is red-purple, proves that this agglutinating matter contains uronic acid; Agglutinating matter after trifluoroacetic acid hydrolysis takes on a red color by the Elson-Morgan reaction soln, proves and wherein contains aminosugar.To sum up contain polysaccharide in agglutinating matter as a result, wherein the content of neutral sugar, acid sugar and aminosugar (massfraction ratio) is than being 16:9:1.
Autoflocculation agglutinating matter that scenedesmus obliquus produces of the present invention, after trifluoroacetic acid hydrolysis is by saponification and acetylize, is adopted to GC-MS analysis list sugar component, and the temperature programming starting temperature is 170 ℃; keep 2min, then rise to 240 ℃ with 2 ℃/min, helium is carrier gas; flow velocity 1mL/min, 200 ℃ of ion sources.Result shows, this agglutinating matter is comprised of glucose sugar, seminose, semi-lactosi, rhamnosyl and fructose, and its mol ratio is 8:5:3:2:1.
(2) molecular weight determination
It is 0.2%(W/V that autoflocculation agglutinating matter that scenedesmus obliquus produces of the present invention and various standard polysaccharide are made respectively to concentration) the aqueous solution, the centrifugal 3min of 16000r/min, supernatant liquor, through the aseptic membrane filtration of 0.22 μ m, adopts and completely encircles input mode.Test condition: adopt the gel exclusion chromatography post: TSK-G4000PW(7.5 * 300) and TSK-G5000PW(7.5 * 300); 40 ℃ of column temperatures; Moving phase: secondary pure water; Flow velocity: 0.5mL/min; RI differential detector.The GPC experimental result shows that separating the agglutinating matter obtained is sterling, and its molecular weight is 1.279 * 10
5da.
Embodiment 6: flocculating property and the application of autoflocculation agglutinating matter that scenedesmus obliquus produces of the present invention
1. the determination of activity of autoflocculation agglutinating matter that scenedesmus obliquus produces
The agglutinating matter that in example 4, purifying obtains is made to its flocculation activity to the non-flocculation microalgae of difference of determination of aqueous solution.Get the non-flocculation grid algae FSP-3 of 10mL OD0.6, non-flocculation limnetic chlorella CMW-11, the micro-plan ball of non-flocculation seawater algae DUT01, add respectively the agglutinating matter that final concentration is 0.6mg/L, at 25 ℃ of agglutinating matters of processing 30min after the same method, carry out flocculation activity mensuration.Result shows, autoflocculation agglutinating matter that scenedesmus obliquus produces of the present invention has throwing out to three kinds of algaes, wherein best to the flocculating effect of non-flocculation grid algae FSP-3, process 5min after flocculation efficiency can reach 85%.
2. the optimum dosimetry of autoflocculation agglutinating matter that scenedesmus obliquus produces
The aqueous solution of the agglutinating matter that in example 4, purifying obtains being made to different concns flocculates non-flocculation grid algae FSP-3 to measure its optimum concentration.Add respectively the agglutinating matter that final concentration is 0,0.1,0.2,0.3,0.4,0.5,0.6,1.0mg/L in the non-flocculation grid algae FSP-3 of 10mL OD 0.6, concussion mixes 30s, surveys immediately each algae liquid and now under 680nm, surveys absorbancy OD680 (t
0); After standing 20min, the algae liquid of getting same liquid level is surveyed it and survey absorbancy OD680 (t under 680nm
1); Calculated flocculation activity according to formula [1].Autoflocculation scenedesmus obliquus of the present invention produces shown in agglutinating matter various dose measurement result (Fig. 6), when flocculant concentration is 0.5mg/L, flocculation efficiency is 75%, flocculant concentration is that flocculant concentration is while being 0.6mg/L, flocculation efficiency can reach 88%, flocculant concentration continues to increase, and flocculation efficiency is significant increasing not, so its optimum dosage is 0.6mg/L.
3. the optimum treatment time of autoflocculation agglutinating matter that scenedesmus obliquus produces is measured
The agglutinating matter that in example 4, purifying obtains is made to the aqueous solution to be processed to be determined as in different time points and flocculates the efficiency of non-flocculation grid algae FSP-3 to measure its optimum treatment time.Add the agglutinating matter that final concentration is 0.6mg/L in the non-flocculation grid algae FSP-3 that is 0.6 in 10mL OD680 value, concussion mixes 30s, surveys immediately each algae liquid and now under 680nm, surveys absorbancy OD680 (t
0); Survey absorbancy OD680 (t in the sampling of 1,3,5,10,20,30,45,60min same method respectively subsequently
1); Calculate respectively the flocculation activity of each time point according to formula [1].Autoflocculation scenedesmus obliquus of the present invention produces shown in agglutinating matter different treatment timing result (Fig. 7), while processing 10min, flocculation efficiency is 56%, while processing 20min, flocculation efficiency is 72%, while processing 30min, flocculation efficiency is 79%, treatment time continues to extend, and flocculation efficiency is significant increasing not, so its optimum is treated to 20min.
4. the optimum pH of autoflocculation agglutinating matter that scenedesmus obliquus produces measures
The agglutinating matter that in example 4, purifying obtains is made to the aqueous solution and flocculated non-flocculation grid algae FSP-3 to measure its optimum pH under different pH.Add the agglutinating matter that final concentration is 0.6mg/L in the non-flocculation grid algae FSP-3 of 10mL OD0.6, regulate respectively pH 4.0-11.0, measure the flocculation efficiency of its non-flocculation grid algae FSP-3 that flocculated according to above-mentioned method, to determine its optimum pH under different pH.Result (Fig. 8) shows, the optimum pH of autoflocculation agglutinating matter that scenedesmus obliquus produces of the present invention is 7.0, and flocculation efficiency now can reach 83%, and in the scope of pH5-8, flocculation activity all surely is held in more than 50%.
5. the optimal temperature of autoflocculation agglutinating matter that scenedesmus obliquus produces is measured
The agglutinating matter that in example 4, purifying obtains is made to the aqueous solution and flocculated non-flocculation grid algae FSP-3 to measure its optimal temperature at different temperature.Add the agglutinating matter that final concentration is 0.6mg/L in the non-flocculation grid algae FSP-3 that is 0.6 in 10mL OD680 value, regulate respectively temperature 20-60 ℃, at 121 ℃ of agglutinating matters of processing 20min after the same method, carry out flocculation activity mensuration in addition.Result (Fig. 9) shows, the optimal temperature of autoflocculation agglutinating matter that scenedesmus obliquus produces of the present invention is extensive, and in the scope of 20-60 ℃, flocculation activity all surely is held in more than 65%; And have good thermostability, after 121 ℃ of processing 20min, flocculation activity still can reach 46%.
6. the impact that different metal ion pair autoflocculation scenedesmus obliquus produces the agglutinating matter flocculation efficiency
The agglutinating matter that in example 4, purifying obtains is made to the aqueous solution non-flocculation grid algae FSP-3 that flocculated, add the different metal ion of different concns in the flocculation reaction system, study its impact on flocculation activity.Add the agglutinating matter that final concentration is 0.6mg/L in the non-flocculation grid algae FSP-3 that is 0.6 in 10mL OD680 value, add respectively Al
3+, Fe
3+, Ca
2+and Mg
2+, various material final concentrations are 50mmol/L, measure flocculation activity.Result shows, the Al of high price
3+, Fe
3+, Ca
2+and Mg
2+the agglutinating matter flocculation activity is had to certain promoter action, but difference is not obvious.
Claims (1)
1. the scenedesmus obliquus (Scenedesmus obliquus) of autoflocculation material is produced in a strain, it is characterized in that: registering on the books of described scenedesmus obliquus is numbered CGMCC No.6567, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, the depositary institution address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preservation date is on September 14th, 2012.
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