CN104031053A - Rapid and efficient extraction method of chlorophylls in large quantity of cyanobacteria samples - Google Patents

Rapid and efficient extraction method of chlorophylls in large quantity of cyanobacteria samples Download PDF

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Publication number
CN104031053A
CN104031053A CN201410289771.7A CN201410289771A CN104031053A CN 104031053 A CN104031053 A CN 104031053A CN 201410289771 A CN201410289771 A CN 201410289771A CN 104031053 A CN104031053 A CN 104031053A
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chlorophyll
extraction
microcystic aeruginosa
chlorophylls
cyanobacteria
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CN104031053B (en
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孙朋飞
赵宇华
王冠
卢丽玲
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Zhejiang University ZJU
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Zhejiang University ZJU
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains four or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources

Abstract

The invention discloses a rapid and efficient extraction method of chlorophylls in a large quantity of cyanobacteria samples. The method comprises the following steps: centrifuging microcystis aeruginosa with the OD (Outer Diameter) 680 of 1.0 in a culture solution and then collecting microcystis aeruginosa strains; respectively treating the microcystis aeruginosa strains in boiling water for 3 minutes and naturally cooling at a room temperature (25 DEG C); adding 4mL of acetone and fully mixing; centrifuging so as to obtain a supernate, i.e., a chlorophyll extracting solution. According to the method, the extraction time of the microcystis aeruginosa is obviously shortened. The method is convenient to operate and high in extraction effect. The method has obvious advantages when being applied to the extraction of a large quantity of cyanobacteria chlorophylls of multiple samples.

Description

The rapidly and efficiently extracting method of blue-green algae sample Determination of Chlorophyll in enormous quantities
Technical field
The invention belongs to biological chemical field, particularly a kind of rapidly and efficiently extracting method of blue-green algae sample Determination of Chlorophyll in enormous quantities.
Background technology
Chlorophyll is the fat-soluble natural pigment that is present in a kind of natural, nontoxic of part blue-green algae and all green plantss etc. and has certain physiological function, and its end-use is extensive, for example food, medication chemistry industry all tool have been widely used.As fat-soluble pigment, chlorophyll is water insoluble, dissolves in the organic solvents such as acetone, ethanol, and therefore above reagent Chang Zuowei extracts chlorophyllous active solvent.
Blue-green algae is photosynthetic oxygen evolution biology the earliest, is a kind of of planktonic algae, and earth surface has been become aerobic environment to huge effect from the atmospheric environment of anaerobic.When often occurring with cell mass form, blue-green algae just easily sees " wawter bloom " that namely we see conventionally.Because part blue-green algae can produce the virose secondary metabolite of tool---algae toxin, therefore for the improvement of the wawter bloom being caused by blue-green algae, become a large problem of current environmental protection circle, and in to the research of blue-green algae, often using chlorophyll content as weighing one of index of large cortical cells biomass.
Current chlorophyllous conventional extraction has: 1. organic solvent extractionprocess.The method extraction time is long, and extraction efficiency is low.2. polymeric adsorbent extraction process.The steps such as whole process comprises organic solvent extraction, and resin absorption and wash-out are concentrated, troublesome poeration and total extraction time are long.3. supercritical CO 2extraction process.The method requirement for experiment condition is high, and total extraction time is long.4. ultrasonic extraction method etc.Although the method chlorophyll extraction efficiency is high, is not suitable for batch samples and extracts.Due to all inconvenience existing in above the whole bag of tricks operating process, the problem simultaneously conscientiously existing in reference laboratory chlorophyll leaching process, is necessary chlorophyllous extracting method to improve.
Summary of the invention
The object of the invention is to for the deficiencies in the prior art, a kind of rapidly and efficiently extracting method of blue-green algae sample Determination of Chlorophyll in enormous quantities is provided.
The object of the invention is to be achieved through the following technical solutions: get OD 680be 1.0 microcystic aeruginosa nutrient solution 5mL, the centrifugal 5min of 12000rpm, collects microcystic aeruginosa thalline; Above-mentioned microcystic aeruginosa thalline is joined in the centrifuge tube that contains 1mL sterilized water, centrifuge tube heats 3min in boiling water, then under room temperature after (25 ℃) naturally cooling, in centrifuge tube, add 4mL acetone fully to mix, the centrifugal 5min of 12000rpm, collect supernatant liquor, obtain chlorophyll extracting solution.
The invention has the beneficial effects as follows:
Blue-green algae chlorophyll extraction time obviously reduces.Blue-green algae chlorophyll, as weighing one of important indicator of blue alga biomass, is one of sign of wawter bloom regulation effect.The minimizing of Determination of Chlorophyll of the present invention extraction time is mainly reflected in the broken and chlorophyllous leaching process of cell walls, and especially in Multi-example chlorophyll leaching process, time decreased effect is more obvious.
Easy to operate, extraction effect is high.First the present invention extracts chlorophyll method by the ultrasonic extraction method to conventional and is optimized, the method of usining after this optimization is as reference, compare and learn with the Boiling bath method extraction chlorophyll effect of improvement, the chlorophyllous amount that the chlorophyllous amount that Boiling bath method extracts is extracted higher than the ultrasonic extraction method of optimizing, and in whole leaching process, requirement for experiment condition is simple, and working method is simpler and more direct.
It is remarkable that the present invention is applied to the large batch of blue-green algae chlorophyll extraction of Multi-example advantage.While being mainly reflected in the extraction of Multi-example chlorophyll, use Boiling bath method fracturing cell walls, compared with ultrasonic method fracturing cell walls, saved a large amount of time, and because Boiling bath method can operate by Multi-example simultaneously, while having reduced cytoclasis, with batch each sample room, owing to testing wait, cause the experimental error that chlorophyll photodissociation causes.Therefore present method is more applicable for the chlorophyllous extraction simultaneously of Multi-example blue-green algae.
concrete steps are as follows:
A rapidly and efficiently extracting method for blue-green algae sample Determination of Chlorophyll in enormous quantities, the method is specially: get OD 680be 1.0 microcystic aeruginosa nutrient solution 5mL, the centrifugal 5min of 12000rpm, collects microcystic aeruginosa thalline; Above-mentioned microcystic aeruginosa thalline is joined in the centrifuge tube that contains 1mL sterilized water, centrifuge tube heats 3min in boiling water, then under room temperature after (25 ℃) naturally cooling, in centrifuge tube, add 4mL acetone fully to mix, the centrifugal 5min of 12000rpm, collect supernatant liquor, obtain chlorophyll extracting solution.
Below in conjunction with embodiment, the invention will be further described.
comparative example:conventional chlorophyllous ultrasonic extraction method
Get 5mL microcystic aeruginosa nutrient solution (OD 680=1.0), the centrifugal 5min of 12000rpm, collects thalline; Above-mentioned microcystic aeruginosa thalline is joined in the centrifuge tube that contains 1mL sterilized water, ultrasonic disruption cell (10s, 10s), fragmentation is for 45 times that total time is 15min respectively.After cytoclasis, in centrifuge tube, add 4mL acetone, after the standing 0.5h of dark sections temperature (25 ℃), the centrifugal 5min of 12000rpm.Get supernatant separately and measure OD 750, OD 663, OD 645, OD 630, by following formula, calculate chlorophyll content ρ (Chla) (μ g/L):
ρ(Chla)?(μg/L)?=11.64×(OD 663－OD 750)?－2.16×(OD 645－OD 750)+0.1×(OD 630－OD 750)
By calculating the chlorophyll concentration extracting by the method, be 3.05 μ g/L.
embodiment 1
Get OD 680be 1.0 microcystic aeruginosa nutrient solution 5mL, the centrifugal 5min of 12000rpm, collects microcystic aeruginosa thalline; Above-mentioned microcystic aeruginosa thalline is joined respectively in 5 centrifuge tubes that contain 1mL sterilized water, 5 centrifuge tubes heat respectively 3min, 5min, 10min, 15min, 20min in boiling water, then under room temperature after (25 ℃) naturally cooling, in 5 centrifuge tubes, add respectively 4mL acetone fully to mix, the centrifugal 5min of 12000rpm, supernatant liquor is chlorophyll extracting solution.Get supernatant separately and measure OD 750, OD 663, OD 645, OD 630, by formula, calculate chlorophyll content ρ (Chla) (μ g/L):
ρ(Chla)?(μg/L)?=11.64×(OD 663－OD 750)?－2.16×(OD 645－OD 750)+0.1×(OD 630－OD 750)。
The different boiling water baths of table 1 chlorophyll extracted amount after the treatment time
Processing mode Boiling water 3min Boiling water 5min Boiling water 8min Boiling water 10min Boiling water 15min
ρ(Chla) (μg/L) 3.14988 3.04912 3.164184 2.851184 1.67192
embodiment 2
Get 5ml microcystic aeruginosa nutrient solution (OD 680=1.0), the centrifugal 5min of 12000rpm, collects thalline; Above-mentioned microcystic aeruginosa thalline is joined respectively in 2 centrifuge tubes that contain 1mL sterilized water, 2 centrifuge tubes are in boiling water after heat treated 3min, adopt respectively ice bath but with room temperature under two kinds of types of cooling of (25 ℃) naturally cooling, in cooling backward two centrifuge tubes, add respectively 4mL acetone fully to mix, the centrifugal 5min of 12000rpm.Get supernatant separately and measure OD 750, OD 663, OD 645, OD 630, by formula, calculate chlorophyll content ρ (Chla) (μ g/L).Chlorophyll extracted amount is in Table 2.
The different type of cooling chlorophyll of table 2 extracted amount
The type of cooling Naturally cooling Ice bath is cooling
ρ(Chla) (μg/L) 3.219464 3.134184
embodiment 3
Get 5mL microcystic aeruginosa nutrient solution (OD 680=1.0), the centrifugal 5min of 12000rpm, collect thalline, above-mentioned microcystic aeruginosa thalline is joined respectively in 7 centrifuge tubes that contain 1mL sterilized water, 7 centrifuge tubes are in boiling water after heat treated 3min, and at room temperature (25 ℃) naturally cooling, adds respectively 4mL acetone fully to mix in cooling backward 7 centrifuge tubes, after dark sections' temperature (25 ℃) difference standing 0h, 0.5h, 1h, 1.5h, 2h, 2.5h, 6h, the centrifugal 5min of 12000rpm.Get supernatant separately and measure OD 750, OD 663, OD 645, OD 630, by formula, calculate chlorophyll content ρ(Chla) (μ g/L).Chlorophyll extracted amount is in Table 3.
Table 3 chlorophyllous extracted amount of different extraction times
Extraction time 0h 0.5h 1.0h 1.5h 2.0h 2.5h 6.0h
ρ(Chla)(μg/L) 3.169361 3.166472 3.058928 2.670952 2.297768 2.005824 1.749136
Embodiment 1-3 to sum up, in boiling water, be 3min heat-up time, the type of cooling is (25 ℃) naturally cooling under room temperature, the standing 0h(in dark place and acetone mix rear directly centrifugal, without standing at dark place), chlorophyllous extraction effect is best, at 5mL microcystic aeruginosa nutrient solution (OD 680=1.0), in, the chlorophyll content ρ (Chla) extracting is 3.15 ~ 3.22 μ g/L.And ultrasonic extraction method of the prior art, at 5mL microcystic aeruginosa nutrient solution (OD 680=1.0) in, the chlorophyll content ρ (Chla) extracting is 3.05 μ g/L, and therefore method extracted amount provided by the invention is higher.In addition, chlorophyll extracting method provided by the invention has greatly shortened extraction time, be mainly reflected in and saved gentle and quiet time of putting of dark sections and cytoclastic time: wherein ultrasonic extraction method need to be at the gentle and quiet .5h of setting to 0 of dark sections, the cytoclasis time is 15min, chlorophyll extracting method provided by the invention is without standing at dark place, and as long as the cytoclasis time is 3min.This advantage is especially embodied in while carrying out the extraction of Multi-example chlorophyll, Boiling bath method can carry out simultaneously, and ultrasonic extraction method need to be carried out one by one when cell walls is broken, only cell walls is on the broken time, ultrasonic extraction method has consumed a large amount of time, as can be seen here, when Multi-example extracts blue-green algae chlorophyll, the advantage that Boiling bath method extracts blue-green algae chlorophyll method is more obvious.

Claims (1)

1. a rapidly and efficiently extracting method for blue-green algae sample Determination of Chlorophyll in enormous quantities, is characterized in that, the method is specially: get OD 680be 1.0 microcystic aeruginosa nutrient solution 5mL, the centrifugal 5min of 12000rpm, collects microcystic aeruginosa thalline; Above-mentioned microcystic aeruginosa thalline is joined in the centrifuge tube that contains 1mL sterilized water, centrifuge tube heats 3min in boiling water, then under room temperature after (25 ℃) naturally cooling, in centrifuge tube, add 4mL acetone fully to mix, the centrifugal 5min of 12000rpm, collect supernatant liquor, obtain chlorophyll extracting solution.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102888353A (en) * 2012-08-06 2013-01-23 常州大学 Algicidal bacteria and method for removing microcystis aeruginosa
CN103196884A (en) * 2013-04-19 2013-07-10 河北科技大学 Method for determining biotoxicity of atrazine by utilizing microcystis aeruginosa

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102888353A (en) * 2012-08-06 2013-01-23 常州大学 Algicidal bacteria and method for removing microcystis aeruginosa
CN103196884A (en) * 2013-04-19 2013-07-10 河北科技大学 Method for determining biotoxicity of atrazine by utilizing microcystis aeruginosa

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GANG PAN, 等: "Removal of cyanobacterial blooms in Taihu Lake using local soils. I. Equilibrium and kinetic screening on the flocculation of Microcystis aeruginosa using commercially available clays and minerals", 《ENVIRONMENTAL POLLUTION》 *
冯青英，等: "地标水中浮游植物叶绿素a提取方法的改进研究", 《安徽农业科学》 *
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