叶绿素a的测量-乙醇提取法

Hydrobiologia485:191–198,2002.©2002Kluwer Academic Publishers.Printed in the Netherlands.191 Chlorophyll-a determination with ethanol–a critical test´Eva P´a pista1,´Eva´Acs2&B´e la Böddi3,∗1Eötvös Lor´a nd University of Science,Doctoral School,P´a zm´a ny P´e ter allee1/A,Budapest H-1117,Hungary2Eötvös Lor´a nd University of Science,Department of Microbiology,P´a zm´a ny P´e ter allee1/C Budapest H-1117, Hungary3Eötvös Lor´a nd University of Science,Department of Plant Anatomy,P´a zm´a ny P´e ter allee1/C,BudapestH-1117,HungaryTel:12660240;E-mail:bbfotos@ludens.elte.hu(∗Author for correspondence)Received2May2001;in revised form30August2002;accepted20August2002Key words:algae,chlorophyll-a determination,ethanol,ISO standard10260(1992)AbstractChlorophyll-a content is widely used as an indicator of the quality of freshwater bodies.Quantification of chlorophyll-a is a routine procedure in the test laboratories of water works,and in research laboratories.Although attempts have been made to standardise the measurement procedure,there are nonetheless many procedures currently in use.This work is focused on a careful re-examination of the ISO:10260,1992standard,which prescribes90%(v/v)ethanol for chlorophyll extraction and measurement.Chlorophyll contents of cultures of the cyanobacterium Synechococcus elongatus Nägeli and the chlorophyte Scenedesmus acutus Meyen were determined by means of a series of concentrations of ethanol/water mixtures which were employed as extracting agents–the water content was gradually decreased from20to0%.The extraction procedure was verified by measuring the amount of retained water after using both water and oil pumps forfiltering the samples.The spectroscopic effects of the presence of water were studied and the molecular background of these spectral phenomena is discussed.The extraction yields obtained with90%ethanol were compared to those obtained with methanol and acetone.On the basis of the calculated error level,improvements to the ISO:10260,1992standard method have been suggested.IntroductionThe chlorophyll(Chl)content of freshwater bodies is a widely accepted indicator of water quality.Research projects on periphyton(Cattaneo,1983;Jonsson, 1987;Robinson&Rushforth,1987;Pantecost,1991) or phytoplankton(Kiss&Genkal,1993;Balogh et al., 1995;Jones,1995;Kiss,1996;Shafik et al.,1997; Skidmore et al.,1998;Kiss et al.,1998)use these characteristics to describe the trophic state(Sumner &Fisher,1979;Vörös&Padisák,1991;Talling, 1993)of the studied system.However,the identific-ation of the alga species,the knowledge of the algal cell number,or the physiological state of cells may also be important in providing a true picture of the water quality or trophic state.A combination of Chl determination and consideration of these other factors may provide an improvement in the reliability and ac-curacy of water quality estimation.Utermöhl(1958) developed a method to determine the individual num-ber of algae with an inverted microscope and Lund et al.(1958)described a procedure to estimate the accuracy and limitations of Utermöhl’s method.If certain taxa are in developing or degrading stages in the studied populations,consideration of the factors above is essential,since certain species produce toxins harmful to both water animals and hu-man(Slatkin et al.,1983;Codd et al.,1992).It has been established that the presence of algae and thus the Chl content indicate the concentration of certain chemicals or the appearance of toxins in the drinking water(Bernhardt&Clasen,1991).Thus,considera-192tion of the Chl content together with the algal species is beneficial in thefield of water treatment technology.There is a large variety of Chl determination meth-ods in hydrobiology.The main variability lies in the choice of the extracting solvent,probably because the extraction yield is different in the case of different alga taxa.Absolute methanol or acetone is often used in basic research.To increase the extraction yield,meth-anol/water mixtures(Jewson et al.,1981;Skidmore et al.,1998)are used or dimethysulfoxide(Lean& Pick,1981)is added(especially if Bacillariophyceae are studied).Pure(Stauber&Jeffrey,1988)or90% acetone(Rosen&Lowe,1984;Levasseur et al.,1993) is used if certain Chlorophyceae species are present.The ISO standard method(ISO10260,1992) has been developed with consideration given to the toxicity of the solvents above and is accepted in freshwater test laboratories in many countries.This method prescribesfiltering(although the conditions are not fully specified)of the water sample followed by Chl extraction with90%ethanol/water(v/v)mix-ture,centrifugation and spectroscopic measurement of the supernatant atfixed wavelengths.The spectro-photometer must be set to zero at750nm and the absorbance is recorded at666nm.Unfortunately,the physico-chemical properties of the90%ethanol/water mixture predict a poor extraction yield in case of nu-merous alga taxa,furthermore the same properties make the results of the spectroscopic Chl determina-tion dubious.Despite of the numerous methods and equations used in the basic research(for example Lichtenthaler,1987;Porra et al.,1989;Wellburn, 1994),the ethanolic extraction is used in recent pub-lications(Thompson et al.,1999;Halling-Sorensen, 2000;Kataura et al.,2000).The extraction yields of the different methods have not been determined and compared to each other and no standard procedure has been generally accepted(Vladkova,2000).Despite these uncertainties,a prerequisite for the accreditation of freshwater test laboratories in several countries is the application of ethanolic extraction.This paper studies the effect of water content in the extracting ethanol on the extraction yield of Chl from Synechococcus and Scenedesmus cultures.The amount of water retained in thefilter after different filtration methods were employed was measured.The absorption spectra of pigment extractions were recor-ded.These results,and the influence of disturbing physical effects(baseline distortion,absorption band diffusion)and chemical effects(Chl-hydrate crystal formation,colloid structures)are studied and dis-cussed.The extraction yields of different solvents is also studied and compared.Materials and methodsAlgaeTo test the Chl determination methods,algal cultures and natural water samples were used.The algal cul-tures were the cyanobacterium Synechococcus elong-atus Nägeli and the chlorophyte Scenedesmus acutus Meyen grown in Allen medium under12L/12D and 20◦C.The natural water sample was collected from a eutrophic pond in which Chlorophyta(mainly Scene-desmus and Kirchneriella species)usually dominate the algal community.Pigment extractionA small volume of the cultures(400–750µl/sample) wasfiltered with an oil pump through glassfibrefilters (MN GF-1;Macherey-Nagel GER).Thefiltration time was60s.Thefilters were immersed into the extraction solutions which were ethanol with varying water con-tent between80%and100%(virtual concentrations, v/v),pure methanol,or acetone.The samples were heated to the boiling point of the ethanol and methanol solvents,then maintained at this temperature for15s before being cooled to room temperature.The samples were centrifuged with MLW T52.1centrifuge for10 min with1000g.The experiments were repeated three times with10parallels in each experiment.The extrac-tion with90%acetone was performed in accordance with the Standard Methods for the Examination of Water and Wastewater(1992).After thefiltration,the filters were immersed in acetone and kept in the dark at4◦C for48h.The samples were then centrifuged as above.FiltrationA water pump(at10000Pa)and an oil pump(Vacu-ubrand ME2C at8000Pa)were used for testing the amount of water retained by thefilters.Then500cm3 of distilled water wasfiltered in each test for30–120 s.The time periods were measured from the moment when the water drained from the upper device of the filtering apparatus to the moment when thefilter sur-face became matted.The amount of the retained water was determined by measuring the mass of thefilter193before and after the experiments.Three experiments were conducted,each comprising10repetitions. Absorption spectroscopyThe absorption spectra were recorded with UNICAM UV/Vis2and SHIMADZU UV-2101PC spectropho-tometers.The spectra were measured between375 and800nm with0.5nm data frequency and a1nm slit.The data were exported into ASCII format for further data processing:baseline correction and cal-culation of average spectra.The software SPSERV V-3.14(Copyright:Csaba Bagyinka;Inst.Biophys. Biol.Res.Cent.Szeged,Hungary)was used.The Chl-a contents were determined with the ISO calculation (ISO:10290,1992).ResultsThe spectroscopic concentration determination is based on equations in which the absorption coefficient refers to readily identifiable and uniform chromophore species.To demonstrate that the water modifies the absorption spectrum of Chl-s in algal extractions,the following experiment was carried out:the chloro-phylls were extracted from a Scenedesmus acutus culture with80%acetone,then transferred into diethyl ether and the traces of water removed with desic-cated Na2SO4.The pigment solution was halved and the diethyl ether was evaporated from both extrac-tions.The pigments were resolved in100%ethanol in one sample and with90%ethanol(v/v)in the other. The comparison of the absorption spectra in the red region(which is used for concentration determina-tion)showed significant differences(Fig.1).In the spectrum of the watered sample,the absorbance has increased in the700–800nm region.A reduction in the maximum absorbance value is also evident,as is a change in the spectral position of the maximum value.These spectral phenomena are more obvious in Figures2A and2B in which the abovementioned details are magnified.The spectral structure formed by the broad maximum around740–745nm indicates that new chromophore species have been formed in the watered sample.In addition to the error sources described above,a reduction in the extraction yield was found in the case of natural water samples.When the Chl was extracted from natural water samples with 90%ethanol using the ISO:10260,1992standard method,a decrease in the absorbance ofapproximately parison of the absorption spectra of pigments extrac-ted from Scenedesmus acutus:1,solvent100(v/v)%ethanol,2, solvent88(v/v)%ethanol.50%was observed.Furthermore,baseline distortions were found in the absorption spectrum,compared with the absorption properties of the extraction in which 100%methanol was used(Fig.3).This baseline dis-tortion proved to be significant.When the baseline was corrected with a computer program and the corrected baseline subsequently subtracted from the spectra,the calculated Chl content values decreased by5–10%. To investigate the reasons for these anomalies,the following experiment was performed.Using the ISO:10260,1992standard method, Chl was extracted from a natural water sample.The chlorophyllous solution was centrifuged and the ex-traction repeated with the precipitate with100%eth-anol.The suspension was boiled,cooled,centrifuged and its absorption spectrum was measured.This ex-periment showed that approximately35%of the Chl content of the alga culture remained in the precipitate after thefirst extraction with90%ethanol.This res-ult illustrates that low extraction efficiency can be a source of basic error in the ISO:10260,1992standard method.In the next series of experiments,we studied the actual water content of the solutions after thefiltering of the water samples.The amount of water retained by thefilter on which the alga cells are collected from the water sample varies,and depends upon the details of the procedure employed.Consequently,immersion of thisfilter into the extracting solution will modify the ethanol/water molar ratio dependent upon the amount of water introduced via thefilter.This experiment was important because thefiltration procedure(vacuum value,pore size offilter,time offiltration)is poorly194Figure2.Details of the absorption spectra of pigments extracted from Synechococcus elongatus:1,solvent100(v/v)%ethanol,2,solvent88 (v/v)%ethanol.(A)Spectral shift and broadening of the absorption band are caused by the presence of water.(B)Formation of micro-crystals caused the appearance of an absorption band at743nm.described in the ISO standard.These measurementshave shown that the amount of retained water wassignificant–it modified the initial90%ethanol/waterconcentration of the extraction solution by3.4–4.1%or1.3–1.5%when using a water-pump or an oil-pump,respectively(Table1).In the next experiment,the molar ratio of wa-ter to ethanol was varied to study the extractionyield of the different solutions when the ISO:10260,1992standard method was used.Chl-s were extractedfrom Synecococcus elongatus and Scenedesmus acu-tus cultures;the extracting agents were ethanol-watersolutions with88–96%(virtual concentration,v/v)ofethanol.To demonstrate the data scattering,the Chl content obtained with90%ethanol was normalised to 100%and the standard error values were calculated. There is no evident trend in the series of the data but this experiment does show a high level of error in the case of each concentration(Fig.4).The extraction yields were compared when100% methanol,90%ethanol,and acetone were used ac-cording to the ISO:10260,1992standard method, and the modified Standard Methods,1992,which was applied in the last two cases only.(In case of the Standard Methods,we did not apply the acidifica-tion because we were not interested in the determ-ination of pheophytins;consequently,we used other equations[Jeffrey&Humphry,1975]for the cal-culations.)The Chl-s were extracted from a natural water sample dominated by Scenedesmus and Kirch-neriella(Chlorophyta)species.Methanolic extraction was found to provide both the highest extraction yield and the lowest standard error value.Normalising this Chl concentration value to100%,the90%ethanol and Figure3.Absorption spectra of pigments extracted from natural water sample collected from an eutrophic pond dominated by green algae:1,solvent100(v/v)%methanol,2,solvent88.5%(v/v) ethanol.the acetone yielded only77%and60%,respectively –furthermore,the error level increased significantly (Table2).DiscussionThe extraction solvent must meet the following re-quirements:it must penetrate the alga cells;it must denature the membranes holding the Chl-s,including the Chl–protein complexes,in such a way that the Chl-s can leave the complex;andfinally it must form a uniform monosolvate complex with the Chl-s.In this case,the chromophore can be characterised with a single extinction coefficient and the Beer-Lambert equation can be used.The ethanol/water mixture can-195 Table1.The amount of retarded water in glassfiberfilters after different times offiltering in case of water-pump and oil pump.Calculating10ml extraction solvent ofethanol90%(v/v),thefinal concentrations are shown in the right columns.Values ofstandard deviation are shownWater pump Oil pumpAverage weight of Modified cc.Average weight of Modified cc.retarded water(g)(%)retarded water(g)(%)30s0.483±0.01185.870.172±0.00788.4860s0.424±0.01286.340.167±0.00888.5290s0.411±0.01586.450.155±0.01088.62120s0.389±0.01786.630.142±0.01288.74Table2.Extraction yields of chlorophylls with different solvents.The data are meanvalues of30measurements;the standard deviation values are indicated90%(v/v)EtOH90%(v/v)Acetone100%(v/v)MeOHE max0.1110.1060.129Chl a concentration18.67±4.3314.65±3.98a24.28±4.53µg/l(EtOH:ethanol;MeOH:methanol).a Using the equation of Jeffrey&Humphrey(1975).Figure4.The relative extraction yields obtained with different ethanolic solvents in which the water content was varied.The ex-traction yield at90(v/v)%(ISO10260standard)was normalised to100%.Thefigure shows results and data scattering offive inde-pendent experiments.Pigments were extracted from Synechococcus elongatus“Cyano”and Scenedesmus acutus“Green”cultures. not meet these requirements–it does not form a suitable,homogenous molecular environment for the Chl molecules.H-bridges connect water and ethanol molecules–the size and amount of aggregates formed this way change in time statistically and depend on numerous factors(e.g.temperature,molar ratio of wa-ter/ethanol,impurities in the solvent,etc.).Due to these molecular interactions,the density of the eth-anol/water solution exhibits a non-linear relationship with respect to concentration(Horwitz,1955).Corres-pondingly,the refraction indices of the ethanol/water solutions have a similarly non-linear relationship with concentration–this has a direct bearing on the spec-troscopic properties(extinction coefficient and absorp-tion band position)of Chl-s(Seely&Jensen,1965). Moreover,in ethanol/water solutions,Chl-s form dif-ferent complexes.Since the central Mg in the por-phyrin ring is coordinatively unsaturated it can accept electron donor molecules or molecule groups.In this way,Chl-monosolvate or Chl-bisolvate complexes are produced in which the coordination number of Mg is 5or6,respectively(Fong,1975;Fong&Koester, 1975;Mukherjee et al.,1978).Both ethanol and wa-ter can be electron donors in forming such complexes. Besides Mg,water can coordinate to other atoms of the porphyrin ring producing a series of solvate com-plexes in various structures.With its positive pole it can coordinate to the non-bonding electron pairs of N-atoms or O-atoms of the porphyrin ring.These in-teractions modify the energy level of the delocalized electron cloud and so change the excitation energy of the molecule,i.e.the position of absorption band will be shifted by several nm(Clarke,1982).In ad-196dition,water,as a bifunctional ligand,can connect two or several Chl molecules thereby forming hy-drated dimers,oligomers or micro-crystals.(The shift of the absorption maximum and the broadening of the absorption band in Figures1and2indicate the formation of several Chl-water or Chl-ethanol com-plexes.The appearance of the absorption band in the 740–745nm region(Fig.2B)indicates the formation of Chl-hydrate micro-crystals(Ballsmither&Katz, 1972).The probability of the appearance and the size(or size distribution)of these crystals depend on the molar ratios of Chl,water and ethanol.Considering the usu-ally low Chl concentration of samples obtained from natural water samples(about10−5M),the number of water molecules is usually several orders higher than that of Chl-s.Therefore,any of the above-described complexes can appear.In addition,in a90%eth-anol/water solution the molar ratio is close to3.At this value,ethanol and water molecules form aggregates of different sizes in which Chl molecules can build.If the size of these complexes reaches a value close to the absorption band of Chl,interference takes place in a manner similar to that which occurs in colloid solu-tions.As a result,the absorption band broadens,the baseline distorts,and the solution will be unsuitable for absorption measurements.The formation of Chl-hydrate micro-crystals can significantly modify the results of Chl content determ-ination.Their absorption maximum is at740–745nm and their band is very broad.When using the ISO 10260method,the spectrophotometer is set to zero at750nm,i.e.close to the absorption maximum of the micro-crystals.Consequently,the amplitude of absorption band of the monomer Chl-a(666nm)is reduced,depending on the degree of micro-crystal formation.The extinction coefficient of the micro-crystals is not known,nor have their exact structures been de-scribed(i.e.the number of Chl molecules built into the crystals).The baseline distortion is,at least partly, due to the formation of the colloid-like structures –ethanol-water-Chl associations.The prevalence of these structures depends upon the molar ratios of the interacting molecules.Therefore,even the small amount of water introduced with thefilter can signi-ficantly change the shape of the absorption spectra. The absorption spectrum of these solutions is super-posed on a light scattering curve,which increases the absorbance value measured at the maximum of Chl-s. The two effects described above may compensate each other,but their contribution is unknown.In light of the fact that the ISO10260standard method prescribes the measurement of the absorbance value at the absorp-tion maximum and the fact that the spectrum is not recorded,the researcher has no information about the above-described spectral phenomena.Another source of uncertainty is the formation of ethanol-water as-sociations that reduce the extraction yield–these complexes can denature the biological membranes and the Chl–protein complexes to a limited extent.These phenomena are not significant in the cases of methanol or acetone.However,acetone cannot be used generally in algological studies–hot methanol may provide better extraction yields depending upon the species present(Iwamura et al.,1970).Compared with methanolic extraction,the yield obtained with acetone was smaller.This solvent either cannot de-nature,i.e.break up all algal cells,or it precipitates the Chl–protein complexes producing stable structures –thus100%solubility of the Chl-s is unattainable. The most effective extracting agent was the methanol, the mobility of which is superior to that of ethanol, it does not form large adducts with water,and it eas-ily denatures both the biological membranes and the Chl–protein complexes.The data presented within this work shows that the application of ethanol(either in90%concentration as in the ISO10260method,or pure)provides dubi-ous results.It is clearly necessary that water quality testing laboratories have simple and fast test meth-ods because of the large quantity of samples to be processed,but the current unreliable methods lead to erroneous data,which can mislead and corrupt asso-ciated environmental research.Further basic research is needed to develop a technique that provides the requisite processing speed and level of test accuracy and reliability.Employment of modern computerised spectroscopy technology provides a good opportunity to develop such a technique,which would suit wide-spread application in water quality testing laboratories.On the basis of the foregoing results and related experiences,we suggest the following:1.The90% ethanol/water solvent should be avoided–100%meth-anol seems to be generally more suitable.(Algologists engaged in basic research traditionally use this.)How-ever,after determining the presence of the alga spe-cies,the method must be varied.The tendency to avoid the use of methanol in the laboratory can be addressed through attention to the appropriate precautions and laboratory conditions.2.The conditions forfiltration must be defined:the type of thefilter,thefiltration197time,and the vacuum value.The use of an oil-pump and a minimum of60sfiltration time are suggested.3. The centrifugation conditions must be defined(10min with1000g seems to be effective)and the pellet must be checked(at least randomly,or in case of unusual samples–alga blooming or new species–regularly).4.The measurement of absorption spectra should be recorded at least in the600–800nm region in order to obtain information about the spectral distortions. 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