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LIPID CLASS AND CARBOHYDRATE CONCENTRATIONS IN MARINE COLLOIDS

By Qingjun Liu B.Se and M.Eng

©

A thesis submitted to the School of Graduate Studies in partial fulmment of the requirements for the degree of

Master of Science

Department of Chemistry Memorial University of Newfoundland

August,1994

St.John's Newfoundland

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.+.

NaliCNlalUbfary orCanada

Acquisitionsand Direcl~des~guisiti~et Bibliographic Services Branch desservcesblbhograptllques 39SWeningtonSt'et!l 39S,rueWelington

~Ji.Olllatio ~:o~~()rltallO)

The author has granted an irrevocablenon-exclusivelicence allowing the National Libraryof Canada to reproduce, loan, distribute or sell copies of his/herthesisby any meansand in any form or format,making thisthesis availableto interested persons.

The author retainsownershipof the copyright in his/herthesis.

Neither the thesis norsubstantial extracts fromit may be printed or otherwise reproduced without his/her permission.

l'auteur a accorde une licence irrevocable et non exclusive permettan!

a

la Biblioth eque nationale du Canada de reproduire,preter,dislribuerau vendredes copiesdesa these de quelque maniere et sous quelque forme que cesoit pour meitre desexemplaires de cette these

a

la disposition des

personnesintel'essees.

L'auteur conservela proprletedu droit d'auleur qui protege sa these.Nila theseni des extraits substantiels de celle-ci ne doivent etre imprlmes ou autrement reproduits sansson autorisation.

Canada

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To LianfangCao

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Cross-flowfiltration(CFF)wasused to concentratemarine colloidsfrom microalgal culturesand surface seawater. Lipid classes in different size fractionswere determinedusing theChrornarcd- Iatroscan thin-layer chromatography withthe name ionizationdetection method. Total carbohydrate concentrationswere determined colo nmetnr ally using methyl henzothiazolinone hydrazone reagent. Extensive determinationsof CFF blanks andrecoveries were conductedforeach lipidclass to optimizeCFF operatingprocedures.The total lipid blankinthe CFFsystemwas approximately 6-7 JAg(-0.5JAM C).Recoveriesfor the major lipidclasses in marine samplesvariedfrom80%to120%.

Bothcollo idal lipid and carbohydrate concentrat ionsinmicroalgalcultures wer e found 10 be120- 140JAg/L.Triacylglycerol(TAG) and polar lipidswere the majorlipid classes incolloids Irorn microalgalcultures. Concentrationsof colloidal(>10.000 dalton)lipidsand carbohydratesin Newfoundlandseawater ranged from0.9 JAg/Lco 8.7 JAg/Landfrom21pg/tto30 JAg/t. respectively.The majorlipid classespresent in coastal seawater colloids wereTAG.free fattyacids.and phospholipids.

Data from both algalcultures and actual seawater samplesshowed that marine colloids are characterizedby having high proportions of free fattyacids.phospholipids andhydrocarbons by comparison withtruly dissolvedmaterial. Thefree fattyacidsmay be producedby microbialdegradation.the phospholipidsare probahlyderived from

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biologicalmembranes.andthehydrocarbonsare likelypreferentiallyadsorbed(Willthe surroundingwater oruo thecolloids.

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AC KNOWLEDGEMENTS

rwould liketoexpressmygratitudeto myco-supe rvisors. Dr.C.C.Parrishand Dr.R.uelleor.forall the support,knowledge.guidance.and advicethatI received during my research andthesis writing.

I would alsolike toexpressspecial thanks[QDr. N.Gogan. Mr.Paul Matthews, Mr.Russel Meschishnick,and Dr.R. Rivkin for theirhelpincollectingseawater samples. to Dr.R.Thompson for usc of spectrophotometer,toDr. P. E.Kepkayfor DOCanalysisand 10 Mr.EnochDaniel.Mr. AchillesLau,Ms.JeanetteStricklandand Ms.Qing Wei for theirwillingness to discuss problemsas theyarose andtosharewith me their expertiseinlaboratory techniques. Special thanks to all myfello w students and friends at M UN for theircooperationand moralsupportduringthis study.Helpprovided bypersonnelinthe general officesand theche mical stores at theOcean SciencesCentre and inthe Chemistry Department is verymuchappreciated.

Astipe nd from Dr.Parrish (fromhisNS ERCresearchgra nt) and Dr.Helleur (from his Departmentof Fisheriesand Oceans grant)is gratefullyacknowledged.I am alsogratefulforsuppo rtthroughateachingassistantship from the ChemistryDepartment or MemorialUniversity or Newfoundland.

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TABLE OFCONTE NTS ABSTRACT

ACKNO WLEDGEM ENTS

TABLEOF CO NTENTS

LIST OF TABLES .. • ..•.•.• • • •... .• • •.••. .•

LIST OF FIGURES. ..

ABBREVIATIONSANDSYM BOLS•. •

..iii

..viii

.. . . •:(iii

2) 25 1.0LITERATUREREVI EW lotMari ne colloids 1.1.1Introduction. . . . . Lt.zCharacrerfsticsofmarine collo ids .." .

1.1 .3Origin ,formationandfate of marinecolloids .. 5

1.1.4Importance ofmarinecolloids.. •. . . ... . . 7

1.1.5Chemicalcompositionofmarinecolloids•.. III 1.1.6 Sam pling of marine colloids.. , 14

1.2 lipids .. • . ..••. . . .. .•. .•••.... .•. ... . III 1.2.1 Introduction ... • • ..• •. .• •.• • ..•. HI 1.2.2GCmethods. •.• •... ...• •.. • . ... ..•. 19

1.2.3 The TLC·FIDmethod. . . . ... • . ... ... .. . .. 21

1.3 Carbohydrates .. . • •. .. •... . .• ..• • • . •. . .•.. .. . .. 22

1.3.1Chromatographicmethods •• • • •••••• . • 1.3.2 Col orimetr icmethods.• •... .•••.. •. 2.0OBJECTIVES •...•..• •.. . ...•. . .•. .. . 28

3.0MATE RIALS ANDMETHODS 31 3.tMat erials ... ... .. ... .. .. 31

3.1.1Chemicals . . .. ... .•.•. ... . . 31

3.1.2Filters . .. . . . ... .... 34

3.2.3Instruments . . .. ... . . . .. . . . .. 35

3.2Methods... .••.. . .. .,... 38

3.2.1Sampling. . . . .... 311

3.2.2Handlingof filters.. . .••. . . .... . 4r 3.2.3Treatment of samples..••.••. 42

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3.2 .4Measurernem oflipid classes.. 3.2.5Measurementof totalcarbohydrates

46 55

4.0 RESULTS ANDDISCUSSION ... .. 60

4. 1Blank fur measurements oflipid classes. 60

4.1.1Chrornarod-tatroscansystemblank. 60

4.1.2Extractingsolvent blank .. , 63

4.1.3Cross-flowfiltrat ionsystemblank 63

4.2Crossnow filtration. 67

4.2 .ICharacteristicsofCFF 67

4.2.2Optimization ofCFFsystem. 69

4.2 .3Massbalance ... . ... ... . . ... 69 4.3Lipid classconcentrations in seawatersamples 73

4.3 . 1 Total lip ids 73

4.3.2Hydrocarbons (He) .,. 76

4.3.3 Wax andsterolesters (WEISE) 81

4.3.4Triacylglycerols (TAG). 82

4.3.5 Free fauyacids(FFA) 82

4.3.6Free fattyalcohols (ALC) 84

4.3.7Sterols(ST) •.• •• • • • . • .. .. ... .. ..•. 55 4.3.8Acetone-mobile polarlip ids(AMPl)• .. 87 4.3.9Phosphol ipids(PL). . .. . , ... 88 4.4Particulate anddissolved lipids inseawater samples .. 89 4.5Lipidclasses and carbohydratesin microalgalcultures 98 4.5 .1Cellularlipid class concentration .. 98 4.5.2 Cellularcarbohydrateconcentrat ion 102

4.6 Carbohydrate in seawatersamples 102

4.6.1Totaldissolved carbohydrates 103

4.6.2Particulatecarbohydrates... ... . . . 106

4.7Marine colloids ... ...108

4.7.1Molecularweight of colloidallipids andcar bohydrates 108 4.7 .2Colloidalcarbohydrateandlipidclass concentrations 110 4.7.3Compositionof colloidallipids . ..114 4.7.4The distribution of lipidclasses . .... .119 5,0CONCLUSIONS ..

REFERENCES APPENDIX..•... . .

...122 ..126 ..142

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LISTOFTABLES

Table Puge

1.1 Percentage of colloidal organic carbon in"dissolv ed" organiccarbon l)

1.2 Cress-flowfiltrationin marine applications 16

3.t Marine lipidclasses and standards used for their ide ntificatio nand

calibrationbyTLC-FID... . J:!

3.2 Microalgalcultureconditions . 39

3.3 StationBRLP4hyd rography 39

3.4 Filtration flow diagram for anlsochrysis galbana (T-1SOlculture

sample. .. 45

3.5 Operatingconditionsforthe latroscan MK-5 TLC-FIDanalyzer 51 3.6 Flowchart fordetermining carbohydrates by the MBTH method. 57 4.1 Total lipid concentra tionsindissolved and particulatefractions ofseawater

in dif ferentlocations.. .. . ... . hi

4.2 Contributio nsto the blank by eachstepin the procedure 62 4.3 Comparison of totalblank (/lg)before filteringculturesampleswhh after

cleaning the CFF system with methanol-water(1:1vlv) 65 4.4 Lipid class concentrations(/lg/L, mean±S.D.,n= 4-8)in unfiltered

seawater samplescollectedin Conception Bay, Newfoundland. 74 4.5 Totalhydrocarbo n concentrat ions(/lg/L) in seawater.Da ta fromthe

literatureare the sum ofdissolved and particulateconcentratio ns. 78 4.6 Total free fattyacid concentrations(ltg/L)in seawater.Data from the

literaturearethe sumof dissolved and particula teconcentratio n.... .. 83 4.7 Total sterol concentrations(J4g/L)in seawater.Data fromthelitera ture are

the sumofdissolved and pa rticu late concentration. 86

viii

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4.8 Particulate(part.) and dissolved (diss.)lipid concentrationsin seawater

collectedinConception Bay in 1991and1993 92

4.9 Linear regressiononlipid class concentrationin 75 um tiltrationversus

ultraflhrate(10K-F) filtrate..•.. 94

4.10 Concentrations(pg/cell,mean±S.D.,n=4-8) andproportion(%)of solvent-extractable lipid classandtotalcarbohydrateco ncentrationsin

microalgal cultures 100

4.11 Dissolvedcarbohydrateconcentrations in filtered seawater 104 4.12 Carbohydrateconcentration(/A-MC/L .mean±S.D.•n= 3·5)in

seawatercollectedfromstation BRLP4in ConceptionBay.Newfoundland ... .. . .. . .. . . ...IDS 4.13 Totallipid concentrations(p.g/L ,mean±S.D.,n= 3-6) and total

carbohydrate concentrations (p.g/L,mean±S.D.,n=4·8) in different sire class particles formicroalgalsamples ...., ..,II I 4.14 Totallipidconcentrations(p.g/L,mean±S.D . ,n= 3·6)and total

carbohydrateconcentrations(Jlg/L,mean±S.D.•n=3·5) in different sire class particles for seawatersamplescollected fromConceptionBay, Newfoundland.•. • ., , , , ... . " ..,.. . 112 A I Flow diagram forthefiltration ofIsochrysisgalbana(T·iso)culture(5,8

x10" cell/mL.0.61div.lday)grown on semicontinuousand N-replete

condition. . 142

A2 Flow diagram forthe filtrationoflsochrysis galbana(T.iso)culture(4.2 x[0"cell/mL.0.90div.lday)grownon semicominuous andN·replete

condition ..••.. ,....•. ..143

A3 Flowdiagramfor [he filtration ofChaetoceros muellericulture (4.4 x 106 cell/mL. 0.24 div.lday)grown on semicontinuous and N-replete condition...,.... .• .... .. . . • .•.•. . ., .. . .,,,, ..144 A4 Flow diagram forthe filtrationof seawater sample collected Conception

Bay,NewfoundlandonMay19,1993 . " . ... ... . .•. . ...145

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AS Flowdiagramforthe filtration of seawater sample collectedConception Bay,Newfoundland on June 14,199] , .,, ,., , , , , , ,lolt;

A6 Flow diagramfor the filtrat ionofseawatersamplecollectedConception

Bay.Newfoundland on August2,1993 ' ,. lol7

A7 Flow diagramfor the filtrationof seawater samplecollected Conception

Bay,Newfoundland onOctober S.1993,, . WI

BI Analytical dam of lipid standard solutionby TLC-FID ,,149 B2 Linear regressionbetweenpeak area(rnv-mm) andlipidclass100ld

~.. . . . ... ... ..Im

B3 Therelationship between absorbency of colourcomplex compound and

standardglucose concentration(pM) ..ISI

B4 Regression betwee n absorbencyandglucosestandard solution(JtM) 152 C( Lipid classconcentrat ions (pg/L ,mean±S.D.. n

=

6-12) in diffcrclll

filtration fractionsof antsocnrystsgafbana (5,8x1(1'cells/mL,0.61 divJday) culture and reco veries(%)on threefillers , .ISJ C2 Lipidclassconce ntrations (lAg/L .mean±S.D.•n

=

6·11)in different

filtrationfractionsof anIsochrysisgalbana (4.2x10"cells/mL. 0.90 divJday)cultureandrecoveries(%)on threefilters , ., 154 C3 Lipid class concentrations(Jtg/L .mean±S.D.,n

=

6-12)indifferent

filtration fractions of aCnaetoceros muelleri(4.4 x 10" cclls/mt..0.24 div.rday)culture andrecoveries(%)on threefilters .. . . .ISS C4 Lipidclass concentrations (Jtg/L ,mean ±S.D., n = 6-12)and

distribution(%)in differentparticlepoolsand"dissolved" fractionofan Isochrysis galbana(5.8 x1(1 cells/roL,0.61div.lday)culture .. . . . 156 C5 Lipid classconcentrations (Jtg/L , mean±S.D. , n"" 6·12) and

distribution(%)in differentparticlepoolsand"dissolved" fractionof an Isochrysis galbana (4,2Itl<tcells/ mL. 0,90div.ld ay)culture 157

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C6 Lipid class concentrations (J..I g/L, mean ±S.D.,n "" 6-12)and distribution(%)in differentpanicle pools and "dissolved"fractionof a Ooetocerosmuelleri(4.4 x10" celts/ml.,0.24div./day)culture. .. IS8 C7 Lipid compositionpercentage (%,mean±S.D.•n "" 6·12)in different

particlepools,the dissolvedfractionand theunfilteredfraction for tsocbrysis ga/balla(5.8x 10" cells/mL ,0.61 div.lday)culture .. .. 159 C8 Lipidcomposkton percentage(%.mean±S.D.• n=6- 12) in different

particle:pools, the dissolvedfraction andthe unfilteredfract ionfor

tsocnrystsgotbana(4.2 x111cells/mL.0.90div./day)culture ., ..160 C9 Lipidcomposition percentage (%. mean±S.D.• n:::6·12)in different

part icle pools,the dissolvedfractionandthe unfilteredfractionfor Chaetocerosmuelleri(4.4x 10" cells/mL,0.24 div./day) culture. ..161 DI Lipid class concentration (ltg/L .mean±S.D"n ::: 6-10) indifferent

filtrationfractionofseawater collectedinConception Bay,Newfoundland in May19,1993andrecover ies (%)on two filters t62 02 Lipid class concentration(ltg/L ,mean±S.D.•n:::6-10)in different

filtrationfraction of seawatercollected inConceptionBay, Newfoundland inJune 24,1993 and recoveries (%)onIWOfilters ...163 D3 Lipidclass concentrati on(ltg/L .mean±S.D. ,n::6-10) indifferent

filtration fraction ofseawatercollectedinConceptionBay, Newfou ndland inAugust2,1993 andrecoveri es(%)ontwo filters .164 D4 Lipidclass concentra tion(J..Ig/L .mean±S.D.,n "" 6·10) in different

filtrationfractionofseawatercollectedin Conception Bay, Newfoundland inOctober5,1993 and recoveries (%)ontwofilters.... . .... 165 05 Lipidclass conce ntrations (J.lg/L, mean ±S.O.. n = 6·10) and

distrib ution(%)indifferentparticle poolsand"dissolved"fractionof seawater collected fromConceptionBayonMay19,1993 166 06 Lipid class concentrations (ltg/L, mean ±S.D. , n :::6-10) and

distributi on(%)indifferen t panicle poolsand"dissolved " fraction of seawater collectedfro mConception Bay onJune 24,1993. . .. . . 167

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07 Lip id class conce ntratio ns(Itg/L .mean ±S.D .•n = 6·10) and distribution(% )indifferentpa rt icle pools and"dissclv cd" fractio nof sea watercollected fro m Conception Ba y on August:!.1993 1M 08 Lip id classco nce ntratio ns (fig/L .mean ±S.D.• n =" 6.10) anti

distribution(%) ill different particle poolsarid"diss olved"fraction of seawatercollected from Co ncept ion BayonOctober5.1993 169 09 Lipid compositionpercentagein differentparticlepools.dissol ved . and

tota llipidinseawa terco llectedfro mConception Bay.Newfou ndland ,on

M~19.lm. . . ... . ....... . IW

010 Lipidcornposulo npercenta gein differentparticle pools .dissolved .and totallipidinseawater coll ectedfro m ConceptionBay.Newfoundland.on

June24.1 993... ,171

Dil Lipidcompositionpercentage in differentparticlepools. dissolved,and totallipidinseawater collect ed from ConceptionBay.Newfoundla nd.on

August2,1993 . In

D12 Lipidcompositio n percenta geindifferentparticle pools. dissolved.and total lipidin seawatercollected from Co nception Bay,Newf oundland , on

October 5.1993 ,173

013 Lipid class concentration (Jlgf L, mean±S.E. M,n=3)in different filtration fractionsofseawatercollectedin Conception Ba y on May19.

June14,August2,1993and mass balance(%)on two fillers 174

xii

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LIST OF FIGURES Figure

3.1 Diagram of MiJliporecross-now filtrationsystem

Page 36 3.2 Diagramsformajor operatingprocedu resin TLC-F IDmethod 37 3.3 Map showing the location of theBRLP4samplingstation.. 40 3.4 TypicalTLC-f[D chromatogramforlipid analysis in seawatersamples.

No internalstandard was added to thissample.... 50 3.5 Calibrat ioncurvesforeachlipid class measuredbyTLC-FID 52 3.6 Chemicalreactionsfor determinationof carbohydrateby theMBTH

method. .. ... . ... . ... . ... 56 3.7 Calibrationcurveforcarbohydrate measur ed bytheMBTH colori metric

method . .... . 59

4.1 Massbalance (%. mean±S.E.M.,n=3-4)oflipid classes in seawater sample for two filters ... ....•.... . . . .•. . . ... 72 4.2 Total lipidconcentrations inseawater... 75 4.3 Concentrationdistributionoftotallipid in particlepools anddissolved

fraction.•... .. ... .•.• . .•.•.. . . .••. • ... 91 4,4 Particulate(3nm-75,um)anddissolved « 3nm)lipidconcentrationsin

surface mixedlayer 93

4.5 Linear regression ofphospholipid concentration in75 ,urn filtrate versus ultrafilt rate (10K-F)fraction. .. .•.•.. ..••.• ..•..•... 9S 4.6 Colloidallipidclass concentratio ns (,ug/L, mean±S,D. ,n=3w5)for

Isochrysis ga/bana(5.8 x 1()6cells/ mL, 0.61div.l day) samp le .•.... . lIS 4.7 Colloidal lipid classconcentrat ion (,ug/L, mean±S.D. n=3-5) (or

Isochrysisgalbana(4.2x1()6cells/mL. 0.90 div.l day)samp le .• •.... 116

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4.8 Colloidallip id class concentra tion(ltg/L.mean±S.D.•n=3-5) for Cnaaoceros mud lt ri(4.4:1t 10"celts/ml.,0.24div.lday)sample .. . . .117 4.9 Lipid class concentration (""gIL.mean±SEM.n=3)in differentsize

fraction for surfaceseawatersamplecollected fromMay10Augusi.

ConceptionBay.NF. . . .... . . . ... . •. . . • .. . . . .... .118 4.10 Lipidclass distribution(%)illparticlepoolsand-dissolved-fraction for

seawatersamplescollected from MaytoAugusi.Conception Bay.NF. .110

xlv

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urn

JIM C 0.45Jlm-F 0.45Jirn-R IOK·F IOK·R

A1E~F

AII:-R ALC AMPL AOU

AVG C.Y.

CFF COC COM DOC

ABBREVIATIONS AND SYMBOLS

carhon-13isotope abundance micrometer

mfcrcmotesof carbonper litre

filtratepassed throughafilter with 0.45Jimporesize retentate ofa tilterwith0.45 Jimporesize filtratepassed througha filterwith 10,000NMWL cut-off retentateof afilter with10,000NMWL cut-off filtratepassed through a GelmanAlEfilter retentare of a GelmanAlEfilter free fattyalcohols acetone-mobilepolarlipid apparent oxygenutilization averagevalue coefficient of variation cross-nowfiltration colloidalorganiccarbon colloidalorganicmatter dissolvedorganic carbon

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Daltonx1000 ketone gaschromatography retemareofa WhatmanGFfCfilter filtratepassed throughaWhatmanCiF/Cfilter

hydrocarbon

highperformance liquidchromatography hightemperaturecatalytic oxidation inside diameter

dissolved organicmatter energy-dispersivex-ra yspectroscopy equiv alentsphericaldiameter

freefatty acid flow injectionanalysis nameionizationdetector

3-mernyl-2-bc.nlOlhiazolioonehydrazonehydrochloride n·..:.osspectroscopy

molecularweight nanometer nM nanomolesperlitre

KET

MBTH MS MW i.d.

kDa DOM

EDS ESD

FFA FIA FlO GC GF/C-R GF/C-F HC HPLC HTCO

NMR nuclear magneticresonance xvl

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NMWL OM pL

roc

'Pm RT S.D.

SE ST TAA TAG TDCHO

TEM TLC UF WCO

nominal molecular weight limit organic matter phosphol ipid particulate organiccarbon revolutionsper minute room temperature standard deviation sterolester sterol total amino acids triacylglycerol

totaldissolved carbohydrateconcentration transmissionelectron microscopy thinlayerchromatography unfilteredsample wetchemicaloxidation

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1.0 LITERA11JREREVIEW

1.1Marinecolloids t.t . tIntrodu ct ion

Colloidsarevery small pan icles withdimensionsbetween InmandIp.mthat exhibit Brownianmotion.Thisdefinition. involvinga wide range(Ifsizes. impliesthe ubiquity of colloidalsystems inthenaturalworld.

Inmarinescience.thedefinitionofthe"d issolved " fractionand the"panjcutate"

fractionisusu all y operational.These definitionsare quite simple.For example.the divis ion betweendissolvedand particulateorganicmatterdepends on whetherthe particles pass through a0.4511mme mbrane.Obviously.such a dclinitioncannot distinguishthetruly dissolved fractio n andthecolloidal fraction. Intact.the colloids!

fractionhasbeenneglec ted inthetraditionaloperationaldefinition.

Inrecentyears, therehasbeenanincreasingawarenessthatmarine colloidsplay an importantroleinbiogeochemical processes .especiallyintheoceaniccarboncycli ng and trace metalscavengingprocesses.Mar ine colloidsarenotonly differcnlfromlarge r panicles (>I~m)butalso fromthe truly dissolvedfraction«I nm).Unlike larger particles.marine collo idsdonotsettle inrespo nse togravitatio nal fletdsand hav ea larger liquid/solid interf ace than largerparticles(>I~m).Onthe otherhand, marine colloids,likedissolved man er.aresubjected 10 thesa metransportationandmix ing processes asthewatermass in whichtheyaresuspended,buttheirbiol ogicalanti chemicalpropertiesmaybevastly different fromthoseof trulydissolvedmatterof

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similargross chemical composition. Therefore. marine colloids have theirown character istics governing their oceanicbiogeochemical processes.

I. J.2 Characteristicsof marinecolloids

From theoperational perspective.marinecolloids include clay minerals.organic detritus.picoplankto n,bacter ia,viruses, andmacromolecules.Colloidal particlesin seawater have recently been foundto be quiteabundant.There are about1O~_1O'particles (sizerange 0.38- 1porn)per mLin the upperlayer (50 m)ofthe ocean (Koikeetat..

1990).Smaller marinecolloids«120 nm in size) are at leastthree ordersofmagnitude moreabundant than larger colloids (sizerange 0.38-1urn: Wells and Goldberg ,1991).

Thesize spectrain the openocean show increasesin colloid numbers withdecreasing size,often nearly logarithmicallyin surfacewaters (Wellsand Goldberg, 1993). Particles smallerthan 120 11m areextremelyabundantin coastal surfacewater,particularlynear the seasonal thermocline.Theyare3-6 orders of magnitudemoreabundantthan either bacteriaorplankton.

Verticalprofileshaveshown that colloids<120 nm insize were highlystratified with a mid-depthmaximum(Wellsand Goldberg, 1991).This distributionis different fromthat reponed for larger(O,38- 1.0 /-lm) colloidsinthe northwestPacific,where the concentrenon sharply decreased withdepth(Koikeet al.,1990).

Thelarge concentrationof marine colloidsin seawaterprovidesalarge solid/liquidinterface.The surface area is 0.2-0.5em' mL-1of seawaterforparticleswith

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size range 0.36-1.0Jim (Koikeetcl.,(990). Thetotalsurfacearea ofsmaJlcrmarine colloids (5- 12011m,10' particlesmL"I)reaches 0.08 em' mL·Jof seawater (Wellsand Goldberg,1992).

Koikeet al,(1990) found>95%of colloidalpanicles (OA- lJimin size) arc non- livingbutseem to be composedlargely of organicmaterial.Thisfinding was confirmed by Wells and Goldbe rg(1991),whenthey examinedcolloida lpart iclesby transmission electronmicroscopy (TEM)andenergy-dispersiveX-rayspectroscopy(EOS). Thelow electron opacity of thesecolloidssuggests that they are composedof largelyorganic materials thoughthey alsomay containsome rnecals such as Fe,AIandCo,TEM examinationalsoindicatedthatmanyof smallercolloids areaggregatesofgranules 2-5 nmin size (Wells and Goldberg,1992).

The residencetimeofcolloidalmatterwas studiedusingnaturallyoccurring !J..-l"h as aninsitutracer(MoranandBuesseier. 1992;Saruschietal.1994). Theresults show that the~)~activityassociatedwithcolloidalmatter (sizerange10k Oa100.2P.1lI)is similarto that associatedwithsmallerparticles(0.2-53pm). Both macromolecular colloidalmatterand smallerparticleshave a shortreside nce time (-<10 days) and a rapid turnoverratein theupperopen ocean.Sanachlet al,(1994) alsoconfirmedthe colloidal pumpingmodelproposedbyHoneymanand Santschi(1989).Thismodel involvesamorephysical ratherthan a biological drivingmechanismforcolloidturnover,

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Colloidalmatterwastho ught to be chemicallyrefrac tory and hiologicallylabile.

DOC valuesobtained by thehigh-te mperature catalyticoxidation(HTCO)methodare usua llyhigherthan those obtained by wet chemical oxidation(W e D)methods.Suzuki (1988) attribu ted pan of the newlyenlarged DOMpool to colloida lmacromo lecules(i.e..

nano metre-sizedl . The extracolloidal organic matter cou ld notbe oxidizedby UV irradi ation orpersulfa tcoxida tion,thus,it was thoughttobe che mically re fractory.On the otherhand.colloida lorgan icmailer was thoughtto bebiologicallylabilebecauseof correlatio nsbetween colloidal DOC andtheapparent oxygenutilization (AOU) (Sugimur aandSuzuki,1988; Suzukiet al..1992:Guoet at..1994).The staiemeru that colloidal mailer isbiologicall y labile could be supporte d indirec tly ordirectly by the followingfacts: theyhave sho rtresidencetimes and rapid turnover rate s(Mora n&

Bucsseter. 1992:Sautschlel at.,1994);theycanbeutilized ind irectlyordirectlyby bacteriaor microzoop lankton (Floodetal.,1992) ;bacterialrespiration andcell number bo th increase when

cae

andbacteria came into close contactthroughsurface coagulatio n(Jo hnsonetai.,1986 :Kepkay andJohnson ,19 88, 198 9;andKepkey.t990a, b).

Koikeet al.(1990)fo und halfof thelarger colloids(:>0.38 nm equivalent spherical diameter(ESD» canpassthroug h a O.I,umNucleporefilter.Thissuggests that marine collo idshavea highlyflexible,amorphousstruct u re.

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Thus. from avariety of perspec tives. marineco lloi ds do havetheir own characteristicsandbehaviourwhich shouldgreatly affect thechemis tryanticyclingof OMandtracemeta ls.

l.t.]Ori gin, formationand fat eofmar inecolloids

Althoughthe frequen cydistribut ionof largeparticles (1- 100 p.rn)varies geographica llyandwith depth (Sheldonet at.•1972).thesimil a ritiesincolloid morphology .abunda nce and size distributionin differe nt ocea nicareas suggestthnthcrc aresomegenera lmecha n ismsto explain theorigin ,formation.and removalofmarine colloids.

Asignifica nt linear correlat ion betweenthe number of submtcrom eirc particles (0.38-1p.m)andbac terialnumbe r indifferentpelagic statio nsaswell as chlo rophyll a indicates a biolog ical origin for these particles. The vertical stra titicatio n (nc ncon servenv ebehaviour)ofsmallercolloids «200 nm) and theirorgan ic nature sugges ttheyare derivedprimarilyfrombiologicalprocesses .

These colloids werethoughttohave abiogenicorigin,buthow werethey for med'!

Marine viruses, bacteria,and picoplankton maycontribute tothese abundant collo ids.

Theycan alsooriginate from releaseand degradation of microo rga nismsand the disruptionof particulatematerials.Monomericormacromolecularmaterialsofbioge nic originmayconde nse into colloidsbyflocc ulatio n (Sholkowitz , 1976),abiolog icalbind ing (Carlsona al. ,1985) ,and photochemicallyinitiated cross-li nking (Harveyetal.,1983).

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They mayalsobefor med bythe collapse of bubbles.and by growthinsiru(Biddanda.

1985;Johnso netat.•1986).At present.thefor matio n mechanismis stillnotclear.

Whatisthe fate of marinecolloids'!Sincemarine colloids donotsink directlyto thesedime nts.only threepathways canbeused to explainthefate ofmarine colloids:

dissolution. directutilization.andaggregation.Dissolutionappearstobethe least likely explanatio nfortheremoval ofmarinecolloidsfrom the wate rcolu mn.Hydrolytic enzymes havebeen assumedtore nder aggregates(>0.5~m )soluble(Smithet at..

1992),butif dissol ut ionwasthedominantmechanismforremovingmari necolloidsfrom thewatercolumn,it would be difficulltoexp lainwhyparticlenumbersincreasewith decreasingsilt.

Thecoagulationamong collo idsto formaggregates.andthen sinkingtosediment or utilizationbymicrozooplanktonappearstobethe principalmechanis m forre moving smallcolloid s(McC avc.19 84; Well sandGoldberg,19921.b).Anaggregae-domiraed mechanismiscons istent with . panicle size distribu tion inwhich panicle number increases nearlylogarithmically withdecreasingsize.McCave(1984)suggested thatthe aggregatio n ratesof colloidsismainlydeterminedby thecollision freque ncyand coalescence efficiencyin Brownianmotion.Johnsonand Kepkay's(1992) studies also highlightthe importanceofcoagulationand formationofaggrega tesbefo re largercolloids can be utilizedeffic ientlybybacteri a.The evidenceshowsmobilebacte riain a turbulent flow arcthe least efficientintheirabilitytointeractwith colloidsin the O.I- I p.msize range, However, fineColloidal matter canbe directly utilized as food by

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microzooptankron. Some studieshave suggested COMischemicallyrefractory but biologicallylabile.Themeanresidencetimeof smallercolloidswasreportedtobeshlvt and similartolarger colloids orlargerparticles(Morgantl al.•1992:Santsclti etal..

1994).All ofthissuggests colloidalmailercanbeutilizedaslarge particlesby microorganisms.Ontheotherhand,itisinterestingthat particle-sizespectraarereponed aslog normaldistributionswitha peakar about0.5 JIm (Lambertttlat..1980: Longhum etal.,1992).Thevalleyncarthcpeakseemsto indicate colloids withO.35 -U.4S~11lslze rangecan beutilized directlybesides beingefficiently utilized after they havetonncd largeraggregates.Th isjudge menthas beensupportedbynewevidence that oikoplcuritls consumecolloidalorganiccarbon insizerangesdowntoabout 0.2p.1Oindiameter (Floodttal.,1992).

Althoughme exactorigin,formation.andremovalmechanismsofmarinecolloids arenotclear atmemoment.the proposed mechanisms oftheirformation and removal are important10help usrecog nize the natureandroleof ma:inecolloidsin the ocean.

1.1.4Importanceof marinecolloids

Traditionally, itwasbelievedthatlargerpan icles(>IJIm)played themost importantrole inmarine carbondyna micsastheysettle out of watercolumn.Because marine colloids donotsink inwatercolumn,theywereconsiderednotto beasimportant as larger panicles. However.as thenatureofmarinecolloidsisbeingrevealed,the

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importanceofmarine colloidsin oceanic biogeochemicalprocessesis gradually being recognized.

Fir.~l l y .alarge quantityof"dissolved"organic carbon is actually presentincolloidal form.In1970.Ogurafound that between8%and12%of total dissolvedorgan iccarbon was inthe sizerangeofO.IJimand0.5urn.Alittlelater.Sharp (1973)alsofounda largeamountofparticulate carbon to be present incolloidal sizerange. Part icleswith arange of3nm to25 omcontainedabout10to30%of thetotalorganic carbonpresent.

Fromtheincreasingcollo idal organic carbon (COC) dataavailable(T a ble1.1). COC (I kDa to1.0Jtm) accounts for at least10%ofDOC,although theamountofDOCand CDC in theoceanisatpresentcontroversia land difficulttocompare becauseof differencesin blank corrections,analytical methLldo!ogy,spatial variability,and use of different filters,and alsobecause of the flexiblenatureof colloids.However ,most of theseindependentinvestigations (Table1.1)indicaterhara significa nt fractionof the

"d issolved-organicmatterin seawaterexists in colloidalform and thatthiscolloidal organic carbonis importantinthe dynamics ofthecarbon cycle.The latestdata(Guoet 01.•1994,BaueretaJ..1994)sho w as muchas45%ofDOCisincolloidalform(>I kDaMW).

As mentionedabove,marine colloids havea largesolldlliquidinterface (Wells and Goldberg,1992). Many interactions occur atinterfaces. Theseinterfacial reactions include adsorptionoftrace organicsand metals{Means and Wijayaratne. 1982}, polymerizationoforganic materialsadsorbedonsurfaces (Degensand Matheja,1967),

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Sit.Rano- P.,...nt...

\"4'

I

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"' . .

0.025-08"",

.

Il-l-U·5" W.5tC..,lraINoIthA!IlIn_coc:-n

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1976 >lkO. 10 - 15 .0-75 Gullol...oc;o

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1979 Z5Qlnlly >2CkO. -O.~ NoIthC...lil\ll

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..

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:

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10kOa -1JJm SugimY,.."dSuzuld>1.8 kO.

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Holli~.I". 10 kOa -l.2 J1'"

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lOkO« -0.2 j11'1l 404-11.5

>Ike. 40-53 50-131

>10ko. 8-14 50-131

0.01-0.71J1fl

Valuucalculatedbuildon ori£!illal(lalll

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coruprcxerfcn oftrace metals withorganic materials(Mooreetal.• 1979).and coagulationto formaggregates (JohnsonandKepkay.1992),Thesereactio ns canproduce macromolec ulesorlargercolloids. Adso rptionat particlesurfaces notonlyinfl uencesthe dist ributio n andconce ntra tionoftracemetalsand biogenicorganics but also theirblo- availability(SugaiandHenrichs.1992).The formation of macromo leculesand aggregates by interfacialactionsis animportant step in thebacterialutilizatio nofDOM(Joh nson andKcpkay,1992),Interfacia lactio ns on surfaces of marinecolloidsare important proc esses fo rtransferringdissolvedsubstances into particulate (>1Jlm)substances (FarleyandMorel.1986;Ho neyma nandSantschl, 1989. 1991).Itis thusapparentthat ma rine colloidscanplay an importan t rolein enrichingparticles withOMand trace metalsandmaybe responsible fortran sportin g contaminants intofood webs,

1.1.5Chemicalcomp ositionof marine colloids

Desp itetheimportance ofmar ine colloi ds in biogeochemical processes. relatively little progr esshas been madeon determ iningthe chemicalstructureand compos ition of colloidal matter.partlybeca useoftheappare ntlyco mplex, macro molec ular natureof co lloidal matter. the difficulties in isolating sufficient unaltered materia l for cha racteriza tion,andthelimitation in analytic al methodology.Limi ted studiesavailable ma inlyfocu s oneleme ntalcomponentssuchasnutrient elements(C.N.P)andtrace metals(AI.Fe,Mn.Cu,l.I4Th.'Be.ZIOPb),

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FromTable1.1.weknow about10-1511ofDOCis present incolloidalform.

If theaverageDOCcoocemrauonin theoceanistaken10be80 11MC.thereisabc'1Jt 200 ,IoIg-400 Ilg of colloidal organicmailer per litre ofseawater.lloUibaughet ot,(19911 investigatedcolloidal (size rangefrom10 kDa to1.211m)anddissolved« 10KO) nutrientconcentrationsin samplesfrom atemperateshoreenvironment(TomalesBay.

California).Theyfoundasignificantamo unt of organic carbon(28%).organicnitrogen (27%)andorganic phosphorus (71%)inGF/C(1.2.11m) filtrateswas presentin L"OlIoid;1I form(10kDa to\,211m).However.only3%ofreactive phosphorusand1%uf ammoniumwas presentincolloidalform.They found no evidence for nitrateplus nitrite or silicate as components ofmarine colloids.Similarly,Baueretat. (1994) fo und colloidalorganicmaterial(>IkOa MW)comprisedapproximately4511of thetotal DOC.TheA"'Cvalueofthiscolloidalfractionwasidenticalwiththose ofthe totalI:>OC whileh''C valuewassignifcntlylower thanthoseof total DOC.Their resultsshow colloidalorganic materialwas N-poor(C/N -15·19) relative to total dissolved organic matter(CIN-8·10).

Moran andMoore(1989)investigatedthedisiribution of colloidal(10kDa-U.45 ,101m)aluminum andorganiccarbon in coastaland openoceanwaters.'They found<5"

Atand<10-15%OC indissolved«10kDa) fractionswerepresentincolloidalform.

Some metals(Fe.Mn,Cu)associated with marine colloidswerestudiedby whhcbousc etai.(1990).Baskaran etal.(1992)proposedthal 80%of This associatedwith colloidal (10 kDato 0,4 j,(m)materialsinseawater samplesfromthe Gulf ofMexico.32%of

II

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lJvrhand8.3% of"Bein the"dissolved"«0.4,lim)fraction of coastalseawater was presentin colloidal(\0 kDatoOA/lm)form(Baskaranand Santschi,1993).

Thebiochemical composition(aminoacids, carbohydrates,and lipids)of colloidal matterhasbeeninvestigated mainlyinestuarineenvironments.Colloida lmatter inthe sizerange1.2nmto0.4 ,lim isolatedfrom ChesapeakeBay was studied bypyrolysis GC/MS(Stglecaal.,1982).The results obtained are consistentwiththe composition of ChesapeakeBayphytoplan kton (approxi mately50%protein .30% carbohydra te,10%

lip id, and 10% nucleotide by dry weight). These results suggest thataquatic microorgani sms are thedom inant sourceof colloidalorganicmaterialin estuarine surface water. Sigl eoet al.(\983)also found that thehydrolysableaminoacidsandassociated ammoniaaccountfor 80% ofthenitrogenthatexistedinestuarinecolloidal(1.2 nm-OA I!m)samp lesfromsurfacewatersthat ranged fro mfresh tobrackish water (12g/kg salinity).Totalcollo idal(1.2 nm-O.4S,lim) carbohydrates,aminoacids, andlipids in estuary wat er fro mChesapeakeBay accountedfor35· 60 %,4·13%and less than 1%, respective ly,of DOC«1.2 nm)(Meansand Wij ayaratne,1984).

Therefore, estuari ne colloids are composed mainly of carbohydra teand proteinaceousmater ials associatedwith differentamountsof clayminerals and trace metals. Whatthenis the compositionofmarinecolloids? Howdoes the salinityaffect the compositionofmarinecolloids?A strong negative correlatio nbetweencolloidalcarbon concentrationsand salinity was found.indicatingtha t the bulkof the colloidal matter in rivers preciparatedoncontact withcoastalseawater,Zsolnay (1979) suggestedthat

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coastalandestuarine colloidalmaterialare composed of different compounds.Coastal colloidal materialhadnosignifICantncorescerceorlightah'iOrplion intheUVand visiblerange.indicatinga paucityof aromatics andotherchromophores(Zsolnay.1979).

Inseawater.colloidalorganicmatter(COM)withMW betwee n3.<kXJandS.WI Daltons was indicated10beprote inaceous by Degens(1970).Itwas foundtllal proteinaceo usmattercancomprise upto60%ofthe colloidal nitrogen and over athird ofthe colloidalcarbon (Sharp,1975).However.recent researchwork (BauerI!till.1;11)4) showedCOMwasNcpoorOMrelativetoDOM.Benneretal. (1992)reponed that colloidal(IkDato 0.2#J.m)polysaccharidesaccount for-50 % ofDOC«IkDa)in surfacewater(10 m)and -25 %ofDOCin deep samples.Thisevidence indica tes that carbohydra te-proteinace ousmaterialsarealso majo rcomponents of marinecollokls apparentlyin disagreementwith theresultsof Maurer (1976) and Zsolnay (1979).M;lUrcr (1976)found thatonlyto-lSI of totalDOCexistsascompounds with a molecular weight>t.OOOand carbohydrateand proteinaceousmatterart:minorpropor lion inthese compounds.

Biogeochem icalprocesses inoceans canbeinfluenced greatlybythe chemical compositio n of marinecolloids.Knowledge of the chemical compositionofco lloidal matter can explain manybiogeochem icalprocesses , asthe chemicalnatureof colloidal matter is an importantfactor in determininghowitwillbeha vein relationto other substances inthe watercolumn. Withthe development of effective samplingtechniques

13

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andnew analytical methods, itshould bepossibleto find outtherelationships amongthe variousprocesses.

1.1.6Samplingof marinecolloids

Studies that assessthequantity, composition.distribution,andcharacterist icsof colloid.'> in aquatic systems require an efficient collection process for colloidal/macromolecular materials, There is a variety of methods (e.g , ultracentrifugation.size-exclusionchromatography.adsorptionandfiltration) to isolate macromolecular/colloidalmaterials.

Wellsetai.(1991)usedultracemrifugationto collect directly on specimengrids fortransmissionelectronmicroscopy(TEM) studies of colloidalnumber,size and distribution inseawater.Itis verydifficult using thismethod,toobtain alarge collection ofmacro molecular/co lloidal materials from seawater.

The conventionaladsorptionmethod for the separationofDOM fromseawater is based ontheadsorptionofacidified DOM ontononionic XADresinsanddepends on an adjustment ofpH.Themaj or shortcomingsofthismethod arelowrecoveryand preferencesforhydrophobic substances,as wellas otherconstituents of DOM.

Unliketheadsorptionmethod ,filtrationis based onthesize ofcolloidsrather than theirchemical properties. Thismethod doesnot dependon theDOMconstituentsof colloids, therefore the filtration method is widely used toobtain a colloidal Imacromolecular fraction. However. traditional ultrafiltrationhasastowfiltration rate

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(on me order of mUh)andthefillers areeasilyclogged.Wilhthe developmentof new Iiltranontechniquessuch as cross-flowfiltration.filtnuionhas become a powerful 1001 .n isolating colloidsfor variousmarinestudies (Table1.2).

Unlike theconventional filtra tion method.theflowin cross-flowfiltration(CFF) is parallelrathermanperpendicula r10thefiller. Tangentialflow Ilushes particlesaway from the filterthusreducingfilterclogg ing.Application of mutnpte filtersis helpfulin increasingthe filtrat ionrate.The filtrationrateof CFFcanreach Uh(Carlsonrttil.•

1985;Whitehouse .1990).Being ableto filterlargevolumesisveryimportantin accuratelydeterminingtrace materials intheaquatic colloidal fractionbecause the usc ofsmallsamples mag nifiesproblemscaused by conta mination of tracematerials.CFF decreases theconvent ional problemoflarger colloids Slayingonthesurfaceof membranes . anditmaintainsanapproximatelyconstantnominalfilter poresize during filtration.These features makeCFFsuitablefor studies of trace materialsinthe colloidal fraclion.

Inthe CFFmethod.colloidal/macro molecular fractions canbeobtained by filteringaprescreenedsample throughultrafilter membranes.Itis obvious thaIisolation of colloidallmacro molecularfracucnsdepe nds onthepreflltration filler and ultra filter membrane. Sheldonelal.(t 969)reported thatallthefillersremovedmanypart icles from suspens ionwhicharemuchsmaller thantheminimalporesire oftheflftcrs.

Therefor e.the poresizeofafilter is nOIagoodindicatorof itseffectivenessfor separatingdifferentsizefractionsof marinepan icles.

IS

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(39)

Afterfurtherstudyingseveraltypes ofmembranesand glass-fibre filters.Sheldon (1972)pointed out thattheaverageminimumsize ofpan icles retainedbysilver membranes(Plo tronic') and pclycarboneremembranes(Nuclepore')wassimilarto the statedporesizewhenrelativel ysmallseawatersampleswithmoderateconcentrations(If panicles werefiltered.However.allcellulose ester membranesretain particles much smallerthan the minimalporesize. Borosilicateglass-fibre filtersalsosuffe r fromthis problem.

Althoughthe glass-fibrefiltershavesome drawbacksas mentionedabove.they stillhavebeen employedwidelybecauseof theirrelatively high flow rates andhigh loadingcapacities (Altabet.1990).Organiccontaminantsonglass-fibretillersarceasily removed by combustionataround 400°C.Combustionof filtersaltersthe effectiveporc size towardthelarger size. In fact,no filtershaveauniformpore size and the specified filterpore size is a meanvalue. On theother hand,theparticle stzcdistribution inthe water columnis continuous.Thedefinitionsofparticulate,colloidal.and trulydissolved fractionarc arbitra ry.Therefore,the isolationof macromolecular/colloidalmatterdepend grc.atlyonthe choice of filters.

Polysclfone ultrafiltermembranesare usuallyemployedto seta division between macromolecular/colloidalmatterand the dissolvedfraction.Carlsonet al.(1985)re- evaluated the uncertaintiesof ultrafiltratio n by measuringamountsof DOMin uhrafiltratesrelative to the originalsolutions.A total of 9Am icon 43mm diameter ultrafiltrationmembranes(nominalMW cut-offrange from500 10 300,000Da)were

17

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examined and31oceanic sampleswereultrafiltered through filterswithporesizes rangingfrom 0.02to 1.2urn.The experiment indicatedthemajorityofultrafiller-derived MWdataarcconsistent with filterpatternsshown previously;earlierreports ofhigh-MW DOMin seawaterwereapparently exaggerated due10proceduralandultrafilterartifacts.

The other problemswithfiltrationinvolveadsorption ofsomedissolved organics.

especiallysur face-active materials(QuinnandMeyers.1971;Gordon and Sutcliffe,1974;

Uno.1976;Abdcl-Moati,1990) andthe release of dissolvedbiochemicals fromcelllysis caused by high filtration pressuresor by thefiller structureitself(Nagataand Kirchman.

1990).Contaminationbyfiltrationmembranes (Norrman,1993) and electrostatic rejectionof polyelectrolyteDOMcan alsobeproblems. Anyresearcher interestedin colloidssho uld be awareof theseproblems.Someprecautionsshouldbe takeninorder toisolate representativecolloidal/macromolecularmaterials.

1.2Lipids 1.2.1Introdu ction

Unlike the hydrophiliccompounds.amino acids,and carbohydrates,marine lipids constitutea group of hydrophobicorganic compoundseasilysoluble inorganicsolvents.

Acharacteristicof thechemicalcomposition of lipidsisthepresence of longalkylchains in the molecules giving them their hydrophobiccharacter. Because of this,lipids may ser ve as a means of transportforhydrophobic pollutantsin the marineenvironment.

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Thecompos lncnand quantityof the various lipid classes inlhe dissolved , colloidal.and particulatefraclions can providectues(0the natureand originoflhe organicmaterialas well as 10ilS role infoodweb dynamicsand contaminanttrans fer.

Due to Ihelowccnceneatjonand complexcomposiuo nof lipids inseewa rer.the methodolog ical difficulliesinvolvedin sampleprocessing,andthelowscusirivhyof analyticalinst rume ntation. reliablequa ntitative data wereunavailable untilthebne1960 ' ~ (Jef fery.1%6;zsclnay,197n.Since then.manyinvesdgatorshavemeasuredrnartrc lipids ,especiallyfanyacids andsterols.by GC·FIDorGC-MSmethods.

1.2.2GCmet hods

GC me thod s necessitate sample bandllng procedur es suc has cxtfaclion, evapo ration.saponlflcauon.andderivatization.Inorder 10 analyzelip idsin seawater. 1·2 Lof seawaterisusually collected and extracted withCHelJ.11pH 2.Ke nnicuu and Jeffrey(198l)used asample-to-solventrareof 70:Iforextraction.Theextractswere passed Ihrougha silicagelcolumnandthreefraclions(al iphatic.eSler/aromaticanda polarfract ion)werecollectedby elutingwithdiffere ntsolvents(hexa ne. benzene and rnelha nol).Each fractionwasconcentrated ina rotary-evapora tor and then analysedby GC witha glasscapillarycolumn coaled withSP·2100(OV- IOO(KennicuuandJeffrey, 1981),Venkatesanetai.(1987) frecdonate dMeOH/CB lCIJextracts into(WOparts (acidicand non-saponifiable)by saponification withO.5NKOHinI:1 MeOHl H,O.The acidicfractionwas reactedwith BFJ/MeOHand themethyl estersofthe fattyac ids were

19

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quantifiedby GC.Hydrocarbons,alcohols, andsterolswere isolatedfromdie non- saponifiablefractionbyTLC (silicagelwithCHJC1Jdevelopment). Alcohols and sterols were trimethylsilylated usingbis·(trimedlylsilyl).trifluoroacetamide. The silylated derivativeswerethen analyzed byGC-FID.Hydrocarbons were passed througha silica gelcolumnto separatealiphaticandaromaticfractions.and these were directlyquantified by GC.A fused silicacolumn(Ourabond 08·5J&W ScientificInc;25 m x0.25 mm;

0.25J.lm film)wasprogrammedfrom35to 290°C at 4°C/minandthen heldisothermally for about2h.Therecoveries forsterols ranged from80%to 92% and forhydrocarbons, alcohols,and fattyacidsfrom44%to 85%. Obviously, the analytical results werenot totally satisfactorybecauseof thelow recoveries.

Gomez·Belinchon(1988)determined hydrocarbonsand fattyacids indissolved andparticulatematterinadeltaic environmentbyGC-FID.Acolumn of25mx0.25 mmi.d.coatedwithSE·54 (filmthickness0.15 J.lrn)was used.Hydrogenwas used as thecarrier gas(SOmU min)andthetemperaturewas programmedfrom60to300°C at 6°Clmin. Before analysiJ.the samplewasprocessedby filtration,extraction. XAO·2 columnadsorption. hydrolysis. and esterificationwithIO~BF~MeOH.The limits of detection(orhydrocarbonsand fattyacidswas0.01nglLand 0.1ngIL.respectively.The recoveries andstandard deviationswerenot mentioned.

Until now,there havebeenfew reportson the analysisof phospholipidsin seawater.However,results frominvestigatorsworkingin arelated matrixmayprovide a veryuseful basis for developinga GC methodthat canbeusedin the analysisoflipid

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profil es inseawater.Kuks is (1975)andMybera oj.(19!W) deter mined theccnreraI}f fatty acids.alcohols.cbolesterci,triglyceride.andphosphol ipid inblood plasmabyOf afterthedephosphoryla tion andtrimethylailylaricnof samples.

At presen t,somelipidsclasses (hydrccarbons ,sterols/alcohols.fauy acids) C3n beanalyzedbyGC.However.mostGC methods necessitatelhe preparat ion\If derivativesforsomelipidclasses(fattyacidsand sterols)andtherracuoreuonoftllher lipid classes.These proceduresareusuallylabour-intensiveand pro ne10cnntamimuiou,

l.2.3TheTLC-FIDmethod

SinceIaeoscanthin-layerchromatographywith flame io nizat ionde«..'Ctio nflU'·

FlO)becameavailablein1970,it hasbeenusedextensivelyfortheanalysis ofsimple and complex lipidsin biologyandmedici ne.Inthe early1980's,TLC-F IDwasusedfor the analysis of lipids in marine samples(Parri shandAckman.1983). This method combines the separationefficienc yof TlCand the detect ion sens ilivity ofFlO.This methodismuchsimplerthan GC-FID.UnlikeaC-FIO.TlC-FIOdoesno tneccsslrare [hepreparanonofvolatilederivatives anditcanbealso usedfor shipboar d analysis (Delmasetal.,1984).

Proc ed uresused for sa mpling, extraction. sto ra ge. concentrat io n.sample application, development, detecnon.andcalibrationby FlOhavebeendescrib edby Parr ish (1987).Seawate rsampleswerescreenedthrough a200pm nylonmeshto remove

21

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larger particles.Thena precombusted (400~C)glass fibre filterwasused tofractionate seawater into dissolvedand particulatemauer.Dissolved lipids were extractedinto dichloromethaneand particulate lipidswereextracted intoCH~C1/MeOH(2:I). Before extraction.hexadecan-f-onewasaddedto eachfractionasan interna l standard.The extractswereconcentratedbyevaporation undera gentle nitrogenflow.Afew microlhresofa co ncentratedextract was spotted ontothe Chromarod using a syringe.

Complexlipid mixturesmaybeseparatedin 3-slep separationswithpartial scanning between developments.Hexane-dimethyl ether-formic acid (H DF99: 1.0:0.1 ) was usually usedasthefirstdeve lopmentsystem(ParrishandAckman.1983).The operating conditionsfor thelarroscan TLC·FIDwasadjustedtomaximize FlO response . Thehydrogen flowrate wasbetween 170 and 190mUmin.theairflow rarewas2000 mUmin,andthesca n speed was between3. 1and 4.2mm/sfor routinelyuse .

A stocksolutionof aneight-componentstandardwasused to make calibration curves.In orderto obtaina precision that was better than 10% coeffic ient of variation (c.V.).extensive calibration wasnecessary,especiallyintrace analyses. Lowerloads tendtogivehigher C.V.values.

1.3 Carbo hydrates

As theprimaryproductofphotosynthesis, carbohydratesareanimportantform of energy storagefor autotropbsas well asfor the heterotrophswhichconsume them.

Car bohydrates maybereleasedinto seawaterby living organismsduring growth periods

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or afterdeath (Magueet01..1980 ).Becauseofthe lowconemof carbcbjdraresin the

\'ery complex seawatermatrix. characterizing carbohydratesis ratherdiffiruh.Isolation and purification Ste-psartusuallyneededbeforeany chromatographicprocedure.with consequentlosses and changesin consuruents.Becauseof these problems.varices analytical methodshave been lnvesngaredinordertodeveloparel iable routine me thod to analyzecarbohydrates inseawater.

1.3.1Chromatographicmethods

Paper chromatographysuffersfromlowsensitivity.Gas chromatographyis a highlysensitivetechniquefor analyzing volatilecompounds.Althoughitisposs ible10 convert non-vo latilecarbohydrates10volatiletrimethylsilylderivativesfModzeskiet al..

1971)oracerylaredderivatives(Sakugawaet01.•1985:Ochialttal.•1988>. desalting and concentration of seawater samplesarerequired before denvenzation canbecarried out.Theseprocedures are laboriousanditis diffICultto avoid contamination.

Partitionchromatography of sugarson anion-exchange resi nsalso has serious shortc omings when appliedtotheanalysisofmar ine samples.There is interferencefrom othercompounds insamples afterpreccnceneaucn.leadingtoarapid deterioration inlhe performanceoftheanion-exchange column(MapperandDegens.1972).Although the chroma tographyoftheboratecomplexes ofcarbohydrates on strong anion-exchange resins with a sensitive colorimetric regent (Cul ' /aspartic acid/sodium 2,2' · bicinchoninnate )overco mestheabovedrawbacks (Mapper andGindter.19731.the

23

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seawatersample stillneedstobe desalted andconcentratedusing ion-exchange.

electrodialysisandfreeze drying(Iyophilisation). Aswith gaschromatographic analyses.

theseparationof traceamounts of carbohydrates from salts in seawaterwas impractical for routine analysis(MopperandGindler,1973).However.because GC canbeused with MS, itwas a veryimportanttool foridentifyingindividualmonosaccharidesand short oligosaccharides.Nineoligosaccharidesin dissolvedfractionof seawaterwere quantified with a rangeof0.5-28~g/LbyGC. GC·MS.andIH_NM R(Sakugawaet ai..1985a. b).

Anemptstoemploy fluorornetriclocating reagentsfor the detectionof the carbohydrates inseawaterafter ion-exchangechromatographyhavenotresulted inany significantimprovementin sensitivityand precision (Mopperet al. ,1980: Mopperand Johnson.1983). HPLC determinationof carbohydrateshas few applicationsbecauseof thelack of a suitablefluorescent reagent for sugars.It is noteworthythat Compianoet at.(1993) analyzedthe monosaccharidecompositionin seawatersamplesbyHPLC and fluorescence detectionafter dansylhydrazineprecolumn derivatization (65°C. 20 minutes) andobtained good precision.Thedetectionlimit var ied from5.5nmol/Lfor rhamnoseto 30 nmot/t,for gtocose.and reproducibilityvariedfrom I%for glucoseto 6%forarabinose.

Becausethe composition of monosaccharides can be analyzedby HPLCmethods, HPLCis a potentialmethod10befully developed. Its development depends on the availab ilityand sensitivityQfsugar-specificfluorochromereagents for highsaltmatrix samples.

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1.3.2Cctorl metrtcmethods

Manyearliercolorimetric methodsemployingN-cthylcarbazoJe.anthrone,lind phenolsulphuricacid(Duboisetof..1956: Gerchakovand Hatcher.1972) have three fundamental problems for measuring totalcarbohydrate concentrations (Burneyet af.•

1977): the calibration curveis differentfor each carbohydrate;some reagents do 110!

detect sugar alcohols;and the useofstrong acids can produceother degradation products affecting detection(Josefssonet af .•(972),

These shortcomings may beovercomeby the methodof Johnsonand Sicburlh (1977). Theymadesome modifications10 minimizecontamination of samples in assaying procedures.The method includesreduction of freemonosaccharidesto sugar alcohols withKBH~or NaBH..pertodateoxidation.and the determinationof derivatives

or

formaldehydewith 3-methyl-2-benzothiazolinon ehydrazone hydrochloride(MBTI-I)and FeCisreagent.The reactionof formaldehyde with MBTHandFeCI)produced a coloured complex , which dissolvedin50%acetone.The molar absorptivityof this complex (43,600pM-Ic m"' at 635 nm) wasnot affectedby changesinsalinity.Themodified proceduredecreased the standard deviationby50%from older methods(from 5.310 2,7 pg/L) and allowed a single analyst to determine 15 instead of 8 samplesinthesame two dayperiod.The limitof detectionwas180 nmol/Lfor formaldehyde when a path length of 1emwas used(Eberhardt and Sieburth, 1985).Analyticalprecisionfor total carbohydrate analyseswas10%(Henrichsand Williams, 1985).

25

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The MOTH methodisthus a good procedurebecauseof itssensitivity andit shows nosalinity dependence,andneeds nopreconccotranonandseparation steps. The otheradvantagesof thismethodare thatboth total dissolved monomericcarbohydrate (MC HO) and totalcarbohydrate concentrations(TCHO)can bedetermined whena hydrolysisstepis usedtohydrolysecombinedcarbohydrate. Hence,this methodhas been widely acceptedbymanylaboratoriestodetermine total carbohydratefractions in seawater.Full details ofthis method anditsmodificationsmaybefound in the following papers(BurneyandSieburth,1977; Johnsonand Sieburth,1977;Johnson et al., 1981;

Henrichsand Williams,1985:Pakulskiand Benner,1992).

However. this method does suffer fromsome shortcomings.Itis a time- consumingmethodandcannotbeusedtodetermine the compositionofmonosaccharides.

Obviously,thismethodcannotdistinguishbetween truemonosaccharides and any substances which possessaterminalglycolgroup.Asaresult,the total dissolved carbo hydrateconcentrationsperhaps do not closely reflect biologicalavailability of carbohydrates(SeniorandChevolor. 1991).As well.the hydrolysisof combined carbohydrateswithdilutedhydrochloricacid maynotbe effectiveenough.Pakulskiand Benner(1992) found thatsulphuricacidwasmore effectivethan dilute hydrochloric acid inhydrolysingoligosaceharides. Forexample,dissolved carbohydratesdete:minedbythe MBT H methodafter10%He lhydrolysis accounted for6-10%oftheDOC , whereas whensulphuric acid was usedthe carbohydrate fraction accountedforsubstantlymore oftheDOC (tQ..28%). Benner etal.(1992)alsoapplieda modified MOTHmethodwith

(49)

HlSO~hydroly sis todeterminethe amountof carbohydra tein the colloidalsize fraction (1-200nm) ofseawatersamples. Theresultsofsucha modified MBTHmethod were similar10the those determined independently using a uC-NMRmethod.Hence. more studiesusing the modified MBTHassay shouldyieldamorereasonableestimate of total carbohydrate contentin seawater.

The onlydrawbackin themodified methodis therequirement that thesamplebe speciallydried before addition of concentrated H2S04,Itwas found thaisamp le evaporation using arotary evaporator resultedinlossofcarbohydrate trappedill the inorganicsalts.Henc e . thisstudydidnot adoptthemodified MBTH methodof Pakulski and Benneret al. (199 2)but. becauseof the lackoftherequired evaporator.the diluted Helhydrolyzed stepwas used.

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2.0 OBJECTIVES

DOMexerts a significant influence on biogeochemicalprocesses. Thedynamics of the DOM pool is a key factor in understanding carbon flux in the ocean. After the report ofhighconcentrations ofDOC(Suzukiet al.,1985, 1988. 1992) and of the high number of colloidal panicles (Koikeet af.•\990;WellandGoldberg,1991)found10 existin seawater. more and more marine scientists realized the importance of marine colloids in biogeochemical cycles inthe oceans.This is particularly true since marinecolloids can aggregateand then can be utilizedor, settle out of Ihe water column(Welland Goldberg, 1993).

Because of lhe large solidlliquid interface,marine colloidswere believed 10 be very important in interfacial processes such as adsorption. polymerization,complexation, and coagulation. These interfacialprocesses playa key factor in the transfer of dissolved substances into particulate maner.

In spite of theimportance of marine colloids,relatively little progresshas been made onthe chemicalcompositionor structure of marinecolloids. At present.less than 35%ofnOMhas been characterized.owning to the llmitations of collecting sufficient amounts of unalteredOMand of the lack of sufficient analyticalmethods. Thus, the characterizationof the chemical composition ofOM,especially of marinecolloids, continues 10 challenge many marinechemists.

(51)

TheoverallobjectiveofthisstudyWMtounderstand furtherthechemjcatreauucs and roles of mari necolloidsin biogeochemical cycles by characterizing thechemical composition of marinecolloidsassociated with differen t pan icle size classes. Toreach thisobjective.CFFwas used to concentratemarinecolloidsfrom microalgalcultureand fromseawatersamplesbecauseCFFcanrapidly filteralarge volume ofseawater and effectivelycollectmarine colloids.Water samples~reobtainedfromlaboratoryalgal culturesin order to developmethodsfor waterswithhigh concentrationsof cul1uidll.

Seawatersampleswere thencollected inatimecoursefollowing the spring phytoplankton bloom in ConceptionBay,Newfoundland.

The relativelyprecisetechniqueof TLC-FIDwasemployed toanalyze the lipid classesbecauseit doesnor necessitatethe prepara tion of volatilederivativesasGCdocs.

Total carbohydrate concentrations inseawater and algal cultures wasdeterminedusing theestablishedand widely used MBTH method.Analyses of boththelipids and thc carbohydrates usingaccuratemeasure mentswere used(i)to evaluatetheeffteiencil...sef CFFbycalculationof the mass balanceon differentfillers.(ii)(0illustrate partitioning oforganics betweentrulydissolvedand colloidalfractions.and(iii)toexplainsome biogeochem icalprocesses.

Thespeci ficobjectiveswere;

(a) to assess cross-now filtrationas ameans of separarlng andconccntratingmarine colloids bystudying:

(I)The blank lipidvalues oftheCFFsystemformeasuringlipid classes;

29

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(2)a mass balance forall the lipidclasses in alltheparticlesize classes:

(b)toapplycross-flowfiltration to collectmari necolloi dspresentinalgalculturesand in localseawatersamples. The two types ofculturesamplesanalyzed camefrom laboratorycultures of phytoplankton(Chaetocerosmuetten. and tsocnrysisgalbana);

(c)to measurelipid....ldcarbohydrateconcentra tionsindifferent filtration fractionsand to apply linearregression to analyzethe data.The resultsof theanalyses of lipid classes andof totalcarbohy drate!'might help explainthe roleof marinecollo ids in biogeochemical cycles.

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3.0 MATER IALS AND METHODS

3.1 Materia is 3.1.1Chemicals Analysisof lipids

For theanalysisof lipids.therepresentativecompoundsinTuble3.1wereused as standardsto identifyandquantify lipid classes.These compoundsarc representative of themarineenvironment.Allofthese compoundwerepurchased from SigmaChemical Co.Inordertomake acalibration curve, a stock standardsolutionofthesecompounds wasmadeinchloroformand storedundernitrogenat·20°C.Becausethereisverylillie ketone presentin most marine samples. bexdecan-f-orewas usuallyusedasan internal standard.Development solvents usedforseparation oflipidclasses artchloroform, formic acid.acetone ,dkthyletherandhexane.

Ana1nis ofcarbohydra tes

Glucosewasused

as

astandard forthe spec rropbxomemc measurementof carbohydrates.The otherreagents include hydrochloricacid.sodiumhydroxide.sodium borchy dn de.periodicacid,sodiumarsenite.3·methyl.2.benzothiarolinonehydrazone hydrochloride(MBTH). ferricchloride,andacetone.Allofthesechemicals were analyticalgradereage nts. MBTHandsodiumborohydridewerekeptinadesiccatorin a refrigerator.

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Tuble 3.1Marinelipid classes and standardsused for theiridentificationand calibration by TLC·FID

Marinelipid Modelcompo und ~

Class Abbreviation Name Formula mg/mL

Aliphatic

He

s-Nonedecane' C\9HoIO 0.5834

hydrocarbon

Sterol ester SE Cholesteryl C41H7~01 0.5200

hexadecanoate'

Ketone KET 3-Hexdecanonel C,&H3ZO 0.8235 Triacylglycerol TAG Glyceryl C' IH93Or, 0.9855

mhexadecenoate'

Freefatty FFA Hexadecanoic C16H1202 0.4074

acid acid'

Free aliphatic ALe l-Hexadecanol' C\&H34O 0.3658 alcohol

Sterol ST Cholesterol1 C27H46O 0.5115

Acetone-mobile AMPL Glyceeyl CI9HnO. 1.0006

polarlipid l-monohexadecanoate-

Phospholipid PL Phosphattdyl C-lllH~NO,P 0.7622

choline!

IStoredin desiccatoratroom temperature 2Storedin desiccator at<0°C

JStored indesiccatorat 0.5°C

(55)

I.Sodium toronydrtdesolution

NaBH~(100mg)wasdissol vedin 5.0mL ofchilleddoublydistilledwater(ahOUI 40c) . Thissolutionwasprepared for immediate use.

2. Periodic acidsotuuon

Periodicacid (0.57g)was dissolvedin100 mLof doublydistilled water.This solution was stored at room temperatur ein the dark.

3,Sodiumarsenitesolution

Sodium arsenite (3,2 5g) was dissolved in100 mL of redistilledwater.The solution is stable indefinit ely.

4.Ferricchloridesolution

FeCI)(5.0g)wasdissolvedin 100mLofdoublydistilled water.The FeCI, solutionwas filtered througha WhatmanGFtCfilterand storedat SoC.

5.MBTHsolution

MBTH(276)mg was dissolved in 10 mL of 0. 1NHCI with warm ing.The solution was filtered through aWhatmanGF/Cfilter,if any precipitate rema ined,and was storedin a clean.amberbottle atroo m temperature.This solut ionwas prepared freshlyeveryday.

33

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3.1.2Flners Gla5-~fibrefillers

Gelman' AlEglassfibrefillers(Cal.No.6[63 1) withnominalporesize I~m (liquidrating 95%).diameter 47mm,and typicalthicknessof450I'm wereused. The manufacturers state thatthey are widelyused forfilteringnatura l water for studiesof colloidaland suspended solids or the "dissolved"fraction becausetheyprovide high flow rates(550 mLmin"cm''),greatwetstrength, andhigh solid-holdingcapacity.

Whatman'GF/C(Ca t.No. 1822.(4 7)glassfibre filters with a nominalparticle retentionof1.2sm.typical thickness of260pm,and a diameterof47mmwerealso used.The manufacturersstatethat theyhavea good flow rate,highloading capacity,and low water absorption (250 mLlml).Theyarewidelyused forthe collection of sus pended particles in potables,naturaland indusuial-wastewaters.

MicmporQlIsand ultrafiltermembrane

Four Mintain'mlcropo rous filter plates (Cat.No. HVLPOMP04) were usedin a cross-flow filtration apparatus, Each plateis constructed by heat-bindingtWO hydrophillic Durapore" filtermembranesto a polyvinylldenefluoride(PVDF) backing.

The nominal pore sizeofthe Durapore'filtermembrane is 0.45 sm.

FourMinitan 'ultrafil tration filterplates(Cal.No.PTGC OMP 04)were also used.Each filler plate consistsof two potysulfore filtermembranes heat-bondedto a polypropylenebacking.The uhrafiltration membranehas ano minal MWcut-off of 10 ,000 Da.The filterareafor each filterplateis60em' ,and four plates wereusually

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