Response of the coccolithophore Emiliania huxleyi to increased CO2 and Fe availability within the plankton food web

! AUTOR: María del Rosario Lorenzo Garrido http://orcid.org/0000-0003-2927-2184 EDITA: Publicaciones y Divulgación Científica. Universidad de Málaga Esta obra está bajo una licencia de Creative Commons ReconocimientoNoComercial-SinObraDerivada 4.0 Internacional: http://creativecommons.org/licenses/by-nc-nd/4.0/legalcode Cualquier parte de esta obra se puede reproducir sin autorización pero con el reconocimiento y atribución de los autores. No se puede hacer uso comercial de la obra y no se puede alterar, transformar o hacer obras derivadas. Esta Tesis Doctoral está depositada en el Repositorio Institucional de la Universidad de Málaga (RIUMA): riuma.uma.es ! Response!of!the!coccolithophore!Emiliania!huxleyi!to! increased!CO2!and!Fe!availability!within!the!plankton! food!web! ! ! ! ! ! ! ! ! ! ! ! ! ! Doctoral!Thesis! María!del!Rosario!Lorenzo!Garrido! ! ! ! ! UNIVERSIDAD!DE!MÁLAGA! FACULTAD!DE!CIENCIAS! DEPARTAMENTO!DE!ECOLOGÍA!Y!GEOLOGÍA! Área!de!Ecología! ! ! Response!of!the!coccolithophore!Emiliania!huxleyi!to! increased!CO2!and!Fe!availability!within!the!plankton! food!web! ! ! ! ! ! Memoria!presentada!para!optar!al!! grado!de!Doctor!en!Ciencias!Ambientales! !por!María!del!Rosario!Lorenzo!Garrido! ! ! ! ! Dirigida!por!María!Segovia!Azcorra! ! ! ! ! ! Esta!Tesis!doctoral!ha!sido!subvencionada!por!el!Ministerio!de!Educación!mediante! una! beca! de! Formación! de! Profesorado! Universitario! (AP2010-5565)! y! por!el! Ministerio!de!Ciencia!e!Innovación!Consejería!de!Innovación,!Ciencia!y!Empresa,! (MICINN)! mediante! el!proyecto! de! investigación! “Interferencia! de! la! disponibilidad!de!hierro!sobre!el!aumento!de!CO2!y!radiación!UV!en!procesos!de! estrés! oxidativo! y! muerte!celular! en! fitoplancton! marino:! consecuencias! en! la! modificación! de! la! diversidad”! (CTM/MAR2010-17216)! cuya! investigadora! principal!es!la!Dra.!María!Segovia!Azcorra.! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Diseño!portada:!Caren!García!Gómez! Imagen!portada:!Ainhoa!López!Segovia! ! Contents! ! ! ! ! ! Chapter!1! General!Introduction!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! Thesis!outline! 1! Chapter!2! Iron!availability!modulates!the!effects!of!future!CO2!levels! within!the!marine!planktonic!food!web! 17! Chapter!3! Effects! of! increased! CO2!and! iron! availability! on! the! carbon!assimilation!and!calcification!during!an!! Emiliania!huxleyi!bloom! 53! Chapter!4! Physiological!stress!response!associated!to!elevated!CO2! and! dissolved! iron! in! a! phytoplankton! community! dominated!by!the!coccolithophore!Emiliania!huxleyi! 85! Chapter!5! Particulate! trace! metal! dynamics! in! response! to! increased!CO2!and!iron!availability!in!a!coastal!mesocosm! experiment! 121! Chapter!6! ! ! ! Interactive! effects! of! CO2,!Fe,!and! UV! radiation! on! primary! production! and! calcification! of! the! coccolithophore! Emiliania! huxleyi:! In! vitro!physiological! response! 6.1! Effects! of!elevated! CO2!in! the! coccolithophore! Emiliania! huxleyi:! Effects! on! growth,! photosynthesis,! calcification!and!spectral!sensitivity!of!photoinhibition! 6.2! Short-term! response! to! the! effects! of! Fe! availability,! increased! CO2!and! UV! radiation! of! Emiliania!huxleyi! ! ! 153! ! ! 183! Chapter!7! General!discussion!and!conclusions! 211! References! ! 221! List!of!Figures!and!Tables! 243! Resumen! ! 247! Abbreviations, ! A:#Anteraxanthin# Al:#Aluminium# BED:#Biological#effective#dose# BEI:#Biological#effective#irradiance# BER:#Base#excision#repair# BUTA:#19’#butanoyloxyfucoxanthin# BWF:#Biological#weighting#function# CAL:#Central#area#length## CAW:#Central#area#width# CCM:#Carbon#concentration#mechanism# Cd:#Cadmium# CFC:#Chlorofluorocarbon# Chl-a:#Chlorophyll#a# Ci#:#Inorganic##carbon# CL-FIA:#Flow#injection#analysis#chemiluminiscence#detection#system# Co:#Cobalt# CO2:#Carbon#dioxide# CO3 2-:#Carbonate#ion# CPD:#Cyclobutane#pyrimidine#dimmer# Cu:#Copper# DD:#Diadinoxanthin#(DD)## dFe:#Dissolved#iron# DBS:#Dextran-bound#sulphanilamide# DFB:#Desferrioxamine#B## DIC:#Dissolved#inorganic#carbon# !General!Introduction!!!!!!!!!!!!!!! ! ! 5! Effects!of!ocean!acidification!on!phytoplankton! The!extent!to!which!phytoplankton!may!respond!to!increased!CO2!(decreased!pH)! is! likely! to! depend! on! the! physiological! mechanisms! of! inorganic! carbon! uptake! and!intracellular!assimilation.!During!photosynthesis,!CO2!is!fixed!in!phytoplankton! by! the! carboxylating! enzyme! Rubisco! (ribulose-1,! 5-biphosphate! carboxylase/oxygenase).! However,! for! most! species,! Rubisco! is! less! than! half! saturated!under!current!CO2!levels!(Riebesell!2004).!Furthermore,!the!dual!role!of! the! Rubisco! as! oxygenase! produces! a! significant! inhibition! of! CO2!fixation.! ! This! limited!CO2!fixation!together!with!Rubisco’s!low!affinity!for!CO2!is!counterbalanced! by!the!operation!of!carbon!concentrating!mechanisms!(CCMs)!that!provide!a!CO2rich!environment!around!the!enzyme,!suppressing!the!oxygenase!and!saturating! the! carboxylase! activity! (Beardall! &! Raven! 2004).! Most! marine! phytoplankton! species! show! little! effect! on! their! photosynthetic! rates! because! their! photosynthesis!is!already!saturated!by!carbon,!due!to!the!presence!of!CCMs!that! actively!take!up!inorganic!carbon,!either!as!CO2!or!HCO3 -!or!both!(Giordano!et!al.! 2005).! Increased! CO2!might! directly! benefit! biomass! production! either! by! enhancing!the!carboxylation!reaction!at!Rubisco!(Raven!et!al.!2005),!by!reducing! CO2!leakage!(Rost!et!al.!2006),!or!by!allowing!a!down-regulation!of!activity!of!the! energy-consuming!CCMs! (Kranz! et! al.!2010).! Raven! (1991)! proposed! that! a! downregulation!of!the!photosynthetic!machinery!in!phytoplankton!could!increase! the!resource!use!efficiency.!On!the!other!hand,!the!predicted!pH!drop!represents! an!increase!in!the!H+!concentrations,!which!may!affect!intracellular!pH,!membrane! potential,!energy!portioning!and,!enzyme!activity!(Flynn!et!al.!2012,!Taylor!et!al.! 2012).!Ocean!acidification!may!reduce!phytoplankton!growth!rates!through!direct! pH! effects!(Berge! et! al.!2010).! Thus,! ocean! acidification!may! potentially! both! stimulate!and!reduce,!due!to!the!increase!in!CO2!concentration!and!through!direct! pH!effects!by!marine!phytoplankton,!respectively.! The!decrease!in!oceanic!pH!caused!by!elevated!CO2!will!impact!calcification! rates.!CaCO3!is!one!of!the!most!common!building!materials!used!in!the!formation! Chapter(1( ! ! 6( of( skeletons,( shells,( and( other( protective( structures( in( the( marine( biota.( Calcification( is(facilitated( by( high( pH( and( high( CO3 2–(concentration.( These( conditions(are(achieved(at(the(site(of(calcification(through(energy-consuming(ion( transport( processes(such( as( for( instance( phosphate( uptake( (Paasche( 2002).( The( energetic( cost( of( calcification( is( thought( to( increase(with( ocean( acidification( causing( a( decrease( in( pH( and( in( the( carbonate( (CO3 2–)( concentration.(This(extra( energy(needed(to(compensate(for(the( changes( in(seawater(carbonate( chemistry( depends( on( the( specific( pathways( employed( in( CaCO3(precipitation,( specific(for( each(taxonomic(group.(A(case(in(point(is(coccolithophores((Prymnesiophyceae),(a( group(of(unicellular(microalgae(growing(in(the(surface(layer(of(the(ocean(and(the( most(productive(calcifying(organisms(in(the(sea((Paasche(2002).(It(is(known(that( intracellular(pH(in(coccolithophores(is(particularly(sensitive(to(changes(in(external( pH((Suffrian(et(al.(2011,(Flynn(et(al.(2012,(Taylor(et(al.(2012).(This(indicates(that( the( direct( negative( effect( of( high( H+(concentration( on( calcification( rates( may( at( some( point( be( overcome( by( increasing( availability( of( HCO3 -(substrate( of( calcification((Bach(et(al.(2013).(Therefore,(the(majority(of(coccolithophores(reduce( their( level( of( calcification( when(growing( at(elevated( CO2((Langer(et(al.(2009,(De( Bodt(et(al.(2010,(Langer(2011,(Richier(et(al.(2011,(Lefebvre(et(al.(2012,(Rokitta(&( Rost(2012)( and(the(fossil(record(suggests(that(eras(with(reduced(CO2(levels((e.g.( glacial(era)(have(favoured(more(heavily(calcified(cells((Beaufort(et(al.(2011).( Increased(CO2(will(alter(competitive(relationships(and(can(result(in(shifts(of( plankton( species( composition( due( to( the( sensitivity( to( carbon( enrichment( that( widely( differs( among( taxa( (Riebesell( &( Tortell( 2011,( Mackey( et( al.( 2015).( Such( changes(have(important(implications(for(marine(organisms,(affecting(physiological( processes((Figure(1.2).(In(turn,(changes(in(the(physiology(of(individuals(alter(the( dynamics(of(their(populations(and(ultimately(may(affect(the(entire(ecosystem.( !General!Introduction!!!!!!!!!!!!!!! ! ! 7! ! Figure! 1.2.! Diagram! about!the! effects! of! ocean! acidification! on! changes! in! seawater! carbon! chemistry,! biochemistry,! the! response! of! the! organism! and! the! ecological! responses.! Diagram! taken!from!Iglesias-Rodriguez!et!al.!2016.! ! Based!on!the!biological!processes!responsible! for! carbon!fixation,! two! biological! carbon! pumps! can! be! distinguished:! (1)! the! organic! carbon! pump,! driven! by! photosynthesis,!and!(2)!the!carbonate!counter!pump,!generated!by!the!formation! of! calcium! carbonate! (CaCO3)! shell! material! by! calcifying! plankton,! calcification.! While! photosynthesis! fixes! CO2!into! organic! carbon,! acting! as! a! net! sink! of! atmospheric! CO2,! calcification! converts! two! HCO3 –! anions! into! one! molecule! of! CaCO3!and!one!molecule!of!CO2,!acting!as!a!net!source!of!CO2!in!the!surface!ocean.! Hence,! the! two! carbon! pumps! reinforce! each! other! in! terms! of! maintaining! a! vertical!dissolved!inorganic!carbon! (DIC)!gradient,! whereas!they!counteract!each! other!with!respect!to!their!impact!on!air–sea!CO2!exchange,!this!process!is!called! “carbonate! counter! pump”! (Rost! &! Riebesell! 2004).! The! relative! strength! of! the! two!biological!carbon!pumps,!represented!by!the!so-called!rain!ratio!(the!ratio!of! particulate!inorganic!to!organic!carbon!in!exported!biogenic!matter),!determines! Chapter(1( ! ! 8( the( flux( of( CO2(between( the( surface( ocean( and( the( overlying( atmosphere.( Attending( to( global( organic( carbon( vertical( fluxes(studied,( the( organic( carbon( pump(clearly(dominates(over(the(carbonate(counter(pump((Rost(&(Riebesell(2004).( Consequently,( the( effect( of( elevated(CO2(on(calcification(and( photosynthesis(could(affect(carbon(export(by(altering(the(“rain(ratio”.(A(reduced( calcification( provides( a( negative( feedback( to( rising( atmospheric( CO2(levels,( contributing(to( the( stabilization( of( the( Earth’s( climate((Hoffman( &( Schellnhuber( 2009).(However,(strong(correlations(between(the(export(fluxes(of(POC(and(mineral( particles,( especially( with( CaCO3,( have(been( found,( the( so-called( “ballast( effect”( (Klaas(&(Archer(2002,(Sanders(et(al.(2010).(Mineral(ballast,(especially(CaCO3,(plays( a(dominant(role(in(carrying(matter(through(the(water(column.(If(the(flux(of(mineral( ballast( declines,( there( will( be( less( transport( of( POC( into( the( deep( ocean( and( a( reduction(of(its(export(fluxes(weakens(the(strength(of(the(biological(carbon(pump.( As( a( result,( the( mentioned( above(“negative( calcification( feedback”( on( rising( atmospheric(CO2(levels( is( counteracted( by(the( reduced( mineral( ballast( transport( (‘‘ballast( feedback’’).(Furthermore,( there( is( another( consequence:( the( organic( matter(is(oxidized(in(shallow(waters(when(mineral-ballast(fluxes(weaken,(hypoxic( zones(start( to( expand( considerably( in( the( oceans( in( our( model( world—with( potentially( harmful( impacts( on( a( variety( of( marine( ecosystems( (Hoffman( &( Schellnhuber(2009).( Changing(the(pH(of(seawater(is(also(expected(to(affect(the(efficiency(of(the( enzymatic( processes( involved( in( acquiring( organic( forms( of( nitrogen( and( phosphorus.(Ocean(acidification(can(also(inhibit(organic(nitrogen(and(phosphorus( acquisition(in(marine(organisms((Hutchins(et(al.(2009).(Thus,(the(biogeochemical( cycling(of(macronutrients(is(predicted(to(be(greatly(affected(by(ocean(acidification( (Hutchins( et( al.(2009).( Concomitantly,( the(distribution( and( speciation( of( trace( metals(in(the(ocean(is(also(affected(by(the(variations(in(pH(due(to(OA((Millero(et(al.( 2009).(Because( most( organic( particles( in( seawater( are( negatively( charged,( their( surface(sites(will(become(less(available(to(adsorb(metals(as(pH(decreases.(These( !General!Introduction!!!!!!!!!!!!!!! ! ! 9! changes! are! expected! to! alter! the! availability! and! toxicity! of! metals! for! marine! organisms.! ! Trace!metals! Trace!metals!(Mn,!Fe,!Co,!Cu,!Zn,!and!Cd)!are!required!for!numerous!processes!in! phytoplankton!and!can!influence!phytoplankton!growth!and!community!structure! (Morel! &! Price!2003).!Trace!metals!are!necessary!for!the!growth!and!survival!of! photosynthetic!organisms!(Price!&!Morel!1990,!Morel!&!Price!2003)!and!the!metal! contents!of!phytoplankton!(metal!quotas)!reflect!biochemical!demands!as!well!as! environmental! availability,! influencing! the! distribution! of! metals! in! the! ocean! (Sunda!2012).! Among! all! the! trace! metals,! iron! (Fe)! is! the! most! important,! required! for! essential!physiological!and!molecular!processes!in!phytoplankton.!Iron!is!cofactor! of! metaloenzymes!and! proteins,! which! are! vital! to! metabolic! processes! such! as! photosynthesis,!respiration,!electron!transport,!nitrate!metabolism,!detoxification! of!reactive!oxygen!species!(ROS)!and!DNA!repair!(Behrenfeld!&!Milligan!2013).!For! example,! most! of! the! cell's! Fe! requirement! (up! to! 80! %)! is! associated! with! photosynthesis! (Raven! 1999),! since! nearly! every! aspect! of! thylakoid! electron! transport!is!Fe!dependent.!Iron!is!required!in!both!photosystems!(2–3!atoms!for! PSII;!12!atoms!for!PSI):!the!cytochrome!b6f!complex!(5!atoms),!which!links!the!two! photosystems,!and!the!ferredoxin!molecule!(2!atoms;!Raven!1999).!This!strong!Fe! contingent!deeply!influences!electron!transport!kinetics.! Iron! is! the! fourth! most! abundant! element! on! Earth! but! its! solubility! is! extremely! low! in! contemporary! well-oxygenated! waters! resulting! in! low! Fe! concentrations!in!many!natural!aquatic!systems!(Norman!et!al.!2014).!In!today’s! oxygenated!world,!surface!ocean!iron!concentrations!may!be!as!low!as!20!pM!(Rue! &!Bruland!1995),!and! phytoplankton!have!responded!to! these!conditions!with! a! diversity! of! mechanisms!for! acquiring! iron! and! economizing!its! cellular! demand.! Iron!exists!in!seawater!in!two!oxidation!states:!soluble!Fe!(II)!and!low!soluble!Fe! Chapter(1( ! ! 10( (III).( Generally,( the( predominant( form( of( Fe( in( seawater( is( the( more( thermodynamically(stable(Fe((III).(The(chemical(form(of(iron(is(defined(by(physical( size(fractions(separated(on(the(basis(of(filtration(methods((Bruland(2001);(hence( these( forms( are( operationally( defined.( The( size( fractions(comprise( particulate( (>0.2( μm)( and( dissolved( fraction( (<0.2( μm).( The( dissolved( fraction( is( divided( in( colloidal(and(soluble.(Several(studies(have(demonstrated(that(the(majority(of(the( dissolved(concentration((mostly(>99%)(is(in(the(form(of(metal(organic(complexes( (Gledhill(&(van(den(Berg(1994,(Rue(&(Bruland(1995).(Fe(binds(to(organic(molecules( called(organic(ligands((OLs)(that(can(be(weak(or(strong(depending(on(the(stability( constant( (i.e.( association( constant( for( the( equilibrium( that( exists( between( the( metal(and( the(organic(ligand).(This(organic(complexation( is(extremely(important( for(maintaining(solubility(and(controlling(the(bioavailability(of(Fe(to(phytoplankton.( The( complexation( and( speciation( of( Fe( can( result(in( Fe( limitation( conditions,( because( the( Fe( bioavailable( concentration( is( below( than( the( requirements( of( phytoplankton((Marchetti(&(Maldonado(2016).(The(bioavailability(may(be(defined( as( the( degree( to( which( a( certain( compound( can( be( accessed( and( utilized( by( an( organism.( Thus,( Fe( stress( due( to( low( Fe( concentration( is( widespread(in( open( waters( and( in( some( coastal( areas( (Behrenfeld( &( Milligan(2009),( affecting( phytoplankton(growth,(physiology(and(production(of(organic(matter(and(biogenic( minerals.(( Ocean(acidification(will(have(an(impact(on(the(thermodynamics(and(kinetics( of(metals(in(seawater.(The(changes(in(the(speciation(of(metals(due(to(the(lower(pH( will( result( in( changes( in( the( behaviour( of( metals( in( seawater.( The( decrease( in( concentration( of( OH-((due( to( the( decrease( in( pH)( and( CO3 2-((due( to( changes( in( carbonate(chemistry)(can(affect(the(solubility,(adsorption,(toxicity(and(the(rates(of( redox(processes((Millero(et(al.(2009).(All(these(changes(could(possibly(affect(the( concentrations(and(chemical(speciation(of(iron,(and(hence(on(iron(availability(to( phytoplankton.( The( decrease(in( pH( will(promote(rates(of(reduction(of( Fe( (III)(to( soluble(Fe((II),(and(retard(the( oxidation( of( Fe((II)(to( Fe( (III)( (Millero( et(al.(2009).( !General!Introduction!!!!!!!!!!!!!!! ! ! 11! Moreover,! many! organic! complexation!reactions! are! sensitive! to! changes! in! pH! (Shi! et! al.!2010).!The! effects! of! ocean! acidification! in! Fe! could! enhance! the! solubility! leading! to! increased! iron! concentration! but! as! mentioned! above! more! than! 99%! of! dissolved! Fe! is! bound! to! strong! organic! ligand.! Shi! et! al.!(2010)! reported! that! the! conditional! stability! constant!of! iron-organic! ligand! complexes! increased!as!pH!decreased!when!the!ligand!was!an!acidic!binding!group.!Thus,!the! changes!in!Fe!availability!will!depend!on!the!nature!of!the!organic!ligands!(Shi!et!al.! 2010).! ! Interactive!effects!between!Fe!and!increased!CO2! Only!a!few!studies!have!been! focused! on! the! interactive! effects! between! ocean! acidification! and! Fe! concentrations.! The! responses! obtained! were!different! depending!on!the!features!of!the!natural!phytoplankton!community.!Additionally,! the! Fe!requirement! is! generally! higher! in! coastal! phytoplankton! species! than! in! oceanic!species,!probably!reflecting!the!environmental!conditions!of!their!habitats! (Sunda!&!Huntsman!1995a).!As!a!result,!the!effects!of!changes!in!iron!availability! due!to!changes!in!CO2!conditions!could!differ!depending!on!the!nature!of!the!iron! demand!of!each!phytoplankton!species.!! The!interactive! effects! of! ocean! acidification! and! Fe! have! been! shown! to! influence!the!phytoplankton!physiology!and!the!changes!in!the!community.!It!has! been! shown! that! under! increased! CO2!concentration! in! different! regions! dominated!by!diatoms!and!in!cultures!of!Thalassiosira)weissflogii)there! were!no! effects!in!primary!production!with!conditions!of!Fe!limitation!(Hoppe!et!al.!2013,! Yoshimura!et!al.!2013,!2014,!Sugie!&!Yoshimura!2016).!Furthermore,!a!negative! (Shi!et!al.!2010,!Sugie!et!al.!2013,!Yoshimura!et!al.!2013,!2014)!or!positive!response! (Hopkinson! et! al.!2010)!have!also! been! observed! under! elevated! CO2!and! ironlimitation! during!different!experiments!in!natural!assemblages!and!in!laboratory! conditions.! The! discrepancies!observed! can! be! due! to! a! number! of! factors,! Chapter(1( ! ! 12( including( differences( in( the( Fe( concentrations,( Fe( speciation( and( the( phytoplankton(community(composition.(( It(is(widely( recognised(that( the( lack(of(knowledge( on(the(interplay(of(the( different( environmental( factors( is( one( of( the( main( constraints( in( our( ability( to( predict( the( functioning( and( composition( of( the( future( ocean.( Therefore,( further( studies(are(essential(to( unravel(the(effects(of(ocean( acidification(on(trace(metal( bioavailability(and(the(synergistic(effects(to(assess(their(impacts(on(phytoplankton( within(the(food(web.(( ! Thesis!outline! Within(the(future(predicted(scenario,(different(stressors(of(global(change(will(vary( simultaneously( (Boyd( &( Doney( 2002).(These(changes( will( affect( phytoplankton,( leading(to(shifts(in(community(composition(and(biomass(and(it(is(known(that(the( responses(obtained(are( in( some( cases( specie-specific.( Due( to( the( importance( of( phytoplankton( in( sustaining( marine( food( webs( and( driving( the( biogeochemical( cycles(of(carbon(and(nutrients,(it(is(key(to(understand(all(the(possible(responses( from( the( different( species( as( a( consequence( of( the( different( changes( in( global( change(driven(factors.(( The( most( important( biogeochemical( cycle( is( the( global( carbon( cycle(and( coccolithophores(play( a( major( role(contributing( with( ca.( 1–10%( to( total( organic( carbon( fixation( and( with( approximately( 50%( to( pelagic( deep( ocean( CaCO3( sediments.( In( particular,( Emiliania'huxleyi(is(the(most( abundant( and( widespread( coccolithophore(in(the(world’s(ocean(with(a(global(distribution(from(the(tropics(to( subarctic(waters(accounting(for(20%–50%(of(the(total(coccolithophore(community( in( most( areas( and( close( to( 100%( in( subpolar( waters( (Mohan( et( al.( 2008).( Coccolithophores(in(general(are(known(to(follow(a(haplo-diplontic(life(cycle,(with( individuals( of( both( stages( being( able( to( propagate( independently( by( mitosis( (Billard(1994).(Emiliania'huxleyi(can(exist(as(calcified,(non-motile,(diploid((2N)(cells( and( non-calcified,( motile,( haploid( (1N)( cells,( with( the( capability( of( unlimited( !General!Introduction!!!!!!!!!!!!!!! ! ! 13! asexual! cell! division! (Green! et! al.! 1996).! A! third! form,! known! to! be! diploid,! nonmotile,!and!uncalcified,!may!be!a!culture!artifact!(Klaveness!1972,!Green!et!al.! 1996).! Furthermore,!E.#huxleyi!produces!extensive!blooms!in!temperate!latitudes! and! subpolar! regions! such! as! the! Northern! Atlantic! (Holligan! et! al.! 1993).!The! blooms! generally! coincide! with! a! combination! of! thermal! stratification! and! high! irradiance! in! phosphate-poor! environment! (Nanning! &! Tyrrell! 1996,! Tyrrell! &! Taylor!1996).!However,!the!temperature!is!not!an!overriding!environmental!factor! and!the!temperature!range!of!maximum!growth!rates!can!oscillate!between!10-26! ºC!(Paasche!2002).!Precisely!due!to!the!high!growth!rates!achieved!under!high!N:P! ratios,! its! capacity! to! grow! under! elevated! irradiances,! its! ability! to! use! organic! compounds! and! the! fact! that!it! can! release! allelochemicals! to! the! surrounding! medium,! this!species! can! outcompete! others! from! the! same! niche! where! it! inhabits,!thriving!along!vast!ocean!regions.! !Emiliania#huxleyi!is!the!keystone!of!the!coccolithophores!and!it!has!been! widely!studied!in!many!different!works,!with!one!or!more!global!change!stressors! either!in!laboratory!or!in!natural!conditions.!The!results!obtained!varied!depending! on! the! differences! between! natural! assemblages! (where! interactions!with! other! plankton!groups!exist)!or!controlled!laboratory!experiments!(where!there!can!be! disparities!due!to!culture!conditions).!The!main!general!response!observed!is!that! ocean!acidification!will!affect!E.#huxleyi.!However,!there!is!a!lack!of!knowledge!on! the! response! to!interactive! effects! of! ocean! acidification! and! dissolved! Fe! at! different!levels!in!this!species.!! The!aim!of!this!thesis!is!to!gain!deeper!insight!in!the!physiological!response! of! E.# huxleyi!to! increased! CO2!and! Fe! availability! within! the! food! web! using! mesocosms!and!also!under!controlled!laboratory!experiments.!Mesocosms!allow! the! use!of!perturbation!studies!in!natural!communities!analysing!the!interactive! effects!of!multiple!stressors!across!multiple!trophic!levels.!On!the!other!hand,!!the! effect! of! increased! CO2!and/or! Fe! levels! modulating!the! response! to! ultraviolet! Chapter(1( ! ! 14( radiation((UVR)(exposure(were(assessed(under(culture(conditions.(Therefore,(this( thesis(addresses(the(following(major(questions:( ( 1. What( is( the( growth( response( of( E.# huxleyi#to( elevated( pCO2( and( changes( in( Fe( availability?(How( the( response( of( E.# huxleyi(to( the( different( pCO2(and( Fe( levels( leads( to( cascading( effects( on( other( members(of(the(plankton(community?(What(is(the(impact(of(E.#huxleyi( behaviour( in( the( global( C-cycle( under( the( above-mentioned( circumstances?( 2. Are( carbon( assimilation( and( calcification(processes(affected(by( increased( CO2(and( Fe( availability(in( E.# huxleyi?( Are( the( variations(in( carbon( assimilation( and( calcification(explained( by( the(changes( observed(in(the(phytoplankton(community?( 3. Does( the( phytoplankton( community( overcome( the( stress( induced( by( increased( CO2(and( Fe(levels?( Are( there( protective,( and/or(defense( mechanisms(involved(in(the(response#of#E.#huxleyi?(( 4. How(the(particulate(trace(metals(change(during(the(bloom(of(E.#huxleyi( due(to(CO2(and(Fe(levels?(Are(the(particulate(trace(metals(are(related( to(E.#huxleyi?( 5. Does( the( effect( of( increased( CO2(and/or( Fe( levels( modulate( the( response(to(UVR(exposure(in(E.#huxleyi#in(culture(conditions?( To(tackle(these(questions,(a(mesocosm(experiment(was(carried(out(in(the( Raunefjord( (Bergen,( Norway)( and( manipulated( to( achieve( combinations( of( ambient(and(increased(pCO2(and(dFe(to(investigate(the(interactive(effects(of(both( stressors( on( functional( plankton( groups.( In( Chapter! 2,( the( increased( CO2( concentrations(as(well(as(the(addition(of(the(siderophore(desferrioxamine(B((DFB)( promoted( the( enhancement( of( dissolved( Fe.( The(response( to( both(factors( (CO2( and(Fe)(was(assessed(for(the(whole(food(web.(A(bloom(of(the(coccolithophore(E.# huxleyi(was(developed(under(ambient(CO2(conditions(and(increased(dissolved(Fe,( Iron%and%CO2%modify%the%plankton%food%web% ! ! 21% 2013).% Specifically,% mesocosm% experiments% allow% directly% analysis% of% the% interactive%effects%of%multiple%stressors%across%multiple%trophic%levels%(Riebesell%et% al.%2010,%Stewart%et%al.%2013,%Riebesell%&%Gatusso%2015).% Here% we% present% the% main% results% from% a% mesocosm% experiment% that% aimed% to% investigate% the% effects% of% future% changes% in% pCO2%and% Fe% availability% during%a%bloom%of%the%coccolithophore%Emiliania'huxleyi%within%a%natural%plankton% community.%Emiliania'huxleyi%is%a%major%primary%producer%in%the%world’s%oceans% and% a% sensitive% phytoplankton% to% elevated% pCO2.% This% species% is% of% paramount% significance%in%the%global%carbon%cycle%since%it%is%responsible%for%a%large%fraction%of% the% ocean% calcium% carbonate% production% and% export% to% the% deep% ocean% contributing% to% the% regulation% of% the% exchange% of% CO2%across% the% oceanatmosphere%interface%(Rost%&%Riebesell%2004).%%To%our%knowledge%this%mesocosm% experiment% is% the% first% to% manipulate% pCO2%and% iron% concentrations% simultaneously,% and% examine% their% combined% effects% and% interactions% on% planktonic%organisms%in%multiple%trophic%levels.%% We%investigated%(a)%the%effects%of%elevated%pCO2%levels%on%dissolved%Fe;%(b)% the%extent%to%which%changes%in%Fe%availability%affect%the%growth%of%E.'huxleyi%and% other% autotrophs% and,% (c)% whether% different% pCO2%and% Fe% levels,% and% their% interaction,% lead% to% cascading% effects% on% other% members% of% the% plankton% community.%% % Materials!and!methods! Experimental!design! A%mesocosm%experiment%was%carried%out%in%the%Raunefjord%(60.39%ºN,%5.32%ºE),%off% Bergen,%Norway,%June%5-27,%2012.%Twelve%mesocosms%(11%m3%each)%were%set-up%in% a% full-factorial% design% with% all% combinations% of% ambient% and% high% pCO2%and% two% treatments% of% dFe% in% three% independent% replicate% mesocosms% per% treatment.% High-density% polyethylene% (HDPE)% mesocosms% were% filled% with% fjord% water% Chapter(2( ! ! 22( pumped( from( 8( m( depth.( They( were( covered( with( low-density( polyethylene( (LDPE)(lids(in(order(to(avoid(pCO2(losses(and(contamination.(Mesocosms(and(their( lids(were(transparent(to(photosynthetically(active(radiation((PAR)(and(ultraviolet( radiation( (UVR).( After( the( first( sampling( day( (0),( the( seawater( of( half( of( the( mesocosms( was(enriched( with( CO2((Schulz( et( al.( 2009)( to( achieve( pCO2( concentrations( corresponding( to( levels( predicted( for( the( year( 2100( (900( µatm,( HC),((IPCC(2013)(and(the(other(half(were(not(manipulated((ca.(390(µatm,(LC).(All( mesocosms(were(continuously(and(gently(mixed(by(using(an(airlift(system((Egge(&( Heimdal(1994).(For(the(CO2(enrichment,(150(L(of(fjord(water(was(aerated(with( pure(CO2(at(a(flow(rate(of(1.5(L(min-1(overnight(and(added(to(each(of(the(high( pCO2((HC)(mesocosms.(To(maintain(the(pCO2(in(the(HC(treatments,(ambient(air( was(mixed(with(pure(CO2(at(a(flow(rate(of(200(mL(min-1(and(the(enriched(mixture( (900(µatm(CO2)(was(pumped(directly(to(the(airlift(system.(LC(treatment(consisted( of(only-ambient(air(similarly(connected.(HEPA(filters(were(placed(between(the(air( pumps(and( the( airlift( system( to( avoid( particulate( contamination.(Mesocosms( were(fertilised(after(initial(sampling((day(0)(by(addition(of(10(µM(nitrate(and(0.3( µM(phosphate( to( induce( a( bloom( of( the( coccolithophorid( E.# huxleyi# (Egge( &( Heimdal(1994).(On(day(7,(half(of(the(mesocosms((3LC(and(3(HC)(were(amended( with(70(nM((final(concentration)(of(the(siderophore(desferrioxamine(B((DFB)(to( promote( changes( in( iron( availability.( The( treatments( were( named( LC-DFB( (control),( LC+DFB,( HC+DFB( and( HC-DFB.( Water( samples( from( each( mesocosm( were(taken(from(2(m(depth(by(gentle(vacuum(pumping(of(25(L(volume(into(acidwashed( carboys( that( were( quickly( transported( to( the( onshore( laboratory.( All( variables(were(analysed(on(a(daily(basis(and/or(every(other(day,(except(otherwise( stated.(( ( pCO2,!DIC,!Ωcalcite,!pH,!alkalinity!and!rain!ratio! pCO2(inlet(flows(in(all(the(mesocosms(were(measured(by(non-dispersive(infrared( analysis( by( using( a( CO2(gas( analyser( (LI-820,( Li-COR).( pCO2,( dissolved( inorganic( Iron%and%CO2%modify%the%plankton%food%web% ! ! 23% carbon%(DIC)%and%the%calcite%saturation%state%(Ωcalcite)%were%calculated%from%daily% measurements% of% pH,% temperature,% salinity% and% total% alkalinity% (TA)% using% the% CO2Calc%software%(Robbins%et%al.%2010).%pH%was%measured%in%all%mesocosms%using% a% pH-meter% (CRISON% Basic% 20+)% calibrated% daily% using% NBS% (National% Bureau% of% Standards)%scale.%Salinity%was%measured%with%a%conductivity-meter%(CRISON%524).% The%accuracy%of%the%pH%meter%and%conductivity%meter%was%±%0.01%pH%units%and%±% 1.5% %% respectively.%TA% was% measured% using% the% classical% Gran’s% potentiometric% method%(Gran%1952).%The%rain%ratio%(RR)%was%calculated%applying%the%models%of% Ridgwell%et%al.%(2007)%and%Hoffmann%&"Schellnhuber%(2009).%! ! Dissolved!iron!(dFe)!! Dissolved% Fe% (dFe)%samples% were% obtained%from% each% mesocosm% every% 4th%day,% filtered%through%0.2%µM%AcroPak%supor%membrane%capsule%filters%(Pall,%USA)%into% LDPE%bottles,%and%immediately%acidified%with%ultra-clean%HCl%(Seastar)%in%a%Class% 100% laminar% flow% hood.% Total% dFe% measurements% were% conducted% by% chemiluminescence%flow%injection%analysis%(CL-FIA,%Waterville%Analytical,%USA)%as% described%in%de%Baar%et%al.%(2008)%and%de%Jong%et%al.%(1998).%All%filters,%sampling% and%filtration%equipment%were%trace%metal%cleaned%using%HCl%and%subsequent%high% purity% water% (MilliQ)% rinses% and% protected% with% double% bags% for% storage% and% transport.% Trace% metal% clean% techniques% were% used% throughout% all% the% process% when% collecting% and% manipulating% samples% for% both% dissolved% and% particulate% metals%analyses.%% ! Chlorophyll!a!(Chl!a)!concentration!and!in!vivo!Chl!a!fluorescence! Water%samples%(750%mL)%were%collected%every%other%day%from%each%mesocosm%to% determine%total%Chlorophyll%a%(Chl" a)%concentration.%Samples%were%filtered%onto% 0.7% µm% GF/F%filters% (Millipore)% and% kept% at% -80ºC% until% analyses.% Chl" a% was% extracted%in%N,N-dimethylformamide%overnight%at%4ºC%in%the%dark.%Chl"a%content% was%determined%spectrophotometrically%and%the%concentrations%were%calculated% Chapter(2( ! ! 24( by(using(Wellburn((1994)(equations.(Optimal(quantum(yield((Fv/Fm)(of(PSII(charge( separation( was( measured( in( 10( min( dark-adapted( samples( by( pulse( amplitude( modulated(fluorometry((Water-PAM,(Waltz,(Effeltrich,(Germany)(as(described(by( Schreiber(et(al.((1986).(After(initial(dark(measurement(and(a(saturation(pulse(to( determine( minimum( fluorescence( (F0)( and( maximum( fluorescence( (Fm),( respectively,(Fv/Fm(was(obtained,(considering(Fv/Fm(as((Fm-F0)/(Fm.(Thus,(Fv(is(the( maximal( variable( fluorescence( of( a( dark-( adapted( sample,( Fm(is( the( maximal( fluorescence( intensity( with( all( PSII( reaction( centres( closed,( F0(the( basal( fluorescence.( High( Fv/Fm(values( indicate( that( cells( are( in( healthy( physiological( condition,(whereas(a(decrease(in(Fv/Fm(indicates(stress(of(any(kind((Foyer(et(al.( 1994,(Beherenfeld(&(Milligan(2013).( ! Plankton!analyses! Phytoplankton+<20+µm+and+bacterioplankton:+Phytoplankton(cells(smaller(than(20( µm( from( each( mesocosm( were( analysed( using( a( FACSCalibur( flow( cytometer( (Becton( Dickinson,( USA)( equipped( with( an( air-cooled( laser( providing(15( mW( at( 488(nm(and(with(a(standard(filter(set-up.(The(trigger(was(set(on(red(fluorescence( and( samples( were( analysed( for( 300( s( at( an( average( flow( rate( of( 56( μL( min–1.( Autotrophic( groups( were( discriminated( on( the( basis( of( the( side-scatter( signal( (SSC)(versus(pigment(autofluorescence((chlorophyll(and(phycoerythrin)(according( to( Marie( et( al.( (1999)( and( Larsen( et( al.( (2001),( and( biovolumes( converted( into( carbon-biomass( according( to( Kana( &( Glibert( (1987),(Menden-Deuer( &( Lessard( (2000)(and(Olenina(et(al.((2006).( Enumeration(of(bacterioplankton(was(performed(according(to(Marie(et(al.((1999).( The(samples(were(fixed(with(glutaraldehyde((0.5%(final(concentration)(for(30(min( at(4°C,(snap(frozen(in(liquid(nitrogen,(stored(at(–80°C(and(stained(with(SYBR(Gold( Nucleic(Acid(Stain((Invitrogen)(for(10(min(at(80°C(in(the(dark(after(thawing(before( counting( using( a( Cytomics( FC( 500( flow( cytometer( (Beckman( Coulter)( equipped( with( a( 20( mW( 488( nm( air-cooled( argon-ion( laser( and( standard( filters.( The( Iron%and%CO2%modify%the%plankton%food%web% ! ! 25% discriminator%was%set%to%green%fluorescence%and%the%samples%were%analysed%for% 60% s% at% a% flow% rate% of% 30% µL% min-1.% Data% were% calibrated% by% measuring% latex% fluorospheres% (1.0% µm% in% diameter;% Polysciences% Inc.)% at% 525% nm% using% CXP% analysis% software.% Bacterial% abundances% were% transformed% into% carbon-biomass% according%to%Lee%&%Fuhrman%(1987)%and%Vrede%et%al.%(2002).% Phytoplankton+ >20+ µm+ and+ microzooplankton-%Live% samples% were% immediately% analysed%with%a%FlowCAM%(Fluid%Imaging%Technologies,%Scarborough,%Maine,%USA)% using%a%4x%objective%and%a%300%µm%flowcell%to%analyse%particles%ranging%from%18%to% 1000%µm%equivalent%spherical%diameter%(ESD)%and%run%in%automatic%imaging%mode% (Jakobsen% et% al.% 2011).% 6.3% mL% of% each% sample% were% analysed% at% flow% rates% adjusted%to%guarantee%that%no%more%than%1%particle%appeared%in%each%frame.%All% the% image% collages% were% post% analysed% to% separate% the% main% taxonomic% groups% (diatoms,% dinoflagellates% and% ciliates).%Abundances%and% particle% sizes% of% these% groups%(Jakobsen%&%Carstensen%2011)%were%then%converted%into%carbon-biomass% using%the%equations%provided%in%Menden-Deuer%&%Lessard%(2000).+ Mesozooplankton%was% sampled% at% the% beginning% and% at% the% end% of% the% experiment.% At% the% beginning,% samples% were% collected% by% filtering% 2.2% m3%water% pumped% through% a% 90% µm% Apstein% plankton% net% (Hydrobios,% Kiel,% Germany)% as% described% in% Nejstgaard% et% al.% (2006).% At% the% end% of% the% experiment,% the% whole% water% column% of% the% mesocosms% were% mixed% with% a% 45% cm% disc% (Striebel% et% al.% 2013),%immediately%sampled%by%two%vertical%90%µm%net%tows%and%preserved%in%4%% borax–buffered%formaldehyde%solution.%Species%composition%and%abundance%was% determined% using% a% dissecting% microscope% and% the% total% mesozooplankton% biomass%was%calculated%according%to%Nejstgaard%et%al.%(2006).%% ! Net!growth!rates! Apparent% net% growth% rates% for% phytoplankton% and% bacterioplankton% were% calculated%according%to%the%logistic%model:% % Chapter(2( ! ! 26( Ln#[(K-N) N] ="Ln"[(K-N0)-!1]#-!μt( ( where(K(refers(to(the(loading(capacity(of(the(mesocosms,(N(is(the(cell(density(at( any(given(time,(N0(is(the(cell(density(at(time(0,(µ(is(the(intrinsic(growth(rate,(and(t( is(the(time((in(days).( ! Iron!requirements! Fe( demand( (mol( Fe( per( L( of( seawater)( for( each( phytoplankton( group( was( calculated(using(published(Fe:C(ratios((µmol(Fe(per(mol(C)(and(the(maximum(C( biomass( achieved( by( each( phytoplankton( group( during( the( experiment.( The( estimates( of( C( biomass( were( calculated( as( described( above.( We(assumed( Fe:C( ratios( (µmol:mol)( published( for( E.! huxleyi((Muggli( &( Harrison( 1996),( diatoms( (Sarthou( et( al.(2005),(Synechococcus!sp.( and(Prasinophytes((Quigg(et(al.(2010),( picoeukaryotes( (Timmermans( et( al.(2005)( and( dinoflagellates( (Marchetti( &( Maldonado(2016).(( ! Irradiance!and!temperature(( Solar( spectral( irradiance( comprising( photosynthetically( active( radiation( wavelengths( (PAR,( 400-700( nm)( ultraviolet( radiation( A( (UVA,( 320-400( nm)( and( ultraviolet( radiation( B( (UVB,( 280-320( nm)( was( recorded( at( 2( m( depth( in( one( mesocosm((5(min.(intervals)(using(a(spectroradiometer(TRIOS(RAMSES((Ramses,( TrioS( GmbH,( Germany).( Because( the( spectroradiometer( developed( a( crust( of( ferric(material(that(interfered(with(all(the(analyses(this(mesocosm(was(excluded( as( a( replicate( and( was( only( used( for( irradiance( measurements.( HOBO( Pendant( Temperature/Light( loggers( (Onset( Computer( Corporation,( Massachusetts,( USA)( were(attached(to(the(airlift(system(in(one(of(each(treatments(at(the(depths(0(m,(1( m,(2(m,(and(3(m(to(check(that(mesocosms(received(the(same(irradiance(as(well(as( to(monitor(water(temperature.( ! Iron%and%CO2%modify%the%plankton%food%web% ! ! 27% Statistical!analyses! Statistical%significance%of%treatment%effects%on%variables%measured%was%analysed% by%performing%Split-Plot%ANOVAs%(also%called%SPANOVA%or%mixed-model%ANOVA)% followed% by% post-hoc% Sidak% or% Tukey% and% Bonferroni% tests,% respectively% (considering%p<0.05%and/or%p<0.01%as%significant).%When%appropriate,%data%were% specifically%tested%for%significant%differences%(p<0.05)%induced%by%the%treatments% by% using% 1% or% 2% Way% ANOVAs% and/or% Student’s% t-tests,% as% well% as%Pearson’s% product-moment% correlations.% All% analyses% were% performed% using% the% GLM% (general% linear% model)% procedure% with% main% effects% (CO2,% dFe),% time% (repeated% measure)% and% all% interactions.%Data% were% previously% checked% for% normality% (by% Saphiro-Wilks’% test),% homoscedasticity% (by% Cochran’s% and% Levene’s% tests)%and% sphericity% (by% Mauchly’s%and/or% Bartlett’s% tests).% Variables% met% all% the% criteria% above% mentioned.% Statistical% analyses% were% performed% by% using% the% software% Statistica%v12%(Statsoft,%Inc.)%and%SPSS%v22%(IBM%statistics).%% % Results!! Experimentally!induced!stressors!(carbonate!system!and!dissolved!iron)! The% experimental% set-up% was% successful% in% that% pCO2%reflected% the% target% concentration%of%900%µatm%in%the%high%CO2%treatment%(HC)%versus%390%µatm%in%the% ambient%CO2%treatment%(LC)%(Figure%2.1a).%pCO2%dropped%significantly%about%2-fold% between% day% 3% and% 10% (Bonferroni,% p<0.001)% as% a% consequence% of% biological% activity%in%both%HC%and%LC%treatments,%but%re-addition%of%CO2-enriched%fjord%water% on%days%3,%7%and%10%(in%HC)%re-established%pCO2%to%1100% µatm.% pH%stabilised% at% around%7.8%in%HC%and%8.1-8.3%in%LC%treatments%(Table%2.1).%Average%alkalinity%was% 2029%(±%60)%µmol%L-1%for%all%mesocosms% and%DIC%remained%stable%after%day% 5%at% 1953%(±%36)%µmol%L-1%in%HC%and%1795%(±%61)%µmol%L-1%in%LC%(Table%2.1).%calcite%was% significantly%higher%in%LC%than%in%HC%(t-test%p<0.05;%Table%2.1).%% To% induce% changes% in% Fe% availability,% 70% nM% of% the% siderophore% DFB% was% added%to%half%of%the%mesocosms.%Even%though%DFB%is%a%strong%Fe%binding%organic% Chapter(2( ! ! 28( ligand( often( used( to( induce( iron( limitation( in( phytoplankton( (Wells( 1999),( DFB( additions( may( also( increase( the( dissolved( Fe( pool( in( environments( with( high( concentrations(of(colloidal(and/or(particulate(Fe,(such(as(fjords((Kuma(et(al.(1995,( Öztürk(et(al.(2002).((In(this(experiment,(the(DFB(addition(increased(dissolved(iron.( Before(DFB(was(added(on(day(7,(the(initial(dFe(concentration(was(5(nM((mean(of( all( mesocosms),( without( significant( differences( between( LC( and( HC( treatments( (Figure(2.1b,(SPANOVA,(p=0.069).(Dissolved(Fe(concentrations(in(the(control((LCDFB)(remained(at(this(level(throughout(the(experiment((SPANOVA(p=0.399,(posthoc(Bonferroni(p<0.001).(The(DFB(amendment(on(day(7,(resulted(in(a(significant( ca.(3-fold(increase(in(dFe(in(both(HC(and(LC(treatments(by(day(17((post-hoc(Sidak( p=0.003( and( p=0.0004,( respectively)( relative( to( the( initial( levels( (post-hoc( Bonferroni(p=0.002).(On(day(17,(the(only(treatment(significantly(different(from( the(rest(in(terms(of(dFe(was(the(control((LC-DFB,(post-hoc(Sidak(p=0.0004),(which( showed( the( lowest( dFe( levels.( At( high( pCO2(without( DFB( addition( (i.e.( HC-DFB( treatment)( dFe( also( increased( ca.3-fold( by( day( 17,( confirming( the( expected( increase(in(iron(solubility(due(to(lowering(pH((Millero(et(al.(2009).(Surprisingly,( dFe(decreased(sharply(between(days(17(and(21.(In(contrast,(the(+DFB(treatments( sustained( high( dFe( throughout( day( 21,( regardless( of( the( pCO2(level( (post-hoc( Sidak,(p0.025).(These(results(suggest(that(Fe(solubility(was(enhanced(by(either( the( addition( of( DFB( and/or( CO2.( Furthermore,( significant( effects( of( pCO2,( DFB,( and( their( interaction( were( observed( on( the( availability( of( dFe( during( the( experiment.(( ( ( ( ( ( ( Iron%and%CO2%modify%the%plankton%food%web% ! ! 29% % % % % % % % % % % % ! ! ! ! ! ! ! ! ! ! ! ! Figure!2.1.%Temporal%development%of%CO2%partial%pressures%(pCO2)%(a)%and%dissolved%iron%(dFe)% (b)%within%the%mesocosms.%Ambient%pCO2%and%ambient%dFe%(black%filled%circle);%ambient%pCO2% and%increased%dFe%(LC+DFB,%open%circle);%increased%pCO2%and%increased%dFe%(HC+DFB,%open% square),% increased% pCO2%and% ambient% dFe% (HC-DFB,% black% filled%square).% Symbols% indicate% means% of% measurements% in% three% independent% mesocosms% (n=3)% except% for% LC-DFB% where% n=2.%Error%bars%indicate%standard%deviations.! Table& 2.1.!Measured!pH,!total!alkalinity!(TA,!µmol!L-1)!and!calculated!dissolved!inorganic!carbon!(DIC,!µmol!L-1)!and!calcite!saturation!estate!(Ω!calcite)!under!the! different!treatments;!LC:!ambient!CO2!(390!µatm);!HC:!increased!CO2!(900!µatm);!-DFB!(ambient!dFe);!+DFB!(increased!dFe).!Note!that!DFB!was!added!on!day!7.! Data!are!means!of!three!independent!mesocosm!bags!±!standard!deviation!(in!brackets),!except!for!LC-DFB!treatment!in!which!n=2.!Significant!differences!between! treatments!within!each!day!are!represented!by!different!letters!(2-Way!ANOVA!followed!by!post-hoc!Tukey!tests,!considering!p<0.05!as!significant).!The!rain!ratio! (RR)!was!calculated!applying!the!models!from!Ridgwell!et!al.!(2007)!and!Hoffmann!&#Schellnhuber!(2009).!The!calculated!RR!represents!the!average!between!the!2! models,!and!standard!deviation!between!brackets.!! & ! pH& Total&Alkalinity&& DIC& Ω&calcite& Rain&ratio& ! LC-& DFB& LC+& DFB& HC+& DFB& HC-DFB& LC-& DFB& LC+& DFB& HC+& DFB& HCDFB& LC-& DFB& LC+& DFB& HC+& DFB& HCDFB& LC-& DFB& LC+& DFB& HC+& DFB& HCDFB& LC-& DFB& LC+& DFB& HC+& DFB& HCDFB& Day!1! 8.04! (0.01)!a! 8.05! (0.02)!a! 7.71! (0.02)!b! 7.70! (0.04)!b! 2100! (20)a! 2176! (40)a! 2096! (25)a! 2140! (34)a! 1933! (6)a! 2002! (44)!ab! 2041! (20)bc! 2086! (25)!c! 2.98! (0.04)!a! 3.15! (0.10)!a! 1.5! (0.08)!b! 1.53! (0.14)!b! 0.75! (0.06)!a! 0.80! (0.08)!a! 0.41! (0.11)!b! 0.41! (0.10)!b! Day!3! 8.06! (0.05)!a! 8.10! (0.02)! 7.77! (0.01)!b! 7.75! (0.01)!b! 2086! (23)!a! 2046! (6)!a! 2060! (35)!a! 2046! (15)!a! 1910! (33)!a! 1861! (11)!a! 1989! (33)!b! 1980! (16)!b! 3.07! (0.25)!a! 3.28! (0.09)!a! 1.68! (0.04)!b! 1.63! (0.04)b! 0.77! (0.07)!a! 0.82! (0.08)!a! 0.45! (0.09)!b! 0.44! (0.09)!b! Day!5! 8.24! (0.02)!a! 8.22! (0.02)!a! 7.66! (0.15)!b! 7.81! (0.02)!b! 2027! (40)!a! 2032! (8)!a! 1956! (108)!a! 1975! (67)!a! 1775! (27)!a! 1793! (14)!a! 1917! (143)!a! 1894! (66)!a! 4.16!! (0.5)!a! 4.22! (0.15)a! 1.30! (0.6)!b! 1.77! (0.09)!b! 1.06! (0.19)!a! 1.03! (0.18)!a! 0.38! (0.10)!b! 0.47! (0.08)!b! Day!10! 8.28! (0.01)!a! 8.3!! (0.03)!a! 7.84! (0.00)!b! 7.86! (0.01)!b! 2045! (48)!a! 2006! (5)!a! 2021! (14)!a! 2013! (15)!a! 1769! (34)!a! 1727! (17)!a! 1915! (16)!b! 1930! (14)!b! 4.49! (0.03)!a! 4.85! (0.21)!a! 1.93! (0.01)!b! 2.01! (0.02)!b! 1.15! (0.25)!a! 1.21! (0.29)!a! 0.51! (0.07)!b! 0.53! (0.06)!b! Day!22! 8.22! (0.06)!a! 8.16! (0.03)!a! 7.77! (0.03)!b! 7.81! (0.02)!b! 2003! (35)!a! 1983! (38)a! 1996! (15)!a! 2006! (12)!a! 1777! (38)!a! 1773! (28)!a! 1926! (7)!b! 1924! (10)!b! 4.08! (0.3)!a! 3.59! (0.29)!a! 1.64! (0.1)!b! 1.80! (0.07)!b! 1.03! (0.18)!a! 0.90! (0.11)!a! 0.44! (0.09)!b! 0.48! (0.08)!b! Iron and CO2 modify the plankton food web 37 After an initial decrease during phase 1, Synechococcus sp. recovered during phase 2 (Figure 2.3b and Supplementary Figure 2.1Sb). The fastest recovery was observed in the LC treatments, with net growth rates of 0.58 d-1 and biomasses of 0.18 µg C L-1 in the LC+DFB mesocosms (Table 2.2, Figure 2.3b). Elevated pCO2 had significant negative effects on Synechococcus sp. from day 12 onwards (posthoc Sidak p=0.018 and Bonferroni p<0.001) while dFe had no effect (p=0.785). In general, biomass values and/or net growth rates of other microorganisms were not significantly affected by changes in pCO2, and/or dFe levels or their interaction during phase 2 (all SPANOVAs, p>0.05). Mesozooplankton was dominated by calanoid copepods and reached high biomass in all treatments from initially 16.5 µg C L-1 to 159-223 µg L-1 at the end of the experiment (Figure 2.4), but there were no significant differences on total mesozooplankton due to treatments (SPANOVA, p>0.05). Figure 2.4. Total mesozooplankton biomass (white bars) and calanoid copepods (black bars) at the beginning and at the end of the experiment. Bars indicate means of measurements in three independent mesocosms (n=3) except for LC-DFB where n=2. Error bars indicate standard deviations. Chapter(2( ( ! 38( Discussion( The( target( CO2(concentrations( were( achieved( in( the( twelve( mesocosms.( The( reproducibility(between(the(triplicates(of(each(treatment(was(high,(allowing(us(to( isolate( and( identify( single( and( interactive( effects( on( the( variables( measured.( Instead(of(our(intention(to(induce(Fe(limitation(with(the(DFB(addition(on(day(7,( we(found(that(DFB(enhanced(dFe(levels,(likely(due(to(an(increase(in(Fe(solubility.( Similar(results(have(also(been( found(in( other(fjord(environments( (Öztürk(et(al.( 2002).( Indeed,( a( decrease( in( particulate( iron( concomitant( to( an( increase( in( dissolved( iron,( up( to( a( significant( 12-fold( change( from( particulate( iron( to( dissolved(iron(in(the(LC+DFB(treatment((Supplemental(Figure(2.2S,(Supplemental( Table(2.2S),(indicates(that(DFB(mediated(the(transfer(of(Fe(from(the(particulate(to( the(soluble(pool.(In(addition,(Fe(solubility(is(enhanced(at(pH(lower(than(7.8((Kuma( et(al.(1996).(Hence,(high(CO2(also(increased(dissolved(Fe(levels(in(our(experiment,( as(previously(observed(in(a(mesocosm(experiment(in(the(same(fjord((Breithbarth( et( al.( 2010).( In( conclusion,( in( fjord( environments( dissolved( Fe( levels( increased( similarly(by(either(the(addition(of(CO2(and/or(DFB.(In(the(high(CO2(treatment((HC),( the( presence( of( the( siderophore( prevented( the( re-precipitation( of( dFe( (Figure( 2.1b)(implying(that(either(increasing(pCO2(or(DFB(may(result(in(high(dissolved(Fe( levels,(therefore(maintaining(Fe(in(the(dissolved(pool.(( These( distinct( chemical( conditions( steered( the( mesocosms( performance( into( two( clearly( differentiated( phases.( In!phase( 1( (day( 0-10)( ambient( dFe( conditions( in( the( mesocosms( were( sufficient( for( all( planktonic( groups( to( meet( their(iron(demand((in(the(range(of(pM,(Supplemental(Table(2.1S)(and(nutrients( were( sufficient( to( sustain( growth( (Supplemental( Figure(2.3S).( Phase( 1( thus( consisted(of( a( phytoplankton( bloom( succession( similar( to( what( has( previously( been( reported( (Paulino( et( al.( 2008)( with( small( autotrophs( being( grazed( by( microzooplankton((data(not(shown),(which(in(turn(were(probably(preyed(upon(by( copepods((Nejstgaard( et(al.(2001).(However,(no(pCO2(effects(were(observed(in( phase( 1( in( any( of( the( functional( groups,( except( for( bacterioplankton.( Iron%and%CO2%modify%the%plankton%food%web% ! ! 39% Heterotrophic% bacteria% reacted% positively% to% HC% treatments% in% phase% 1% and% had% higher% net% growth% rates% at% increased% pCO2%levels,% possibly% due% to% enhanced% available% DOM,% in% agreement% with% previous% studies% (Endres% et% al.% 2014)% (Supplemental% Figure%2.3S).% The% reverse% situation% in% phase% 2% (days% 12-18)% may% reflect% changes% in% bacterial% community% composition,% but% further% studies% are% needed.%% In% phase% 2% (day% 11-22),% the%coccolithophore% E.# huxleyi,% and% the% cyanobacterium%Synechococcus#sp.%were%both%affected%by%the%different%pCO2%and% Fe%conditions.%The%effects%of%increased%pCO2%on%E.#huxleyi%are%in%agreement%with% previous%mesocosms%studies%(Riebesell%et%al.%2007),%showing%that%elevated%pCO2% had%a%detrimental%impact%on%the%net%growth%of%E.#huxleyi,%while%at%ambient%pCO2% and% high% N:P% ratios,% E.# huxleyi%dominates% the% phytoplankton% communities% (Paasche%2002).%%% The%most%remarkable%results%in%our%experiment%were%the%massive%increase% in%E.#huxleyi%biomass%and%its%growth%rate%in%the%LC+DFB%treatment%(up%to%20-fold% higher% biomass% and% 2-fold% faster% growth% rate,% Figure% 2.3a,% Table% 2.2)% (cell% numbers%also%increased%several%orders%of%magnitude%relative%to%other%autotrophic% groups).%It%is%not%surprising%that%under%ambient%CO2%E.#huxleyi%flourished%due%to%its% ability%to% exploit%efficiently% organic%nutrients,%use% ammonium%instead%of%nitrate% and%have%an%efficient%alkaline%phosphatase%(shown%in%Supplemental%Figure%2.3S,% and%reflected%in%Paasche%(2002)).%However,%the%beneficial%effect%of%DFB%is%more% cryptic.% Our% results% point% to% that% the% DFB% addition% actually% increased% dFe,% resulting%in%higher%biomass.%The%insufficiency%of%the%ambient%dFe%to%support%the%E.# huxleyi%biomass% can% be% demonstrated% by%calculating% the% iron% demand% of% the% bloom%relative%to%the%concentration%of%dFe%in%the%control%(LC-FB).%Our%estimated% Fe% demand% to% sustain% E.# huxleyi%abundance% in% LC+DFB% was% ~6-10% nM% (Supplementary% Table% 2.2S),% a% dFe% concentration% measured% in% all% treatments% except% in% the% control% mesocosms% with% ambient% pCO2%and% no% DFB% (LC-DFB,% dFe% concentration%of%4.5%nM).%Therefore%in%the%control%treatment,#the%cells#were%most% Chapter(2( ( ! 40( likely(experiencing(Fe(limitation((i.e.(the(rate(of(dFe(supply(is(slower(than(that(of( iron( demand( by( the( coccolithophore),( which( was( affecting( their( growth( rates( (Table(2.2).(The(faster(growth(rates(observed(in(the(presence(of(DFB(imply(that(Fe( bound(to(DFB(is(bioavailable(to(E.#huxleyi,(as(previously(demonstrated(by(Lis(et(al.( (2015).( ! ! Figure!2.5.!SEM(photographs(from(treatment(LC+DFB((A)(with(the(decline(of(Skeletonema( sp(and(before(the(bloom(of(Emiliania#huxleyi((day(4)(and((B)(during(the(bloom(of(Emiliania# huxleyi((day(18).( ( In(addition(to(slower(net(growth(rates(and(reduced(Chl(a,(significant(lower(Fv/Fm( values(were(observed(in(the(LC-DFB(with(respect(to(the(LC+DFB(treatment(from( days(11-22((Figure(2.2b),(in(accordance(with(values(observed(in(E.#huxleyi(under( Fe(limitation(at( the( same( light(conditions(than(in(our(experiment((Honey(et(al.( 2013).(This(combination(of(symptoms(is(typical(of(Fe(deficient(algae((Behrenfeld( &( Milligan( 2013),( offering( further( support( for( the( Fe( limited( condition( of( E.# huxleyi# in#the( control.( The( observed( E.# huxleyi# biomass(as( well( as( Chl( a(do( however( increase( slightly( in( the( control( (LC-DFB)( in( spite( of( assumed( iron( limitation.( It( can( be( considered( that( dFe( should( decrease( under( these( circumstances.(However,(Fe(demand(estimated(for(the(control(is(0.8(to(1nM((data( not(shown).(The(drawdown(of(such(iron(concentration(by(E.#huxleyi(in(the(control( ! A! B! Iron%and%CO2%modify%the%plankton%food%web% ! ! 41% is%within%the%error%range%of%our%dFe%measurement,%thus%dFe%uptake%by%the%cells,% might%not%have%been%evident.%On%the%other%hand,%biomass%can%increase,%and%cells% can%still%be%iron%limited.%In%this%case,%limited%cells%show%slower%net%growth%rates% than% Fe-replete% cells% (Sunda% &% Huntsman% 1995),% in% full% agreement% with% our% results.% Fe%availability%often%limits%primary%productivity%in%open-ocean%waters%and% in% some% coastal% upwelling% regions% (de% Baar% &% Boyd% 2000),% but% Fe% limitation% in% fjord%environments%is%less%common%(Öztürk%et%al.%2002).%Fe%bioavailability%depends% not%only%on%dissolved%Fe%concentrations,%but%also%on%Fe%speciation.%Although%E.# huxleyi%net%growth%rates%were%higher%in%the%LC%than%in%the%HC%treatments,%high% biomass%values%in%LC%were%not%sustained%at%in#situ%dFe%concentrations%(i.e.%-DFB% treatment).%It%could%be%argued%that%the%positive%effect%of%the%DFB%addition%on%the% biomass%of%E.#huxleyi%is%due%to%DFB’s%ability%to%buffer%high%concentrations%of%toxic% metals% in% the% fjord.% A% previous% study% in% the% Raunefjord% documented% the% production%of%strong%organic%ligands%for%copper%(Cu)%by%Synechococcus%sp.%after%a% rain%event%that%resulted%in%significant%input%of%dissolved%Cu%(dCu)%levels%from%~7% nM% to% 16% nM% (Muller% et% al.% 2005).% During% our% experiment,% dCu% concentrations% were%typical%of%this%fjord,%not%excessively%high,%and%did%not%change%throughout%the% experiment% (~7-9% nM,% Chapter% 5).% Given% the% low% affinity% of% DFB% for% divalent% metals,% it%is%unlikely% that%the% DFB%addition%affected%Cu%speciation%or%any%other% divalent%metal,%such%as%Zn.%% The% only% mesocosm% treatment% where% E.# huxleyi# did% not% dominate% the% phytoplankton%community%was%in%the%HC-DFB%treatment.%DFB%was%added%in%17.5fold%excess%to%the%fjord%dFe%concentration,%therefore%we%assume%that%all%dFe%was% complexed%to%DFB.%Our%results%suggest%that%E.#huxleyi%is%able%to%utilise%DFB-bound% Fe%(FeDFB).%Indeed,%E.#huxleyi%has%been%shown%to%produce%organic%complexes%with% high%affinity%for%Fe%(Boye%&%Van%den%Berg%2000)%and%to%be%able%to%acquire%Fe%from% organic%Fe%complexes%(Hartnett%et%al.%2012),%including%Fe-DFB%(Shaked%&%Lis%2012,% Lis% et% al.% 2015).% Most% likely,% E.# huxleyi%was% able% to% access% Fe% bound% to% DFB% by% Chapter(2( ( ! 42( means( of( a( reductive( pathway( (Maldonado( &( Price( 2001),( one( of( the( most( prevalent( Fe( acquisition( mechanisms( in( phytoplankton.( The( fact( that( E.# huxleyi( biomass( in( the( HC( treatments( was( significantly( higher( in( presence( than( in( the( absence(of(DFB(further(suggests(that(the(cells(were(able(to(cope(better(with(the( unfavourable(effects(of(ocean(acidification(when(they(were(not(simultaneously(Fe( limited.( The( negative( effects( OA( has( on( calcifying( algae( is( caused( by( external( acidification( reducing( the( [H+]( electrochemical( gradient,( impairing( the( cellular( passive([H+](outflow(that(is(a(by-product(of(the(calcification(process((Taylor(et(al.( 2012).(In(addition,(maintaining(a(constant(intracellular(pH(is(energetically(costly( and(OA(likely(affect(the(cellular(energy(demands((Taylor(et(al.(2012).(Metabolism( drives(traits(that(determine(fitness,(growth(and(survival(of(populations((Dell(et(al.( 2011),( thus( increased( dFe( during( our( experiment( may( have( helped( the( cells( to( meet(the(extra(metabolic(demands(imposed(by(the(decrease(in(pH,(allowing(them( to(sustain(growth.( Synechococcus(sp.(was(also(negatively(affected(by(increased(pCO2(leading( to(reduced(net(growth(and(biomass(in(the(HC(treatment(during(phase(2(of(this( experiment( in( agreement( with( Paulino( et( al.( (2008),( but( in( contrast( to( another( study( from( the( Raunefjord( locality( (Larsen( pers.( comm.).( The( estimated( Fe( demand( for( Synechococcus(sp.( net( growth( was( ~2( pM,( well( below( the( dFe( concentrations( in( the( control( (LC-DFB).( This( suggests( that( Fe( concentration( did( not( influence( the( net( growth( of( Synechococcus(sp.( and#niche( differences( in( Cyanobacteria# Fe-metabolism( are( well( documented( (Desai( et( al.( 2015).( The( harmful(pCO2(effects(on(this(species(are(not(well(understood;(though(it(has(been( hypothesised(that(high(pCO2(might(decrease(photosynthetic(efficiency(and(light( saturation(constants(or(affect(nutrient(availability(or(competition(with(other(taxa( (Mackey( et( al.( 2015),( being( perhaps( more( sensitive( to( grazing( pressure.( Therefore,( we( do( not( know( at( present( whether( the( increased( pCO2(effects( on( Synechococcus(sp.(are(indirect.( Iron%and%CO2%modify%the%plankton%food%web% ! ! 43% The% consequences% of% the% interactive% effects% of% pCO2%and% Fe% availability% on% E.# huxleyi%can% be% critical% to% C-cycling% and% marine% ecosystems.%Within% the% future% predicted% climate% scenario% for% coastal% ecosystems,% particulate% and/or% colloidal% iron% might% become% more% solubilized% by% lower% pH% (Sunda% &% Huntsman% 1995,% Breithbarth% et% al.% 2010),% by% natural% organic% ligands,% and/or% by% increased% ultraviolet% radiation,% which% mediates% photo-solubilisation% (Kuma% et% al.% 1996,% Hassler%&%Schoeman%2009).%Given%the%variability%of%responses%to%increased%pCO2% observed% in% E.# huxleyi# (Hutchings% 2011,% Riebesell% &% Tortell% 2011,%Meyer% &% Riebesell%2015)%some%strains%that%are%held-back%by%iron%limitation%might%become% more% abundant,% gaining% a% competitive% advantage% through% their% low% stringent% requirements% for% nutrients% and% high% growth% under% photoinhibitory% conditions% (Paasche%2002)%at%increased%Fe%availability.%According%to%the%results%shown%here,% the% deleterious% effect% of% OA% on% the% development% of% E.# huxleyi%blooms% will% be% more% relevant% in% high% Fe% environments% than% in% Fe-limited% ones.% The% loss% of% C% production%of%E.#huxleyi%(estimated%as%the%areas%below%the%curves%in%Figure%2.3a)% due%to%OA%was%~92%%%in%enhanced%Fe%availability%conditions,%and%~70%%%in%ambient% Fe%conditions.%The%potential%benefit%of%higher%Fe%availability%in%an%acidified%ocean% will%be%overridden%by%the%decrease%in%pH%itself.%However,%increased%dFe%by%DFB%in% high-pCO2-seawater% enhanced% E.# huxleyi%C% production% by% ~60%.% Fe-favoured% strains% of%E.# huxleyi# could% outcompete% other% phytoplankton% and% influence% the% counterbalance%between%the%carbonate%pump%and%the%organic%carbon%pump%(i.e.% the%rain%ratio,%RR)%(Rost%&%Riebesell%2004).%RR%is%the%ratio%of%particulate%inorganic% to%organic%carbon%in%exported%biogenic%matter%(calcite:POC%or%PIC:POC),%which%is% used% as% a% proxy% for% calcification% vs.% photosynthesis% (Hutchings% 2011).% In% our% experiment,%RRs% were% significantly%higher%in%LC%than% in%HC%treatments%(average% 0.9% vs.% 0.4% respectively,% Table% 2.1)% in% close% agreement% with% a% wide% number% of% experiments% (Meyer% &% Riebesell% 2015).% Furthermore,% Fe% limitation% has% been% shown% to% decrease% PIC:POC% ratios% (Muggli% &% Harrison% 1996)% and% CaCO3% production%rates%(Schulz%et%al.%2004)%in%cultures%of%E.#huxleyi,%while%the%opposite% Chapter(2( ( ! 44( has(been(shown(under(P(and(N(limitation((Paasche(2002).(PIC:(POC(in(our(study( increased(significantly((c.a.(4-fold)(during(phase(2(in(LC+DFB(relative(to(all(other( treatments((Chapter(3),(implying(that(the(increase(in(dFe(enhanced(calcification,( in( agreement( with( a( former( study( (Muggli( &( Harrison( 1996).( Sustained( growth( and(possibly(calcification(under(future(increased(pCO2(levels(might(be(mediated( by(strains(that( are( favoured(by( high-dFe.(If(so,(the(PIC(ballast( effect(in(surface( oceans((Sanders(et(al.(2010)(would(not(be(affected((calcite(saturation(state(above( 1,(as(shown(in(Table(2.1)(and(the(downward(POC(flux(would(continue(and(directly( influence(the(RR.( In(summary,(this(study(demonstrates(that(Fe(concentrations(may(control( phytoplankton( community( structure( in( coastal( ecosystems( and( that( ocean( acidification(can(enhance(Fe(bioavailability.(This(is(contrary(to(the(idea(that(Fe(is( rarely( limiting( in( fjord( and/or( estuarine( environments,( and( that( OA( might( decrease( or( does( not( change( Fe( availability( depending( on( the( organic( ligands( present.( Moreover,( as( previously( reported,( E.# huxleyi(might( be( negatively( affected(in(a(near(future(scenario(by(increased(pCO2(levels.(However,(our(study( indicates( that( some( E.# huxleyi(strains( might( have( higher( Fe( requirements( than( initially(thought.(Thus,(in(areas(with(high(total(Fe(concentrations((particulate(and( dissolved(Fe),(the(detrimental(effects(of(increased(pCO2(on(these(strains(can(be( partially(mitigated(by(enhanced(dFe,(possibly(inducing(cascading(effects(on(food( web(dynamics,(carbon(export,(and(the(rain(ratio,(finally(affecting(the(exchange(of( CO2(across(the(ocean-atmosphere(interface.(The(interactive(effects(of(pCO2(and( Fe( observed( in( our( mesocosm( study( highlight( the( importance( of( examining( multiple(stressors(simultaneously(combined(in(natural(communities.(Investigating( how(multiple(drivers,(competition,(acclimation(and(adaptation(interact(at(longer( experimental(times(and(affect(natural(plankton(communities(is(essential(to(better( predict(how(marine(ecosystems(will(respond(to(future(changes.( ! ! ! Iron%and%CO2%modify%the%plankton%food%web% ! ! 45% Acknowledgements!! This%work%was%funded%by%CTM/MAR%2010-17216%research%grant%from%the%Spanish%Ministry% for%Science%and%Innovation%(Spain)%to%MS.%MRL%and%CI%were%funded%by%FPU%grants%from%the% Ministry% for% Education% (Spain).% MAM% and% JAF% were% supported% by% Grant% no.% 228224,% Transnational% Access,% from% the% EU% FP7-INFRA-2008-1% MESOAQUA% (Network% of% leading% MESOcosm%facilities%to%advance%the%studies%of%future%AQUAtic%ecosystems%from%the%Arctic% to%the%Mediterranean).%AL%was%supported%by%the%EU-ERC%grant%250254%(MINOS)%and%the% RCN%project%no.%225956/E10%(MicroPolar:%Processes%and%Players%in%Arctic%Marine%Pelagic% Food%Webs%–%Biogeochemistry,%Environment%and%Climate%Change).%We%thank%Jay%T%Cullen% for% hosting% MRL% to% analyse% dFe% at% his% laboratory,% Iole% di% Capua% for% counting% mesozooplankton,% and% the% Spanish% Institute% for% Oceanography% (IEO-Fuengirola,% Málaga)% for% silicic% acid% analyses.% We% also% thank% Jens% C% Nejstgaard% and% Hans% H% Jakobsen% for% discussions%and%suggestions,%and%the%MBS%staff%for%logistic%support.% ! ! Chapter(2( ( ! 46( Supplemental*Information* * Methods* Total*iron*analyses*(tFe)** Total(iron(concentration(consists(of(both(dissolved(and(particulate(iron(pools((dFe( and(PFe).(Methodology(for(dFe(analyses(is(described(in(M&M(section(in(the(main( document.( For( particulate( iron( analyses( (PFe)( seawater( samples( (1-3.5( L)( were( gently(filtered(onto(0.45(µm(Supor(®-450(filters.(Filters(were(precleaned(with(10%( trace(metal(hydrochloric(acid((Fisher,(trace(metal(grade)(at(60ºC(overnight,(rinsed( with(Milli-Q(water,(dried(and(stored(until(further(analysis.(Filters(were(digested(in( 7(mL(acid-washed(Teflon(vials((Teflon,(Rochester,(NY,(USA)((pre-cleaned(with(10%( trace(metal(hydrochloric(acid(and(nitric(acid(-trace(metal(grade-(at(70(ºC(during(23( days( each( step).( Samples( were( digested( in( 3( mL( of( HNO3(and( 0.5( mL( of( HF( (Fisher,(trace(metal(grade)(for(1(h(at(200(ºC(in(closed(vials(and(HF(was(evaporated( afterwards( at( the( same( conditions.( One-and-a-half( mL( of( HNO3(was( to( the( samples(and(incubated(at(150(ºC(overnight.(Samples(were(then(mixed(with(2.25( mL(of(HClO4((Fisher,(Optima(grade)(and(heated(for(4(h(at(200(ºC.(After(complete( digestion,(samples(were(evaporated(at(200ºC(until(dry,(dissolved(in(1%(HNO3with( 1( ppb( indium( as( internal( standard,( and( analyzed( by( using( a( high-resolution( inductively( coupled( plasma-mas( spectrometer( (HR-ICPMS,( Element( XR,( Thermo( Scientific).( Filter( blanks( were( subjected( to( the( same( process( than( samples( and( blank( values( were( subtracted( from( sample( measurements.( Trace( metal( clean( techniques( were( used( throughout( all( the( process( when( collecting( and( manipulating(samples(for(both(dissolved(and(particulate(metals(analyses.( ! Inorganic*and*organic*nutrients* Inorganic( nutrient( concentrations( were( analysed( in( a( QuAAtro( AQ2( AACE( autoanalyser((Seal(Analytical(Ltd,(Fareham,(UK)(following(the(methods(described( by( Grashoff( et( al.( (1983).( Fifty( ml( water( samples( from( each( mesocosm( were( Chapter(3( ( Effects' of' increased' CO2'and' iron' availability'on'the'carbon'assimilation'and' calcification' during' an!Emiliania! huxleyi' bloom'( ( M(Rosario(Lorenzo*,(Concepción(Iñiguez,(Jorun(K(Egge,(Aud(Larsen,( Stella(A(Berger,(Candela(García-Gómez,(María(Segovia( Manuscript+for+submission+ Chapter(3( ! 54( Abstract(( The( potential( interactive( effects( of( ocean( acidification( and( iron( availability( on( phytoplankton(ecophysiological(processes(are(still(unknown.(We(conducted(an(in# situ(mesocosm( experiment( to( investigate( the( single( and( combined( effects( of( increased( CO2(and( iron( availability( on( photosynthetic( carbon( assimilation( processes( including( acquisition(and( fixation,(and( calcification( during( a( bloom( of( the( coccolithophore( Emiliania# huxleyi.( Phytoplankton( cellular( stoichiometry( (C:N( ratio),(a(key(factor(in(biogeochemical(cycling(of(nutrients,(was(also(included(in(our( study.(We(observed(that(inorganic(carbon((Ci)(acquisition(was(unaffected(by(CO2( and(iron(availability(and(that(the(main(inorganic(carbon( source(used(was(HCO3 -.( Carbon( fixation(instead( was( negatively( affected( by( high(CO2(levels(whereas( increased( iron( availability( promoted(highest( values( of( carbon( fixation( and( calcification(under(present(CO2(conditions.(Although(Ci(acquisition(was(unaffected( by( CO2(and( iron,( the( different( treatments( had( an( effect( on( Ci(accumulation( as( shown( by( particulate( organic( matter( (POM)( concentrations(showing( the( same( response( as( carbon( fixation( rates.( The( accumulation( ratio( of( POC( to( PON( was( significantly( different( between( treatments(showing( an( imbalance( between( CO2( assimilation(caused(by(the(nutrient(depletion.(The(differences(observed(in(carbon( fixation(were(due(to(the(negative(impact(of(increased(CO2(in(E.#huxleyi.(Such(main( effect(was(more(significant(than(positive(effects(of(increased(Fe(availability.(Thus,( our( results( suggest( that( ocean( acidification( might( decrease( inorganic( carbon( mineralisation( and( organic( carbon( production( under( iron-replete( conditions( affecting( the( biological( carbon( pump( and( biogeochemical( processes( in( coastal( ecosystems.( Key( words:( Ocean( acidification,( dissolved( iron,( desferoxamine-B,( calcification,( carbon( concentrating( mechanisms,( carbon( fixation,( primary( productivity,( elemental(composition,(stoichiometry,(mesocosm(experiment,(Emiliania#huxleyi( ( !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Changes!in!CO2!and!Fe!affect!carbon!processes!in!E.!huxleyi!!! ! 55! Introduction! Oceans! have! absorbed! one-third! of! the! CO2! released! into! the! atmosphere! as! a! result! of! anthropogenic! activities,! producing! a! decrease! in! pH! of! 0.1! since! preindustrial!times!and!changes!in!the!dissolved!inorganic!carbon!(Ci)!equilibrium! due! to! CO2!dissolution! in! seawater! (ocean! acidification,! OA)! (IPCC!2013).! Additionally,!the!atmospheric!CO2!concentration!is!expected!to!reach!more!than! 900!µatm!by!2100!and!pH!to!decrease!from!0.06!to!0.32!(IPCC!2013).!Thus,!HCO3 -! concentrations!will!increase!by!17%!and!CO3 2-!concentrations!will!be!reduced!by! 50%,! altering! the! calcium! carbonate! saturation! state! (Doney! et! al.! 2009).! OA! is! then! expected! to! have! direct! impacts! on! photosynthesis,! calcification! and! elemental! composition! of! marine! phytoplankton,! leading! to! changes! in! phytoplankton!communities!(Riebesell!&!Tortell!2011,!Mackey!et!al.!2015).!! Marine! phytoplankton! contributes! to! half! of! the! world’s! total! primary! productivity,!sustaining!marine!food!webs!and!driving!the!biogeochemical!cycles! of! carbon! (C)!and! nutrients! (Field! 1998).! The! responses! of! phytoplankton! to! a! changing! environment! are!essential! to! understand! future! variations! in! marine! ecosystems.!The! effect!of! elevated!pCO2!levels! on!photosynthesis!and! growth! is! species-specific;! some! species! benefit,! while! others! show! no! response! or! even! inhibition!within!future!scenarios!(reviewed!in!Riebesell!&!Tortell!2011,!Mackey!et! al.!2015).!The!sensitivity!of!phytoplankton!to!increased!pCO2!seems!to!be!related! to!the!mode!of!Ci!acquisition!and!most!species!are!able!to!regulate!such!carbon! acquisition! by! means! of! CO2!concentrating! mechanisms! (CCMs)! (Beardall! &! Giordano!2002,!Rost!et!al.!2003,! Giordano!et!al.!2005).!CCMs!act!increasing!the! efficiency! of! net! C! fixation! by! concentrating! CO2! in! the! vicinity! of! Rubisco,! suppressing!the!oxygenase!activity!and!saturating!the!carboxilase!activity!(Beardall! &!Raven!2004).!The!efficiency!and!regulation!of!CCM!differs!among!phytoplankton! species!and!functional!groups!(Colman!et!al.!2002,!Giordano!et!al.!2005).!! Chapter(3( ! 56( Changing(carbonate(chemistry(in(seawater(due(to(decrease(in(pH(is(likely(to(affect( calcification(rates( of( calcifying(organisms.( Coccolithophores( are( one(of(the(most( important( groups( of( calcifiers( in( today’s( oceans( producing( calcium( carbonate( plates( (coccoliths),( and( accounting( for( 1–10%( to( marine( primary( production.( Furthermore,( coccolithophores(contribute( with( ~50%( to( the( pelagic( CaCO3( deposition(in(sediments(in(parallel(with(particulate(organic(carbon((POC)(export(to( the( deep( ocean( (called( the( ballast( effect)( (Paasche( 2002).( The( best-studied( coccolithophorid( is( Emiliania' huxleyi,( which( plays( a( fundamental( role( in( the( dynamics( of( marine( food( webs( and( CO2(sequestration.( Marine( biogenic( calcification((CaCO3(formation)(is(known(to(decay(with(decreasing(seawater(pH(in( different( strains( of( coccolithophores( (Riebesell( &( Tortell( 2011;( Rokitta( &( Rost( 2012).( However,( no( changes( in( calcification( or( in( the( ratio( of( CaCO3(to( organic( matter(production((PIC:POC)(at(elevated(pCO2,(as(well(as(stimulating(effects(of(OA( on(calcification(have(also(been(reported((Iglesias-Rodriguez(et(al.(2008,(Langer(et( al.(2009,(Rickaby(et(al.(2010);(possibly(due(to(differences(in(experimental(design(or( data(normalization((Riebesell(et(al.(2008,(Field(et(al.(2011).( The(elemental(composition(of(phytoplankton(could(be(affected(by(future( high(CO2(conditions,(because(the(increase(in(CO2(levels(has(the(potential(to(change( the(cycling(of(important(elements((Hutchins(et(al.(2009).(The(interaction(between( changes( in( CO2(and( other( physicochemical( factors(could(influence(the(biological( effects( of( ocean( acidification( (Boyd( &( Hutchins( 2012,(Boyd( et( al.( 2015).( The( bioavailability(of(trace(metals(is(affected(by(changing(carbonate(chemistry,(which( affects( pH-sensitive( coordination( chemistry( with( the( trace( metals( (Millero( et( al.( 2009).( Among( them,( Fe(is( an( essential( trace( element( for( phytoplankton( growth( due(to(its(key(role(in(metabolic(processes(such(as(photosynthesis,(respiration(and( N-assimilation( (Behrenfeld( &( Milligan( 2013).( Importantly,( its( availability( is( regulated(by(changes(in(pH((Millero(et(al.(2009,(Shi(et(al.(2010).(At(elevated(CO2( conditions,( the( Fe-availability( depends( on( many( factors( including( dissolved( Fe( concentrations,(Fe(speciation,(organic(ligands(and(their(interactions((Sunda(2010,( !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Changes!in!CO2!and!Fe!affect!carbon!processes!in!E.!huxleyi!!! ! 57! Shaked! &! Lis! 2012,!Chapter! 2).! Despite! that! phytoplankton! production! in! the! oceans!is!often!limited!by! nitrogen!and/or!Fe!(Saito! et! al.! 2008);! little! is! known! concerning!the!effect!of!CO2!on!phytoplankton!ecophysiology!under!changes!in!Fe! availability.! The! response! to! increased! CO2!in! the! elemental! composition! and! primary!production!obtained!depended!on!the!sensitivity!of!each!phytoplankton! community! to! changes! in! Fe!availability! (Hoppe! et! al.! 2013,!Sugie! et! al.! 2013,! Yoshimura!et!al.!2013,!2014).! To! unravel! the! single! and! interactive! effects! of! increased! pCO2!and! Fe! availability! on! the! mechanisms! of! carbon! assimilation! and! calcification,! an! Emiliania!huxleyi!bloom!was!induced!in!a!mesocosm!experiment.!The!aim!of!the! present!study!was!to!analyse!(i)!whether!increased!CO2!and!Fe!availability!changes! the!preference!of!the!Ci!source!and!the!mechanisms!involved!in!inorganic!carbon! acquisition!within!the!phytoplankton!community;!(ii)!whether!carbon!fixation!and! calcification! are! affected! under! different! Fe! and! CO2!levels;! and! (iii)! whether! variations!in!carbon!assimilation!and!/or!calcification!explains!the!shifts!observed! in!the!phytoplankton!community!during!the!experiment.!This!experimental!study! provides! new! insights! on! the! knowledge! of! carbon! metabolism! within! a! coastal! phytoplankton!community!exposed!to!future!environmental!scenarios.!! ! ! ! Chapter(3( ! 58( Materials)and)methods) Experimental)set-up) The(experimental(work(was(carried(out(in(June(2012(in(the(Raunefjord,(off(Bergen,( Norway,(as(described(in(detail(in(Chapter(2.(Briefly,(12(polyethylene(mesocosms( (~11(m3)(covered(by(lids((both(transparent(to(PAR(and(UVR)(were(filled(with(fjord( water( from( 8(m( depth( and( manipulated( to( achieve( two( different( CO2(levels( corresponding(to(the(present((390(ppm,(LC)(and(to(levels(predicted(for(year(2100( (900( ppm,( HC).( Specific( CO2( concentrations,( as( well( as( CO2(inlet( flows( in( all( the( mesocosms,(were(measured(by(non-dispersive(infrared(analysis(by(using(a(Li-Cor( (LI-820)(CO2(gas(analyser((Li-COR,(Nebraska,(USA)(and(CO2(AirSense-310(sensors( (Digital( Control( Systems,( Inc,( USA).( CO2( concentration( in( the( mesocosms( was( calculated(from(pH(and(total(alkalinity(measurements(using(the(CO2(SYS(software( (Robbins( et( al.(2010).( Iron( was( manipulated(by( addition( of( the( siderophore( desferrioxamine(B((DFB)(on(day(7(to(promote(two(different(Fe(conditions((+DFB,( high(dissolved(iron;(and(–DFB,(ambient(dissolved(Fe).(Dissolved(Fe(concentration( increased( ~3-fold( with( respect( to( the( control( as( a( result( of( both( factors( manipulation.( The( multifactorial,( experimental( design( comprised( triplicate( mesocosms(per(treatment(and(the(combinations( of(high(and(ambient( pCO2(and( dFe(levels(resulting(in(a(total(of(12(mesocosms:(3x(LC-DFB((control),(3x(LC+DFB,(3x( HC+DFB( and( 3x( HC-DFB.( Nitrate( (10( μM)( and( phosphate( (0.3( μM( final( concentrations)(were(added(once(at(the(beginning(of(the(experiment(to(induce(a( bloom(of(the(coccolithophorid(Emiliania(huxleyi.(( ( Isotope)disequilibrium) The(isotope(disequilibrium(assay((ID)(was(used(to(determine(the(relative(fraction( of(HCO3 -(and( CO2(uptake(in(concentrated( cell(suspensions( of(the(phytoplankton( community(according(to(the(method(described(in(detail(in(Elzenga(et(al.((2000);( Martin( &( Tortell( (2006)( and( Tortell( et( al.( (2008).( Briefly,( short-term( cellular( 14C( accumulation(was(monitored(during(a(transient(disequilibrium(between(14CO2(and( !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Changes!in!CO2!and!Fe!affect!carbon!processes!in!E.!huxleyi!!! ! 59! H14CO3 -! in! solution.! Following! the! addition! of! a! 14CO2-rich! spike! (pH! 7)! to! cell! suspensions! at! pH! 8.5,! the! specific! activity! of! CO2!is! initially! high,! decaying! exponentially! to! an! equilibrium! value,! whereas! the! specific! activity! of! H14CO3 - /14CO3 -2!remains! nearly! constant! during! the! experiment.! Therefore,! the! time! course!of!14C!accumulation!reflects!the!specific!activity!of!CO2!and!HCO3 -.!Cells!that! transport! only! HCO3 -!should! exhibit! a! linear! time! course! of! 14C! accumulation,! assuming! a! constant! rate! of! 14C! fixation.! Contrary,! cells!that! transport! only! CO2! should! exhibit! a! negative! slope! curve,! because! rates! of! 14C! uptake! will! be! high! initially!and!decrease!over!time.! Samples!(0.5-3!L)!were!concentrated!by!gentle!filtration!under!low!pressure! on!0.8!µm,!47-mm!polycarbonate!membranes!to!a!final!volume!of!20-30!mL!at!the! same!fjord!temperature!in!a!temperature!controlled!room.!Chlorophyll!a!(Chl!a)! content!in!the!concentrates!was!measured!by!extraction!in!90%!acetone!overnight! and! determined! fluorometrically! using! a! Turner! fluorometer! 10-AU! (Turner! BioSystems,!CA,!USA).!Final!Chl!a!concentration!in!cell!concentrates!ranged!from! 0.2!to!400!µg!L-1.!During!the!filtration!procedure,!cells!were!kept!in!suspension,! while!the!medium!was!gradually!exchanged!with!pH!8.5!fjord!water!buffered!with! 20!mM!Bicine.!Concentrated!cell!suspensions!(4!mL!sub-sample)!were!transferred! into!a!20-mL!glass!cuvette,!acclimated!for!15!min!to!150-µmol!photons!m–2!s–1!at! 11!ºC!in!a!custom-made!transparent!Plexiglas®!container,!and!20!µCi!14C!spike!pH! 7.0!in!50!mM!HEPES!was!injected.!Two!hundred!µL!subsamples!were!withdrawn!at! short!intervals!(between!5!s!and!10!min)!and!dispensed!into!1!mL!of!50%!HCl!to! stop! C! fixation! and! remove! unassimilated! inorganic! carbon.! Experiments! were! carried!out!in! the! presence! and! absence!of!the! membrane-impermeable!carbon! anhydrase!(CA)!inhibitor!dextra-bound!sulphanilamide!(DBS,!Ramidus!AB,!Sweden)! to!examine!the!role!of!extracellular!CA!(eCA).!The!inhibitor!was!added!to!a!final! concentration!of!100!µM!at!least!15!min!prior!to!the!assays.! For! quantitative! interpretation,!14C! disequilibrium! data! were! fitted! according!to!the!equations!presented!in!Martin!&!Tortell!(2006)!using!a!Marqand- Chapter(3( ! 60( Levenberg( non-linear( regression( algorithm( in( SigmaPlot( 11((Systat( Software,( Chicago,(USA).(Two(physiological(parameters(were(obtained:(the(fraction(of(HCO3 -( uptake((f"HCO3 -)(and(the(rate(of(extracellular(CO2:HCO3 -(interconversion((α’).(The( fraction( of( HCO3 -(uptake,( f,( is( the( fraction( of( photosynthetically( fixed( C( derived( from(HCO3 -(and(the(relative(eCA(activity(is(estimated(by(a(catalytic(enhancement( factor,(which(is(defined(as((α’:α),(where(α’(is(obtained(in(control(experiments(and( α( is( the( uncatalyzed( thermodynamic( rate( (obtained( in( the( inhibitor(treatment).( The( results( are( expressed( as( catalytic( enhancement( factors((in( the( inhibitor( treatment).(Thus,(a(catalytic(enhancement(factor(of(1(indicates(no(detectable(eCA( activity.( ( Carbon'fixation'rates' Carbon( fixation( rates( were( estimated( by( using( in-vitro(short-term( 14C( uptake( assays( of( concentrated( cell( suspensions( according( to( Steemann-Nielsen( (1952).( 0.5–3(L(of(seawater(were(pre-concentrated(in(20-30(ml(as(mentioned(above.(All( incubations( were( carried( out( in( 20-ml( glass( vials( in( a( custom-made( transparent( plexiglas( container,( continuously( homogenized(by( a( magnetic( stirrer( at( ambient( fjord( conditions( (10-11(ºC( and( 150( µmol(photons(m-2( s-1(provided(by(white(light( LED-lamps).(Prior(to(the(addition(of(H14CO3,(samples(were(pre-acclimated(for(15( min(to(the(above-mentioned(conditions,(thereafter(inoculated(with(10(µCi(H14CO3 -( (final(concentration)(and(incubated(for(30(min.(Dark(controls(were(run(under(the( same(conditions.(After(incubation,(4N(HCl(was(added(to(stop(the(reaction(and(to( remove(excess(DI14C.((Samples(were(transferred(to(20(ml(scintillation(vials(and(left( open(in(the(fume(hood(over(night(to(remove(excess(DI14C.(The(scintillation(cocktail( (Ultima(Gold,(Perkin(Elmer,(USA)(was(added(and(the(radioactivity(of(each(sample( was(measured(using(a(Packard(Tri(Carb(Liquid(Scintillation(Analyser,(model(1900(A( (Perkin(Elmer,(USA).(( ' ' !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Changes!in!CO2!and!Fe!affect!carbon!processes!in!E.!huxleyi!!! ! 61! Particulate!Matter! Total! particulate! carbon! (TPC)! and! particulate! organic! nitrogen! (PON)!were! measured! on! a! C:H:N! elemental! analyser! (Perkin-Elmer! 2400! CHN)!from! 0.5-1! L! samples!gently!filtered!onto!pre-combusted!(12!h,!500!ºC)!and!dried!at!65!ºC!GF/F! filters! (Whatman).! For! determination! of! particulate! organic! carbon! (POC),! filters! were!fumed!with!saturated!HCl.!Particulate!inorganic!carbon!(PIC)!was!assessed!as! the! difference! between! TPC! and! POC.! Water! samples! (0.15-0.5! L)! were! filtered! gently!onto!0.8µm!polycarbonate!filters,!in!triplicates,!for!analysis!of!particulate!Ca.! Dry! filters! was! measured! by! wavelength! dispersive! X-ray! fluorescence! (WDXRF)! (S4!Pioneer,!Bruker-AXS,!Karlsruhe,!Germany)!according!to!Paulino!et!al.!(2013).!! ! Statistical!analyses! Data! were! checked! for! normality! (by! Shapiro-Wilks’! test),! homoscedasticity! (by! Levene’s!test)!and!sphericity!(by!Mauchly’s!test).!All!data!met!the!requirements!to! perform!parametric!tests.!Statistical!significance!of!treatment!effects!was!carried! out! using! Split-Plot! ANOVA! followed! by! post-hoc! Sidak! and! Bonferroni! tests! (considering!p<0.05!as!significant).!All!analyses!were!performed!using!the!General! Linear! Model! (GLM)! procedure! with! main! effects,! time! (repeated! measure)! and! interaction! terms.!Some! of! the! data! were! specifically! tested! for! significance! of! differences! (p<0.05)! promoted! by! the! treatments! using! ANCOVA.! Statistical! analyses!were!carried!out!using!SPSS!v22!(IBM!statistics)!and!the!Systat!statistical! package!included!in!Sigmaplot!12!(Systat!Software,!Chicago,!USA).!! ! Chapter(3( ! 62( Results' Most(of(the(variables(analysed(in(this(study(followed(a(general(trend(that(showed( no(effects(of(treatments((CO2(and(DFB)(during(the(first(10(days((phase(1)(of(the( experiment,(but(that(responded(to(the(treatments(during(the(last(12(days((phase( 2)((see(Chapter(2(for(details(on(the(plankton(community(behaviour).( ( HCO3 -'was'the'main'Ci'source'and'was'not'affected'by'Fe'and'pCO2''' The( fraction( of( HCO3 -(uptake( (fHCO3 -)( derived( from( 14C( accumulation( curves( is( presented(in(Table(3.1(for(control(experiments(without(DBS(addition((a),(and(for( the(DBS(treated(experiments((b).(The(results(point(out(that(HCO3 -(was(the(major( source( of( inorganic( C( uptake( for( photosynthesis( in( all( treatments( over( time( as( indicated(by(fHCO3 -(values(obtained(for(the(control.(The(average(fHCO3 -(was(0.88(±( 0.03( suggesting( that( ~90%(of(fixed(C(was(derived(from( extracellular(HCO3 -.( The( values(of(fHCO3 -(in(DBS-treated(cells(were(slightly(lower(and(ranged(from(0.81(to( 0.91(with(an(average(of(0.85(±(0.03.(Neither(in(the(control(nor(the(DBS( treated( assays(did(we(observe(significant(differences(in(fHCO3 -(between(treatments((Table( 3.1).( There( were( no( significant( differences( between( treatments( during( the( experiment(in(fHCO3 -(of(the(control(or(the(DBS(treated(assays((Table(3.1).(Model( fits(to(the(time(course(of(14C(accumulation(showed(slight(differences(between(the( control( and( DBS-treated( samples( in( the( HC( treatments,( presented( as(increased( curvature(in(the(presence(of(the(eCA(inhibitor.(Both(curves((with(and(without(DBS)( were(clearly(significantly(different(from(the(theoretical(uptake(curve(based(only(on( CO2(incorporation.( This( difference( between( curves( supported( that( direct( HCO3 -( transport( accounted( for( the( majority( of( C( uptake( by( the( phytoplankton( assemblage(for(all(treatments((Figure(3.1).( ' ' !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Changes!in!CO2!and!Fe!affect!carbon!processes!in!E.!huxleyi!!! ! 69! The!POC:PON!ratio!significantly!decreased!with!increasing!pCO2!(independent!of! DFB! addition)! and! was! positively! affected! by! dFe! (Table! 3.2).! In! order! to! determine!the!effect!of!the!treatments!on!POM!net!production!during!phase!2,! ΔPOC! and! ΔPON! were! calculated! for! each! treatment! by! subtracting! the! concentrations!on!day!11!from!the!result!obtained!at!the!end!of!the!experiment.! Calculated!ΔPOC!and!ΔPON!in!phase!2!were!significantly!higher!in!LC!than!in!HC! treatments! (p<0.001).! Similarly,! the! ratio! ΔPOC:ΔPON! was! significantly! affected! by!CO2!(p<0.001)!and!the!slopes!of!regressions!for!HC!and!LC!were!significantly! different!(ANCOVA,!p=0.037,!data!not!shown).!! Changes! in! PIC/POC! ratios! (indicators! for! calcification)! were! driven! by! changes! in! POC! values! in! all! treatments! (Figure! 3.5).! However,! there! were! also! changes! due! to! variations! in! PIC! in! LC+DFB! for! which! the! highest! values! were! obtained!(average!PIC/POC!was!0.7).!Thus!PIC/POC!was!affected!by!both!CO2,!DFB,! their!interaction!and!over!time.!When!calcification!was!assessed!as!particulate!Ca! per!POC!(comprising!the!particulate!calcium!in!attached!and!detached!coccoliths! and! based! on! day-to-day! pairs! of! Ca! and! C! determinations),! the! Ca/POC! ratio! showed!differences!due!to!the!treatments!during!phase!2!with!highest!values!(an! increase! of! 5~folds! per! day)! measured! in! LC+DFB! treatment! (Figure! 3.5).! In! agreement!with!particulate!Ca!concentration!(Supplemental!Figure!3.S2),!Ca/POC! ratio!was!higher!in!LC+DFB!treatment!and!showed!a!linear!increase!over!time.!At! the! end! of! the! experiment,! HC! treatments! resulted! in! the! lowest! ratios! (~0.06)! compared!to!the!control!(~4-fold!higher)!and!LC+DFB!(~10-fold!higher)!with!the! same!significant!differences!as!PIC/POC!ratio!(Table!3.3).!! Furthermore,!changes!in!TPC,!POC!and!Ca!concentration!were!assessed!as! POM!net!production!during!the!dominance!of!Emiliania!huxleyi!in!each!treatment! considering!for!the!LC+DFB!treatment!the!period!between!day!11!and!21!and!for! the! other! treatments! the! period! between! day! 16! and! 21! (when! E.! huxleyi! dominates!or!presented!higher!cell!concentration).!! ! Chapter(3( ! 70( ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Figure!3.5.(Temporal(development(of(molar(inorganic(C:organic(C((a)(and(molar(Ca:organic( C(ratios((b)(in(all(the(treatments(during(the( experiment.(Ambient(pCO2(and( ambient(dFe( (LC-DFB,(l);(ambient(pCO2(and(increased(dFe(((LC+DFB,(¡);(increased(pCO2(and(increased( dFe((HC+DFB,(¨),(increased(pCO2(and(ambient(dFe((HC-DFB,(n).(Symbols(indicate(means(of( measurements(in(three(independent(mesocosms((n=3)(except(for(LC-DFB(where(n=2.(Error( bars(indicate(standard(deviations.( ! ! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Changes!in!CO2!and!Fe!affect!carbon!processes!in!E.!huxleyi!!! ! 71! TPC! production! showed! significant! differences! due! to! carbon,! DFB! and! the! interaction!of!both!factors.!The!lowest!values!were!observed!for!HC!treatments.! For!the!LC!treatments,!the!mean!rate!of!the!control!(LC-DFB)!was!9.43!±!5.26!µmol! L-1! d-1!and! for! the! LC+DFB! treatment! 13.22! ±! 0.71! µmol! L-1! d-1.! The! mean! of! Ca! production! was! similar! in! HC! conditions,! but! increased! in! LC! +DFB! and! control! conditions,! respectively.! Ca! production! presented! significant! difference! due! to! both!factors!and!the!interaction.!POC!production!only!showed!differences!due!to! CO2!main!factor!(p<0.05).! ! ! Table!3.3.!Statistical!analyses!(Split-Plot!ANOVA!followed!by!post-hoc!Sidak)!of!the!effects!of!CO2,!DFB,! and! their! interaction,! on! the! variables! analysed! during! the! development! of! a! bloom! of! the! coccolithophore!Emiliania!huxleyi!in!the!different!treatments.!!! ! ! CO2! DFB! Interaction! Time! f"HCO3 -!! (control)" 0.827! 0.08! 0.953! 0.310! f"HCO3 -!! (DBS-treatment)! 0.246! 0.635! 0.026! 0.078! eCA!! catalytic! enhancement! 0.659! 0.282! 0.435! <0.001! Carbon!fixation-! per!Volume! <0.001! <0.001! 0.001! <0.001! Carbon!fixation-! per!Chl!a!content! 0.298! 0.255! 0.053! <0.001! PON! concentration! 0.007! 0.006! 0.262! <0.001! POC! concentration! <0.001! 0.001! 0.002! 0.039! PIC! concentration! <0.001! <0.001! <0.001! <0.001! POC:PON! ratio! <0.001! 0.182! 0.012! 0.001! PIC:POC! ratio! <0.001! <0.001! <0.001! <0.001! Ca:POC! ratio! <0.001! 0.001! 0.001! <0.001! ! ! ! Chapter(3( ! 72( ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Figure!3.6.(Total(particulate(carbon((TPC)(production(during(E.#huxleyi(dominance(between( d16(and(d21,(except(for(LC+DFB( treatment(from( d11(until( d21((a);( organic(carbon( (POC)( production( (b);( and( particulate( calcium( (Ca)( production( (c)( for( the( different( treatments:( ambient(pCO2(and(ambient(dFe((LC-DFB,(black);(ambient(pCO2(and(increased(dFe(((LC+DFB,( dark(gray);(increased(pCO2(and(increased(dFe((HC+DFB,(light(shading),(increased(pCO2(and( ambient( dFe( (HC-DFB,( white).( Data( correspond( to( means( of( measurements( in( three( independent(mesocosms((n=3)(except(for(LC-DFB(where(n=2.(Error(bars(indicate(standard( deviations.! TPC production (mmol L -1 d -1 ) LC-DFB LC+DFB HC+DFB HC-DFB 0 5 10 15 20 POC production (mmol L -1 d -1 ) LC-DFB LC+DFB HC+DFB HC-DFB 0 2 4 6 8 10 Ca production (mmol L -1 d -1 ) LC-DFB LC+DFB HC+DFB HC-DFB 0 2 4 6 8 LC-DFB LC+DFB HC+DFB HC-DFB a b c !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Changes!in!CO2!and!Fe!affect!carbon!processes!in!E.!huxleyi!!! ! 73! Discussion! Our! mesocosm! experiment! demonstrated!that! carbon! cycling! processes! are! affected!by! changes! in!Fe!availability! and! CO2!concentration! during! a! mesocosm! experiment.! During! this! experiment,! a! phytoplankton! bloom! dominated! by! the! coccolithophorid! Emiliania! huxleyi! occurred! in! the! treatment! with! ambient! CO2! and!increased!dFe!(LC+DFB).!In!addition!to!the!CO2!and!Fe!manipulation,!nutrients! (nitrate! and! phosphate)! were! added! to! the! mesocosms! at! the! beginning! of! the! experiment! to! promote! the! growth! of! Emiliania! huxleyi.! The! increased! Fe! availability! showed! a! positive! effect! on! carbon! assimilation,! incorporation! and! fixation,!and!calcification!processes.!This!is!in!agreement!with!the!results!obtained! in!Chapter!2,!suggesting!that!the!cells!were!Fe-limited!in!the!control!(LC-DFB).!! Rising!global!CO2!is!changing!the!carbonate!chemistry!of!seawater!(IPCC! 2013).! Since! increased! CO2!concentrations! are! known! to! affect! several! carbon! assimilation! processes! (Mackey! et! al.! 2015)!it! can! be! expected! that!the! photosynthetic! inorganic! carbon! uptake! and! the! membrane-bound! extracellular! CA!(eCA)!activity!from!phytoplankton!will!also!be!influenced.!The!major!source!of! inorganic! carbon! uptake! by! photosynthetic! C! fixation! in! the! present! experiment! was! HCO3 -,! which! is! the! common! carbon! source! in! many! phytoplankton! communities!in!a!variety!of!oceanic!regions!(Tortell!et!al.!2010).!Our!data!are!in! agreement!with!previous!studies!showing!that!E.!huxleyi!had!moderate!affinities! for! Ci!and! that! HCO3 -! appeared! as! the! primary! Ci!source! (Herfort! et! al.! 2003,! Rokitta!&!Rost!2012,!Stojkovic!et!al.!2013).!Changes!in!Fe!availability!and!primary! productivity!in!natural!phytoplankton!assemblages!do!not!seem!to!influence!HCO3 -! utilisation!nor!do!changes!in!pCO2!in!laboratory!cultures!or!natural!communities! (Cassar!et!al.!2004,!Martin!&!Tortell!2006,!Tortell!et!al.!2010,!2013).!!Our!results! congruently! show! that! the! different! communities! that! developed! during! the! experiment! constitutively! expressed! HCO3 -! transporters! without! differences! between! treatments! (Table! 3.2).! This! apparent! insensitivity! to! increased! CO2!of! HCO3 -!uptake! affinity! obtained! may! derive! from! instantaneous! pH! effects! Chapter(3( ! 74( (Kottmeier( et( al.( 2014),( because( of( the( measurements( are( performed( under( stabilized( pH( conditions( that( often( do( not( mimic( environmental( situations( but( constitutively(express(HCO3 -(transporters.(The(presence(of(HCO3 -(transporters(can( indicate( the( capacity( of( phytoplankton( to( directly( use( HCO3 -(when(CO2(becomes( limiting(under(blooming(conditions,(when(pH(values(increase(up(to(8.5(or(higher( than(natural(levels((Kottmeier(et(al.(2014).( The( isotope( disequilibrium( results(also( allowed( us( to( estimate( the( eCAcatalyzed( enhancement( of( HCO3 --CO2.( Highest( levels( of( eCA( activity( were( found( when( long( chain-forming( diatoms( were( most( abundant( (days( 0( to(10,( phase( 1),( contributing( to( elevated(biomasses( at( the( beginning( of( the( experiment( (Supplemental( Figure(3.S1).( However,( there( was( low( or( inexistent( eCA( activity( when(E.#huxleyi(dominated(the(community(at(the(end(of(the(experiment((days(1122,(phase(2).(The(low(eCA(levels(indicate(the(dominance(of(direct(HCO3 -(transport( as(a(mode(of(inorganic(C(uptake((accounting(for(~85%(of(total(inorganic(C(uptake).( The(low( eCA( activity( showed( by( E.# huxleyi(and( the( HCO3 -(utilisation( suggest( an( important( role( of( bicarbonate( transporters( that( could( be( strain-specific.( The( discovery( of( E.# huxleyi(pangenome( (Read( et( al.( 2013)(explains( the( different( metabolic-response( repertoires( observed( depending( on( the( environmental( conditions( (e.g.( CO2(and( Fe).( Even( though( the( genome( analysis( shows( that( E.# huxleyi(has(nine(putative(CAs,(none(of(them(have(been(identified(as(external(ones( and,(gene(expression(data(indicate(that(the(up-regulation(of(these(genes(occur(at( CO2(concentrations(below(those(currently(experienced((380(and(900(ppm)((Bach( et( al.( 2013).( Yet,( the( core( genome( of( E.# huxleyi(contains( low-affinity( DIC( transporters((Read(et(al.(2013).(Comparing(the(molecular(data(with(the(high(HCO3 −( uptake( capacity( and( eCA( expression( observed( in( our( physiological( assays,( our( results( indicate( the( physiological( potential( of( this( E.# huxleyi(strain( to( maintain( efficient(inorganic(C(acquisition(under(blooming(conditions.(( Neither(HCO3 −(transport(nor(eCA(activity(was(related(to(the(partial(pressure( of(CO2((pCO2)(as(shown(previously((Tortell(et(al.(2010).(Tortell(et(al.((2013)(suggest( !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Changes!in!CO2!and!Fe!affect!carbon!processes!in!E.!huxleyi!!! ! 75! a! higher! and! potentially! CO2-dependent! activity! for! intracellular! CA,! which! acts! dehydrating!HCO3 −!to!deliver!CO2!at!the!site!of!C!fixation!by!Rubisco.!In!addition,! the! observed! lack! of! CO2-dependent! regulation! of! eCA! during! phase! 1! could! be! explained! by! interespecific! differences! in! the! CO2-dependent! regulation! of! inorganic!C!uptake!in!diatoms!(Rost!et!al.!2003,!Trimborn!et!al.!2009).!High!eCA! activity! has! been! reported! for!diatoms! compared! to! other! groups! of! phytoplankton!(nanoflagellates!and!haptophytes)!in!natural!assemblages!(Martin! &!Tortell!2006,!Tortell!et!al.!2010).!The!differences!in!eCA!activity!observed!during! our! experiment! could! be! attributed! to! the! differences! in! the!taxonomic! composition!of!the!treatments!(Table!3.2,!Supplemental!Figure!3S.1).! Although! Ci!acquisition! was! unaffected! by! CO2!and! Fe,! the! different! treatments!did!effect!on!Ci!accumulation!as!shown!by!particulate!organic!matter! (POM)!concentrations!(Figure!3.4,!Table!3.1)!and!carbon!fixation!rates!(Table!3.3)! showing! the! same! trend! that!the! development! of! the! bloom! (Figure! 3.2,! Supplemental!Figure!3S.1).!Increased!Fe!availability!at!ambient!CO2!concentration! (LC+DFB)!enhanced!carbon!fixation!and!promoted!the!E.!huxleyi!bloom.!In!contrast,! the! carbon! fixation! rates! were! similar! and! lower! in! HC! treatments! and! in! the! control!(LC-DFB)!(Figure!3.4).!In!accordance,!elevated!CO2!did!not!have!any!effect! on! carbon! fixation! rates! in! previous! mesocosm! experiments! carried! out! in! the! same!fjord!(Delille!et!al.!2005,!Egge!et!al.!2009).!However,!other!studies!presented! different!responses! of! primary! production! to! high! CO2!such! as! an! increase,! a! decrease! or! no! response! (Riebesell! &! Tortell! 2011,!Mackey! et! al.! 2015).! This! chapter! and! chapter! 2!suggested! that! the! results! obtained! under! ambient! conditions! (control)! are! due! to! E.! huxleyi!cells! experiencing! a! constrained! metabolism!because!Fe!was!limiting! net!growth!rates!and!biomass!increases.!In! such!nutrient-limiting!scenario,!cells!may!down-regulate!their!maximum!C!fixation! capacity.! Thereby,! there! are! no! differences! between! control! (LC-DFB)! and! HC! treatments! during! the! experiment! until! day! 19! when! the! carbon! fixation! rate! increase!as!well!as!POC!concentrations!(Figure!3.3!&!3.4)!and!the!growth!rate!of!E.! Chapter(3( ! 76( huxleyi((Chapter(2),(following(the(trend(of(the(treatment(LC+DFB(treatment.(The( carbon(fixation(rates(obtained(should(approach(to(gross(primary(production(since( short( incubations(reflect( the( total( cellular( capacity( for( C( fixation( (gross( primary( production),( while( longer-term( rates( include( a( significant( contribution( of( respiration(and(other(C-loss(processes((Tortell(et(al.(2010).(Thus,(the(gross(carbon( fixation( rates( could( not( reflect( the( differences( observed( in( POC( concentrations( between(HC(treatments(and(the(control((LC-DFB(treatment).(( Organic(matter((OM)(dynamics(are(mainly(driven(by(biological(processes( and(ultimately(reflected(the(dynamics(of(phytoplankton(photosynthesis.(Both(POC( and(PON(productions(were(significantly(lower(at(high(CO2(levels(compared(to(the( ambient(CO2(conditions(and(the(highest(values(were(obtained(in(treatments(with( increased(dFe((Figure(3.4(&(3.5).(These(changes(in(OM(production(were(most(likely( related(to(shifts(within(the(phytoplankton(community((Supplemental(Figure(3S.1)( responding(to(diverse(environmental(conditions.(On(the(other(hand,(an(alternative( and/or(concomitant(explanation(for(the(differences(observed(in(POC(production( between( LC( and( HC( treatments( could( be( that( the( phytoplankton( physiology( is( affected.( Cytoplasmic( pH( changes( produced( by( perturbations( in( the( cells’( microenvironment(by(high(pCO2((Suffrian(et(al.(2011,(Flynn(et(al.(2012,(Taylor(et(al.( 2012)(can(alter(key(physiological(processes(in(coccolithophores((Rokitta(et(al.(2012,( Muller( &( Nisbet( 2014,( Holtz( et( al.( 2015).( While( possible( positive( effects( of( increased(Fe(availability(on(aiding(cells(physiology(in(HC(treatments(were(observed( in( E.( huxleyi( (Chapter(2),(the( negative( impact( of( high( pCO2(might(become(more( significant( in( organic( carbon( production( (Figure( 3.4).( Moreover,( organic( carbon( production(has(been(shown(to(differ(depending(on(Fe(availability(under(high(CO2( conditions,( e.g.( increased( CO2( concentration( had( no( influence( on( C( production( under(Fe(limiting(conditions,(whereas(C(production(reached(at(high(CO2(levels(with( Fe( enrichment( (Feng( et( al.( 2010,( Hoppe( et( al.( 2013).( The( difference( in( such( responses(could(be(attributed(to(environmental(conditions,(which(strongly(affect( !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Changes!in!CO2!and!Fe!affect!carbon!processes!in!E.!huxleyi!!! ! 77! phytoplankton! community! composition! and! metabolism! (Richier! et! al.! 2014,! Yoshimura!et!al.!2014b).!! In! this! study,! the! accumulation! ratio! of! POC! to! PON! was! significantly! different! between! treatments! (Table! 3.1)! with! differences! also! due! to! the! interaction!with!Fe!availability!as!previously!shown!(Yoshimura!et!al.!2014b)!when! the! nutrients! (nitrate,! phosphate,! silicic! acid)! were! exhausted! or! at! low! concentrations.! Nutrient! depletion! can! cause! an! imbalance! between! CO2! assimilation,! changes! in! the! stoichiometric! composition! and! altered! biogeochemical!pathways!through!the!microbial!food!web!(Bellerby!et!al.!2008).! The!comparison!between!C!assimilation!relative!to!N!uptake!and!the!Redfield!C:N! ratio!can!indicate!carbon!overconsumption!(Toggweiler!1993)!and!this!imbalance! in!carbon!and!nitrogen!assimilation!has!been!related!to!nutrient!limitation!of!the! cell! (Schartau! et! al.! 2007).! Elevated! C:N! of! accumulated! POM! in! N-limited! conditions! was! also! observed! in! response! to! high! CO2!in! natural! communities! (Losh! et! al.! 2012).! In! contrast,! constant! C:N! of! net! produced! POM! among! the! different!CO2!conditions!has!been!reported!(Kim!et!al.!2006,!Riebesell!et!al.!2007,! Engel! et! al.! 2008,! Feng! et! al.! 2010,! Czerny! et! al.! 2013),! suggesting! that! phytoplankton! communities! presented! constant! C:N! under! nutrient-replete! conditions!and!this!ratio!did!not!change!significantly!due!to!pCO2!variations!and!Fe! additions!when!the!nutrients!were!not!exhausted!(Sugie!et!al.!2013a,!Yoshimura! et!al.!2014).!It!has!been!attributed!that!increases!in!POC:PON!ratios!under!nutrient! deficiency!is!due!to!preferential!PON!degradation,!intracellular!increase!of!the!C:N! ratio!or!an!excess!of!carbon!fixation!(Engel!2002,!Taucher!et!al.!2012).!Therefore,! the!ratios!obtained!could!be!due!to!excess!in!carbon!fixation.! In!accordance!with!the!model!proposed!for!E.!huxleyi!by!Muller!&!Nisbet! (2014)!both!net!inorganic!carbon!and!organic!carbon!productions!were!affected!by! ocean! acidification! (Figure! 3.5).! Coccolithophores! are! potentially! affected! by! pH! through!effects!on!calcification!and!the!physiological!effects!due!to!elevated!CO2! concentration!(Bach! et! al.! 2011).! Additionally,! we!demonstrated!that!interactive! Chapter(3( ! 78( effects(between(CO2(and(Fe(directly(affected(calcification(in(E.#huxleyi(during(the( bloom( development.( It( is( important( to( consider( the( different( phytoplankton( groups(and(their(biomass(contribution(present(in(each(treatment(with(respect(to(E.# huxleyi#(Supplemental(Figure(3S.1).(The(PIC:POC(ratio(reflected(the(ratio(of(carbon( deposited(in(coccoliths(to(carbon(converted(to(organic(form(by(photosynthesis(as( Ca:POC(ratio.(Schulz(et(al.((2004)(demonstrated(that(the(growth(and(calcification( rate( of( E.# huxleyi(were( reduced(under(low(Fe(conditions.(Comparing(the( results( obtained( in( LC( treatments( between( ambient( (-DFB)( and( increased( Fe( concentrations( (+DFB),( a( decrease( in( the( growth( rates( was( observed( in( –DFB( in( this( experiment( (Chapter( 2).( Such( a( decay( also( occurred( in( the( PIC:POC( and( Ca:POC(ratios,(and(Ca(production((Figure(3.4(&(3.5).(On(the(other(hand,(high(CO2( affected( calcification( negatively( by( reducing( the( overall( production( of( CaCO3( during( the( E.# huxleyi(bloom( (Figure( 3.5).( The( decrease( in( PIC:POC( and( Ca:POC( ratios(are(attributed(to(a(decrease(in(calcification,(as(well(as(an(increase(in(organic( carbon( production( but( could( also( be( due( to( the( net( specific( growth( rate( of( E.# huxleyi.( The( growth( rate( dropped( off( with( increased( CO2(and( increased( in( CO2( conditions(and(increased(Fe(and(this(decrease(might(have(influenced(differences( in( net( PIC( accumulation( (Figure( 3.4).( Despite( intraspecific( variability,( negative( effects( of( OA( on( calcification( and( on( the( cellular( ratios( were( observed( for( the( dominant( bloomer( in( our( system,( E.# huxleyi,(supportive( of( previous( studies( (Riebesell(&(Tortell(2011,(Meyer(&(Riebesell(2015),(even(though(some(strains(of(E.# huxleyi(appear( to( be( less( sensitive( to( ocean( acidification( (Langer( et( al.( 2009,( Beaufort( et( al.( 2011).( Diminished( calcification,( and( a( decreased( PIC/POC( ratio,( could(lead(to(a(negative(feedback(in(atmospheric(CO2(concentration((Zondervan(et( al.(2001).!But(it(could(affect(the(ballast(effect((i.e.(the(strong(correlations(between( the(export(fluxes(of(POC(and(mineral(particles),(affecting(the(transport(of(POC(into( the( deep( ocean( and( contributing( with( an( increase( of( CO2(concentration( counterbalancing(the(possible(negative(feedback.(Therefore,(our(results(are(thus( highly(ecologically(relevant(in(view(of(the(current(global(changes.( Chapter(4( ( Physiological+ stress+ response+ associated+ to+ elevated+ CO2+and+ dissolved+ iron+ in+ a+ phytoplankton+community+dominated+by+ the+coccolithophore+Emiliania'huxleyi.+ María( Segovia,( M(Rosario( Lorenzo*,( Candela( García-Gómez,(( Concepción(Íñiguez( Manuscript+for+submission+ + + Chapter(4( ! 86( Abstract( In(the( present( work( we( investigated( the( physiological( response( of( the( phytoplankton(community(and(in(particular,(the(response(of(the(coccolithophore( Emiliania' huxleyi(to( combined( effects( of( increased( and( ambient( CO2(and( iron( concentrations,( during( a( mesocosm( experiment.( Changes( in( both( factors( promoted( a( shift( in( the( phytoplankton( community( structure( by( day( 10( of( the( experiment,(leading(to(2(well(distinct(phases((phase(1(and(2,(before(and(after(day( 10,( respectively).( We( report( on( the( main( physiological( responses( occurring( in( phase( 2,( when( a( massive( bloom( of( the( coccolithophore( Emiliania' huxleyi( developed.(Pigments(performance(was(higher(under(increased(iron(levels,(leading( to(a(better(functioning(of(the(electron(transport(chain(that(was(modulated(by(CO2( levels.( Thus,( high( Fe( also(promoted( a( significant( increase( in( Fv/Fm(and( in( the( photosynthetic(efficiency(with(respect(to(the(control(treatment((ambient(CO2(and( ambient(Fe).(Strikingly,(cell(death(was(only(detected(during(the(community(shift( (between( days( 10-12)( and( was( mostly( affected( by( high( CO2(levels.( The( accumulation(of(reactive(oxygen(species((ROS)(decreased(under(elevated(Fe,(thus,( the( general( oxidative( stress( was( very( low( in( general,( pointing( to( a( more( active( metabolism( rather( than( to( a( stressed( metabolism.( DNA( lesions,( caused( by( ultraviolet(radiation( (UVR)( and( excess( irradiance,( were( minimised( by( high( Fe( levels.(Emiliania'huxleyi(has(been(reported(not(to(have(high(Fe(requirements(for( growth.(However,(we(demonstrate(in(this(experiment(that(Fe(is(essential(for(DNA( repair,( to( overcome( oxidative( stress( and( to( keep( the( photosynthetic( machinery( functional(in(the(studied(E.'huxleyi(strain.( ( Key(words( CO2,( iron,( stress( response,( phytoplankton( community,(Emiliania' huxleyi,( photosynthesis,( thylakoidal( electron( transport( chain,( pigments,( cell( death,( oxidative(stress,(DNA(damage/repair.( !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Phytoplankton!stress!under!increased!CO2!and!dissolved!iron! ! ! 87! Introduction! Anthropogenic! activities! are! resulting! in! a! progressive! enhancement! of! atmospheric!CO2!towards!levels!predicted!to!reach!around!900!µatm!by!the!year! 2100!(IPCC!2013),!are!decreasing!ocean!pH!(ocean!acidification,!OA)!and!!also!the! calcium!carbonate!saturation!state!(Doney!et!al.!2009,!Beardall!et!al.!2009)!at!an! unprecedented! rate.! In! addition,! concomitant! global! warming! may! enhance! stratification!reducing!nutrient!availability!and!irradiance!will!be!increased!due!to! a!narrower!thermocline!(Boyd!&!Doney!2002).!A!quarter!of!the!CO2!emitted!to!the! atmosphere!by!anthropogenic!activities!since!the!beginning!of!the! industrial!era! has! been! absorbed! by! the! oceans.! However,! most! of! the! anthropogenic! CO2! remains!above!the!permanent!thermocline!and!up!to!30%!remains!in!the!upper! 200! metres! of! the! water! column! unequivocally! affecting! physiological! processes! (Sabine!et!al.!2004).!! OA! also! affects! biogeochemical! processes! and! highly! impacts! trace! metal! solubility! and! speciation.! Trace! metals! are! required! for! numerous! physiological! processes!in!phytoplankton.!Among!them,!Fe!is!the!most!essential!trace!metal!for! phytoplankton! for! crucial! physiological! processes! controlling! phytoplankton! growth! by! means! of! nitrate! assimilation! and! increased! photosynthesis! (Beherenfeld! &! Milligan! 2013).! Therefore,! changes! in! Fe! bioavailability! have! profound! consequences! in! biologically! mediated! responses,! ultimately! affecting! carbon! fluxes! in! marine! systems! (Marchetti! &! Maldonado! 2016).! Iron! is! usually! complexed! to! hydroxides! and! so! the! decrease! in! pH! will! change! Fe! speciation,! increasing!the!solubility!of!Fe!(III)!and!preventing!Fe!(II)!from!binding!to!carbonate! (Millero!et!al.!2009)!due!to!the!carbonate!ion!will!be!reduced!by!54%!at!low!pH! (Beardall! et! al.!2009).! Additionally,!the! increased!CO2!concentration!in!seawater! and!thus,!the!decrease!in!seawater!pH,!increase!Fe!solubility!(Millero!et!al.!2009),! but!such!increase!depends!on!the!nature!of!strong!organic!Fe!ligands!(Shi!et!al.! 2010).!!It!is!widely!known!that!elevated!CO2!also!affects!phytoplankton!(Mackey!et! al.!2015).!Field!experiments!have!shown!mixed!responses!to!alteration!in!CO2!and! Chapter(4( ! 88( Fe(concentrations(within(natural(phytoplankton(assemblages(from(HNLC(regions.( Fe(availability(increased(as(a(result(of(CO2(enrichment(in(a(mesocosm(experiment( in(the(same(fjord(where(this(experiment(was(carried(out((Breithbart(et(al.(2010).(In( contrast,(decreases(in(Fe(uptake(rates,(Fe(availability(and(growth(of(phytoplankton( with(increasing(CO2(levels(were(observed(in(the(North(Atlantic(and(the(Bering(Sea( (reviewed(in(Marchetti(&(Maldonado(2016).(( Coccolithophores( is( one( of( the( taxa( most( affected( by( CO2((Riebesell( &( Tortell(2011).(The(majority(of(coccolithophores(reduce(their(level(of(calcification( when( growing( at( increased( CO2(and( the( fossil( record( suggests( that( eras( with( reduced(CO2(levels((e.g.(glacial(maxima)(have(favoured(more(heavily(calcified(cells( (Beaufort(et(al.(2011).(Elevated(CO2(may(act(as(stressor(in(coccolithophores(due(to( problems( related( to( calcification( (Mackey( et( al.( 2015),( leading( to( subsequent( adding(effects(of(other(stressors,(such(as(excess(of(PAR(and(UVR(irradiances,(that( are(able(to(exert(harming(effects(in(cells(with(insufficient(calcium(carbonate((Gao( et(al.(2009,(Xu(et(al.(2011).(In(Chapter(2,(we(showed(in(this(very(experiment(that( the( coccolithophore( Emiliania' huxleyi(was( negatively( affected( by( increased( CO2( and(ambient(iron(conditions,(but(its(biomass(massively(increased(at(ambient(CO2( and(increased(dFe,(suggesting(in'situ(iron(limitation.(However,(increased(dissolved( Fe( partially( mitigated( the( negative( effect( of( elevated( pCO2(indicating( that( E.' huxleyi(was( able( to( acclimate( better( to( ocean( acidification( when( Fe( was( high.( Therefore,(the(underlying(physiological(response(to(acclimation(to(stress(must(be( very(tightly(regulated(by(iron(in(the(studied(natural(strain.(Fe(is(required(for(many( processes;(hence(it(is(involved(in(a(plethora(of(cell(functions(including(carbon((C)( and(nitrogen((N)(fixation,(nitrate(and(nitrite(reduction,(chlorophyll(synthesis,(and( the( electron( transport( chains( of( respiration( and( photosynthesis.( Fe( is( also( incorporated( into( several( enzymes( used( by( cells( to( deal( with( reactive( oxygen( species( (ROS)( (Twining( &( Baines( 2013),( and( in( some( of( the( enzymes( that( participate(in(DNA(repair((Lukianova(&(Davis(2005,(Morita(et(al.(2010).( !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Phytoplankton!stress!under!increased!CO2!and!dissolved!iron! ! ! 89! Despite!the!considerable!contribution!of!marine!phytoplankton!to!global!climate! and!biogeochemical!cycles,!many!aspects!of!the!physiology!of!these!organisms!in! future! global! change-ocean! biology! relationships! are! poorly! understood.! Thus,! experimental!designs!aiming!to!unravel!the!relevance!of!the!interactive!effects!of! global!change!drivers,!contribute!to!reduce!the!lack!of!knowledge!on!the!interplay! of!the!different!environmental!factors,!which!is!one!of!the!main!constraints!in!our! ability!to!predict!the!functioning!and!composition!of!the!future!ocean.! Here!we!present! the!results!from!a!mesocosm!experiment! that!aimed!to! investigate! the! effects! of! future! changes! in! pCO2!and! Fe! availability! on! the! physiological! stress! response! during! a! bloom! of! the! coccolithophore! Emiliania' huxleyi!within! a! natural! plankton! community.! Emiliania' huxleyi!is! the! keystone! species! of! the! coccolithophores!(Paasche! 2002).! It! is! a! globally! relevant!species,! playing!a!major!role!in!the!global!carbon!cycle!by!regulating!the!exchange!of!CO2! across! the! ocean-atmosphere! interface! through! photosynthesis! and! calcium! carbonate! precipitation!(Rost! &! Riebesell! 2004)!and,! it! has! also! been! demonstrated! that! it! is! a! sensitive! phytoplankton! to! elevated! pCO2! (Riebesell! &! Tortell!2011).!The!objective!of!this!work!was:!i)!examine!the!effects!of!increased! CO2!and!iron!levels!in!the!stress!response!of!the!different!phytoplankton!groups;!ii)! analyse!the!effect!of!increased!CO2!and!dissolved!Fe!in!the!thylakoidal!transport! chain! and! accessory! pigments;! iii)! to! determine! the! impact! of! the! response! by! means!of!cell!viability,!oxidative!stress!and!DNA!damage!analyses.!This!study!is!the! first! to! analyse! the! stress! physiological! response! of! different! phytoplankton! species! in! a! mesocosm! experiment! where! CO2!and! Fe! concentrations! were! simultaneously!manipulated.! ! Chapter(4( ! 90( Material(and(methods( Experimental(design( A(mesocosm(experiment(was(carried(out(in(the(Raunefjord((60.39(ºN,(5.32(ºE),(off( Bergen,( Norway.( Twelve( mesocosms( (11( m3(each)( were( set-up( in( a( full-factorial( design(with(all(combinations(of(ambient(and(high(pCO2(and(two(treatments(of(dFe( in( three( independent( replicate( mesocosms( per( treatment.( High-density( polyethylene((HDPE)(mesocosms(were(filled(with( fjord(water(pumped(from(8( m( depth.( They( were( covered( with( low-density( polyethylene( (LDPE)(lids( in( order( to( avoid(pCO2(losses(and(contamination.(Mesocosms(and(their(lids(were(transparent( to( PAR( and( UVR.( After( the( first( sampling( day( (0),( the( seawater( of( half( of( the( mesocosms( was( enriched( with( CO2((Schulz( et( al.( 2009)( to( achieve( pCO2( concentrations(corresponding(to(levels(predicted(for(the(year(2100((900(µatm,(HC),( (IPCC(2013)( and( the( other( half( were( not( manipulated( (ca.( 390( µatm,( LC).( All( mesocosms(were(continuously(and(gently(mixed(by(using(an(airlift(system((Egge(&( Heimdal( 1994).( For( the( CO2(enrichment,( 150( L( of( fjord( water( was( aerated(with( pure(CO2(at(a(flow(rate(of(1.5(L(min-1(overnight(and(added(to(each(of(the(high(pCO2( (HC)( mesocosms.( To( maintain( the( pCO2(in( the( HC( treatments,( ambient( air( was( mixed(with(pure(CO2(at(a(flow(rate(of(200(mL(min-1(and(the(enriched(mixture((900( µatm( CO2)( was( pumped( directly( to( the( airlift(system.( LC( treatment( consisted( of( only-ambient( air( similarly( connected.( HEPA( filters( were( placed( between( the( air( pumps(and(the(airlift(system(to(avoid(particulate(contamination.(Mesocosms(were( fertilised(after(the(initial(sampling((day(0)(by(addition(of(10(µM(nitrate(and(0.3(µM( phosphate( to( induce( a( bloom( of( the( coccolithophorid( Emiliania' huxleyi' (Egge( &( Heimdal(1994).(On(day(7,(half(of(the(mesocosms((3LC(and(3(HC)(were(amended( with(70( nM( (final(concentration)( of(the(siderophore(desferrioxamine( B( (DFB)(to( promote(changes(in(iron(availability.(Dissolved(iron((dFe)(concentration(increased( ~3-fold(with(respect(to(the(control(as(a(result(of(both(factors(manipulation.(The( treatments( were( named( LC-DFB( (control),( LC+DFB,( HC+DFB( and( HC-DFB.( Water( samples( from( each( mesocosm( were( taken( from( 2( m( depth( by( gentle( vacuum( !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Phytoplankton!stress!under!increased!CO2!and!dissolved!iron! ! ! 91! pumping!of!25!L!volume!into!acid-washed!carboys!that!were!quickly!transported! to! the! onshore! laboratory.! All! variables! were! analysed! on! a! daily! basis! and/or! every!other!day,!except!otherwise!stated.!! ! Irradiance!and!temperature! Solar! spectral! irradiance! comprising! photosynthetically! active! radiation! wavelengths!(PAR,!400-700!nm)!ultraviolet!A!(UVA,!320-400nm)!and!ultraviolet!B! (UVB,!280-320!nm)!was!recorded!at!2!m!depth!in!M3!(LC-DFB),!every!1!h!every!day,! by!using!a!TRIOS!RAMSES!(Ramses,!TrioS!GmbH,!Germany)!spectroradiometer!that! was!deployed!in!the!M3!mesocosm.!For!a!basic!control!of!all!of!the!mesocosms! receiving! the! same! irradiance! at! the! surface! and! monitoring! of! temperature,! HOBO! Pendant! Temperature/Light! loggers! (Onset! Computer! Corporation,! Massachusetts,!USA)!were!attached!to!the!airlift!system!at!0,!1,!2!and!3!m!depth! and! also! at! surface! in! air.! The! equivalent! biologically! effective! irradiances! were! calculated! using! the! biological! weighting! functions! (BWFs)! for! chloroplast! inhibition!(Jones!&!Kok!1966),!general!plant!damage!(Caldwell!1971),!DNA!damage! (Setlow!1974),!inhibition!of!phytoplankton!photosynthesis!(Cullen!et!al.!1992)!and! inhibition! of! photosynthesis! in! Antarctic! phytoplankton! (Cullen! &! Neale! 1997).! These!data!are!summarized!in!Table!4.1.! ! Pigments!concentration! For! this! purpose,! samples! (1! to! 2! L)! were! collected! from! each! mesocosm! and! gently!filtered!through!GF/F!filters.!Samples! were! snap! frozen! in! liquid! nitrogen! and! kept! at! −80°C! until! analysis.!Pigments! were! extracted! using! N,Ndimethylformamide!(DMF)!overnight!at!4!º!C.!Chlorophyll!a!(Chl!a)!concentration! was! determined! spectrophotometrically! and! the! ! concentration! was! calculated! according! to! Wellburn! (1994).!Lutein! (Lut),! neoxanthin! (Neo),! violaxanthin! (V),! anteraxanthin! (A),! zeaxanthin! (Z),! chlorophyll! c2$ (Chl$ c2),!chlorophyll! c3!(Chl$ c3),! peridinin! (PERI),! fucoxanthin! (FUCO),! 19’-butanoyloxyfucoxanthin! (BUTA),! 19’– Chapter(4( ! 92( hexanoyloxyfucoxanthin( (HEXA),( diadinoxanthin( (DD),( diatoxanthin( (DT),( prasinoxanthin( (PRAS),( Pheophorbide((Pheo)( and( chlorophyllide( a((Chlide)( were( determined(by(HPLC(as(described(in(Lubian(&(Montero((1998).( ( In#vivo!chlorophyll!a!(Chl!a)!fluorescence! Optimal(quantum(yield((Fv/Fm)( of(PSII(was(measured(in(20(min(dark-adapted( samples(by(pulse(amplitude(modulated(fluorometry((Water-PAM,(Waltz,(Effeltrich,( Germany).(Rapid(light(curves((RLCs)(were(constructed(according(to(Figueroa(et(al." (2009)(and(fitted(to(the(model(of(Eileers(&(Peters((1986)(to(obtain(the(initial(slope( (αETR)(and(maximal(electron(relative(transport(rate((rETRmax).(The(light(saturation( parameter((Ek)(was(derived(from(rETRmax(and(α.((( ( Cell!viability! Cell(viability(was(assessed(using(the(nucleic(acid(stain(SYTOX(Green((Invitrogen,( Oregon,(USA)(according(to(Segovia(&(Berges((2009).(SYTOX(Green(only(stains(the( nucleic(acid(of(cells(that(have(compromised(plasma(membranes.(Green(staining(of( the(cell(nucleus(indicates(a(dead(cell,(before(the(cell(loses(its(integrity(and(lyses( (Veldhuis(et(al.(2001)(while(viable(cells(are(not(stained(and(fluoresce(in(red.( Samples(from(each(mesocosm((1-2(L)(were(concentrated(by(gentle(filtration(under( low(pressure(on(0.8(µm,(47-mm(polycarbonate(membranes(to(a(final(volume(of(15( mL(at(10ºC.(Chlorophyll(a((Chl(a)( content(in(the(concentrates(was(measured(by( extraction(in(90%(acetone(overnight(and(determined(fluorometrically(using( a( Turner(fluorometer(10-AU((Turner(BioSystems,(CA,(USA).((Final(Chl(a(concentration( in(cell(concentrates(ranged(from(0.2(to(400(µg( L-1.(SYTOX-Green((5(µM(final( concentration)(was(added(to(1(mL(of(the(concentrated(cell(suspensions(from(the( natural(phytoplankton(community.(Samples(were(incubated(at(10(°C((natural( temperature(condition(in(the(fjord)(in(darkness(for( 60( min.(Fluorescence(was( quantified(in(a(NIKON(epifluorescence(microscope(at(an(excitation(of(490(nm(and( an(emission(of(525(nm.(Between(30-50(fields(of(view((FOVs)(were(analysed(per( !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Phytoplankton!stress!under!increased!CO2!and!dissolved!iron! ! ! 93! sample!(N≥600).!Positive!controls!consisting!of!100%!killed!cells!were!run!in! parallel!and!obtained!by!pre-treating!the!samples!with!1%!glutaraldehyde!(final! concentration)!for!2!h!at!4!°C.!! ! General!oxidative!stress! The!intracellular!accumulation!of!reactive!oxygen!species!(ROS)!was!assayed!in! concentrated!cell!suspensions!from!the!natural!phytoplankton!community!(as! above)!by!using!a!modification!of!the!method!used!by!Segovia!&!Berges!(2009)! described!in!Bouchard!et!al.! (2013).!Shortly,!cells!were!incubated!with!10μM! carboxy-H2DFFDA!(Invitrogen,!Oregon,!USA)!(final!concentration)!at!10!°C!for!90! min!in!darkness.!Fluorescence!was!quantified!in!a!NIKON!epifluorescence! microscope!at!an!excitation!of!490!nm!and!an!emission!of!525!nm.!Between!30-50! fields!of!view!(FOVs)!were!analysed!per!sample!(N≥600).!!! ! DNA!Damage! DNA! damage! was! assessed! according! to! García-Gómez! et! al.! (2012).! For! this! purpose,! samples! (1! to! 2! L)! were! collected! from! each! mesocosm! and! gently! filtered!through!0.8!μm!polycarbonate!filters.!Samples!were!snap!frozen!in!liquid! nitrogen! and! kept! at! −80°C! until! analysis.! For! analysis,! DNA! was! extracted! and! quantified! from! the! filters;! 15! ng! of! DNA! were! used! from! each! sample! for! immunodetection! of! CPDs! with! a! monoclonal! anti-CPD! antibody! (H3,! Affitech).! Possitive! controls! consisted! of! UVR-radiated! DNA! from! lambda! phage! and! Dunaliella( tertiolecta!(García-Gomez! et! al.! 2014).! Negative! controls! consisted! of! the! same! former! DNA! sample! species! without! radiation.! ! Unspecific! crossreactivity! controls! were! carried! out!by! incubating! the! membranes!with! only! the! secondary! antibody! in! the! absence! of! the! primary! antibody.! The! signal! was! detected! by! chemiluminescence! (ECL,! GE! healthcare,! Buckinghamshire,! UK)! and! the!intensity!of!cross-reactions!quantified!in!a!Gel!Logic!Image!Analyser!(EastmanKodak,!Rochester,!NY,!USA).!! Chapter(4( ! 94( Statistical(analyses( Data( were( checked( for( normality( (by( Saphiro-Wilks’( test),( homoscedasticity( (by( Cochran’s(and(Levene’s(tests)(and(sphericity((by(Mauchly’s(and/or(Bartlett’s(tests).( Variables( met( all( criteria( to( perform( parametric( tests.( Statistical( significance( of( treatment( effects( on( variables( was( performed( by( using( Split-Plot( ANOVA( (also( called(SPANOVA(or(mixed-model(ANOVA)(followed(by(post-hoc(Sidak(or(Tukey(and( Bonferroni( tests,( respectively( (considering( p<0.05( and/or( p<0.01( as( significant).( When( appropriate,( data( were( specifically( tested( for( significant( differences( (p<( 0.05)(induced(by(the(treatments(by(using(1(or(2(Way(ANOVAs(and/or(Student’s(ttests,( as( well( as( Pearson’s( product-moment( correlations.( All( analyses( were( performed(using(the(GLM((general(linear(model)(procedure(with(main(effects((CO2,( dFe),( time( (repeated( measure)( and( all( interactions.(Statistical( analyses( were( performed(by(using(the(software(Statistica(v12((Statsoft,(Inc.)(and(SPSS(v22((IBM( statistics).(( ( Results( Irradiance(and(Temperature( Meteorological(conditions(during(the(experiment(were(stable(with(75%(clear(sky( days(and(25%(partly(cloud(covered(days.(Average(midday(range((solar(noon(±(2(h)( irradiance(in(the(mesocosms(at(2(m(depth(was(276.2((±(45)(µmol(photons(m-2s-1( PAR;(1.61((±(0.98)(Wm-2(UVA;(and(0.01((±(0.01)(W(m-2(UVB.((The(midday(average( irradiance( varied( around( this( mean( on( days( with( cloud( cover.( Experimental( irradiances( and( corresponding( weighted( irradiances( (i.e.( biological( effective( irradiance,( BEI),( as( well( as( the( biological( effective( dose( (BED)( calculated( for( the( control( mesocosms( are( shown( in( Figure( 4.1( and( Table( 4.1.( Irradiance( and( transmittance( through( polyethylene( mesocosms( and( lids( are( shown( in( Supplemental( Table-4.2S.( The( water( temperature( in( the( mesocosms( during( the( experiment( varied( between( 10-11°C( corresponding( to( the( surrounding( fjord( temperature.((( !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Phytoplankton!stress!under!increased!CO2!and!dissolved!iron! ! ! 101! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Figure! 4.3.! Ratios!of! (a)! diatoxanthin! and! diadinoxantin! to! total! fucoxanthins! (DT+DD/! TFUCO)!and!(b)!19’-hexanoyloxyfucoxanthin!to!total!fucoxanthins!(HEXA/!TFUCO).!Ambient! pCO2!and!ambient!dFe!(LC-DFB,!black);!ambient!pCO2!and!increased!dFe!(LC+DFB,!white);! increased!pCO2!and!increased!dFe!(HC+DFB,!striped),!increased!pCO2!and!ambient!dFe!(HCDFB,! grey).! Data! are!means! of! measurements! in! three! independent! mesocosms! (n=3)! except!for!LC-DFB!where!n=2.!Error!bars!indicate!standard!deviations.! ! Chapter(4( ! 102( ! ! ! ! Figure! 4.4.! Temporal(development(of(the(photosynthetic(parameters:((a)(Fv/Fm,((b)(α,((c)( rETRmax(and( Ek.!Ambient( pCO2(and( ambient( dFe( (black( filled( circle);( ambient( pCO2(and( increased( dFe( (LC+DFB,( open(circle);( increased( pCO2(and( increased( dFe( (HC+DFB,( open( square),(increased(pCO2(and(ambient( dFe((HC-DFB,(black(filled(square).( Symbols( indicate( means(of(measurements(in(three(independent(mesocosms((n=3)(except(for(LC-DFB(where( n=2.(Error(bars(indicate(standard(deviations.! ! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Phytoplankton!stress!under!increased!CO2!and!dissolved!iron! ! ! 103! Photosynthetic!parameters!were!dependent!on!pCO2!and!dFe!concentration! During! phase! 1! (days! 1-10)! there! were! no! significant! differences! between! treatments!in!any!of!the!photosynthetic!parameters!(Figure!4.4).!However,!in!day! 10,!a!transitory!decrease!in!Fv/Fm!(Figure!4.4a)!and!in!the!photosynthetic!efficiency! (α)!(Figure!4.4b)!were!observed,!in!parallel!with!an!increase!in!rETRmax!(Figure!4.4c)! and!Ek!(Figure!4.4d)!in!all!the!treatments.!Between!days!11-22!(phase!2),!Fv/Fm!was! significantly!higher!in!LC!than!in!HC!treatments.!In!LC,!high!dFe!(+DFB)!promoted!a! significant! increase! in! Fv/Fm!with! respect! to! the! control! (LC-DFB)! (Table! 4.2).! In! contrast,!in!the!HC!treatments!Fv/Fm!was!higher!in!the!absence!of!DFB.!Both!pCO2! and!DFB!factors,!and! their! interaction! had! significant! effects! on! Fv/Fm!during!all! this! period.! The! photosynthetic! efficiency! (α)! matched!the! Fv/Fm!trend! and! LC! showed!higher!values!than!in!HC.!DFB!addition!had!a!significant!positive!effect!on! LC!treatments!promoting!a!higher!efficiency,!while!it!did!not!exert!any!potential! benefit! in! HC! (Table! 4.2).! Pmax!and! Ek!did! not! present! significant! differences! between!treatments!in!phase!2.! ! Cell! death! was! affected! by! high! CO2!and! general! oxidative! stress! decreased! under!high!dFe! Cell!death!was!not!detected!during!the!whole!experiment!except!for!days!11!and! 14!(Figure!4.5a).!!On!day!11,!20!%!cells!showed!SYTOX-positive!green!fluorescence,! therefore! pointing! to! the! complete! lost!of!viability! under!increased!pCO2.! In! LC,! there! were! 4-fold! less! green! fluorescent-labelled! cells! indicating! that! indeed! HC! had! a! significant! negative! effect! on! part! of! the! phytoplankton! population! when! compared!to!LC!(Table!4.2).!The!percentage!of!SYTOX-positive!cells!decreased!10fold!to!threshold!levels!by!day!14.!Green!fluorescence!emitted!by!the!cells!due!to! general!oxidative!stress!was!significantly!higher!in!HC!than!in!LC!at!the!beginning! of!the!experiment,!declining!by!60%!between!days!3!and!7.!As!it!was!observed!for! cell!death,!ca.!20%!of!the!cells!showed!symptoms!of!oxidative!stress! on!day! 11! (Figure!4.5b).!However,!in!this!case,!the!percentage!of!green!fluorescent!labelled! Chapter(4( ! 104( cells( was( significantly( higher( at( ambient( dFe( (-DFB( treatments)( than( in(high-dFe( grown(cells((+DFB),(and(independent(on(the(pCO2(levels((Table(4.2).( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ! ! Figure! 4.5.! Temporal( development( of!percentage( of( cells( SYTOX-Green( labelled( (a)( and( percentage(of(cells(c-H2DFFDA-green(labelled((b).!Ambient(pCO2(and(ambient(dFe((black(filled( circle);(ambient(pCO2(and(increased(dFe((LC+DFB,(open(circle);(increased(pCO2(and(increased( dFe( (HC+DFB,( open( square),( increased( pCO2(and( ambient( dFe( (HC-DFB,( black( filled(square).( Symbols(indicate(means(of(measurements(in(three(independent(mesocosms((n=3)(except(for( LC-DFB(where(n=2.(Error(bars(indicate(standard(deviations.! !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Phytoplankton!stress!under!increased!CO2!and!dissolved!iron! ! ! 105! DNA!damage!was!minimised!by!increased!dFe!in!Emiliania'huxleyi! In!phase!1,!cyclobutane!pyrimidine!dimers!(CPDs)!formation!significantly!increased! 4-fold!between!days!1!and!3!(Figure!4.6),!to!remain!steady!and!with!no!differences! between!treatments!up!to!day!9!(Table!4.2).!CPDs!sharply!increased!in!those!cells! subjected! to! high! dFe! (+DFB)! in! day! 9,! while! the! treatment! showing!less! DNA! damage!was!HC-DFB.!CPDs!content!was!drastically!reduced!between!days!9!and!11.! As!phase!2!progressed,!the!treatments!that!exhibited!significant!increased!levels! of! accumulated! DNA! damage! were! those! at! ambient! dFe! (-DFB)! (Table! 4.2).! By! contrast,! the! treatments! with! +DFB! presented! significant! lower! CPDs! concentration!than!the!control,!regardless!of!pCO2.! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Figure! 4.6.! Temporal! development! of! cyclobutane! pyrimidine! dymers! (CPDs)! within! the! mesocosms.!Ambient!pCO2!and!ambient!dFe!(LC-DFB,!black);!ambient!pCO2!and!increased! dFe!(LC+DFB,!white);!increased!pCO2!and!increased!dFe!(HC+DFB,!striped),!increased!pCO2! and!ambient!dFe!(HC-DFB,!grey).!Data!are!means!of!measurements!in!three!independent! mesocosms! (n=3)! except! for! LC-DFB! where! n=2.! Error! bars! indicate! standard! deviations. Table& 4.2.!Statistical!analyses!(Split-plot!ANOVA)!of!the!effects!of!CO2,!DFB,! and!their!interaction,!as! well!as!the!effect!of!time,!on!the!variables!analysed!in!the!different!treatments:!LC,!ambient!CO2!(390! μatm);!HC,!increased!CO2!(900!μatm);!-DFB,!(ambient!dFe);!+DFB,!(increased!dFe)!considering!p<0.05!as! significant.!! Factor& CO2& DFB& Time& CO2xDFB& CO2xDFBxTime& Fv/Fm& <0.001& <0.001& <0.001& <0.05& <0.001& α& <0.01& ns& <0.005& <0.05& ns& Ek& ns& ns& ns& ns& ns& rETRmax& <0.05& ns& ns& ns& ns& Chl&c3& <0.001& <0.001& <0.05& <0.05& <0.05& Chl&c2& <0.01& ns& <0.001& ns& ns& PERI& <0.05& ns& <0.001& ns& ns& BUTA& <0.01& ns& <0.01& ns& ns& FUCO& <0.001& ns& <0.01& ns& ns& HEXA& <0.001& <0.001& <0.001& <0.001& <0.001& DD& <0.001& <0.001& <0.01& <0.01& ns& DT& <0.05& ns& <0.001& <0.05& <0.01& Zea& <0.001& ns& <0.05& ns& ns& PRAS& <0.01& ns& <0.001& ns& ns& HEXA/FUCO& ns& <0.001& <0.001& <0.05& <0.001& DT+DD/TFUCO& ns& ns& <0.05& <0.001& <0.001& Cell&death& <0.01& ns& <0.001& ns& ns& Oxidative&stress& <0.01& ns ns ns& <0.05& DNA&damage& ns& <0.001& <0.001& <0.05& <0.05& !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Phytoplankton!stress!under!increased!CO2!and!dissolved!iron! ! ! 107! Discussion!! In!the!present!work!we!studied!the!physiological!processes!that!interact!from!the! molecular!to!the!organism!level,!in!order!to!understand!the!final!response!of!the! phytoplankton!community!and!in!particular,!the!response!of!the!coccolithophore! Emiliania' huxleyi!to! the! interactive! manipulation! of! CO2!levels! and! iron! bioavailability.!The!reproducibility!between!the!triplicates!of!each!treatment!was! high,! allowing! us! to! isolate! and! identify! single! and! interactive! effects! on! the! variables!measured.'We!assessed!the!behaviour!of!the!variables!in!relationship!to! the! different! functional! phytoplankton! groups! during! three! important! time! periods!along!the!experiment!(for!details!see!Chapter!2):!i)!phase!1!(days!0-10),! characterised!by!the!absence!of!factors!effects!on!the!phytoplankton!community! and! consequently! the! lack! of! response,! ii)! shift-point! (day! 10-11),! when! the! phytoplankton!community!shifted!due!to!mid-term!effects!of!increased!CO2!and! dissolved! iron! (dFe)! levels,! and! iii)! phase! 2! (days! 11-22),! distinguished! by! an! evident! response! of! the! community! ! to! treatment! effects! in! the! long-term.! A! bloom! of! the! coccolithophore! E.' huxleyi!was! developed! in! this! phase! under! ambient!CO2!and!increased!dFe!(LC!+DFB),!outcompeting!the!rest!of!the!groups.! This!bloom!was!not!observed!neither!in!the!control!at!ambient!CO2!and!dFe!(LCDFB)!nor!in!the!high!CO2!conditions,!although!E.'huxleyi'was!the!most!abundant! species!in!all!treatments!except!in!HC-DFB!(Supplemental!Figure!4.2S).!In!Chapter! 2,! we! demonstrated! that! Fe! concentrations! may! control! phytoplankton! community!structure!in!coastal!ecosystems,!that!ocean!acidification!can!enhance! Fe!bioavailability!and,!that!in!areas!with!high!total!Fe!concentrations!(particulate! and!dissolved!Fe),!the!detrimental!effects!of!increased!pCO2!on!E.'huxleyi!can!be! partially!mitigated!by!enhanced!dFe!depending!on!the!cellular!strain.!Therefore,! consequences! of! the! interactive! effects! of! pCO2!and! Fe! availability!on! E.' huxleyi! can!be!critical!to!C-cycling!and!marine!ecosystems.!! From!the!physiological!point!of!view,!Fe!plays!a!basic!major!role!in!plankton! ecology!since!every!aspect!of!thylakoid!electron!transport!and!accessory!pigments! Chapter(4( ! 108( is(Fe(dependent.(A(decline(in(cellular(pigment(content(seems(a(general(response( to( Fe( stress( (Beherenfeld( &( Milligan(2009).( The( response( showed( by( the( community(when(dFe(concentration(increased(demonstrated(that(E.#huxleyi#was( experiencing(Fe(limitation((the(highest(growth(rates(were(presented(by(cells(in(the( LC+DFB(treatment(whilst(the(lowest(Chl(a(values(were(shown(in(those(treatments( with( ambient( iron( and( high( HC)( (Chapter( 2;( Supplemental( Figure( 4.1S).( The( concentration(of(pigments(occurred(during(phytoplankton(community(succession( (Figure( 4.2)( as( widely( reported( in( other( experiments( (Suffrian(et( al.(2008,( Polimene(2012).(Pigment(signatures(are(used(to(determine(the(contribution(of(the( distinct(taxonomic(groups(in(natural(assemblages.(Accordingly,(the(distribution(of( pigments( in( our( experiment( is( in( agreement( with( the( flow( cytometry( analysis( performed( in( this( experiment((Supplemental( Figure( 4.1S).( Pigments( changed( specifically(in(days(10-11,(in(parallel(with(a(community(shift(that(was(observed(in( phase(2((Supplemental(Figure(4.1S).(Xanthophylls,(fucoxanthins(and(chlorophylls( corresponding( to( Cyanophyta,( Heterokontophyta,( Haptophyta,( Dinophyta( and( Chlorophyta((Takaichi(2011)(were(present(in(the(mesocosms((Figure(4.2).(( However,( what( determines(phytoplankton(species(fitness,(succession( and( distribution( are( the( physiological( trade-offs( between( light( harvesting,( photoacclimation(or(photoprotection(and(dissipation(of(excess(energy,(in(which(all( pigments( participate.( In( phase( 2,( chlorophylls( c2(and( c3(were( the( two( pigments( that(showed(the(highest(concentrations(and(closely(followed(the(same(trend(than( E.# huxleyi(biomass( (Supplemental( Figure( 4.1S).( They( are( considered( secondary( chlorophylls( especially( found( in( light-harvesting( complexes( (LHCs).( ( Due( to( the( distribution(in(LHCs,(the(function(of(Chl(c(in(photosynthesis(has(been(described(as( an( enhancement( of( light( absorption,( particularly( in( the( blue( wavelength( (Mizoguchi(et(al.(2011).(The(DD-DT(cycle(was(probably(active,(playing(a(key(role( being( the( main( driver( for(non-photochemical( quenching( (NPQ)((i.e.( the( nonradiative( dissipation( of( excess( of( energy( reaching( the( photosynthetic( apparatus,( Demmig-Adams(et(al.(1996).(However,(the(VAZ(cycle(was(most(likely(not(active,( !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Phytoplankton!stress!under!increased!CO2!and!dissolved!iron! ! ! 109! because! neither! violaxanthin,! nor! anteraxanthin! were!detected,! and! only! zeaxanthin!accumulated!in!phase!2!(Figure!4.2).!HEXA,!PRAS,!and!PERI!peaked!in! phase! 2! in! the! treatments! with! highest! biomass! accumulation! (LC+DFB).! These! pigments! posses! light-harvesting! functions! and! have! been! shown! to! be! highly! efficient!transferring!energy!to!chlorophylls,!which!is!an!essential!step!to!ensure! an!efficient!utilization!of!the!available!light!by!absorbing!photons!at!wavelengths! not! “covered”! by! chlorophyll.! All! the! pigments! were! significantly! negatively! affected!by!CO2!levels!and!most!of!them!positively!by!dFe!(Table!4.2).!! In!multispecific!bloom!situations,!the!ratio!of!DT+DD!to!total!fucoxanthins! (DT+DD/TFUCO)!can!be!used!as!an!indicator!of!the!cellular!physiological!status!of! E.# huxleyi!(Stolte! et! al.!2000),! instead! of! using! the! ratio! DT+DD/CHLA! that! also! accounts!for!other!taxonomic!groups!present.!Increased!HEXA/TFUCO!rates!have! been!observed!in!nutrient-limited!E.#huxleyi!as!a!consequence!of!reduced!growth! (a!similar!nutrient!scenario!than!in!this!experiment,!see!Chapter!2).!We!observed! the! highest! HEXA/TFUCO! ratios! (mol:mol)! under! LC+DFB,! followed! by! HC+DFB! treatments! (Figure! 4.3),! always! well! above! the! control! (LC-DFB)! and! HC–DFB! treatment.! HEXA!has! been! shown! to! also! have! light-harvesting! functions! in! E.# huxleyi!(Stolte!et!al.!2000);!hence,!HEXA!is!most!likely!increasing!the!antenna,!and! possibly! the! number! of! reaction! centres.! This! points! out! to! a! constrained! physiological!status!of!the!cells!at!ambient!dFe!concentration!(LC-DFB),!and!to!a! benefit!of!high!dFe!effects!for!the!cell!under!HC!(Chapter!2).!In!agreement,! the! (DT+DD)/TFUCO!ratios! were!higher!in!the!+DFB!condition!respect! to! the! control! (Figure! 4.3)! and! regardless! pCO2.! Under! this! condition,! a! lower! (DT+DD)/TFUCO! (mol:mol)! ratio! implies! a! dilution! of! the! DD-DT! cycle,! meaning! that! the! cells’! capacity!for!non-photochemical!quenching!(NPQ)!is!decreased,!thus!being!prone! to! photodamage.! Indeed,! it! is! known! that! Fe-limited! cells! show! a! high! susceptibility!to!photooxidation!(Behrenfeld!&!Milligan!2009).!! The!above-mentioned!pigments!performance!was!higher!under!increased! iron!levels,!leading!to!a!better!functioning!of!the!electron!transport!chain!although! Chapter(4( ! 110( CO2(could( modulate( this( effect( (Table( 4.2).( Photosynthetic( parameters( were( significantly(affected(by(both(CO2(and(iron(levels(in(phase(2.(It(is(worth(mention( that(in( day( 10,( a( transitory( decrease( was( observed( in( both( Fv/Fm(and( photosynthetic( efficiency( (α).( At( the( same( time,( rETRmax((Pmax)( and( Ek(increased( (due( to( a( lowered( α)( (Figure( 4.4).( This( corresponded( to( the( community( shift( in( which( some( groups( completely( declined,( while(others’( growth( rates( increased.( During(days(11-22(high(dFe((+DFB)(promoted(a(significant(increase(in(Fv/Fm(with( respect(to(the(control((LC-DFB)(and(α(at(the(expense(of(a(constant(rETRmax.(Fv/Fm( values( and( growth( rates( are( in( agreement( with( those( observed( for( E.# huxleyi( cultures(grown(under(different(Fe(conditions(at(ambient(CO2((Honey(et(al.(2013).( The( Fv/Fm(decay( under( iron( stress( is( a( widely( observed( response( (Behrenfeld( &( Milligan(2013)(and(so(the(electron(transport(chains(are(the(primary(control(on(cell( growth(under(low(iron(conditions.(This(explains(that(under(elevated(dFe(levels,(the( increases(in(pigments(ratios(and(photosynthetic(parameters(values(lead(to(higher( E.#huxleyi(growth(rates((Chapter(2).(Fv/Fm(and(α(were(significantly(lower(in(HC(than( in(LC(treatments(affecting(the(growth(rates.(The(inability(to(sustain(high(operative( electron(transport(rates(and(cell(division(at(increased(CO2,(is(most(likely(due(the( inability( of( the( cells( to( regulate( the( internal( pH.( Maintaining( a( constant( intracellular( pH( is( energetically( costly( and( OA( likely( affect( the( cellular( energy( demands((Taylor(et(al.(2012).(( It( is( expected( that( a( stressed( cell( metabolism( (by( both( the( lack( of( iron( needed(to(meet(the(cell(quotas(or(by(an(excess(of(CO2(i.e.(lower(pH)(will(produce( reactive(oxygen(species((ROS:(singlet(oxygen,(hydroxyl(and(peroxyl(radicals(among( others),( directly( by( the( harming( effects( of( excess( light(reaching( the( cell( due( to( affected(pigments((by(decreasing(the(size(of(the(sink(for(electrons(produced(in(the( light( reactions( of( photosynthesis),( or( indirectly,( due( to( chemical,( physical,( or( photosensitized( reactions( inside( and( outside( the( cells( (Lesser(2006).( This( can( finally(lead(into(cell(death((Segovia(et(al.(2009,(Bouchard(et(al.(2013,(Sobrino(et(al.( 2014).(Strikingly,(cell(death(was(only(detected(during(the(shift(in(the(community( !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Phytoplankton!stress!under!increased!CO2!and!dissolved!iron! ! ! 117! Hypothetical!proteins! ! Mitochondrial!manganese!superoxide!dismutase! 17267818! MRP!protein! 17269531! NAD(P)H!dehydrogenase! 19046606! NAD(P)H!dehydrogenase! 19046606! NADH-cytochrome!B5!reductase! 17284685! NADPH-cytochrome!P450!reductase! 17280251! NADPH-cytochrome!P450!reductase! 17280251! N-glycosylase/DNA!lyase! 17277269! Peroxiredoxin! 19046203! Peroxiredoxin! 17264933! Photosystem!I!subunit!III! 3562420! Photosystem!I!subunit!III! 3562420! Polyketide!synthase! 17257969! Putative!ABC!transporter! 17280191! Putative!Cry-DASH!cryptochrome! 17261457! Putative!Cry-DASH!cryptochrome! 17257259! Putative!cytochrome!b5!reductase! 17286410! Putative!glutathione-S-transferase! 17287011! Putative!mitochondrial!J-type!chaperone! 17263969! Putative!mitochondrial!J-type!chaperone! 17259500! Putative!mitochondrial!J-type!chaperone! 17263969! Putative!mitochondrial!J-type!chaperone! 17259500! Superoxide!dismutase,!Cu-Zn! 17260763! Superoxide!dismutase,!manganese! 17276920! Thioredoxin! 17285654! Thioredoxin!reductase! 17279611! Thioredoxin!reductase! 17250239! ! ! ! Chapter(4( ! 118( Supplemental* Table* 4.2S.* Irradiance( (%( referred( to( sun)( through( the( polyethylene( mesocosm(material( (a)(and(mesocosm(lids(transmittance((%(referred(to( lamp)(measured( with(SMS-500((Ocean(Optics(LLC,(USA)(between(200-800(nm.(Lamp(refers(to(a(Qpanel(lamp( used(for(UVA(and(UVB(control.(MES(refers(to(mesocosms(lids.( (a)( %*Transmittance* Mesocosm* PAR** 73.33( UVA*(320-400*nm)* 53.95( UVB*(280-320*nm)* 67.07( (b)( %*Transmittance* Mesocosm*lid* PAR** 90.45( UVA*(320-400*nm)* 83.64( UVB*(280-320*nm)* 80.92( ( ( ( Supplemental* Figure* 4.1S.(Temporal( development( of( phytoplankton,( microzooplankton( and( heterotrophic( bacterioplankton( biomasses( (µgC( L-1)( in( the( mesocosms( exposed( to( different(CO2(and(dFe(treatments.((a)(Emiliania'huxleyi((5-10(µm);((b)(Synechococcus(sp.,( (0.6-2(µm);((c)(picoeukaryotes((0.1-2(µm);((d)(small(nanoeukaryotes((Prasinophytes,(small( Haptophytes,(2-7(µm);((e)(big(nanoeukaryotes((small(single(celled(diatoms(and(flagellated( forms,( 6-20( µm);( (f)( Diatoms( (chain( forming( Skeletonema'costatum,( 20->500µm);( (g)( Dinoflagellates( (20-200( µm);( (h)( Ciliates( (20-200( µm);( (i)( heterotrophic( bacterioplankton( (0.2-0.7( µm).( Symbols( (as( in( Figure( 2.1)( indicate( means( of( measurements( in( three( independent(mesocosms((n=3)(except(for(LC-DFB(where(n=2.(Error(bars(indicate(standard( deviations.( ( ! Chapter(5( ( Particulate* trace* metal* dynamics* in* response* to* increased* CO2*and* iron* availability* in* a* coastal* mesocosm* experiment* ( M(Rosario(Lorenzo*,(María(Segovia,(Maria(T(Maldonado( Manuscript+for+submission+ Chapter(5( ! ! 122( Abstract!( Rising( concentrations( of( atmospheric( carbon( dioxide( are( causing( acidification( of( the(oceans,(which(will(undoubtingly(influence(marine(processes(and(trace(metal( biogeochemistry.( The( importance( of( trace( metals( for( marine(ecosystem( functioning(has(been(reported,(but(the(combined(impacts(of(high(CO2(and(changes( in( trace( metal( availability( on( plankton( remain( largely( unknown.( A( mesocosm( experiment( was( performed( in( the( Raunefjord( (Norway)( to( study( changes( in( the( trace( metal( concentrations( of( particles( during( a( bloom!dominated( by( the( coccolithophore( Emiliania! huxleyi(under( different( CO2(and( iron( conditions.( We( employed( a( full-factorial( design,( comprising( of( all( combinations( of( ambient( and( high(pCO2,(as(well(as(dissolved(iron((dFe).(Particulate(metal(concentrations((Fe,(Cu,( Zn,( Co,( Mn,( Cd,( Mo,( Ti( and( Pb)( were( determined( by( high-resolution( inductively( coupled(plasma(mass(spectrometry((HR-ICPMS).(This(work(also(aimed(to(examine( the(source(of(particulate(metals,(either(biogenic(or(lithogenic,(and(the(evolution(of( both(during(the(experiment.(To(estimate(biogenic(metal(concentrations(from(bulk( particle(measurements(we(compared:(i)(the(P-normalized(quotas(with(published( ratios,(and(ii)(the(concentrations(of(particulate(trace(metals(in(the(presence(and( absence( of( an( oxalate-EDTA( wash.( The( two( methods( were( in( agreement.( Our( results( also( demonstrate( that( particulate( Ti( and( Fe( concentrations( were( dominated(by(lithogenic(material(in(the(fjord.(In(contrast,(particulate(Cu,(Co,(Mn,( Zn,( Mo( and( Cd(concentrations( were( correlated( with( P( concentrations( and( phytoplankton( biomass,( emphasizing( their( strong( biogenic( component.( Furthermore,( ocean( acidification( changed( the( relative( concentrations( of( particulate(metals;(a(result(mainly(driven(by(the(effects(of(ocean(acidification(on( the( growth( of( different( phytoplankton( phyla.( This( study( is( the( first( to( combine( trace( metal( analyses( of( particles( in( a( controlled( mesocosm( experiment( with( manipulation(of(CO2(and(Fe(concentrations(using(natural(assemblages(of(marine( phytoplankton.!! Trace&metal&dynamics&in&response&to&increased&CO2&and&iron&availability&&& ! ! 123& Introduction! Marine&phytoplankton&contribute&to&half&of&the&world’s&total&primary&productivity,& sustaining&marine&food&webs&and&driving&the&biogeochemical&cycles&of&carbon&and& nutrients&(Field&et&al.&1998).&Annually,&phytoplankton&incorporates&approximately& 45& to& 50& billion& metric& tons& of& inorganic& carbon& (Field& et& al.&1998),& removing& a& quarter&of&CO2&emitted&to&the&atmosphere&by&anthropogenic&activities&(Canadell&et& al.& 2007).& Yet,& as& a& result& of& anthropogenic& CO2&emissions,& the& atmospheric& CO2& concentration& has& increased& by& 40%& since& pre-industrial& times,& producing& rapid& changes& in& the& global& climate& system& (IPCC&2013).& The& dissolution& of& anthropogenic&CO2&in&seawater,&causes&shifts&in&the&carbonate&chemical&speciation,& and&leads&to&ocean&acidification.&Marine&ecosystems&are&sensitive&to&changes&in&pH& because&pH&strongly&affects&chemical&and&physiological&reactions&(Hoffman&et&al.& 2012).& Increased& CO2&in& seawater& is& expected& to& enhance& or& diminish& phytoplankton&productivity&(Mackey&et&al.&2015),&to&decline&CaCO3&production&in& most& planktonic& calcifiers& (Riebsell& && Tortell& 2011),& and/or& to& inhibit& organic& nitrogen& and& phosphorus& acquisition& (Hutchins& et& al.&2009).& Thus,& the& biogeochemical& cycling&of& nutrients&is& predicted&to&be&greatly&affected&by& ocean& acidification&(Hutchins&et&al.&2009).&Variations&in&pH&will&also&affect&the&distribution& and&speciation&of&trace&metals&in&the&ocean&(Millero&et&al.&2009).& Trace&metals,&including&Fe,&Zn,&Mn,&Cu,&Co,&Cd&and&Mo,&are&essential&for& biological& functions& (e.g.& photosynthesis,& respiration& and& macronutrient& assimilation).& The&availability&of&metals&can&influence&phytoplankton&growth&and& community& structure& (Morel& && Price& 2003).& In& turn,& plankton& may& control& the& distribution,&chemical&speciation,&and&cycling&of&metals&in&the&ocean&(Sunda&2012).& The& effects& of& ocean& acidification& on& inorganic& metal& speciation& will& be& more& pronounced&for&metals&that&form&strong&complexes&with&carbonates&(e.g.&copper)& or&hydroxides&(e.g.,&iron&and&aluminium).&In&contrast,&metals&that&are&mainly&in&the& free&ionic&form&(e.g.,&manganese&or&cobalt)&or&those&that&form&stable&complexes& with&chlorides&(e.g.,&copper&and&cadmium)&will¬&be&greatly&affected.&Thus,&pH& Chapter(5( ! ! 124( mediated( changes( in( trace( metal( concentrations( and( speciation( could( possibly( affect( the( limitation( and/or( toxicity( of( metals( to( marine( plankton( (Millero( et( al.( 2009).( Iron( is( the( most( essential( trace( metal( for( phytoplankton( for( crucial( physiological( processes,( such( as( photosynthesis,( respiration,( and( nitrate( assimilation((Behrenfeld(&(Milligan(2013).(The(decrease(in(seawater(pH(has(been( suggested(to(promote(higher(Fe(solubility((Millero(et(al.(2009),(but(may(result(in( unchanged(or(lower(Fe(bioavailability,(depending(of(the(nature(of(strong(organic( Fe( ligands( (Shi( et( al.(2010).( Consequently,( changes( in( iron( bioavailability( due( to( ocean(acidification(may(affect(positively(or(negatively(ocean(productivity(and(CO2( drawdown.( Copper( is( an( essential( micronutrient( but( may( be( toxic( at( high( concentrations.(An(increase(in(free(cupric(ion(concentrations(in(coastal(areas(due( to( ocean( acidification( (Millero( et( al.(2009)( could( result( in( negative( effects( on( marine(phytoplankton.(From(the(open-ocean(to(coastal(areas,(the(concentration( of(metals(differ,(as(well(as(the(trace(metal(requirements(of(phytoplankton((Sunda( &( Huntsman( 1995a),( and( the( tolerance( to( metal( toxicity.( Thus,( for( example,( changes(in(pH(may(promote(an(increase(in(Cu(toxicity(in(coastal(phytoplankton,(or( enhance(Fe(limitation(in(the(open(ocean.((Given(that(trace(metals(are(essential(for( phytoplankton(productivity,(and(that(are(actively(internalized(during(growth,(it(is( important( to( study( the( impacts( of( ocean( acidification( in( trace( metal( concentrations(in(ecologically(significant(plankton(species.(( A( bloom( of( the( coccolithophorid( Emiliania' huxleyi(was( induced( in( a( mesocosm(experiment(to(examine(the(interactive(effects(of(increased(CO2(and/or( dissolved( iron( in( its( growth( and( physiology.( Emiliania' huxleyi(is( the( most( cosmopolitan(and(abundant(coccolithophore(in(the(modern(ocean((Paasche(2002).( Coccolithophores(play(a(key(role(in(the(global(carbon(cycle(because(they(produce( photosynthetically(organic(carbon,(as(well(as(particulate(inorganic(carbon(through( calcification.(These(two(processes(foster(the(sinking(of(particulate(organic(carbon( to( the( deep( ocean( (carbon( export).( However,( OA( will( affect( the( abundance( of( Trace&metal&dynamics&in&response&to&increased&CO2&and&iron&availability&&& ! ! 125& coccolithophores& and& the& rates& of& calcification& and& organic& carbon& fixation& (Zondervan& et& al.&2007).& The& aim& of& the& present& study& was& to:& i)& analyse& the& changes&in&particulate&trace&metals&concentrations&during&the&bloom&of&E.#huxleyi;& ii)&examine&the&effects&of&increased&CO2&and&iron&levels&in&particulate&trace&metals& concentrations;& and& iii)& estimate& the& origin& (lithogenic& or& biotic)& of& the& bloom& particulate&trace&metals.&& & Materials!and!methods! Experimental!set-up! The&experimental&work&was&carried&out&in&June&2012&in&the&Raunefjord,&off&Bergen,& Norway&as&described&in&Chapter&2.&Twelve&mesocosms&(11&m3&each)&were&set-up& in&a&full-factorial&design&with&all&combinations&of&ambient&and&high&pCO2&and&dFe& in&three&independent&replicate&mesocosms.&The&mesocosms&were&covered&by&lids& (both&transparent&to&PAR&and&UVR)&and&filled&with&fjord&water&from&8&m&depth.& We&achieved&two&CO2&levels&corresponding&to&present&(390&μatm,&LC)&and&those& predicted&for&2100&(900&μatm,&HC)&by&adding&different&quantities&of&pure&CO2&gas& (Schulz&et&al.&2009).&The&specific&CO2&concentration&and&the&CO2&inlet&flows&in&the& mesocosms&were&measured&by&non-dispersive&infrared&analysis&by&using&a&Li-Cor& (LI-820)&CO2&gas&analyser&(Li-COR,&Nebraska,&USA)&and&CO2&AirSense-310&sensors& (Digital& Control& Systems,& Inc,& USA).& CO2& concentrations& in& the& mesocosms& were& calculated&from&pH&and&total&alkalinity&measurements&using&the&CO2&SYS&software& (Robbins& et& al.&2010).& Iron& was& also& manipulated& by& addition& of& 70& nM& of& the& siderophore& desferrioxamine& B& (DFB)& to& half& of& the& mesocosms&on& day& 7,& promoting& two& different& iron& conditions& (+DFB,& high& dissolved& iron;& and& –DFB,& ambient& dissolved& iron).& Dissolved& iron& concentration& increased& ~3-fold& with& respect&to&the&control&as&a&result&of&increased&CO2&and&the&addition&of&DFB.&At&the& beginning& of& the& experiment,& nitrate& (10& μM)& and& phosphate& (0.3& μM& final& concentrations)& were& manipulated& to& induce& a& bloom& of& the& coccolithophorid& Emiliania#huxleyi.#The&multifactorial,&experimental&design&comprised&of&triplicate& Chapter(5( ! ! 126( mesocosms(per(treatment(and(the(combinations(of(high(and(ambient(pCO2(and( dFe(levels,(resulting(in(a(total(of(12(mesocosms:(3x(LC-DFB((control),(3x(LC+DFB,( 3x(HC+DFB(and(3x(HC-DFB.(Water(samples(from(each(mesocosm(were(taken(from( 2(m(depth(by(gentle(vacuum(pumping(of(25(L(volume(into(acid-washed(carboys( that( were( quickly( transported( to( the( onshore( laboratory.( The( biological( and( chemical( parameters( analysed( were( cell( abundance( and( composition,( dissolved( Fe(and(Cu(concentrations,(nutrient(concentrations((nitrate,(phosphate,(silicic(acid( and(ammonium)(and(particulate(trace(metal(concentrations.(( ( Dissolved!copper!! Low( density( polyethylene( (LDPE)( bottles( were( cleaned( using( 1%( alkaline( soap( solution(for(one(week,(then(filled(with(6(M(trace(metal(grade(HCl(and(submerged( in(a(2(M(HCl(bath(for(one(month,(after(that(they(were(filled(with(1(M(trace(metal( grade( HCl( (Fisher( Chemicals)( for( one( more( month( and( then( were( kept( double( bagged.( In( between( each( acid( treatment,( the( bottles( were( rinsed( with( Milli-Q( water((Millipore;(hereafter(referred(to(as(MQ).(Before(sampling,(the(bottles(were( rinsed( three( times( with( filtered( seawater.( Samples( were( collected( from( each( mesocosm,(filtered(through(0.2(µM(AcroPak(Supor(membrane(capsule(filters(into( the(trace(metal(clean(LDPE(bottles,(and(acidified(with(ultra-clean(HCl((Seastar)(in( a(Class(100(laminar(flow(hood.(Total(dissolved(Cu(concentrations(were(measured( following(Zamzow(et(al.((1998)(using(a(flow(injection(analysis(chemiluminescence( detection(system((CL-FIA,(Waterville(Analytical).( ( Particulate!metals! Sampling-(All(equipment(used(during(this(study(was(rigorously(acid-washed(under( trace(metal(clean(conditions,(including(using(dedicated(clean(areas.(Filters(were( precleaned(with(10%(trace(metal(hydrochloric(acid((Fisher,(trace(metal(grade)(at( 60( ºC( overnight( and( were( rinsed( with( Milli-Q( H2O.( Seawater( samples( (1-3.5( L)( were(filtered(gently(onto(0.45(µm(acid(washed(Supor®-450(filters(on(days(12,(17( ! ! Table!5.4.!The!concentration!of!particulate!metals!in!seawater!(nmol!L-1)!in!the!different!treatments!after!oxalate-wash;!LC:!ambient!CO2!(390!μatm);!HC:!increased! CO2!(900!μatm);!-DFB!(ambient!dFe);!+DFB!(increased!dFe)!during!the!development!of!a!bloom!of!Emiliania'huxleyi.!Data!are!averages!of!replicate!mesocoms!and! standard! deviations! are! shown! in! brackets.! The! percentage! (%)! indicates! the! mean! quantity! of! metal! remaining! after! the! oxalate! wash.! Statistically! significant! differences!are!indicated!with!asterisk!(*!if!p<0.05;!**!if!p<0.01!and!***!if!p<0.001;!ns:!not!significant).! ! Treatment! Al! Ti! P! Fe! Cu! Co!(·10)! Zn! Cd!(·100)! Mn! Mo! Pb!(·10)! d12' ! ! ! ! ! ! ! ! ! ! ! ! ! LC-DFB! 11.6!(2.8)! 1.32!(0.34)! 117!(3.27)! 12.52!(0.78)! 0.16!(0.03)! 0.07!(0.00)! 1.92!(0.86)! 0.09!(0.06)! 0.15!(0.02)! 0.02!(0.00)! 0.10!(0.00)! ! LC+DFB! 28.3!(12)! 4.49!(1.91)! 258!(46.1)! 14.67!(3.35)! 0.23!(0.08)! 0.19!(0.00)! 7.16!(1.29)! 0.51!(0.14)! 0.41!(0.06)! 0.03!(0.01)! 0.20!(0.11)! ! HC+DFB! 15.9!(2.3)! 2.52!(0.66)! 139!(14.2)! 8.05!(1.08)! 0.22!(0.06)! 0.09!(0.01)! 2.39!(0.93)! 0.20!(0.09)! 0.21!(0.07)! 0.03!(0.01)! 0.11!(0.06)! ! HC-DFB! 11.6!(8.8)! 1.66!(0.68)! 178!(66.3)! 9.79!(3.75)! 0.19!(0.08)! 0.08!(0.03)! 2.84!(0.52)! 0.22!(0.06)! 0.28!(0.08)! 0.02!(0.01)! 0.19!(0.08)! d17' ! ! ! ! ! ! ! ! ! ! ! ! ! LC-DFB! 6.42!(2.9)! 0.85!(0.35)! 97!(41.6)! 1.23!(0.56)! 0.11!(0.07)! 0.09!(0.05)! 2.86!(1.45)! 0.26!(0.09)! 0.18!(0.07)! 0.03!(0.00)! 0.09!(0.05)! ! LC+DFB! 7.53!(4.7)! 1.85!(0.63)! 245!(136)! 1.28!(0.68)! 0.24!(0.11)! 0.22!(0.08)! 12.1!(3.78)! 1.20!(0.69)! 0.54!(0.29)! 0.05!(0.03)! 0.18!(0.09)! ! HC+DFB! 4.48!(0.2)! 1.29!(0.01)! 131!(5.31)! 1.55!(0.19)! 0.14!(0.01)! 0.07!(0.00)! 3.03!(0.90)! 0.21!(0.06)! 0.14!(0.02)! 0.03!(0.00)! 0.10!(0.05)! ! HC-DFB! 12.8!(2.7)! 1.98!(0.74)! 233!(162)! 5.31!(0.99)! 0.29!(0.11)! 0.18!(0.06)! 5.03!(3.06)! 0.35!(0.13)! 0.43!(0.33)! 0.06!(0.01)! 0.24!(0.02)! d21' ! ! ! ! ! ! ! ! ! ! ! ! ! LC-DFB! 13.9!(3.2)! 1.54!(0.48)! 257!(20.9)! 3.76!(0.75)! 0.29!(0.06)! 0.26!(0.01)! 8.59!(0.69)! 0.74!(0.31)! 0.35!(0.04)! 0.05!(0.02)! 0.21!(0.08)! ! LC+DFB! 4.36!(0.4)! 1.01!(0.41)! 253!(47.6)! 2.04!(0.63)! 0.23!(0.02)! 0.20!(0.01)! 14.3!(1.32)! 0.67!(0.09)! 0.43!(0.05)! 0.05!(0.01)! 0.09!(0.02)! ! HC+DFB! 2.49!(0.9)! 0.62!(0.17)! 79!(19.6)! 0.33!(0.07)! 0.11!(0.02)! 0.07!(0.03)! 2.36!(1.38)! 0.09!(0.06)! 0.09!(0.03)! 0.01!(0.00)! 0.03!(0.01)! ! HC-DFB! 2.56!(1.2)! 0.98!(0.30)! 74!(20.7)! 1.03!(0.18)! 0.12!(0.03)! 0.05!(0.01)! 1.01!(0.35)! 0.05!(0.02)! 0.07!(0.01)! 0.02!(0.00)! 0.13!(0.01)! %!! ! ns! ns! 80*! 75*! 60*! 70*! 55**! 45***! 55**! 35***! 30***! ns:'not'significant;'*''p<0.05;'**'p<0.01;'***'p<0.001' ! ! Table!5.5.!Statistical!analyses!(Split-plot!ANOVA)!of!the!effects!of!CO2,!DFB,!and!their!interaction,!as!well!as!the!effect!of!time,!on!the!concentrations!of!particulate! metals!in!seawater!(A)!and!on!the!oxalate-washed!concentrations!of!particulate!metals!in!seawater!(B)!in!the!different!treatments!LC:!ambient!CO2!(390!μatm);!HC:! increased!CO2!(900!μatm);!-DFB!(ambient!dFe);!+DFB!(increased!dFe)!during!the!development!of!a!bloom!of!Emiliania'huxleyi.!Statistically!significant!differences!are! indicated!with!asterisk!(*!if!p<0.05;!**!if!p<0.01!and!***!if!p<0.001;!ns:!not!significant).! (A)! Factor! Al! Ti! P! Fe! Cu! Co! Zn! Cd! Mn! Mo! Pb! Carbon! ns! ns! **! *! ns! **! ***! ***! **! ***! ns! DFB! ns! ns! *! ns! ns! *! **! ns! *! *! ns! Carbon!x!DFB! ns! *! **! ns! ns! *! **! *! **! **! ns! Time! ns! ns! ns! ***! *! ***! ***! ***! ***! ***! **! !! (B)! Factor! Al! Ti! P! Fe! Cu! Co! Zn! Cd! Mn! Mo! Pb! ! ! ! ! ! ! ! ! ! ! ! ! Carbon! ns! ns! ns! ns! ns! ***! ***! ***! *! *! ns! DFB! ns! *! ns! ns! ns! ns! *! ns! ns! ns! ns! Carbon!x!DFB! ns! ns! *! ns! *! ns! *! *! **! *! ns! Time! **! ***! **! ***! ns! *! *! *! ns! **! *! 'ns:'not'significant;'*''p<0.05;'**'p<0.01;'***'p<0.001' Trace metal dynamics in response to increased CO2 and iron availability 135 After oxalate-wash P, Fe, Co, Zn Mn and Mo were less affected by increased CO2 and DFB addition The effects of CO2 and DFB on particulate metal concentrations were different for the oxalate-treated and untreated samples. All the elements, except Mn and Cu, showed significant differences over time (Table 5.5). Cobalt concentrations were negatively affected by high CO2 levels. In contrast, DFB only induced changes in the particulate concentrations of Zn and Ti. Zinc concentrations showed a strong interaction between CO2 and DFB, and the highest concentrations of this metal occurred in the LC+DFB treatment. Fe and Ti were from abiotic origin The Fe:P and Ti:P ratios were relatively similar to crustal ratios. In addition, significant positive correlations were observed between Me:P and Al:P for Fe and Ti (Figure 5.1). These results suggest that particulate Fe and Ti were determined by lithogenic sources. Iron:P and Ti:P were not significantly affected by increased CO2 and/or DFB addition, but showed significant differences over time (Table 5.7). Samples washed with oxalate-EDTA solution showed significant differences in Fe concentrations due to DFB addition. Furthermore, there was no significant relationship between particulate Fe and Ti concentrations (with or without oxalate wash) with either the total plankton (phytoplankton and microzooplankton) or E. huxleyi biomass (Table 5.6). ! ! ! Table!5.6.!The$relationship$(Pearson$correlations,$p<0.05)$between!particulate$metals$concentrations$(nmol$L-1)$and$the$biomass$(μgC$L-1)$of$Emiliania'huxleyi$and$ total$cells$(phytoplankton$and$microzooplankton)$obtained$from$Chapter$2$during$the$bloom.$$ $ $ P! Fe! Cu! Co! Zn! Cd! Mn! Mo! Pb! Ti! (A)$Samples$without$treatment! E.'huxleyi$ Correlation$ coefficient$ 0.622$ ns$ 0.614$ 0.756$ 0.747$ 0.818$ 0.686$ 0.825$ ns$ ns$ $ P-value$ 0.003$ 0.003$ 7.35·10-5$ 1.01·10-4$ 6.02·10-6$ 5.93·10-4$ 4.20·10-6$ $ $ Total$cells$ Correlation$ coefficient$ 0.641$ ns$ 0.51$ 0.644$ 0.889$ 0.802$ 0.598$ 0.53$ ns$ ns$ $ P-value$ 0.002$ 0.02$ 1.62·10-3$ 7.03·10-8$ 1.23·10-5$ 4.18·10-3$ 1.35·10-2$ $ $ (B)$Oxalate-washed$samples$ E.'huxleyi$ Correlation$ coefficient$ 0.647$ ns$ 0.637$ 0.770$ 0.758$ 0.826$ 0.702$ 0.819$ ns$ ns$ $ P-value$ 0.002$ 0.002$ 4.52·10-5$ 6.79·10-5$ 3.92·10-6$ 3.86·10-4$ 5.68·10-6$ $ $ Total$cells$ Correlation$ coefficient$ 0.513$ ns$ 0.569$ 0.656$ 0.886$ 0.809$ 0.605$ 0.513$ ns$ ns$ $ P-value$ 0.02$ 0.009$ 1.25·10-3$ 9.53·10-8$ 8.98·10-6$ 3.68·10-3$ 0.0175$ $ $ ! ! Table! 5.7.!P-values!for!the!effects!of!CO2,!DFB,!their!interaction!and!time!on!the!P-normalized!metal!quotes!(A)!and!on!the!oxalate-washed!concentrations!of!Pnormalized!metal!quotes!(B)!during!the!development!of!the!bloom!of!Emiliania'huxleyi.!Statistically!significant!differences!are!indicated!with!asterisk!(*!if!p<0.05;!**! if!p<0.01!and!***!if!p<0.001).!! A)! Factor! Fe:P! Cu:P! Co:P! Zn:P! Cd:P! Mn:P! Mo:P! Pb:P! Ti:P! ! ! ! ! ! ! ! ! ! ! Carbon! ns! *! ***! **! ns! *! ns! ns! ns! DFB! ns! ns! ns! ns! ns! ns! ns! ns! ns! Carbon!x!DFB! ns! ns! ns! ns! ns! ns! ns! ns! ns! Time! ***! ***! ***! ***! ns! ns! ns! ns! ***! ! B)! Factor! Fe:P! Cu:P! Co:P! Zn:P! Cd:P! Mn:P! Mo:P! Pb:P! Ti:P! ! ! ! ! ! ! ! ! ! ! Carbon! ns! **! ***! **! ***! **! ns! ns! ns! DFB! *! *! **! ns! **! **! ns! ns! ns! Carbon!x!DFB! ns! ns! ns! ns! *! **! ns! ns! ns! Time! ***! *! ***! ***! ***! ns! ***! ns! **! ns:'not'significant;'*'p<0.05;'**'p<0.01;'***'p<0.001' ! ! Chapter 5 138 Co, Cu, Zn, Cd, Mn and Mo were associated with phytoplankton The comparison of the P-normalized quotas with Al:P showed no correlations with Co, Cu, Zn, Cd, Mn and Mo (Figure 5.1), indicating that these particulate metals were not from lithogenic origin. Compared to trace metal ratios in marine plankton assemblages (Me:P, Ho 2006), our measured ratios were similar for Cu, higher for Mn and Zn, and lower for Co and Cd (Figure 5.1). Furthermore, the metal ratios we measured (Me:P) were higher for Cu, Zn and Mo, and lower for Mn, Co and Cd, than the ratios published for E. huxleyi (Figure 5.1). Copper, Co and Zn:P ratios changed significantly over time (Table 5.7). Copper, Co, Zn and Mn:P ratios showed significant effects due to increased CO2 (Table 5.7). DFB did not affect Me:P ratios of these elements. In contrast, the oxalate-EDTA washed metal ratios (Me:P) for Cu and Co were significantly influenced by DFB (Table 5.7). The oxalate washed samples also presented a significant effect of CO2 on the Cu:P, Co:P, and Zn:P ratios. An interaction between the effects of CO2 and DFB was detected in Cd:P and Mn:P ratios (Table 5.7). The concentrations of these metals also showed significant correlations with the biomass of E. huxleyi and that of total plankton cells (p<0.05) in both untreated or treated oxalate-washed samples (Table 5.6). Figure 5.1. Comparison of P-normalized metal quotas (mmol:mol P) of particles from different treatments; LC: ambient CO2 (390 μatm); HC: increased CO2 (900 μatm); -DFB (ambient dFe); +DFB (increased dFe) during the development of a bloom of Emiliania huxleyi. The x-axis parallel solid and dotted lines represent the average metal quotas obtained from marine plankton assemblages (Ho 2006) and from cultures of Emiliania huxleyi (Ho et al. 2003) and, respectively. The symbol  denotes the average metal: Al in crustal material (Taylor 1964). (A) Fe:P, (B) Cu:P, (C) Co:P, (D) Zn:P, (E) Cd:P, (F) Mn:P, (G) Mo:P, (H) Ti:P, (I) Pb:P. 10 100 1 10 100 A) Fe:P 10 100 0.001 0.01 0.1 1 10 B) Cu:P 10 100 0.001 0.01 0.1 1 C) Co:P 10 100 0.001 0.01 0.1 1 10 100 D) Zn:P 10 100 0.00001 0.0001 0.001 0.01 0.1 1 E) Cd:P 10 100 0.01 0.1 1 10 F) Mn:P 10 100 0.00001 0.0001 0.001 0.01 0.1 1 10 G) Mo:P P:Al (mmol mol-1) 10 100 0.1 1 10 100 H) Ti:P 10 100 0.0001 0.001 0.01 0.1 1 10 I) Pb:P LCDFB LC+DFB HC+ DFB HC-DFB Cr u stal ratio Chapter 5 140 Discussion The purpose of this study was to investigate particulate trace metal concentrations in response to increased CO2 and iron availability in a coastal mesocosm experiment dominated by a bloom of the coccolithophore Emiliania huxleyi. This work also aimed to examine the source of particulate metals, either biogenic or lithogenic, and the evolution of both fractions due to experimental increased iron and/or CO2 levels. Furthermore, we compared two common approaches to estimate biogenic metal concentrations from bulk particle measurements: comparing the P-normalized quotas with the ratios published for natural assemblages and for E. huxleyi cultures (Ho et al. 2003, Ho 2006), as well as with crustal ratios (Taylor 1964). To assess the biogenic fraction of the particulate metals, we also compared the concentrations of particulate trace metals in the presence and absence of an oxalate-EDTA wash, which removes extracellular Fe and P (Sanudo-Wilhelmy et al. 2004, Tovar-Sanchez et al. 2003). The results of the two methods used to calculate biogenic metal concentrations from bulk particle measurements, were in agreement. This study is the first to combine trace metal analyses of particles in a controlled mesocosm experiment with manipulation of CO2 and Fe levels using natural assemblages of marine phytoplankton. Our results demonstrate that in the studied fjord, particulate Ti and Fe concentrations were dominated by lithogenic material. In contrast, particulate Cu, Co, Mn, Zn, Mo and Cd concentrations were correlated with P concentrations, as well as phytoplankton biomass, suggesting their strong biogenic influence (Table 6). The concentrations of these biogenic metals in our E. huxleyi bloom were ranked as: Zn > Cu ≈ Mn > Mo > Co > Cd. The total particulate and biogenic metal concentrations were affected by CO2 and/or Fe levels, but not all metals were equally affected. Changes in CO2 had the most significant effect on particulate Fe concentrations. In contrast, the effects of CO2 on the Me:P ratios were only measured in Co, Cu, Zn, and Mn. Trace&metal&dynamics&in&response&to&increased&CO2&and&iron&availability&&& ! ! 141& Efficacy! of! the! oxalate-EDTA! wash! removing! lithogenic! trace! metals! from! particles! The&oxalate–EDTA&reagent&was&developed&to&remove&surface-adsorbed&Fe& prior& to& particulate& trace& metal& analyses& (Tovar-Sanchez& et& al.&2003).& This& reagent& works& primarily& through& ligand-promoted& dissolution& via& complexation& both& to& EDTA&and&oxalate&(Tovar-Sanchez&et&al.&2003,&Tang&&&Morel&2006).&In&this&study,& the& oxalate& wash& significantly& decreased& the& concentration& of& all& particulate& metals,&with&the&exception&of&Al&and&Ti&(Table&5.4),&as&observed&by&Rauschenberg& && Twining& (2015).& In& general,& the& concentrations& of& Fe& and& Co& in& the& particles& were& decreased& the& least& (by& ~25%),& while& Mo& and& Pb& concentrations& were& decreased& the& most& (by& ~70%)& by& the& oxalate& wash.& The& concentrations& of& particulate& Cu,& Zn,& Cd& and& Mn& were&reduced& by& 50%& by& the& oxalate& wash.& As& shown& previously& (Sanudo-Wilhelmy& et& al.&2004),& the& oxalate& reagent& also& removed&extracellular&P&(by&~20%).&Compared&to&Rauschenberg&&&Twining&(2015),& the&estimates&of&the&biogenic&fraction,&after&the&oxalate&wash,&were&in&agreement& for&Co,&Cu&and&P,&and&lower&for&Fe,&Mn,&Zn&and&Cd&concentrations.&The&efficacy&of& the&oxalate&wash&to&dissolve&Fe,&and&other&metals,&from&lithogenic&particles&is¬& well& constrained& (Frew& et& al.&2006,& Rauschenberg& && Twining& 2015,& King& et& al.& 2012).&Therefore,&the&results&obtained&after&the&oxalate-EDTA&wash&are&hard&to& interpret&because&we&do¬&know&whether&the&removed&metal&fraction&is&a)&only& lithogenic;&b)&mainly&lithogenic&but&some&biogenic&fraction&is&also&removed,&or&c)& whether& metals& absorbed& onto& particles& are& equally& labile& to& the& wash& on& biogenic&and&lithogenic&particles.&&However,&some&of&the&trends&we&observed&[eg.& higher& Me& concentrations& in& the& LC+DFB& treatments& (Table& 5.4);& or& positive& correlations&between&phytoplankton&biomass&and&Me&concentrations&(Table&5.6)& were& identical& for& the& oxalate-EDTA& washed& and& non-washed& particles.& Thus,& below&we&focus&our&discussion&on&the&non-oxalate&wash&results.&& ! Chapter(5( ! ! 142( Particulate! elements! (P,! Cu,! Co,! Zn,! Cd,! Mn! and! Mo)! were! mainly! associated! with!phytoplankton! Certain(particulate(elements((P,(Cu,(Co,(Zn,(Cd,(Mn(and(Mo)(were(clearly(biogenic.( Three(lines(of(evidence(are(presented(in(support(of(this.(First,(the(total(biomass(of( phytoplankton( exhibited( a( significant( positive( correlation( with( particulate( P( (Table( 5.6),( showing( that(most( particulate(P(was(biogenic,(as( shown(previously( (Ho(et(al.(2007,(Ho(et(al.(2009).(The(concentrations(of(Cu,(Co,(Zn,(Cd,(Mn(and(Mo( also( exhibited( positive( significant( correlations( with( the( biomass( of( total( cells( (phytoplankton( and( microzooplankton)( or( E.# huxleyi((Table( 5.6),( indicating( that( these( particulate( metals( were( also( associated( with( phytoplankton.( Second,( the( Me:P(ratios(are(not(similar(to(crustal(ratios.(Third,(the(Me:P(ratios(we(measured( in( the( particles( are( similar( to( those( of( natural( phytoplankton( assemblages( (Ho( 2006)(and(of(Emiliania#huxleyi(cultures((Ho(et(al.(2003).( The(concentrations(of(biogenic(metals(in(the(Emiliania#huxleyi(bloom(that( we(studied(were(ranked(as:(Zn(>(Cu(≈(Mn(>(Mo(>(Co(>(Cd((Table(5.3),(similar(to( those( reported( in( indigenous( phytoplankton( populations( by( Twining( &( Baines( (2013;(Fe(≈(Zn(>(Mn(≈(Ni(≈(Cu(≫(Co(≈(Cd).(Particulate(Zn(concentrations(were( especially( high( in( the( LC+DFB( treatment,( where( the( highest( E.# huxleyi(biomass( was( observed.( The( only( treatment( where( E.#huxleyi(did( not( dominate( the( community(was(the(HC-DFB.(In(this(treatment,(the(particulate(trace(metal(ranking( was(the(same,(but(their(concentrations(were(higher(than(those(in(HC+DFB(on(day( 17.( ( At( the( end( of( the( experiment,( the( trace( metal( concentrations( in( both( HC( treatments( were( comparable( and,( similar( effect( of( both( factors( (increased( CO2( and(the(addition(of(DFB)(was(observed((lower(values(in(HC(treatments,(Table(5.3).(( The(coccolithophorid(Emiliania#huxleyi(is(well(known(for(its(high(Zn(cellular( requirements((Sunda(2012).(This(essential(metal(serves(as(a(cofactor(in(enzymes( involved( in( biochemical( processes,( such( as( alkaline( phosphatase,( RNA( polymerase,( or( superoxide( dismutase( (Cu/Zn-SOD).( An( increase( in( dissolved( Zn( levels(can(stimulate(the(growth(of(E.#huxleyi#in(natural(communities#but(its(effect( List%of%figures%and%tables% ! ! 245% Table& 5.1S.%P-values%for% the%effects%of% CO2,% DFB,%their% interaction% and%time% on% the%Alnormalized%metal& % 149& Table& 5.2S.%Identified% Zn-Metalloproteins% genes%in% the% genome% of%Emiliania( huxleyi((Read%et%al.%2013),%encoding%for%the%corresponding%following%proteins% % 150& Figure&5.1S.&The%partition%coefficients%in%the%different%treatments%at%the%beginning%and% the%end%of%the%bloom& % 151% Figure&5.2S.&Comparison%of%Al-normalized%metal%quotas%(mmol:mol%Al)%of%particles%from% different%treatments& % 151% & CHAPTER&6.1& 153& & Table& 6.1.1.& Filter% configuration% for% the% 12% spectral% treatment% groups% of% the% photoinhibitron%& % 160% Figure& 6.1.1.& Graphical% illustrations% of% the% implied% dependence% of% repair% and% damage% rates%for%the%E,%T%and%Emax%models& % 162% Figure& 6.1.2.& Measured% parameters% on% coccoliths%DL:% distal% shield% length,%DW:% distal% shield%width,%CAL:%central%area%length%and%CAW:%central%area%width& % 164% Table&6.1.2.%Physiological%variables%of%Emiliania(huxleyi%grown%under%ambient%CO2%(400% ppm)%and%high%CO2%(800%ppm)& % 165% Figure&6.1.3.&Cellular%absorbance%of%UVR%and%PAR%measured%as%Chl%a%specific%absorption% (a*[λ]%m2%mg%Chla-1)& % 166% Table& 6.1.3.& Difference%in%AICs%calculated%for%E,%T% and%Emax%model%fits%to%experimental% data%on%the%response%of%E.(huxleyi(photosynthesis%to%UV%+%PAR%exposure& % 167% Figure&6.1.4.&The%panels%illustrate%the%observed%(points)%vs.%fitted%(lines)%results%for%three% BWF/P-E%models,%the%E,%T%%and%Emax%models& % 169% Table& 6.1.4.%Fitted% parameters% for% the% T% model,% mean% ±% standard% errors% for% n% ≥% 3% experiments%under%each%condition%ambient%and%high%CO2%concentration& % 169% Figure&6.1.5.&BWFs%for%the%inhibition%of%photosynthesis%by%UVR%(ԑ%[λ],%[mW%m-2]-1)%of%E.( huxleyi%under%ambient%and%increased%CO2%concentrations& % 169% Figure& 6.1.6.& PIC%production% rate,% POC% production%rate% and% PIC:POC% productivity% ratio% during%the%exponential%phase& % 170% Figure& 6.1.7.& Representative% scanning% electron% microscope% (SEM)% images% of% Emiliania( huxleyi%cells%and%free%coccoliths& % 172% Table& 6.1.5.& Morphometric% analysis% of% detached% coccoliths% and% coccospheres% of% Emiliania(huxleyi%grown%under%ambient%CO2%(400%ppm)%and%high%CO2%(800%ppm)& % 173% Figure& 6.1.8.& Biological%weighting%functions%for% the%inhibition%of%photosynthesis%by%UV% ((ԑ[λ],%[mW%m-2]-1)%data%from%different%phytoplanktonic%species& % 179% & CHAPTER&6.2& 183& & Table&6.2.1.%Nomenclature%of%the%different%treatments%used%in%the%experiment% & % % 189% Figure&6.2.1.%Specific%growth%rates%of%Emiliania(huxleyi%cultures%exposed%to%the%different% irradiance,%CO2%and%Fe%treatments& % 193% Figure& 6.2.2.%Percentage% of% dead% cells% based% on% SYTOX% Green% staining% of% Emiliania( huxleyi%cultures%exposed%to%the%different%irradiance,%CO2%and%Fe%treatments& % 194% Figure& 6.2.3.%Percentage%of%cell%viability%based%on%FDA%green%fluorescence%emission%of% Emiliania(huxleyi%cultures%exposed%to%the%different%irradiance,%CO2%and%Fe%treatments& % 195% Figure& 6.2.4.%Changes% in% the% optimum% quantum% yield% (Fv/Fm)% in% cultures% of% Emiliania( huxleyi%exposed%to%the%different%irradiance,%CO2%and%Fe%treatments& % 196% Table&6.2.2.%Photosynthetic%parameters%of%Emiliania(huxleyi%calculated%from%chlorophyll% a%fluorescence%measurements%after%4%days%of%cultures%at%different%conditions& % 197% ! 246$ Figure'6.2.5.$Changes$in$photosynthetic$production$of$particulate$organic$carbon$(POC)$ of$Emiliania'huxleyi$cultures$exposed$to$the$different$irradiance,$CO2$and$Fe$treatments' $ 198$ Figure'6.2.6.$Changes$in$photosynthetic$production$of$dissolved$organic$carbon$(DOC)$of$ Emiliania'huxleyi$cultures$exposed$to$the$different$irradiance,$CO2$and$Fe$treatments' $ 199$ Table'6.2.3.'Percentage$of$DO14C$extracellular$release$(PER)$of$dissolved$organic$carbon$ of$Emiliania'huxleyi' $ 200$ Figure'6.2.7.$Calcification$rates$of$Emiliania'huxleyi$after$4$days$of$cultures$exposed$to$ the$different$irradiance,$CO2$and$Fe$treatments' $ 201$ Figure'6.2.1S.'Seawater$carbonate$system$during$the$experiment' $ 209$ ' $ ' $ ' $ ' $ ' $ ' $ ' $ ' $ ' $ ' $ ' $ ' $ ' $ ' ' ! Resumen! ' ' ! !' Resumen''''''''''''''' ! ! 247' Resumen' Las' emisiones' de' dióxido' de' carbón' (CO2)' producidas' por' las' actividades' humanas'derivadas'del'uso'de'combustibles'fósiles''y'los'distintos'usos'de'la' Tierra' han' causado' un' considerable' aumento' de' la' concentración' de' CO2' atmosférico' desde' el' inicio' de' la' revolución' industrial.' La' concentración' ha' aumentado'de'280'a'400'µatm.'Los'océanos'han'absorbido'aproximadamente' un'30%'de'las'emisiones'de'carbono'antropogénicas'durante'los'últimos'200' años.' Además,' las' predicciones' establecen' que' la' concentración' de' CO2'se' duplicará'con'respecto'a'la'concentración'actual'en'el'peor'de'los'escenarios' posibles'hacia'finales'de'este'siglo.'Como'consecuencia'de'la'disolución'del'CO2' en'el'agua'de'mar,'el'pH'disminuirá.' Este'proceso' se' denomina' acidificación' oceánica.'Cuando'el'CO2'se'disuelve'en'el'agua,'éste'reacciona'con'el'agua'y'se' forma' ácido'carbónico'(H2CO3)'que'es'una'forma'inestable'y'rápidamente'se' disocia'en'bicarbonato'(HCO3 -)'y'en'iones'libres'de'hidrógeno'(H+).'El'aumento' de'H+'conlleva'una'disminución'del'pH,'aumentando'la'acidez'de'los'sistemas' acuáticos.'Los'modelos'que'se'han'propuesto'establecen'un'aumento'adicional' a'dicha'disminución,'que'de'hecho,'ya'se'ha'producido'desde'el'comienzo'de'la' era' industrial.' Este' aumento'sería' de' 0.06' a' 0.32' unidades' de' pH' en' la' disminución' global,' y' la' variación' final' dependerá' de' las' futuras' emisiones.' Además,'la'incorporación'de'CO2'en'los'océanos'conllevará'una'disminución'de' los'iones'carbonato'(CO3 2-)'y'de'los'niveles'de'saturación'del'carbonato'cálcico' (CaCO3)' en' los' minerales' calcita' y' aragonito.'La' acidificación' de' los' océanos' potencialmente'afectará' de' forma' severa' los' organismos'marinos.' El' fitoplancton'marino'contribuye'con'la'mitad'de'la'producción'primaria'global,' siendo' la' base' de' las' redes' tróficas' marinas' y' de' los' ciclos' biogeoquímicos,' especialmente' del' carbono' y' otros' nutrientes.'Además,' dicho'fitoplancton' marino'incorpora'aproximadamente'de'45'a'50'billones'de'toneladas'métricas' de'carbono'inorgánico'de'forma'anual,'retirando'un'cuarto'del'CO2'emitido'a'la' atmósfera'derivado'de'las'actividades'de'origen'humano.'Por'ello,'la'respuesta' ! 248$ del$fitoplancton$a$la$acidificación$de$los$océanos$es$clave$para$comprender$los$ futuros$cambios$globales$en$los$ecosistemas$marinos.$ La$posible$respuesta$del$fitoplancton$al$incremento$de$la$concentración$ de$CO2$en$los$océanos$y$con$ello,$a$la$disminución$del$pH,$es$dependiente$de$ los$ mecanismos$ fisiológicos$ de$ incorporación$ de$ carbono$ inorgánico$ y$ su$ asimilación$dentro$de$la$célula.$Durante$la$fotosíntesis,$el$CO2$es$fijado$por$la$ enzima$ rubisco$ (Ribulosa-1,5-bifosfato$ carboxilasa-oxigenasa).$ La$ enzima$ Rubisco$ con$ las$ concentraciones$ actuales$ de$ CO2$presenta$ una$ saturación$ inferior$a$la$mitad$en$la$mayoría$de$las$especies.$Por$otra$parte,$esta$enzima$ presenta$ tanto$ actividad$ enzimática$ carboxilasa$ para$ la$ fijación$ de$ carbono$ como$ actividad$ oxigenasa.$ La$ actividad$ oxigenasa$ produce$ una$ disminución$ significativa$ de$ la$ fijación$ de$ CO2$por$ parte$ de$ la$ enzima$Rubisco.$ Esta$ limitación$de$la$fijación$de$CO2$junto$con$la$baja$afinidad$de$la$Rubisco$por$el$ CO2$es$ compensada$ por$ la$ actividad$ de$ mecanismos$ concentradores$ de$ carbono$(CCMs).$Los$CCMs$aumentan$la$concentración$de$CO2$alrededor$de$la$ enzima.$ De$ este$ modo,$ se$suprime$ la$ actividad$ oxigenasa$ y$ aumenta$ la$ actividad$carboxilasa$de$la$Rubisco.$La$mayoría$de$las$especies$de$fitoplancton$ marino$ en$ principio$ presentarían$ poco$ efecto$ en$ sus$tasas$ de$ fotosíntesis,$ porque$ésta$ya$estaría$saturada$de$CO2$gracias$a$la$actividad$de$los$CCMs$que$ activamente$ incorporan$ carbono$ inorgánico,$ $CO2,$ bicarbonato$ o$ incluso$ ambos.$ El$ aumento$ de$ la$ concentración$ de$ CO2$debería$ beneficiar$ directamente$ a$ la$ producción$ de$ biomasa$ $por$ el$ aumento$ de$ la$ reacción$ carboxilasa$ en$ la$ enzima$ rubisco$y$ por$la$ reducción$ de$ las$ pérdidas$ de$ CO2$ debido$ a$las$ diferencias$ de$ concentración$ entre$ los$ medios.$ Además,$ con$ el$ aumento$ de$ la$ concentración$ de$ CO2$se$ produciría$ una$ reducción$ de$ la$ actividad$de$los$CCMs$que$consumen$mucha$energía.$Se$ha$propuesto$que$una$ disminución$ en$la$ regulación$ de$ la$ maquinaria$ fotosintética$ al$ disminuir$ la$ actividad$ de$ los$ CCMs$ podría$ aumentar$ el$ uso$ eficiente$de$ la$ energía$del$ fitoplancton.$Por$otra$parte,$la$bajada$de$pH$que$se$ha$predicho$representa$un$ Resumen''''''''''''''' ! ! 249' aumento' en' las' concentraciones' de' los' iones' de' hidrógeno'libres' (H+)' que' pueden'afectar'el'pH'del'interior'de'las'células,'el'potencial'de'membrana'y'la' actividad' de' las' enzimas.' Debido' al' efecto' de' la' disminución' del' pH' en' las' células,' la' acidificación' de' los' océanos' podría' reducir' el' crecimiento' del' fitoplancton' marino.' Por' ello,' el' efecto' de' la' acidificación' de' los' océanos' presenta'un'doble'rol.'Por'una'parte,'el'aumento'de'la'concentración'de'CO2' favorecería'la'fotosíntesis'y'por'otra'parte,'la'disminución'de'pH'que'afectaría' a'los'organismos'reduciendo'la'producción'primaria.' La'disminución'de'pH'producida'por'el'aumento'de'la'concentración'de' CO2'también' podría' afectar' las' tasas' de' calcificación' de' los' organismos.' La' calcita'es'uno'de'los'materiales'más'comunes'que'se'utiliza'en'la'formación'de' esqueletos,' conchas' y' otras' estructuras' protectoras' de' organismos.' La' calcificación' es' facilitada' por' un' aumento' en' pH' y' un' aumento' en' la' concentración' de' carbonato.' Estas' condiciones' se' alcanzan' en' las' zonas' de' calcificación'mediante'procesos'de'transporte'de'iones'que'consumen'energía' como' puede' ser' la' incorporación' de' fosfato.' El' coste' energético' de' la' calcificación' aumentaría' debido' a' la' disminución' del' pH' y' de' las' concentraciones' de' carbonato.' Esta' energía' necesaria' extra' se' utilizaría'para' compensar' los' cambios' químicos' que' se' producirían' en' el' agua' de' mar' y' dependería'del'proceso'de'calcificación'de'cada'grupo'taxonómico.'' Con' respecto' a' lo' anteriormente' dicho,'los' cocolitofóridos'son' especialmente'importantes.'Pertenecen'a'un'grupo'de'microalgas'unicelulares' que'habitan'en'la'capa'superficial'de'los'océanos'y'que'son'los'organismos'que' tienen'mayores'tasas'de'calcificación.'El'pH'intracelular'de'los'cocolitofóridos' es'muy'sensible'a'cualquier'cambio'externo'de'pH.'Por'ello,'la'mayoría'de'los' cocolitofóridos' reducen'sus' tasas' de' calcificación' cuando' se' realizan' simulaciones'de'las'concentraciones'de'CO2'que'se'alcanzarán'a'final'de'siglo.' El'aumento'de'la'concentración'de'CO2'alterará'las'relaciones'de'competencia' ! 250$ entre$ especies$produciendo$ cambios$ en$ la$ composición$ fitoplantónica$de$ las$ comunidades,$ debido$ a$ que$ la$ sensibilidad$ al$ aumento$ de$ CO2$varía$ entre$ especies.$Todos$estos$cambios$alterarán$los$procesos$fisiológicos$afectando$a$la$ dinámica$de$las$poblaciones$y$de$este$modo,$a$todo$el$ecosistema.$$ Según$los$procesos$biológicos$de$incorporación$de$CO2$se$definen$dos$ tipos$de$bombas$biológicas$de$carbono.$Por$un$lado$está$la$bomba$de$carbono$ inorgánico$basada$en$la$fotosíntesis,$y$por$otro$lado,$la$bomba$de$carbonato,$ generada$por$la$formación$de$carbonato$cálcico$en$las$estructuras$externas$del$ plancton$ que$ calcifica.$Mientras$ que$ la$ fotosíntesis$ fija$ CO2$en$ carbono$ orgánico$(la$ llamada$ “carbon' pump”),$ actuando$ como$ un$ sumidero$ de$ CO2$ atmosférico,$la$calcificación$convierte$2$aniones$bicarbonato$en$una$molécula$ de$carbonato$cálcico$y$en$otra$molécula$de$CO2,$actuando$como$una$fuente$de$ CO2$de$los$océanos$($denominada$“carbonate'counter-pump”).$Son$por$lo$tanto,$$ dos$ bombas$ relacionadas$ entre$ sí$ en$ términos$ de$ alteración$ del$ carbono$ inorgánico$ disuelto$ (DIC),$ que$ además$ se$contrarrestan$ la$ una$ a$ la$ otra$ alterando$el$ impacto$ de$ cambios$ en$ concentración$ de$ CO2$entre$ el$ mar$ y$ la$ atmósfera.$La$relación$entre$las$dos$bombas$se$denomina$“rain'ratio”,$que$es$ el$ratio$entre$el$carbono$particulado$inorgánico$(PIC)$y$el$carbono$particulado$ orgánico$(POC).$ Este$ ratio$ determina$ el$ flujo$ de$ CO2$entre$ la$ atmósfera$ y$ el$ océano.$Los$estudios$existentes$del$flujo$global$de$carbono$orgánico,$indican$ que$ la$ bomba$ de$ carbono$ es$ superior$ a$ la$ bomba$ de$ carbonato,$ pero$ el$ aumento$ de$ la$ concentración$ de$ CO2$y$ su$ efecto$ en$ la$ fotosíntesis$ y$ en$ la$ calcificación$ podría$ alterar$ el$ “rain$ ratio”.$ Una$ reducción$ en$ la$ calcificación$ produce$ una$ retroalimentación$ negativa$ al$ aumento$ de$ la$ concentración$ de$ CO2,$es$decir,$se$reduciría$la$concentración$de$CO2$liberada$a$la$atmósfera.$Pero$ se$sabe$que$hay$una$fuerte$relación$entre$el$carbono$orgánico$particulado$que$ migra$al$fondo$de$los$océanos$con$el$carbono$inorgánico$que$se$produce$por$la$ calcificación.$ Este$ proceso$ se$ denomina$ “ballast' effect”.$ Si$ se$ altera$ la$ producción$de$carbonato$cálcico,$afectaría$a$la$migración$de$carbono$orgánico$ Resumen''''''''''''''' ! ! 251' que' se' retira' de' las' capas' altas.' El' POC' se' quedaría' en' las' capas' altas,' reduciéndose'y'aumentando'de'este'modo'la'concentración'de'CO2.'El'efecto' global' sería' negativo' debido' al'aumento' de' la' concentración' de' CO2,' que' pasaría'a'la'atmosfera'disminuyendo'la'capacidad'de'los'océanos'de'retirar'CO2.' Por'otra'parte,'los'cambios' de' pH' debido'a' la'acidificación' oceánica'pueden' afectar'a'los'metales'trazas.'Concretamente,'se'afectaría'a'la'concentración'y' el'tipo'de'especie'predominante'debido'a'la'sensibilidad'a'los'cambios'de'pH.' El' efecto' final' sería' que' los' cambios' originados' por' la' disminución' de' pH' afectarían'a'la'disponibilidad'de'los'metales'y'a'los'niveles'de'toxicidad'de'los' distintos'organismos'marinos.' Los' metales' traza' (Mn,' Fe,' Co,' Cu,' Zn,' y' Cd)'son' necesarios' para' numerosos'procesos'en'fitoplancton' y'pueden'influenciar'el'crecimiento'y'la' estructura' de' la' comunidad' planctónica.' Además,' los' metales' traza' son' necesarios' para' el' crecimiento' y' la' supervivencia' de' los' organismos' fotosintéticos.'El'contenido'celular'de'los'metales'en'el'fitoplancton'reflejan'la' demanda'bioquímica/fisiológica'de'las'células'además'de'la'disponibilidad'en' el' ambiente' de' los' mismos.' Esto' altera' la' distribución' de' los' metales' en' el' océano.' Entre' todos' los' metales' traza,' el' hierro' (Fe)' es' el' más' importante' porque'es'requerido'para'procesos'fisiológicos'y'moleculares'esenciales.'El'Fe' es' un' cofactor' de' metaloenzimas' y' proteínas' que' son' esenciales' para' la' fotosíntesis,'respiración,'transporte'de'electrones,'metabolismo'del'nitrato,'la' detoxificación' de' especies' reactivas' de' oxígeno' y' la' reparación' del' ADN.' Aunque'sea'el'cuarto'elemento'más'abundante'de'la'Tierra,'su'solubilidad'es' muy' baja' en' agua' tan' oxigenadas,' lo' que' produce' que' se' presenten'bajas' concentraciones'de'Fe'en'la'mayoría'de'los'sistemas'acuáticos.'El'fitoplancton' presenta'distintos'mecanismos'para'incorporar'el'Fe'y'para'que'su'uso'no'sea' excesivo.'El'Fe'existe'en'2'estados'de'oxidación:'Fe(II)'que'es'muy'soluble'y'Fe' (III)' que' es' menos' soluble.' Generalmente,' la' forma' predominante' que' es' la' más'estable'es'la'de'Fe(III).'Además'el'Fe'se'clasifica'en'2'tipos'de'fracciones' ! 252# basado# en# los# sistemas# de# filtración:# Fe#particulado# (>0.2# μm)# #y# Fe#disuelto# (<0.2#μm).#Además,#la#fracción#de#Fe#disuelto#se#divide#en#coloidal#y#soluble.# Distintos# estudios# han# demostrado# que# la# mayoría# de# la# fracción# disuelta# (>99%)# está# formando# parte# de# compuestos# orgánicos#con# metales.# El# Fe#se# une# a# moléculas# orgánicas# denominadas# ligandos# orgánicos# que# pueden# ser# fuertes# o# débiles# en# función# de# la# constante# de# estabilidad#(constante# de# asociación#para#el#equilibrio#que#existe#entre#el#metal#y#el#ligando#orgánico).# Los#complejos#de#coordinación#orgánicos#formados#son#importantes#debido#a# que#influyen#en#la#solubilidad#y#controlan#la#disponibilidad#del#Fe#por#parte#del# fitoplancton.# Los# compuestos# formados# con# las# distintas# especies# del# Fe# pueden# resultar# en# condiciones# de# limitación# de# Fe,# porque# el# Fe#disponible# para#los#organismos#es#inferior#a#las#necesidades#que#presentan#los#organismos.# La# biodisponiblidad# de# un# metal# se# define# como# el# grado# al# que# cierto# compuesto,#en#este#caso#el#Fe,#es#accesible#y#lo#puede#utilizar#un#organismo.# Por# ello,# las# condiciones# de# estrés# debido# a# la# concentración# baja# de#Fe#son# muy#extendidas#tanto#en#océanos#como#en#ciertas#zonas#costeras,#afectando#al# crecimiento,# fisiología# y# producción# de# materia# orgánica# por# parte# del# fitoplancton.#La#acidificación#oceánica#afectará#la#termodinámica#y#cinética#de# los#metales,#además#de#la#especiación#de#los#distintos#metales.#La#disminución# de# la# concentración# de# OH-#debido# a# la# disminución# del# pH# y# de# el# ion# carbonato# debido# a# todos# los# cambios# del# sistema# carbónico-carbonato# afectarán#tanto#a#la#solubilidad#como#a#la#adsorción,#toxicidad#y#las#reacciones# de# oxidación# y# reducción# de# los# metales.# Todos# estos# cambios# posiblemente# afectarán#a#la#concentración#y#a#la#química#de#especiación#del#Fe#y#con#ello,#a#la# disponibilidad#del#Fe#para#las#distintas#especies#de#fitoplancton.#La#acidificación# oceánica#puede#aumentar#la#solubilidad#del#Fe#aumentando#la#concentración# del#mismo,#pero#como#el#Fe#disuelto#mayoritariamente#está#unido#a#ligandos# orgánicos,#el#resultado#final#de#la#concentración#biodisponible#dependerá#de#la# naturaleza#del#ligando.## Resumen''''''''''''''' ! ! 253' Solo' unos' pocos' estudios' se' han' centrado' en' el' estudio' de' los' efectos' interactivos'entre'la'acidificación'oceánica'y'la'concentración'del'Fe.'Además,' las' respuestas' obtenidas' han' dependido' en' parte' de' las' características' de' la' comunidad'fitoplantónica.'Las'necesidades'de'Fe'por'parte'de'una'comunidad' determinada' están' relacionadas' con' las' condiciones' ambientales' de' dicha' comunidad.' De' forma' general,' las' especies' costeras' de' fitoplancton' tienen' necesidades'superiores'de'ciertos'metales'que'las'especies'oceánicas.'Por'ello,' el'efecto'de'los'cambios'en'la'disponibilidad'de'Fe'debido'a'los'cambios'en'la' concentración' del' CO2' dependerán' de' las' demandas' de' Fe'de' las' distintas' especies'de'fitoplancton.'' La' interacción' de' los' efectos' de' la' acidificación' oceánica' y' de' la' concentración'de'Fe'han'mostrado'que'influyen'en'la'fisiología'del'fitoplancton' y' que' producen' cambios' en' la' comunidad' fitoplantónica.' Las' respuestas' obtenidas' han' sido' diferentes' en' función' de' la' región' (en' experimentos' en' condiciones'naturales)'y'de'la'especie'que'se'haya'estudiado'(en'experimentos' de'laboratorio).'Además'influye'la'concentración'del'Fe,''la'especiación'del'Fe'y' la' composición' de' la' comunidad' fitoplantónica.' En' un' futuro' escenario'de' cambio'global,'los'distintos'factores'variarán'simultáneamente.'Considerando' la' importancia' del' fitoplancton' como' base' de' las' cadenas' tróficas' y' en' los' ciclos'biogeoquímicos'de'los'distintos'nutrientes,'es'necesario'entender'todas' las'posibles'respuestas'por'parte'de'las'distintas'especias'como'consecuencia' del'cambio'climático.'El'ciclo'biogeoquímico'más'importante'es'el'del'carbono' y'el'grupo'de'fitoplancton'compuesto'por'los'cocolitofóridos,'se'caracterizan' por'contribuir'con'aproximadamente'entre' 1-10' %'del'carbono' total'fijado'y' con' la' mitad'de' la' calcita' producida' que' migra' hacia' los' sedimentos.' En' particular,'la'especie'Emiliania'huxleyi'se'caracteriza'por'ser'la'especie'de'los' cocolitofóridos'que'se'distribuye'de'forma'más'amplia'y'que'además,'es'la'más' estudiada.' Se' caracteriza'por' ser' una' especie' formadora' de' “blooms”.' Los' resultados'obtenidos'con'esta'especie,'E.'huxleyi,'son'dependientes'de'la'zona' ! 260$ unido$al$quelante$DFB$(FeDFB)$como$se$había$observado$previamente$en$otros$ estudios.$Por$ello,$las$células$del$tratamiento$de$concentraciones$elevadas$de$ CO2$con$ la$ adición$ del$ quelante$ DFB$ presentaron$ una$ mayor$ facilidad$para$ sobrellevar$ el$ gasto$ energético$ metabólico$ extra$ debido$ a$ la$ disminución$ del$ pH,$en$comparación$con$el$otro$tratamiento$de$concentraciones$elevadas$de$ CO2$que$ no$ presentan$ la$ adición$ del$ quelante.$ Esto$ le$ permitió$ también$ sobrellevar$mejor$los$efectos$negativos$de$la$acidificación$oceánica$y$mantener$ las$tasas$de$crecimiento.$La$respuesta$al$estrés$fisiológico$$de$la$comunidad$y$ en$ particular,$ de$ E.# huxleyi#fue$ afectado$ por$ las$ condiciones$ de$ CO2$y$ Fe$ disuelto.$Los$mecanismos$protectores$(la$composición$y$concentración$de$los$ pigmentos)$ se$ afectaron$de$ forma$ negativa$ por$ el$ incremento$ en$ la$ concentración$ de$ CO2$pero$ el$ efecto$ negativo$ fue$ contrarrestado$ por$ el$ aumento$ de$ la$ concentración$ de$ Fe$disuelto.$ Este$ resultado$ destaca$ que$ las$ células$ en$ condiciones$ de$ baja$ concentración$ de$ Fe$muestran$ una$ mayor$ susceptibilidad$ a$ la$ fotoinhibición,$ destacando$ que$ el$ estado$ fisiológico$ de$ estas$células$limitadas$fueron$mejoradas$con$el$aumento$de$la$concentración$ de$ Fe$disuelto.$ Los$ mismos$ resultados$ se$ observaron$en$ la$ cadena$ transportadora$de$electrones$donde$se$destaca$el$efecto$positivo$del$aumento$ de$ la$ concentración$ de$ Fe$modulando$ el$ efecto$ negativo$ del$ aumento$ de$ la$ concentración$ de$ CO2.$ La$ razón$ más$ probable$ de$ esta$ disminución$ en$la$ capacidad$ de$ los$ mecanismos$ protectores$de$ las$ células,$ al$ igual$ que$ en$ los$ procesos$ anteriores,$ sería$ como$ se$ ha$ mencionado$ anteriormente$ por$la$ disminución$del$pH.$Además,$el$$estrés$oxidativo$y$el$daño$en$el$ADN$fueron$ minimizados$también$debido$a$el$aumento$de$la$concentración$del$Fe$disuelto.$ Por$ello,$el$Fe$sería$necesario$para$la$reparación$del$ADN$por$daños$derivados$ del$exceso$de$irradiancia$y$para$superar$el$estrés$oxidativo$generado$por$los$ factores$de$estudio$(CO2$y$Fe),$asegurando$la$viabilidad$celular$y$el$crecimiento$ de$ E.# huxleyi.$ El$ Fe$ sería$ necesario$ para$ mantener$ los$ requerimientos$ energéticos$impuestos$por$las$proteínas$relacionadas$en$estos$procesos,$y$que$ Resumen''''''''''''''' ! ! 261' la'concentración'de'Fe'necesaria'no'es'suplida'en'condiciones'ambientales'o'la' energía' se' invierte' en' sobrellevar' el' efecto'negativo' de' la' acidificación' oceánica'en'la'fisiología'celular.' Este' estudio' demuestra' que' los' efectos' negativos' del' aumento' de' la' concentración' de' CO2'en' estas' cepas' de' E.# huxleyi'pueden' ser' parcialmente' mitigadas' por' el' aumento' de' la' concentración' de' Fe'disuelto' en' áreas' con' concentraciones' totales' de' Fe' elevadas' (tanto' la' concentración' particulada' como'disuelta),'posiblemente'teniendo'efectos'derivados'en'la'dinámica'de'las' redes'tróficas,'en'la'exportación'del'carbono'al'fondo'de'los'océanos,'en'el'rain# ratio'que'relaciona'el'carbono'particulado'inorgánico'y'orgánico'y'finalmente' afectaría' al' intercambio' de' la' concentración' de' CO2'entre' el' océano' y' la' atmosfera.'' Estos' resultados' establecen' que' son' necesarios' los' estudios' que' consideran'los'efectos'combinados'de'distintos'factores'derivados'del'cambio' global'para'poder'entender'las'futuras'respuestas'de'los'ecosistemas.'Por'esta' razón,'además'del'estudio'en'condiciones'naturales'utilizando'mesocosmos'se' realizaron' una' serie' de' experimentos' en' condiciones' controladas' de' cultivo' con' una' cepa' costera' de' E.# huxleyi,' aislada' en'Noruega.#En' estos' estudios,' además'de'considerar'variaciones'de'la'concentración'de'CO2'y'Fe'disuelto'se' consideró'un'tercer'factor'que'sería'la'exposición'a'radiación'ultravioleta'(RUV).' Además,' se' analizó'la' interacción' de' la' exposición' a' la' RUV' con' distintos' niveles' de' los'otros' factores' (CO2'y' Fe).'En' primer' lugar,' se' estimó' la' sensibilidad'a'la'exposición'de'la'RUV'de'los'cultivos'en'dos'condiciones'de'CO2,' ambiental'y'a'una'concentración'elevada'de'CO2.'La'concentración'fue'de'800' ppm,' el' doble' de' la'concentración'actual.' Se' estableció' mediante' modelos' matemáticos'la' sensibilidad' de' la' tasa' de' fotosíntesis' de' la' cepa' en' ambas' condiciones'de'CO2'analizando'el'efecto'de'la'RUV'en'cada'longitud'de'onda.' Se' analizaron' distintos' modelos' posibles' que' analizan'la'inhibición' de' la' fotosíntesis'por'la'RUV'y'se'estableció'el'modelo'más'adecuado'en'función'del' ! 262# ajuste# estadístico# entre# lo# observado# y# lo# que# se# predice.# Este# modelo# está# caracterizado#porque#hay#un#umbral#a#partir#cual#se#produce#la#inhibición#de#la# fotosíntesis#pero#que#por#debajo#de#este#umbral#no#hay#un#efecto#negativo#en# la#tasa#de#fotosíntesis.#Nuestros#resultados#indicaron#que#no#hay#diferencias#en# la# sensibilidad# a# la# exposición# de# la# RUV# en# la# tasa# de# fotosíntesis# en# las# distintas#condiciones#de#CO2#estudiadas#(400#y#800#ppm)#para#esta#cepa#de#E.# huxleyi.# Por# otra# parte,# se# realizó# otro#experimento# donde# se# estudiaba# el# efecto#de#la#concentración#del#hierro#disuelto#en#los#procesos#fisiológicos#de#E.# huxleyi#en#las#dos#condiciones#de#CO2.#Se#estudió#la#inhibición#de#los#procesos# fisiológicos# a# la# exposición# de# RUV# en# comparación# con# cultivos# expuestos# únicamente# a# la# radiación# PAR# en# condiciones# ambientales# de# CO2.##Los# resultados# mostraron#que# las# concentraciones# de# Fe# reducidas# presentan# restricciones# en# la# capacidad# de# sobrellevar# las# condiciones# de# estrés# por# la# exposición#a#la#RUV#en#esta#especie.#Además,#el#estrés#fue#disminuido#en#las# condiciones#de#CO2##para#finales#de#este#siglo#bajo#la#exposición#de#RUV#como# se#observa#incluso#en#los#cultivos#con#la#menor#concentración#de#Fe#disuelto;# observándose#un#aumento#de#la#producción#de#carbono#orgánico#particulado,# de# las# tasas# de# transporte# de# electrones# y# las# tasas# de# calcificación.# Esto# demuestra#que#E.#huxleyi#es#capaz#de#sobrellevar#mejor#el#estrés#generado#por# la#exposición#de#la#RUV#en#condiciones#de#CO2#futuras,#y#que#el#Fe#presenta#es# el#factor#principal#en#controlar#la#respuesta#en#todos#los#procesos#fisiológicos.## # # Conclusiones*************** ! ! 263* Conclusiones* * 1. Emiliania' huxleyi*estaba* limitada* por* hierro* en* condiciones* ambientales*de*CO2*y*de*hierro*disuelto*en*el*fiordo*estudiado*y*en*el* tratamiento*control.* * 2. El* sideróforo* deferoxamina* B* (DFB)* y* una* elevada* concentración* de* CO2*aumentó*la*concentración*de*hierro*disuelto*en*el*fiordo.* * 3. El* aumento* de* CO2*afectó* negativamente* a* Emiliania'huxleyi*y* a* Synechococcus* sp.* mientras* que* no* afectó* al* resto* de* la* comunidad* planctónica.* * 4. El*aumento*del*hierro*disuelto*aumento*el*crecimiento*y*la*biomasa* de*E.'huxleyi*a*condiciones*ambientales,*mitigando*el*efecto*negativo* del*alto*CO2.* * 5. Emiliania'huxleyi*presenta*transportadores*de*bicarbonato*debido*a*la* baja*actividad*de*la*enzima*anhidrasa*carbónica*externa*y*el*uso*del* bicarbonato*como*fuente*de*carbono*inorgánico.* * 6. Los* procesos* de* asimilación* de* carbono* aumentaron*a*altas* concentraciones* de* hierro*disuelto* y*a*concentraciones* ambientales* de* CO2*y* por* el* contrario,* fueron* afectados* negativamente* por* *las* concentraciones*de*CO2*elevadas.* * 7. La*calcificación*disminuyó*en*concentraciones*de*CO2*elevadas*y*no*se* vio*afectada* por* la* concentración* de* hierro.* Como* consecuencia,* el* “rain* ratio”* disminuyó* en* condiciones* de* elevado* CO2* Conclusiones* ! 264* independientemente* del*nivel* de* Fe,* afectando* directamente* la* bomba*biológica*de*carbono.* * 8. El* aumento* de* CO2*tuvo* efectos* negativos* en* la* concentración* de* pigmentos* y* en* la* cadena* transportadora* de* electrones* tilacoidal,* siendo*contrarrestado*por*el*aumento*de*la*concentración*de*hierro.* * 9. El*hierro*es*necesario*para*la*reparación*de*ADN*y*para*controlar*la* formación*de*especies*reactivas*de*oxígeno*(ROS).* * 10. Todos*los*metales*trazas*particulados*analizados*son*de*procedencia* biótica*con*la*excepción*del*hierro*y*titanio.* * 11. El* aumento* de* la* concentración* de* CO2*cambió* las* concentraciones* celulares* de* los* metales* traza* particulados* debido* a* su* efecto* en* el* crecimiento* de* los* distintos* grupos* de* fitoplancton.* Además,* dichas* concentraciones*fueron* moduladas* por* la* concentración* de* hierro* disuelto.* * 12. La* exposición* a* la* radiación* ultravioleta* (RUV)* de* Emiliania' huxleyi* tuvo* el* mismo* efecto* en* la* fotosíntesis* en* las* distintas* concentraciones*de*CO2.* * 13. La* sensibilidad* de* Emiliania' huxleyi*a* la* radiación* ultravioleta* (RUV)** en*las*distintas*condiciones*de*CO2*es*dependiente*de*los*niveles*de* hierro.** * Conclusiones*************** ! ! 265* 14. Las*altas*concentraciones*de*CO2*inducen*a*una*menor*sensibilidad*de* Emiliania' huxleyi*a* la* radiación* ultravioleta*independientemente* de* los*distintos*niveles*de*hierro.* * * * Agradecimientos.. ! ! ! 267! Después! de! más! de! 4! años! de! trabajo,! llega! el! momento! de! dar! las! gracias! a! todas!las!personas!que!me!han!ayudado!y!apoyado!durante!este!camino.!A!pesar!de! todo! el!esfuerzo,!me! siento! muy! afortunada!y! orgullosa! de!haber!podido! realizar!la! tesis!doctoral!por!todo!el!conocimiento!y!todas!las!experiencias!que!he!vivido,!pero! sobre!todo!por!las!personas!que!me!han!acompañado!en!esta!andadura.! En!primer!lugar,!me!gustaría!darle!las!gracias!a!mi!directora!de!tesis,!Prof.!Dra.!María! Segovia,! por!todo! el! conocimiento!que! ha! compartido!conmigo!durante! estos!años,!! por!su!apoyo!pero!especialmente!por!su!confianza!desde!el!principio.!Además,!gracias! por!darme!la!oportunidad!de!trabajar!con!investigadores!de!otros!sitios!que!tanto!me! ha!enriquecido!como!científica!y!tantos!lugares!me!ha!permitido!conocer.!Si!tuviera!un! giratiempo!volvería!a!comenzar!esta!aventura.!! Gracias!a!todos!los!miembros!del!Área!de!Ecología!por!la!disponibilidad!a!ayudarme!en! todo! momento.! En! especial,! al! Prof.! Dr.! Francisco! J.! L.! Gordillo! por! todas! sus! aportaciones! a! mi! trabajo! y! por! brindarme! la! oportunidad! de! colaborar! en! su! investigación.!! Sin! duda,! este! trabajo! no! hubiera! sido! posible! sin! todos! los! integrantes! de! “Phytostress”.! Muchísimas! gracias! por! vuestra! colaboración! y! entrega! durante! el! experimento!pero!también!por!los!momentos!de!diversión!compartidos!en!el!fiordo.! Gracias!Prof.!Dra.!Jorun!Egge!por!tu!ayuda.!! Muchísimas!gracias!Prof.!Dra.!Maria!T.!Maldonado!por!todo!lo!que!me!has!enseñado!a! lo!largo!de!estos!años!tanto!en!España!como!en!Canadá,!por!la!hospitalidad!cuando! trabajé!en!tu!laboratorio!y!sobre!todo,!por!hacerme!sentir!en!casa!a!pesar!de!estar!tan! lejos!de!la!mía.! No! hay! dos! sin! tres! estancias,! así! que! por! último,! gracias! Dr.! Patrick! J.! Neale! por! enseñarme! tanto! en! tan! sólo! dos! meses! y! porque! a! pesar! de! todos! los! problemas! siempre! me! mostrabas! el! camino! para! encontrar! la! solución.! Gracias! Dra.!Cristina! Sobrino!por!ser!mi!guía!de!la!producción!primaria!desde!que!nos!conocimos.! Gracias!a!mis!dos!evaluadoras!externas,!Prof.!Angela!Wulff!y!Dra.!Paraskevi!Pitta,!por! vuestras!aportaciones!de!forma! desinteresada!a!esta!tesis!y!por!todas! las!molestias! burocráticas!que!hemos!tenido!que!pasar.!! De!lo!mejor!de!trabajar!en!Ecología!es!compartir!este!proceso!con!mis!compañeros!de! la!sala!de!becarios!que!me!han!ayudado!tanto.!Gracias!a!todos!los!que!habéis!formado! y!aún!formáis!parte!de!ella!por!hacerlo!todo!tan!especial.!Gracias!a!mis!compañeras!a! las!que!he!pasado!a!llamar!amigas!por!quererme!tanto!y!demostrármelo!siempre,!por! vuestra! ayuda! a! cualquier! hora,! en! cualquier! lugar! y! por! cualquier! medio! y! por! ser! imprescindibles!en!mi!vida.!! ! 268$ En$especial,$gracias$a$Candela$por$todos$los$experimentos$compartidos,$por$todos$tus$ consejos$a$lo$largo$de$este$tiempo$y$por$tu$ayuda$desinteresada$con$la$tesis.$Gracias$ Conchi$porque$tu$amistad$es$un$regalo,$gracias$por$todo$lo$que$hemos$vivido$juntas$en$ nuestras$ estancias$ noruegas$ pero$ sobre$ todo$ por$ lo$ que$ nos$ queda$ por$ compartir.$ ¿Podemos(buscar(ya(al(oso?$ Desde$hace$muchos$años$tengo$la$suerte$de$tener$como$amiga$a$Raquel$y$etapa$tras$ etapa$siempre$seguimos$juntas.$Gracias$por$formar$parte$de$mi$vida$y$por$permitirme$ formar$parte$de$la$tuya$desde$hace$tanto.$ Es$un$privilegio$tener$tantos$amigos$para$compartir$los$buenos$y$malos$momentos,$sin$ ellos$hubiera$sido$difícil$llegar$hasta$el$final.$Gracias$Laura$por$todo$tu$apoyo$y$amistad$ ya$ que$ la$ distancia$ no$ es$ un$ impedimento$ para$ nosotras$ y$ gracias$ por$ todo$ lo$ que$ compartes$conmigo.$Gracias$a$La(Orden$por$las$escapadas$por$tierras$inglesas,$por$las$ vivencias$ compartidas$ y$ por$ las$ que$ quedan$ por$ compartir.$ A$ mis$ mamuchis(porque$ con$vosotros$la$vida$es$más$divertida,$gracias$por$vuestro$apoyo$y$por$tener$siempre$un$ brindis$ pendiente$ que$ haga$ que$ nos$ reunamos.$ Por$ último,$ gracias$ a$ mis$ nuevos$ amigos$que$me$han$adoptado$en$estos$últimos$años,$por$darme$siempre$ánimos$y$por$ las$barbacoas$compartidas.$En$especial,$a$los$nuevos$papis$y$a$Carla$por$todo$vuestro$ cariño.$$ Dejando$lo$más$importante$ para$ el$final,$ me$ gustaría$ agradecer$ a$mi$ familia$ todo$ el$ apoyo$que$he$recibido$durante$este$tiempo.$Gracias$por$perdonarme$las$ausencias$y$ por$todos$los$ánimos$durante$este$tiempo.$Gracias$abuela$por$ser$un$ejemplo$de$vida,$ ojalá$la$sigamos$compartiendo$muchos$años$juntas.$Gracias$tita$Inés$por$todo$tu$cariño$ de$forma$tan$desinteresada$y$gracias$Marta$por$ser$más$hermana$que$prima.$Gracias$a$ mis$ padres$ por$ ser$ los$ pilares$ de$ mi$ vida,$ por$ toda$ vuestra$confianza$ y$ apoyo$ pero$ sobre$todo$por$vuestro$amor$incondicional.$Sin$vosotros$esto$no$hubiera$sido$posible.$ Gracias$ a$ mis$hermanos,$Edu$ y$ Mariló,$ por$quererme$ y$enseñarme$ tanto$desde$que$ tengo$ uso$ de$ razón.$ Gracias$ por$ compartir$ nuestras$ vidas,$ me$ siento$ afortunada$ de$ saber$que$siempre$os$tendré.$También$gracias$a$mis$cuñados$y$a$mi$nueva$familia$por$ acompañarme$y$animarme$durante$todo$este$tiempo.$$ Por$último,$gracias$Noel$por$quererme$tanto$y$ayudarme$siempre,$por$darme$la$calma$ en$los$ momentos$más$difíciles$y$ por$hacerme$reír$ cada$día.$ Me$faltan$palabras$para$ agradecerte$todo$lo$que$haces$por$mí$cada$día$pero$sé$que$tengo$todo$una$vida$para$ decírtelas.$Gracias$por$ser$mi$compañero$de$vida.$$ ( Esta(tesis(está(dedicada(a(mis(padres,(a(mis(hermanos(y(a(Noel(( $ !