Final Revised Paper - Biogeosciences

16 Pages·2012·1.17 MB·English

Biogeosciences,9,2973–2988,2012
Biogeosciences
www.biogeosciences.net/9/2973/2012/
doi:10.5194/bg-9-2973-2012
©Author(s)2012. CCAttribution3.0License.
Response of Nodularia spumigena to pCO – Part 1:
2
Growth, production and nitrogen cycling
N.Wannicke1,2,S.Endres3,4,A.Engel3,4,H.-P.Grossart2,5,M.Nausch1,J.Unger1,andM.Voss1
1LeibnizInstituteforBalticSeaResearch,Seestrasse15,18119Rostock,Germany
2LeibnizInstituteofFreshwaterEcologyandInlandFishery,AlteFischerhu¨tte2,16775Stechlin,Germany
3AlfredWegenerInstituteforPolarandMarineResearch,AmHandelshafen12,27570Bremerhaven,Germany
4HelmholtzInstituteforOceanResearch(GEOMAR),Du¨sternbrookerWeg20,24105Kiel,Germany
5PotsdamUniversity,InstituteforBiochemistryandBiology,AmNeuenPalais10,14469Potsdam,Germany
Correspondenceto:N.Wannicke([email protected])
Received:1February2012–PublishedinBiogeosciencesDiscuss.:6March2012
Revised:26June2012–Accepted:28June2012–Published:6August2012
Abstract. Heterocystous cyanobacteria of the genus Nodu- suggest that rising pCO stimulates the growth of hetero-
2
lariaformextensivebloomsintheBalticSeaandcontribute cystous diazotrophic cyanobacteria, in a similar way as re-
substantially to the total annual primary production. More- portedforthenon-heterocystousdiazotrophTrichodesmium.
over, they dispense a large fraction of new nitrogen to the Implications for biogeochemical cycling and food web dy-
ecosystem when inorganic nitrogen concentration in sum- namics, as well as ecological and socio-economical aspects
mer is low. Thus, it is of ecological importance to know intheBalticSeaarediscussed.
how Nodularia will react to future environmental changes,
in particular to increasing carbon dioxide (CO ) concentra-
2
tionsandwhatconsequencestheremightariseforcyclingof
organicmatterintheBalticSea.Here,wedeterminedcarbon 1 Introduction
(C)anddinitrogen(N )fixationrates,growth,elementalstoi-
2
In summer, the heterocystous diazotrophic cyanobacteria of
chiometryofparticulateorganicmatterandnitrogenturnover
the genus Nodularia form extensive blooms in the open
inbatchculturesoftheheterocystouscyanobacteriumNodu-
Baltic Sea with more than 200mgm−3 wet weight (Was-
lariaspumigenaunderlow(median315µatm),mid(median
mund, 1997), along with cyanobacteria of the genus Aph-
353µatm),andhigh(median548µatm)CO concentrations.
2
anizomenon. These blooms are usually promoted by low
Our results demonstrate an overall stimulating effect of ris-
ing pCO on C and N fixation, as well as on cell growth. nitrogen-to-phosphorus ratios in the surface waters (e.g.
2 2
AnincreaseinpCO duringincubationdays0to9resulted Niemisto¨ et al., 1989; Nausch et al., 2008; Raateoja et al.,
2
in an elevation in growth rate by 84±38% (low vs. high 2011), exhibiting an average annually primary production
pCO ) and 40±25% (mid vs. high pCO ), as well as in rateof∼21molCm−2yr−1 intheBalticProper(Wasmund
2 2
N fixation by 93±35% and 38±1%, respectively. C up- et al., 2001b). The capacity of community N2 fixation in
2
the Baltic Sea is comparable to nitrogen inputs from the
takeratesshowedhighstandarddeviationswithintreatments
land and atmosphere (e.g. Larsson et al., 2001; Wasmund
andinbetweensamplingdays.Nevertheless,Cfixationinthe
highpCO treatmentwaselevatedcomparedtotheothertwo et al., 2001b, 2005b). Annual N2 fixation rates during a
2
moderatebloomintheBalticProperwereaveragedto101–
treatmentsby97%(highvs.low)and44%(highvs.mid)at
263mmolNm−2yr−1(Wasmundetal.,2001a).
day0andday3,butthiseffectdiminishedafterwards.Addi-
A significant fraction of the newly fixed nitrogen can be
tionally,elevationincarbontonitrogenandnitrogentophos-
directlyreleasedbycyanobacteria,therebydispensing35to
phorusratiosoftheparticulatebiomassformed(POC:POP
and PON:POP) was observed at high pCO . Our findings 80% of nitrogen into the surrounding environment (Wan-
2
nicke et al., 2009; Ploug et al., 2011). Nitrogen fixed by
PublishedbyCopernicusPublicationsonbehalfoftheEuropeanGeosciencesUnion.
2974 N.Wannickeetal.: ResponseofNodulariaspumigenatopCO
2
diazotrophs can be transferred to lower food web levels via anoveralldetrimentaleffectofrisingpCO onthecellsas-
2
dissolved organic matter (Ohlendieck et al., 2000) and to sociatedwithadecreaseingrowthandproduction.Theysug-
highertrophiclevelsbygrazingdirectlyoncyanobacteriaor gested that this pattern could be typical for heterocystous
indirectlyviathemicrobialloop(Engstro¨m-Ostetal.,2011). cyanobacteriacomparedtonon-heterocystouscyanobacteria
The extra load of nitrogen thus increases overall ecosys- of the genus Trichodesmium and potentially relate to physi-
tem productivity and meets 20 to 90% of the nitrogen re- ological and structural dissimilarities of both cyanobacteria
quirements for community primary production during sum- groups. Even less information is available on the coupling
mer blooms (So¨rensson and Sahlsten, 1987; Larsson et al., of fluxes of carbon, nitrogen and phosphorus in relation to
2001; Wasmund et al., 2005a). Aggregation and sedimenta- pCO mediatedbyheterocystouscyanobacteria.
2
tionofnetprimaryproductionaccountsfor∼30upto72% Thepurposeofthisstudywastoexaminetherelationship
ofbiomasslossfromtheuppermixedlayer,whichisacon- betweenpCO anddiazotrophicgrowthofNodulariaspumi-
2
siderable proportion of the seasonal sinking flux (Lignell et gena and the related fluxes of carbon, nitrogen and phos-
al., 1993; Heiskanen and Leppa¨nen, 1995). This process is phorus.CulturesofBalticSeaNodulariaspumigenaisolates
supported by the formation of aggregates including Nodu- were grown in a batch mode at three different pCO lev-
2
lariafilaments(Engeletal.,2002). els (low, medium and high), which were supposed to simu-
To date, it is not well understood and barely investigated latingglacial(180µatm),presentdayvalues(380µatm)and
how future changes in climate caused by anthropogenic el- values projected for the year 2100 (780µatm), respectively.
evationofatmosphericCO concentrationwillaffectNodu- Here,wepresentdataongrowthandproductionparameters,
2
laria performance and their potential to alter biogeochem- as well as N fixation and nitrogen turnover in response to
2
ical fluxes. At present, an atmospheric partial pressure of increasing pCO . Carbon cycling and extracellular enzyme
2
CO (pCO ) of 380ppm prevails, elevated by 27% com- activities, as well as phosphorous cycling and utilization of
2 2
pared to pre-industrial times of 280ppm. This accounts for dissolved organic phosphorous (DOP), will be presented in
the highest levels since approximately half a million years two companion publications (Endres et al., 2012; Unger et
(e.g.Lu¨thietal.,2008).WithatmosphericCO dissolvingin al.,2012).
2
seawater,itisexpectedthatcurrentpCO intheoceanswill
2
nearly double to 780µatm by 2100, and lower the oceans’
pH by about 0.35 units (IPCC, 2007), assuming that emis- 2 Materialandmethods
sions will carry on at the present rate. This has severe im-
plications for marine phytoplankton, as they appear to di- 2.1 Cultureconditionanddesignofthebatchculture
rectly respond to increasing pCO2 by altering their physi- experiment
ology (e.g. Riebesell et al., 2007), relative abundance (e.g.
Tortelletal.,2002),andbiogeography(e.g.BoydandDoney, Theexperimentalset-upweappliedcanbedividedintothree
2002). Additionally, unicellular marine cyanobacteria such parts:firstly,thepreparationofagedseawater,secondly,cul-
as Synechococcus and Prochlorococcus can show species- turingoftheparentcultureanditsacclimationtothepCO
2
specificresponsestoincreasingpCO (e.g.Fuetal.,2008). treatments,andthirdly,inoculationwithpre-acclimatedcul-
2
Several studies demonstrate that elevated pCO supports C tures,amendmentwithDIPandtheactualexperimentalrun.
2
andN fixation,aswellasgrowthratesinthenon-heterocyst Thethreeindividualstepstakenaredescribedindetailbelow
2
diazotrophTrichodesmium(Hutchinsetal.,2007;Levitanet andillustratedinFig.1.
al.,2007;BarceloseRamosetal.,2007).Ithasbeenhypoth- Thefirstpartbeganwithsamplingof1000litreofseawa-
esizedthatthesetrendsarefacilitatedbychangesinactivity terintheopenBalticSea(54.22749◦N,12.1748◦E,salinity
of the carbon concentrating mechanism (CCM) and modi- of 9.1psu), four months before the start of the experiment.
fied protein activity (Levitan et al., 2010a, b; Kranz et al., Plankton growth was allowed for three months, resulting in
2011) of the enzyme ribulose-1,5-bisphosphate carboxylase agedseawaterwithlowconcentrationindissolvedinorganic
oxygenase(RUBISCO),resultinginadecreaseofenergyand nutrients.Afterthisgrowthphase,sterilisationwasachieved
nutrientdemandofthecellathighpCO .TheenzymeRU- by UV light treatment and 0.2µm filtration under a clean
2
BISCO has a naturally low affinity to CO . Subsequently, bench. Concentrations of inorganic nutrients in this seawa-
2
energy saved can be relocated to other metabolic processes ter(DINandDIP)werebelowthedetectionlimit.
suchasN fixation.Butexperimentaldatasofararenotable The second part included the growth of three litre par-
2
to prove this hypothesis on a gene expression level and no ent culture of the heterocystic cyanobacterium N. spumi-
publication is available to verify this notion for heterocys- gena, which was isolated by L. Stal and coworkers (NIOO)
touscyanobacteria. from the Baltic Sea and maintained since 2000 at the Leib-
Ingeneral,thereislittleknowledgeontheresponseofhet- niz Institute for Baltic Sea Research in batch cultures in
erocystous cyanobacteria to pCO . Czerny et al. (2009) di- f/2mediumfreeofanycombinedinorganicNcompounds.
2
rectlyaddressedtheeffectsofdifferentpCO conditionson Three weeks prior to the start of the experiment, an ax-
2
growthandCfixationofthegenusNodulariaandobserved enic parent culture was transferred to sterile filtered aged
Biogeosciences,9,2973–2988,2012 www.biogeosciences.net/9/2973/2012/
N.Wannickeetal.: ResponseofNodulariaspumigenatopCO 2975
F01 2

Fig.1.Schematicoverviewoftheexperimentalset-upandtimeflowofthesinglestepstakenfrompreparation(−132days)totheendofthe
experiment(+15days).Seetextfordetailedinformation.
BalticSeawateramendedwith0.3µmoll−1PO everyweek routinelymixedbymanuallyshakingtopreventadhesionof
4
to assure an exponential growth phase. The parent culture cyanobacteriatothewallsoftheculturevessels.
was cultured at 15◦C in a walk-in incubation chamber un- Acclimation of the N. spumigena parent culture to the
der 16:8h light:dark cycle (cool, white fluorescent light- different pCO level started three days before the begin-
2
ing, 100µmolphotonsm−2s−1). One week before the start ning of the experiment. The parent culture was separated
of the acclimation period, the parent culture was still in a into three pre- cultures, one litre for each CO treatment,
2
stationary growth phase and removed from the walk-in in- and acclimated to the target pCO by aeration with pre-
2
cubationchambertoaclimatecontrolledroom.There,tem- mixed gases (Linde gas). Three levels of pCO were used,
2
perature was increased to 23◦C (representing typical sum- i.e. 180µatm representing glacial conditions, 380µatm rep-
mer temperatures at the Baltic Sea water surface) and light resenting present day and 780µatm representing year 2100
supply raised to 200µmolphotonsm−2s−1 (light: dark cy- conditions (Boer et al., 2000). During the course of the ex-
cle of 16:8h, cool, white fluorescent lighting). The chosen periment (including the acclimation perio3d8; 18 days), aera-
light intensity w as rather low compared to light intensities tion took place in the early afternoon (02:00p.m.) for one
experienced by cyanobacteria in summer time in the Baltic hourperdayinordertoavoidcontinuouslyhighturbulence
Sea (ca. 1000µmolphotonsm−2s−1). Higher light intensi- thatcouldpotentiallyharmtheintegrityofthecyanobacteria
ties (>200µmolphotonsm−2s−1) were not achievable by filaments.
using fluorescent light and would have led to an unwanted Aeration,however,wasnotsufficienttoyieldequilibration
large increase in experimental temperature. Cultures were withthepre-mixedgases,i.e.thecalculatedpCO (fromCT
2
andpH)valuesinthepre-cultureswere402µatm,422µatm
www.biogeosciences.net/9/2973/2012/ Biogeosciences,9,2973–2988,2012
2976 N.Wannickeetal.: ResponseofNodulariaspumigenatopCO
2
and 548µatm for the glacial, present-day and future pCO 2000 (Hitachi-Europe GmBH, Krefeld, Germany) accord-
2
treatments, respectively. For this reason, we re-defined the ing to Grasshoff et al. (1983). The detection limits were
threepCO levelsbasedonthetruepCO determinedduring 0.02µmoll−1 for DIP, 0.05µmoll−1 for ammonium and
2 2
theexperiment.ThesearethelowpCO treatment(median 0.05µmoll−1 for NO . A subsample of 100ml was filtered
2 3
315µatm),mediumpCO treatments(median398µatm)and ontoWhatmanGF/Ffiltersforchlorophyllaanalysis,imme-
2
highpCO treatment(median548µatm)pCO treatment. diatelyaftersampling.Filterswerestoredinliquidnitrogen
2 2
The third part started with the inoculation of each exper- or at −80◦C and were extracted with 96% ethanol for at
imental bottle. After three days of pre-acclimation, chloro- least 3h. Chlorophyll a fluorescence was measured with a
phyll a concentrations were determined in the three pre- TURNER fluorometer (10-AU-005) at an excitation wave-
cultures,yielding28µgchlal−1 inthelowpCO treatment, length of 450nm and an emission of 670nm (HELCOM,
2
27µgchl al−1 in the mid and 38µgchlal−1 in the high 2005).Chlorophyllaconcentrationswerecalculatedaccord-
pCO scenario. In order to inoculate the same quantity, ingtothemethodofJeffreyandWelschmeyer(1997).
2
0.8µgchl al−1, we added 296ml to replicate bottles of the
lowpCO treatment,308mltothemidpCO treatmentand 2.2.3 Nodulariafilamentandbacteriacellcounts
2 2
221mltoreplicatebottlesofthehighpCO treatment.Het-
2
erotrophicbacteriacellscountsatthestartoftheexperiment Subsamples of 50ml were taken for phytoplankton analy-
were below the blank value of 1000cellsl−1. Overall, bac- sis (preserved with acetic Lugol’s (KI/I2) solution to 1%
terial biomass during the course of the experiments (18d, fixation) and counted using an inverted microscope (Leica)
15d+3daysacclimation)neverexceeded1%ofcyanobac- (Utermo¨hl,1958)at100×magnification.Celllengthanddi-
terialbiomass. ameterweremeasuredusingamicrometereyepieceandcon-
AfterinoculationwithN.spumigena,eachofthe36bottles verted to biovolume assuming the geometrical approxima-
wasamendedwithphosphateto0.35µmoll−1 attime0and tionofacylinder.
atday3. Bacteria were counted using a flow cytometer (Facs Cal-
FoursamplingtimepointswerechosenforthethreepCO ibur, Becton Dickinson) and the protocol of Gasol and
2
treatments (time 0, +3, +9 and +15 days, Fig. 1) with three del Giorgio (2000). Four ml samples were preserved with
replicate bottles harvested at each time point. Each bottle 100µl formaldehyde (1% v/v final concentration), shock
contained 10litres of aged and sterile filtered seawater that frozen in liquid nitrogen and stored at −70◦C until mea-
hadbeenaeratedwithpremixedgasesforthreedaysinpar- surement. A stock solution of SYBR GREEN (Molecular
allel with the pre-cultures. One replicate bottle of the low Probes) was prepared by dilution of 1µl dye with 49µl
pCO treatmentatday9wasomittedinthedatacompilation DMSO. Three µl potassium citrate solution, 10µl of the
2
duetoinaccurateinoculationwithDIP.Samplesweretaken dyestocksolutionand10µlfluoresbritemicrospheres(Poly-
between08:00and09:00LTbeforedailyaeration. sciences) were added to 300µl of the thawed sample and
incubated for 30min in darkness. Cell counting was done
2.2 Analyticalmethods at a medium flow rate and calculations were performed us-
ingthesoftwareprogram“CellQuestPro”.Meanabundance
2.2.1 Carbonatechemistry of heterotrophic bacteria was 4.45±2.28×105cellsl−1
(low pCO ), 2.38±2.09×105cellsl−1 (mid pCO ) and
2 2
The pH was measured with an electrode (Knick Mikro- 4.80±2.82×105cellsl−1(highpCO ).
2
prozessor pH Meter 761 with Typ SE 100 glass electrode),
calibrated directly before measurement with standard NBS 2.2.4 Dissolvedorganicmatter(DON,DOC,DOP)
buffer.ValuesofpHaregivenrelativetothetotalscale.
Totalcarbon(C )wasanalyseddirectlyaftersamplingus- For analysis of dissolved organic carbon (DOC) and to-
T
ingthecolorimetricSOMMAsystemaccordingtoJohnsonet tal dissolved nitrogen (TDN), subsamples were filtered
al.(1993).Thesystemwascalibratedwithcarbonreference through pre-combusted GF/F filters, collected in 20ml pre-
material provided by A. Dickson (University of California, combusted(8h,500◦C)glassampoules,acidifiedwith80µl
SanDiego)andyieldedaprecisionofabout±2µmolkg−1. of 85% phosphoric acid and stored at 2–5◦C in a refrig-
TotalalkalinityandpCO werecalculatedusingCO2SYS erator. TDN and DOC concentrations were determined si-
2
(Lewisetal.,1998)paralleltoC ,pH,salinityandtempera- multaneouslybyhightemperaturecatalyticoxidationwitha
T
ture. Shimadzu TOC-VCSH analyser. In the auto sampler, 18ml
of sample volume plus 9ml of ultrapure (Type 1) water (in
2.2.2 Nutrientandchlorophyllaanalysis pre-combustedvials)wereacidifiedwith50µlHCl(1M)and
spargedwithoxygen(150mlmin−1)for6mintoremoveall
Dissolved inorganic nutrients (NH+, NO− and PO3−) inorganic C. 100µl sample volume was injected directly on
4 3 4
were determined colorimetrically from 60ml filtered sub- the catalyst (heated to 720◦C). Detection of the generated
samples (combusted GF/F) using a spectrophotometer U CO was performed with an infrared detector. Final DOC
2
Biogeosciences,9,2973–2988,2012 www.biogeosciences.net/9/2973/2012/
N.Wannickeetal.: ResponseofNodulariaspumigenatopCO 2977
2
concentrations were average values of quadruplicate mea- 2.3 Statisticalanalysis
surements.Ifthecoefficientofvariationexceeded0.1%,up
to 4 additional analyses were performed and outliers were Statistical analyses were done using the software SPSS 9.0
eliminated.TotalNwasquantifiedbyachemiluminescence and Sigma Plot 10. The effect of the pCO2 treatment on
detector(gasflowoxygen:0.6lmin−1).Afterevery8thsam- biological and chemical variables was tested by analysis of
ple,onestandardforqualitycontrolandoneblankwasmea- variance of data (ANOVA, t-test). Dependencies of growth
sured.ValuesofTDNwerecorrectedfornitrate,andammo- andproductionparametersfromotherenvironmentalparam-
nium,andthereafterreferredtoasDON. etersweretestedusingPearson’scorrelation“stepwise”mul-
Subsamples (40ml) for the determination of total (TP) tiple regression analysis. Prior to ANOVA and correlation
and dissolved phosphorus (DP) were stored at −20◦C un- analysis,dataweretestedfornormalityandhomogeneityof
til processing either unfiltered (for TP) or filtered through variancesusingWilk-ShapiroandLevene’stests.Linearre-
pre-combusted(450◦C,4h)WhatmanGF/Ffilters(forDP). gressionanalysiswasappliedtocalculategrowthratesfrom
Thawedsampleswereoxidizedwithanalkalineperoxodisul- changesinnaturallogarithmtransformedfilament/cellnum-
fatesolution(Grasshoffetal.,1983)inamicrowave(MarsX- bers,PON,POC,aswellaschlorophyllavalues.
press, CEM) to convert organic phosphorus into DIP. The
subsequent DIP determination was done using a 10cm-
cuvette reducing the detection limit to 0.01µmoll−1. Dis- 3 Results
solvedorganicphosphorus(DOP)wascalculatedasthedif-
3.1 Carbonatechemistry
ference between dissolved phosphorus (DP) and dissolved
inorganicphosphorous(DIP),detectedasdescribedabove. Throughout the study, the pCO treatments were different
2
with respect to pH and total carbon (C ), as well as calcu-
T
2.2.5 Particulateorganicmatteranalysis
lated total alkalinity (A ) and pCO (Table 1). The pCO
T 2 2
(PON,POC,POP)
treatments differed significantly in pH and C between mid
T
and high pCO treatment, as well as between the low and
Stable N and C isotope ratios (δ15N-PON, δ13C-POC), as 2
high pCO treatment (p≤0.001, n=12, Supplement Ta-
well as PON and POC concentration were measured by 2
ble S2). Ranges of calculated values for the single treat-
means of flash combustion in a Carlo Erba EA 1108 at
1020◦CandaThermoFinniganDeltaSmass-spectrometer. ments were for the low pCO2 treatment 248.5–498.6µatm
with a median of 315.7µatm (mean value 340±80µatm),
Filters containing particle samples were trimmed, sectioned
for the mid pCO treatment 286.5–571.1µatm with a me-
and then loaded into tin capsules and pelletised for isotopic 2
dianof353.3µatm(meanvalue398±104µatm)andforthe
analysis. Particulate organic phosphorus (POP) was calcu-
high pCO treatment 395.2–630.4µatm with a median of
latedasthedifferencebetweentotalanddissolvedphospho- 2
548.8µatm(meanvalue508±89µatm).Thelargedeviation
rus.
inpCO withinthetreatmentsoccurredpartlybecauseCO
2 2
wasconsumedduringphotosynthesis.ThecalculatedpCO
2.2.6 Isotopicanalysisandratesmeasurements 2
was significantly different between all three pCO set-ups
(primaryproduction,N fixation) 2
2
(p≤0.001,n=12,SupplementTableS2).
ThestableNandCisotoperatiosmeasuredforeachsample
3.2 Inorganicnutrients
were corrected for values obtained from standards with de-
fined N and C isotopic compositions (International Atomic
There were no significant differences in concentrations
Energy Agency IAEA: IAEA-N1, IAEA-N2, NBS 22 and
of inorganic nutrients between pCO treatments (Sup-
IAEA-CH-6)bymeansofmassbalance.Valuesarereported 2
plement Table S2). Dissolved inorganic phosphate was
relative to atmospheric N (δ15N) and VPDB (δ13C-Vienna
2
depleted in all treatments after three days of incuba-
Peedee belemnite). The analytical precision for both stable
tion (Table 2). DIP amended on day 3 was again be-
isotope ratios was ±0.2‰. Calibration material for C and
low the detection limit at day 9. Throughout the experi-
Nanalysiswasacetanilide(Merck).N fixationactivitywas
2
ment, mean concentration of dissolved inorganic nitrogen
measuredusingthe15N-N assay,Cfixationusingthe13C-
2 (DIN=NO−+NO ) was 0.26±0.1µmoll−1 in the low
NaHCO assay.Tracerincubationswereterminatedbygen- 3 2
3 pCO treatment,0.13±0.1µmoll−1 inthemidpCO treat-
tle vacuum filtration (<200mbar) through pre-combusted 2 2
GF/Ffilters.Thesefiltersweredriedat60◦Candstoredfor mentand0.1±0.1µmoll−1inthehighpCO2treatment(Ta-
ble2),whereasammoniumwasnotdetectable.Duetotheup-
isotopic analysis. Rates were calculated using the approach
take of nutrients during cell growth, an inverse relationship
of Montoya et al. (1996). Incubation time for rate measure-
was observed between DIP and abundance, chlorophyll a,
ments was 6 h, guaranteeing a sufficient dissolution of the
15Ngasintheincubationbottle(methodconsideration,Mohr PON and POP (R2=−0.567, −0.686 and −0.599, 0.359,
respectively,p≤0.05andp≤0.01,n=12,SupplementTa-
etal.,2010).
bleS1).
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2978 N.Wannickeetal.: ResponseofNodulariaspumigenatopCO
2
Table1.Carbonatesystemvariablesforthefoursamplingtimepoints.pHandtotalcarbon(CT)weremeasured,totalalkalinity(AT)and
pCO2inseawaterwerecalculatedfrompHandCTusingCO2SYS(Lewisetal.,1998).Valuesaremeansandstandarddeviationsofthree
replicates (except one replicate bottle of the 180ppm treatment at day 9). Samples were taken between 08:00 and 09:00LT, after daily
aeration.
Date pCO2Target Incubation pH CT AT pCO2
[µatm] Time[d] Totalscale [µmolkg−1] [µmolkg−1] [µatm]
29March2010 180 0 8.02±0.02 1651.9±8.9 1724.2±6.4 472.9±29.9
380 0 7.95±0.01 1656.8±3.6 1713.4±2.8 561.0±8.7
780 0 7.93±0.03 1676.7±2.7 1730.3±2.6 590.7±35.3
1April2010 180 3 8.16±0.03 1607.4±10.2 1709.7±3.2 330.1±26.3
380 3 8.09±0.03 1617.5±11.4 1703.5±3.5 388.8±36.0
780 3 7.94±0.02 1667.9±1.9 1720.5±5.1 576.8±23.3
7April2010 180 9 8.22±0.04 1570.4±14.6 1690.8±5.0 276.7±27.1
380 9 8.18±0.03 1581.1±2.8 1690.9±5.9 305.3±20.3
780 9 8.07±0.02 1620.7±5.3 1702.3±3.8 412.2±16.9
13April2010 180 15 8.20±0.04 1568.0±11.4 1683.3±22.6 291.8±29.3
380 15 8.14±0.01 1595.9±4.9 1696.7±2.0 339.6±12.5
780 15 8.03±0.06 1626.9±21.1 1702.2±6.7 452.9±82.2
Table2.Abioticandbioticvariablesforthefoursamplingtimepoints.Valuesaremeansandstandarddeviationsofthreereplicates(except
onereplicatebottleofthe180ppmtreatmentatday9).
Date pCO2 PO34− DIN Chlorophylla Bacterialabundance DOC DON DOP
treatment [µM] [µmoll−1] [µgl−1] [105l−1] [µmoll−1] [µmoll−1] [µmoll−1]
29March2010 low 0.29±0.02 0.22±0.31 0.74±0.08 4.31±2.54 306.5±18.6 16.02±0.07 0.34±0.04
medium 0.32±0.08 0.28±0.16 0.87±0.09 5.61±2.22 295.6±23.9 18.02±1.17 0.33±0.06
high 0.34±0.02 0.22±0.02 0.71±0.04 3.14±0.22 330.1±76.3 17.67±1.74 0.38±0.06
1April2010 low 0.05±0.02 0.41±0.35 3.04±0.12 2.68±0.82 270.6±8.7 15.61±0.01 0.27±0.02
medium 0.03±0.01 0.24±0.03 4.04±0.79 4.95±1.68 297.8±8.5 15.35±0.14 0.24±0.02
high 0.04±0 0.22±0.02 3.46±0.07 4.33±1.65 296.8±1.6 16.18±1.56 0.28±0.07
7April2010 low 0.01±0.01 0.28±0.27 3.38±2.27 5.14±1.56 308.7±19.6 15.44±1.79 0.27±0.08
medium 0.02±0.02 0.20±0.12 5.15±0.23 5.41±2.39 319.9±17.2 15.91±0.82 0.25±0.09
high 0.01±0 0.14±0.15 7.27±0.42 5.34±3.85 322.6±6.1 15.81±0.44 0.23±0.04
13April2010 low 0.03±0.01 0.20±0.04 1.49±0.28 6.62±3.22 318.7±26.8 14.65±0.74 0.24±0.07
medium 0.02±0.01 0.21±0.15 3.05±0.45 2.20±0.33 323.7±16.4 15.13±0.49 0.19±0.02
high 0.02±0.01 0.10±0 4.41±1.09 6.10±5.24 306.0±15.8 16.79±0.41 0.21±0.04
3.3 Dissolvedorganicmatter(DOM) DON concentrations were 15±1.0µmoll−1 in the low,
16±1.3µmoll−1 in the mid and 17±1.2µmoll−1 in the
high pCO treatment. They were reduced in the low com-
2
DOMconcentrationandstoichiometrydidnotdiffersignifi-
pared to high pCO level, indicating a higher accumula-
2
cantlybetweenthetreatments.
tion of DON at high pCO . From day 3 onwards, DON
DOC concentrations were 303±26µmoll−1 in the concentration decreased by20.3µmoll−1, 0.11µmoll−1 and
low pCO2 treatment, 309±21µmoll−1 in the mid and 0.9µmoll−1, respectively. Nevertheless, it has to be kept in
313±36µmoll−1 in the high pCO treatment. During the
2 mindthatcalculateddifferencesinconcentrationwereofthe
first3daysoftheexperiment,DOCconcentrationdecreased
samemagnitudeasstandarddeviationofthesinglemeasure-
inthelowandhighpCO treatmentby35and33µmoll−1,
2 ments and have to be considered carefully. DON showed a
respectively, while it increased in the mid pCO treatment
2 significantly negative correlation with PON, POP and pH
by2µmoll−1.Fromthereon,untiltheendoftheexperiment (R2=−0.351,−0.574and−0.619,p<0.05andp≤0.01,
at day 15, concentrations of DOC increased by 24µmoll−1 n=12, Supplement Table S1) and positive ones with C
inthelowpCO treatment,by13µmoll−1inthemidpCO
2 2
treatmentandby5µmoll−1inthehighpCO treatment.
2
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NF0.2 Wannickeetal.: ResponseofNodulariaspumigenatopCO 2979
2
to the low pCO treatment (p=0.0001, n=50, data not
2
shown). From thereon, heterocyst number per filament de-
creased significantly in all treatments, while differences be-
tweenthetreatmentswerenolongerstatisticallysignificant.
3.4.2 Chlorophylla
Chlorophyll a increased over time in all pCO treatments
2
(Table2).Highestmeanchlorophyllavaluesoccurredinthe
highpCO treatment(3.96±0.42µgl−1),mediumvaluesat
2
themidpCO treatment(3.28±0.72µgl−1),andlowestval-
2
ues at low pCO levels (2.28±0.79µgl−1). Nevertheless,
2
only differences between the high pCO treatment and the
2
lowpCO treatment were statistically significant according
2
tothet-test(p=0.009,n=12).
Fig.2.ChangesinabundancewithtimeforthethreepCO2 treat-
mentslabelledwiththepartialpressureofpremixedgaswithwhich
thecultureswereaerated(lowpCO2 –whitebars;mediumpCO2 3.4.3 Concentrationandstoichiometryofparticulate
–greybars;highpCO2 –blackbars).Barsrepresentmeanvalues organicmatter(POM)
ofthreereplicateswithrespectivestandarddeviation.
ConcentrationsofPOC,PONandPOPincreasedinallpCO
2
fixation,PO34−,pCO2andCT(R2=0.557,0.599and0.622, treatments, but most pronounced at the high pCO2 level
p≤0.01,n=12,SupplementTableS1). (Fig. 3). POC and PON concentration differed significantly
DOP concentrations were 0.3±0.1µmoll−1 in the low, between pCO2 treatments, with highest concentrations be-
0.27±0.1µmoll−1 in the mid and 0.27±0.08µmoll−1 in ingobservedathighpCO2(Table2).Normalizedtofilament
thehighpCO treatment. abundance,however,POC,PON,andPOPwerelowestinthe
2
Mean values for DOC:DON ratios were 20±3 (low high pCO2 treatment (Fig. 3). Thereby, differences in POC
pCO ),19±2(midpCO )and19±2(highpCO ).Mean contentperfilamentwerestatisticallysignificantforthemid
2 2 2
DOC:DOPratioswere1094±383(lowpCO2),1249±421 vs.thehighpCO2 level(p=0.05,n=12,datanotshown),
( mid pCO2) and 1243±378 (high pCO2). Ratios of39 butnotfortheothercombinationsofpCO2 levels.PONand
DON:DOPwere57±14(lowpCO ),64±17(midpCO ) POPperfilamentdifferedsignificantlybetweenthelowand
2 2
and66±19(highpCO2). highpCO2(p=0.05andp=0.01,n=12,datanotshown)
and between mid and high pCO (p=0.05 and p=0.01,
2
3.4 ResponsesofNodularia respectively,n=12,datanotshown).
Box-plots of POM elemental composition demonstrate
3.4.1 Nodulariaabundance an elevation in all treatments relative to Redfield ratios for
POC:POP and PON:POP, but near Redfield stoichiometry
There was a steady increase in the abundance of Nodularia forPOC:PON(Fig.4).
filaments in all pCO treatments until day 9 (Fig. 2). Af- Elemental ratios decreased with pCO (R2=−0.552,
2 2
terwards, abundance remained in a stationary phase. Nodu- −0.653 and −0.634, respectively, p≤0.01, n=12, Sup-
lariaabundancecorrelatedsignificantlypositivewithchloro- plement Table S1), as well as with biomass specific C fix-
phylla,POC,PONandPOP(R2=074,0.83,0.88and0.88, ation(R2=−0.708,−0.732and−0.711,respectively,p≤
p<0.01,n=12). 0.01,n=12,SupplementTableS1)andDIP(R2=−0.633,
Mean filament length in the high pCO treatment in- −0.653and−0.634,respectively,p<0.01,n=12,Supple-
2
creasedfrom74±28µm(day0)to88±38µm(day9),but mentTableS1).Thismightimplyamorebalancedincorpo-
thistrendwasnotstatisticallysignificant.Filamentlengthin rationofC,NandPathigherpCO ifweassumeincorpo-
2
thelowpCO treatmentandmidpCO treatmentsincreased rationofnutrientsaccordingtoRedfieldratios.
2 2
from 67±24µm to 117±53µm, and from 71±29µm to POC:PON, POC:POP and PON:POP increased with
100±50µm,respectively.Differencesinfilamentlengthbe- abundance of Nodularia filaments (R2=0.751, 0.795 and
tween treatments were not statistically significant, but fila- 0.789,respectively,p≤0.01,n=12,SupplementTableS1),
ments were slightly shorter at the highest pCO by ∼23%. chlorophyll a (R2=0.823, 0.883 and 0.829, respectively,
2
On day 3, there was a significant higher number of hete- p<0.01, n=12, Supplement Table S1) and pH (R2=
rocysts per filament in the high pCO treatment compared 0.529,0.556and0.529,respectively,p≤0.01,n=12,Sup-
2
to the mid pCO (p=0.005, n=50, data not shown) and plementTableS1).
2
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2980 F03 N.Wannickeetal.: ResponseofNodulariaspumigenatopCO2
Fig.3.Timedependedvariationinparticulateorganiccarbon(POC),particulateorganicnitrogen(PON),particulateorganicphosphorous
(POP)pervolume(a,c,e)andperfilament(b,d,f)forthethreepCO2treatments(lowpCO2,whitecircles;mediumpCO2,greycircles;
highpCO2,blackcircles).Valuesaremeansandstandarddeviationsofthreereplicates.
3.4.4 Growthrates pCO and 35±31nmolCµmolPOC−1h−1 at high
2
pCO . Mean values of N fixation, excluding day
2 2
Growth rates calculated for the exponential growth phase 3, were 0.32±0.18nmolNµmolPOC−1h−1 at low
(days0–9)fromchangesinPOCandPONwerelowerthan pCO , 0.36±0.21nmolNµmolPOC−1h−1 at mid
2
growth rates derived from abundance and chlorophyll a in pCO and 0.48±0.20nmolNµmolPOC−1h−1 at high
2
the low pCO2 treatment, while they were equal in the mid pCO2. Mean values of N2 fixation of day 3 were
and high pCO2 treatment (Fig. 5). Compiled growth rates 2.56±0.44nmolNµmolPOC−1h−1 at low pCO2,
basedonallparametersweresignificantlydifferentbetween 3.89±0.12nmolNµmolPOC−1h−1 at mid pCO and
2
thepCO2treatments(p<0.05andp=0.001,n=12,Sup- 5.36±0.32 nmol NµmolPOC−1h−1 at high pCO2. C up-
plementTableS2),withthehighestgrowthrateathighpCO2 takeratesshowedhighstandarddeviationswithintreatments
(0.212±0.018d−1) and in between sampling days. Nevertheless, C fixation
in the high pCO treatment was elevated compared to the
2
3.4.5 CandN2fixation othertwotreatmentsby97%(highvs.low)and44%(high
vs. mid) at day 0 and day 3, but this effect d4i0m inished
Biomass specific C and N fixation rates decreased with
2 afterwards.
incubation time in all pCO treatments (Fig. 6). Mean
2 Statistically significant differences between pCO treat-
2
values of C fixation averaged over the sampling pe-
ments according to ANOVA and Tukey’s post hoc test
riod of 15 days were 21±15nmolCµmolPOC−1h−1
were observed for biomass specific C and N fixation
at low pCO , 30±19nmolCµmolPOC−1h−1 at mid 2
2
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F04
N.Wannickeetal.: ResponseofNodulariaspumigenatopCO 2981
2

Fig.4.Boxplo t(n=12)ofparticulateorganicmatterstoichiometryandstoichiometryofcarbontonitrogenfixedforthethreepCO2treat-
ments(lowpCO2,mediumpCO2,highpCO2).(a)Molarratioofparticulateorganiccarbontoparticulateorganicnitrogen(POC:PON),
(b) atom percent ratio of carbon and nitrogen fixed, (c) molar ratio of particulate organic carbon to particulate organic phosphorous
(POC:POP),(d)molarratioofparticulateorganicnitrogentoparticulateorganicphosphorous(PON:POP).DottedlinesrepresentRed-
fieldstoichiometry.Dasheddottedlinerepresentsmeanvalues.
F05
Redfield ratio and yielded maximum values at mid pCO
2
(C:N=16:1)andlowestvaluesathighpCO (C:N=9.6)
2
(Fig.4).
4 Discussion
4.1 GrowthandproductionunderdifferentpCO
2
conditions
In this study we assessed the response of Nodularia spumi-
genatochangesinpCO .Growthratesintermsofbiomass
2
increase calculated for days 0 to 9 were highest in the
high pCO scenario (508±89µatm). They were statisti-
2
callysignificantelevatedby40±25%relativetomidpCO
Fig. 5. Calculated growth rates (µ) per day based on changes 2
(398±104µatm) and by even 84±38% relative to low
in abundance, chlorophyll a (Chl a), particulate organic nitrogen
pCO (340±80µatm) (p=0.001, n=12). In the same
(PON) and particulate organic carbon (POC) for the three pCO2 2
treatments(lowpCO2,opencircles;mediumpCO2,greycircles; timeperiod(days0–9),N2fixationseemedtobemorestimu-
highpCO2,blackcircles).Symbolsrepresentmeansof12values latedbyhighpCO2thanCuptake(Fig.6).Biomassspecific
withstandarddeviations. CfixationathighpCO increasedby9±44%comparedto
2
mid pCO and by 60±60% relative to low pCO . C up-
2 2
takeratesshowedhighstandarddeviationswithintreatments
rates (p≤0.05 and p≤0.001, Supplement Table S2). C andinbetweensamplingdays.Nevertheless,Cfixationinthe
and N fixation increased significantly with pCO (R2= highpCO treatmentwaselevatedcomparedtotheothertwo
2 2 2
0.747 and 0.362, p≤0.01 and p≤0.05, n=12, Supple- treatmentsby97%(highvs.low)and44%(highvs.mid)at
mentTableS1).RatiosofC :N werehigherthanthe day0andday3,butthiseffectdiminishedafterwards.
fixed fixed
41
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42
2982 N.Wannickeetal.: ResponseofNodulariaspumigenatopCO
F06 2
N fixationrate,suggestingthatitcouldbeusedasanindi-
2
catorforN fixationcapacity.
2
If this tendency in morphology is repeatedly observed in
future studies, it might have implications for grazing on fil-
amentous cyanobacteria by zooplankton. Shorter filaments
might not provide sufficient morphological grazing resis-
tance, apart from chemical resistance. Moreover, Chan et
al.(2004)demonstratedthatinthepresenceofgrazers,hete-
rocystsshowedadeclineinN fixationratesby40%.Byre-
2
ducingfilamentlength,zooplanktongrazingmayacttopre-
empt cyanobacteria blooms by suppressing N fixation and
2
cyanobacterialgrowth.
The simulative effect of high pCO disappeared during
2
thetimecourseofourexperimentfromday9onwards,along
with a complete exhaustion of the inorganic P pool. Never-
theless,theDOPpoolwasexhaustedtoagreaterproportion
in the high pCO treatment along with a higher P concen-
2
tration per filament, indicating a more efficient P usage at
highpCO .FurtherinvestigationofthedifferentDOPcom-
2
ponentsandPturnoverwillbediscussedintwocompanion
manuscripts(Endresetal.,2012;Ungeretal.,2012).
InourstudygrowthratesincreasedwithincreasingpCO ,
2
despiteDIPlimitation,indicatingastimulatingeffectofDIC
availability.Thissuggestsaco-limitationbyCandPinour
experimentalset-upatlowandmidpCO conditions,which
2
mightbeappliedtotheBalticSeainsummer,aswell.Adefi-
ciencyinDIPseemstobepartlycounter-balancedbyexcess
C,whichisopposingtheconceptofLiebig’slawofonlyone
limiting nutrient, which has already been noted by, e.g. Ar-
rigo(2005)andHutchinsetal.(2007).
The first and only study available so far reporting the
response of Nodularia growth and primary production to
changing pCO conditions was published by Czerny et
2
al. (2009), who hypothesized a detrimental effect of high
pCO onNodulariagrowth.
2
BothstudiesinvestigatingNodulariaperformance,Czerny
e t al. (2009) and ours used culture conditions that favoured
the formation of single filaments without visible formation
 Fig.6.ChangesinbiomassspecificC(nonfilledbarcharts),N2fix- oflargeraggregates.Czernyetal.continuouslyrotatedtheir
ation(filledbarcharts)andcorrespondingmolarPOC:PONratios
incubation bottles using a plankton wheel, representing a
(scatterplots)versusincubationtimeforthethreepCO2treatments
closedDICmanipulationset-up.Weusedslightagitationby
lowpCO2,mediumpCO2,highpCO2.Barsandscatterrepresent
manually rotating the bottles once a day and aeration at a
meanvaluesofthreereplicateswithrespectivestandarddeviation.
low flow-rate, representing an open DIC manipulation. The
different methods43u sed might partly explain the observed
opposing trends. Czerny et al. (2009) adjusted the pH by
acid/basemanipulation, whichchangestotal alkalinity(TA)
ElevationinN fixationatthehighestpCO wasaccom- atconstantdissolvedinorganiccarbon(DIC).Concentrations
2 2
paniedbyahighernumberofheterocystsperfilament.This of DIC, HCO−, and CO3− in their study might have been
3 2
went along with a shortening of filaments towards the end lower than their actual target values, because seawater pH
oftheexperiment(notstatisticallysignificant),becausefila- controlstherelativeproportionofthecarbonatespeciesand
−
mentstendtobecomemoreinstable,fragileandbreakmore induces a lower percentage increase in HCO compared to
3
easily. Nevertheless, heterocyst frequency declined over the areductionofpHachievedby,e.g.aerationorbyco-adding
course of the incubation in all treatments. Heterocyst fre- carbonate ions along with acid (e.g. NaHCO , e.g. Gattuso
3
quency in Nodularia (Lindahl et al., 1980) and Aphani- andLavigne,2009).Thisfactmighthavedampenedapossi-
zomenon (Riddolls, 1985) has been shown to correlate with blepositivepCO effect.
2
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