Gross photosynthesis and lake community metabolism during the spring

phytoplankton bloom

David J. Suggett1

Department of Biological Sciences, University of Essex, Colchester, CO4 3SQ, United Kingdom

Stephen C. MaberlyLake Ecosystem Group, CEH, Lancaster Environment Centre, Library Avenue, Bailrigg, Lancaster, LA1 4AP,United Kingdom

Richard J. GeiderDepartment of Biological Sciences, University of Essex, Colchester, CO4 3SQ, United Kingdom

Abstract

Daily productivity determinations of linear photosynthetic electron transfer and of net and gross inorganicCO2 uptake were determined in situ throughout a 6-week sampling period of the spring phytoplankton bloom inEsthwaite Water in the English Lake District. Photosynthetic electron transfer rates, expressed as gross O2

evolution, were determined from fast repetition rate (FRR) fluorometry and discrete laboratory measurements ofthe photosynthetic unit size. These gross O2 evolution determinations were also made free from contemporaneousdark-adapted FRR measurements and the need for sample blanks. Net and gross CO2 uptake was determinedfrom changes in total inorganic carbon calculated from in situ pH measurements. Two phases to the bloom wereobserved. An initial bloom was dominated by the diatom Asterionella formosa, while a secondary phase wascharacterized by an increase in flagellates and cyanobacteria. For both phases, daily FRR-based gross O2

evolution and pH-based gross CO2 uptake were tightly coupled, suggesting that daily gross O2 production wasdriving daily changes in CO2 assimilation and thus in community metabolism. This is the first investigation toshow closely related rates of gross O2 production and CO2 uptake measured in situ.

Daily phytoplankton production is determined routinelyin aquatic studies to examine ecosystem energetics.Typically, daily production is estimated from discreteincubations of natural material. However, this approachmay give rise to considerable error because realisticenvironmental conditions may not be reproduced in theincubation bottles (Wilhelm et al. 2004). Various tech-niques are now used to provide direct, in situ measures ofprimary productivity. These include diel O2 (Gelda andEffler 2002) and CO2 (Maberly 1996) exchange, and fastrepetition rate (FRR) fluorescence (Kolber et al. 1998;Suggett et al. 2001; Raateoja et al. 2004). Direct in situ O2

and CO2 measurements yield a measure of the net dailyplanktonic community metabolism during the day and ofnet community respiration at night (del Giorgio et al. 1999;Cole et al. 2000), whereas FRR fluorescence yields theinstantaneous gross phytoplankton photochemical activity(Falkowski and Kolber 1995; Gorbunov et al. 2001). Wecombined these techniques to examine the transfer ofenergy from photons to carbon dioxide fixation in situ.

Continuous in situ measurements of FRR fluorescenceand pH were used to estimate daily phytoplanktonproductivity at a single depth throughout a 6-weeksampling period of the 2003 spring bloom in EsthwaiteWater in the English Lake District. We describe continuousFRR-based productivity determinations that are free fromadditional dark-adapted fluorescence and sample blankmeasurements and incorporate photosynthetic unit sizemeasurements from natural samples. Here we show that thegross photosynthesis measured by FRR and gross dailycommunity productivity estimated from diel changes ofTCO2 covaried in two phases of the spring bloom. The firstphase was almost exclusively dominated by the diatomAsterionella formosa, whereas the second phase wascharacterized by increased numbers of flagellates, inparticular Rhodomonas spp. and Chrysochromulina parva,and the cyanobacterium Anabaena circinalis. Our resultsdemonstrate tight coupling of gross photosynthetic electrontransfer rates with gross community metabolism. Differentphotosynthetic quotients were observed for the two phasesof the spring bloom corresponding with the change inphytoplankton community structure.

Methods

Sampling was conducted from 28 February to 14 April2003 in Esthwaite Water in the English Lake District(54u229N 2u599W). During these 6.5 weeks, we experiencedtwo distinct phases of the spring bloom, an initial bloom inweeks 1–2.5 (phase 1) and a secondary bloom of lower

1 Corresponding author (dsuggett@essex.ac.uk).

AcknowledgmentsWe thank Mark Moore for providing assistance with high-

performance liquid chromatography pigment measurements andMitzi de Ville and Julie Parker for assistance with field sampling.Insightful comments from Marcel Babin, Mark Moore, and twoanonymous referees considerably improved this paper. TheNational Environmental Research Council U.K. supported thiswork (NER/A/S/2000/01237).

Limnol. Oceanogr., 51(5), 2006, 2064–2076

E 2006, by the American Society of Limnology and Oceanography, Inc.

2064

magnitude in weeks 4–6.5 (phase 2). These two phases willbe referred to throughout.

FRR fluorescence and photosynthetic unit size—A Chel-sea Instruments FASTtracka FRR fluorometer (SN 182039)was attached to a mooring at a depth of 1 m. A 2p collectormeasuring downwelling photosynthetically active radia-tion, Eod (PAR) (400–700 nm), was integrated with theFRR fluorometer and positioned at the same depth as theexcitation-emission optical windows. A second PAR sensorwas mounted on the mooring, ,2 m above the watersurface, to continually log incident PAR. Chlorophylla (Chl a) concentration can typically exceed 20–30 mg m23

in the spring, yielding fluorescence levels that saturate thephotomultiplier tube of the FASTtracka even when the gainis set to the minimum sensitivity. Therefore, the FASTtracka

FRR fluorometer was set to a gain of 1, and a neutraldensity filter was placed over the window between thesample and photomultiplier tube throughout the samplingperiod. Use of a neutral density filter did not significantlyalter the variable fluorescence signal when tested withvarious algal species in the laboratory (data not shown).

FRR fluorescence was acquired continuously for bothopen (exposed to ambient light) and enclosed (dark)chambers at hourly intervals, except during weeks 1–2,when intermittent battery failure resulted in a sparserecord. The fluorometer was serviced twice a week to cleanoptical windows, replace neutral density filters, downloaddata, and change the battery. Surface-water samples werecollected during each service and returned to the laboratoryfor immediate measurements of photosynthetic unit size,pigments, and optical absorption. The underwater spectrallight field at a depth of 1 m was measured every 2 weekswith a Macam 9050 spectroradiometer. Each spectrum wasrecorded between 400 and 700 nm at a 1-nm interval.

Each FRR acquisition consisted of 15 single-turnoverexcitation sequences set according to Suggett et al. (2003,2004). Hourly acquisitions were processed by v4 software(Sam Laney pers. comm.) to generate values of minimumand maximum fluorescence and the effective absorptioncross section (Table 1). Connectivity was also retrievedfrom the v4 software but varied only slightly (0.28–0.32,dimensionless) throughout the investigation. Fluorescenceparameters were adjusted for instrument-specific response,

Table 1. Symbols, terms and definitions (derivations). Fluorescence parameters follow Kromkamp and Forster (2003) and Suggett etal. (2003). All fluorescence parameters are in instrument units unless stated otherwise. PSII and RCII are photosystem II and reactioncenters of PSII, respectively.

Symbol Term Definition or derivation

Fo Minimum fluorescence under dark adaptationF o9 Minimum fluorescence under ambient light

F o9 FRR Minimum fluorescence under ambient light in the FRRenclosed chamber

F9 Steady-state fluorescence in the FRR open chamberFm Maximum fluorescence under dark adaptationF m9 Maximum fluorescence under ambient lightf ‘‘Background’’ fluorescenceFv/Fm Maximum PSII photochemical efficiency under dark adaptation

(dimensionless)5 (Fm 2 Fo)/Fm

F v9/F m9 Maximum PSII photochemical efficiency under ambient light(dimensionless)

5 (F m9 2 F o9)/F m9

F q9/F m9 PSII photochemical efficiency under ambient light (dimensionless) 5 (F m92 F9)/F m9

QOXA : QTOTAL

APSII efficiency factor or the ratio of effective to maximum

trapping efficiency (dimensionless)Proportion of QA molecules that are

oxidized 5 (F m9 2 F9)/(Fm 2 F FRRo )

sPSII PSII effective absorption cross section under dark adaptation(A2 photon21)

Rate of QA oxidation and thus of closureof PSII reaction centers

s PSII9 PSII effective absorption cross section under ambient light ineither FRR chamber (A2 photon21)

sabsPSII9 Spectrally adjusted PSII effective absorption cross section

under ambient light in either FRR chamber (A2 photon21)Eq. 3

nO2 Reciprocal of the Emerson and Arnold number (mol O2

[mol Chl a]21)Single-turnover flash-induced O2

evolution per unit Chl aEo, Eod (PAR) Incident irradiance, incident downwelling photosynthetically

active radiation (mmol photons m22 s21)

P chlO2

FRR-based O2 evolution rate per unit Chl a(mol O2 [g Chl a]21 h21),

Eq. 4

PPGROSS

O2Daily integrated FRR-based O2 evolution rate per unit volume

(mol O2 m23)Eq. 5

TCO2 Total concentration of inorganic carbon as free CO2, HCO 23 ,

and CO 223 (mol CO2 m23)

Eq. 6

PPNET

CO2 ,P

PGROSSCO2

pH-based daily net, gross CO2 uptake per unit volume (molCO2 m23)

Eq. 7, 9

Spring bloom photosynthesis 2065

scatter, and baseline functions during v4 processingessentially as described by Laney (2003).

Under dark-adapted conditions, the minimum andmaximum fluorescence and maximum effective absorption,termed Fo, Fm, and sPSII (Table 1), will be equal for the twoFRR chambers, provided they are intercalibrated. Dark-adapted fluorescence is altered to a new steady state byactivation of photochemical and nonphotochemical path-ways under ambient light. Consequently, different fluores-cence parameters are obtained under ambient light, themajority of which are denoted by a prime (9) symbol.Maximum fluorescence and the effective absorption crosssection under ambient light are termed F m9 and s PSII9,respectively, for both FRR chambers (Table 1). We onlyused the s PSII9 from the enclosed chamber because po-tential scattering of contaminating ambient red light wasvastly reduced, thus improving the signal-to-noise ratio toyield a more robust fitting procedure used in calculatings PSII9 . Minimum fluorescence under ambient light is equalto the steady-state fluorescence in the FRR open chamber,termed F9 (Table 1), but not in the FRR enclosed chamber.Here, we term the minimum fluorescence under ambientlight in the enclosed chamber F o9 FRR (Table 1). The PSIIphotochemical efficiency was calculated as (Fm 2 Fo)/Fm

(5Fv/Fm) or (F m9 2 F9)/F m9 (5F q9/F m9) for dark-adaptedand steady-state (ambient light) conditions, respectively(Table 1). Maximum and minimum fluorescence yields andhence values of PSII photochemical efficiency were thenadjusted for the proportion of nonactive backgroundfluorescence.

Sample blanks were calculated from separate laboratoryFRR measurements on filtrates of discrete dark-adaptedsamples (Suggett et al. 2004; Moore et al. 2005) to yield theproportion of nonactive Chl a fluorescence ( f ). These FRRmeasurements were made with a second FRR fluorometer(SN 182010) that had been intercalibrated against thefluorometer used in situ and corrected for instrument-specific functions as above. f remained relatively constantthroughout the sampling period, ,0.04 6 0.003 (mean 6standard error) instrument units. Consequently, the per-centage contribution of nonactive fluorescence to measuredFm and Fo was highest from water samples with the lowestfluorescence yields (Fig. 1). The relationship between f andmeasured Fm or Fo was used to provide an approximateblank correction for FRR fluorescence yields recorded insitu because continuous blank measurements could not bemade. A ‘‘true’’ Fm and Fo would only be measured in situunder darkness when fluorescence quenching has com-pletely relaxed to reach a dark-adapted state. Therefore, thedaily blank factor was applied from knowledge of the meanFm and Fo the night before and after each diurnal phase.

To determine photosynthetic unit size, 4 liters of surfacewater were filtered onto 0.2-mm Nuclepore filters witha gentle vacuum controlled by a hand pump. Filtrationtypically lasted 30–45 min. The filters were washed witha small amount of filtered lake water to resuspend thealgae. Suspensions yielded final concentrations of 0.7–1.6 gChl a m23, ,40–160 times initial concentrations. FRRfluorescence measurements were made before and afterconcentration. Any samples with a significantly lower

blank-corrected value of Fv/Fm after concentration wereassumed to have been adversely affected by the procedureand were discarded. Less than 10% of prepared sampleswere discarded from throughout the entire samplingperiod. Photosynthetic unit size was determined as de-scribed previously (Suggett et al. 2003, 2004) with a single-turnover flash approach that provides a measure of O2

evolved by the functional PSII reaction centers. Chla divided by the oxygen flash yield is the Emerson andArnold number (Falkowski and Raven 1997), termedPSUO2 (mol Chl a (mol O2)21). The photosynthetic unitsize is a quarter of the Emerson and Arnold number underthe assumption that four electron transfer events arerequired for each O2 evolved. We used the reciprocal ofPSUO2, termed here as nO2 (mol O2 (mol Chl a)21), whichtherefore differs from the strict definition of photosyntheticunit size.

Pigments, absorption, and microscopy—Four aliquots of250–500 mL from surface-water samples and two aliquotsof 1–2 mL from the concentrated surface water used formeasuring nO2 were each filtered onto GF/F filters anddivided for pigment and spectral absorption measurements.Filters were stored at 270uC until analysis. Pigments wereextracted into 90% acetone and analyzed according to themethod of Barlow et al. (1997) on a Thermoseparationsproduct high-performance liquid chromatographer. Chla was identified by retention time and quantified from the

Fig. 1. Relationship between FRR fluorescence yields mea-sured on dark-adapted samples and dark-adapted filtrate blanks.Blank fluorescence ( f ) was expressed as the percentage of theabsolute fluorescence yield of both Fo and Fm. A single functionwas fit to all data: fluorescence blank 5 24.5924 ln(fluorescenceyield) + 5.815 (r2 5 0.875, n 5 56, p , 0.001).

2066 Suggett et al.

diode-array spectra by comparison with a pigment stan-dard.

Absorption spectra were measured between 760 and380 nm with a Hitachi U-3000 spectrophotometer fittedwith a 60-mm integrating sphere, following the methodof Ferrari and Tassan (1999). A wavelength-independentpathlength amplification coefficient obtained from mixedcultures of diatoms, dinoflagellates, chlorophytes, prasino-phytes, and cyanobacteria (Cleveland and Wiedemann1993) was applied to all samples. Absorption spectra werenormalized to corresponding values of Chl a, aChl (m2 (mgChl a)21).

Phytoplankton species composition was determined onsamples preserved in Lugol iodine and counted in a Lundcounting chamber with an inverted microscope (Lund1962).

Calculation of gross photosynthesis from FRR fluores-cence—FRR-based gross O2 production, p chl

O2(mol O2 (gChl a)21 h21), was calculated by using an approachmodified from Kolber and Falkowski (1993) as the productof incident PAR (Eo (PAR), mmol photons m22 s21, 400–700 nm), the effective absorption cross section underambient light (s PSII9, A2 photon21), the reciprocal of theEmerson and Arnold number (nO2, mol O2 (mol Chl a)21)and the proportion of primary quinone acceptor molecules,Qa, that are oxidized (termed here QOX

A : QTOTALA , dimen-

sionless) (Table 1),

P chlO2 ~ Eod (PAR):sPSII

’:QOXA : QTOTAL

A:nO2

:0:0243 ð1Þ

It is assumed that Eo (PAR) < Eod (PAR), which is true towithin less than 10% (Kirk 1994).

Here, the rate with which excitons are trapped byfunctional PSII reaction centers (RCIIs) is given by theproduct of the absorption cross section, s PSII9, and Eod.Because charge separation can only occur at open reactioncenters, the rate of exciton trapping is multiplied by theproportion of total reaction centers that are open,termedQOX

A : QTOTALA , where QA is the primary electron

acceptor in RCII, and Q OXA is the QA that are oxidized. To

obtain the Chl a–specific photosynthesis rate, we multiply

the reaction center specific rate of stable charge separation,

which is given by the product of the first three terms on the

right-hand side of Eq. 1, with the ratio of O2 evolved by

a single-turnover saturating flash to Chl a (nO2). Each

saturating flash provides enough energy, and is of short

enough duration, to stimulate charge separation at all

RCIIs in a sample once, and only once. It is the most

commonly used method for estimating photosynthetic unit

size.For Eq. 1 to balance, sPSII must be expressed with units

of A2 (photon)21. The use of these units, rather than of A2

(RCII)21, is appropriate because the FRR fluorescencetechnique (Kolber et al. 1998) yields a sPSII thatcharacterizes the initial slope of a plot of the fluorescenceratio (F 2 Fo)/(Fm 2 Fo) versus the cumulative photondose. The fluorescence ratio is dimensionless, and the units

for photon dose are (photons A22). Thus, the FRRfluorescence method provides measurements of sPSII thathave units of A2 (photon)21.

The units for Eod (PAR) and nO2 are mmol photons m22

s21 and mol O2 (mol Chl a)21, respectively, whileQOX

A : QTOTALA is dimensionless. The constant 0.0243 ac-

counts for the conversion of Eod (PAR) from mmol photonsm22 s21 to mol photons m22 h21, s PSII9 from A2

(photon)21 to m2 (mol photons)21 and nO2 from mol O2

(mol Chl a)21 to mol O2 (g Chl a)21.Our measurements of the effective absorption cross

section are made at steady state (i.e., phytoplanktonacclimated to ambient light conditions). Therefore, s PSII9in Eq. 1 accounts for nonphotochemical excitation quench-ing that leads to alterations of the PSII photochemicalefficiency (Suggett et al. 2003). QOX

A : QTOTALA describes the

ratio of effective to maximum trapping efficiency ofphotons by PSII reactions centers and accounts forphotochemical alterations to the PSII photochemicalefficiency. We estimated QOX

A : QTOTALA from measurements

made in the open and enclosed FRR fluorometer chambers

(Table 1, Fig 2B) as

QOXA : QTOTAL

A ~(Fm

0{F 0)=(Fm0{f )OPEN chamber

(Fm0{Fo

0FRR)=(Fm

0{f )ENCLOSED chamberð2Þ

The extent of photochemical quenching, QOXA : QTOTAL

A is

assumed to be given by the ratio (F m9 2 F9)/(F m9 2 F o9),

which is now commonly referred to as F 9q/F 9

v (Suggett et al.

2003; Baker and Oxborough 2005) or qP (Kromkamp and

Forster 2003). However, our estimate of QOXA : QTOTAL

A

(Eq. 2) differs from estimates simply based on measured

F q9/F v9 with a correction for f. Specifically, Eq. 2 accounts

for the proportion of background fluorescence (f) inherentto the absolute fluorescence yield measurements. Two

conditions must be met for F o9 FRR to equal F o9. First, the

two FRR chambers must be intercalibrated and thus

directly comparable. This condition was met because the

FRR enclosed chamber is designed to hold cells over

a period of milliseconds to enable relaxation of most

photochemical dissipation without significant changes in

nonphotochemical dissipation (Falkowski and Kolber1995). Additionally, the two sampling areas share a com-

mon photomultiplier tube for fluorescence detection. We

verified that the fluorescence yields from the two FRR

chambers were not significantly different when cells were in

a completely dark-adapted state before dawn (data not

shown). Second, the brief period of time within the FRR

enclosed chamber is sufficient to fully relax photochemical

but not nonphotochemical dissipatory pathways. Thisassumption may not always be met because QA reoxidation

may be slow in the absence of supplementary far red light,

which is not provided by the FASTtracka FRR fluorometer.

In addition, some relaxation of nonphotochemical quench-

ing is possible even under brief dark exposure.Our approach is desirable for in situ productivity

determinations because it does not require additional

Spring bloom photosynthesis 2067

fully dark-adapted fluorescence measurements that areinherent to some other determinations (Kromkamp andForster 2003). Furthermore, use of Eq. 2 eliminates theneed for a sample blank because values of F m9 and fshould be identical in the light and dark chambers andthus cancel out. We did not observe any statisticaldifference between values of F m9, as measured directlyin situ and thus including f, from the two FRRchambers during daylight hours throughout the 6-weeksampling period (Bartlett’s type II regression slopeand intercept not significantly different from 1 and 0,respectively, p , 0.001, n 5 441, data not shown),

verifying the above assertion. Consequently, variabilityof F q9/F v9, will be from relative changes in the mini-mum fluorescence between the two chambers and fromabsolute changes in maximum fluorescence of bothchambers.

Measured values of s PSII9 are weighted to the narrowblue FRR excitation waveband. These were corrected to aneffective rate of light absorption for the excitationexperienced in situ, termed here as s abs

PSII9. Specifically,s PSII9 was adjusted by using optical absorption coefficientsthat were spectrally weighted to the incident spectralirradiance, Eo, of FRR, achl (FRR), and in situ, achl (in

Fig. 2. Diel variability of fluorescence-based and pH-based productivity parameters.(A) Photosynthetically active photon flux density, Eod (PAR) (mmol photons m22 s21) measured2 m above water (open squares) and 1 m below the water surface (solid squares). (B)Photochemical efficiency (dimensionless) from the open (open circles) and enclosed (solidcircles) FRR chamber, and the proportion of Qa that are oxidized (QOX

A : QTOTALA , dimension-

less), determined as the ratio of photochemical efficiency measured from the open chamber tothat from the enclosed chamber (open diamonds) (Eq. 2). (C) Effective absorption, designateds PSII9 during the day and sPSII at night (A2 photon21) from the FRR enclosed chamber (opencircles), and nonphotochemical quenching (s PSII9 :sPSII, open diamonds). For calculation ofs PSII9:sPSII, the mean from all sPSII measurements the night before and after each diurnal phasewas used (arrow 5 473 A2 photon21) (see main text). (D) Volume-specific FRR-based gross O2

production (PGROSSO2 ~ Pchl

O2:Chl a) with units of mol O2 m23 h21 (solid circles), and the

cumulative integral of P GROSSO2 throughout the day (mol O2 m23) (open circles). The sum of

P GROSSO2 at the end of the daylight phase is equal to the total daily integrated O2 production,PPGROSS

O2 . (E) Temperature (uC, and pH); (F) Total inorganic carbon (TCO2, mol CO2 m23)(solid triangles). The cumulative hourly loss of (2) TCO2 between dawn and dusk is equal to thedaily integrated net CO2 uptake (P NET

CO2 , mol CO2 m23). Also shown are TCO2 values aftercorrecting for the respiration rate (5daily integrated gross CO2 uptake [P GROSS

CO2 , mol CO2

(m)23], open triangles). Respiration, R, was calculated as the mean rate of increase in TCO2

from the night before and after each diurnal phase was used (see main text). Note that in thisexample, net CO2 uptake is not observed during daylight hours. Example shown was from20:00 h GMT 31 March to 05:00 h GMT 02 April 2003.

2068 Suggett et al.

situ), excitation (Babin et al. 1996; Suggett et al. 2001;

Raateoja et al. 2004),

sabsPSII0~

sPSII

9

achl(FRR)

� �:achl(in situ) ð3Þ

where

achl ~X700

400

achl(l):Eo(l)

!Dl=

X700

400

EO (l)Dl

Therefore, P chlO2 (mol O2 (g Chl a)21 h21) was finally

calculated as

PchlO2 ~ Eod (PAR): sabs

PSII’: nO2:QOX

A : QTOTALA

:0:0243 ð4Þ

Volume-specific daily integrated gross O2 production,PPGROSS

O2 (mol O2 m23), was determined from the product

of Chl a and integral of P chlO2 throughout the day (Fig. 2D),

XPGROSS

O2 ~Xdusk

dawn

PchlaO2 (t)Dt

!:Chl a ð5Þ

where t 5 1 h. For the most accurate determination ofPPGROSS

O2 , daily measurements of nO2, spectrally adjusted

in situ s PSII9, and Chl a concentration must be known.

Unfortunately, we could only collect these data every 2–

3 d. Therefore, we applied a series of corrections as follows.First, discrete nO2 measurements were compared with

corresponding in situ FRR fluorescence-based determina-tions of the factor Fv/Fo, which can be used to describe theproportion of PSII reaction centers that are functional(Babin et al. 1996) (Fig. 3A). nO2 was linearly related to theblank-corrected Fv/Fo 5 (Fm 2 Fo)/(Fo 2 f ) for both phase1 and 2, but followed a different regression equation foreach phase (Fig. 3A, Table 2). The high correlationcoefficients indicate that (Fm 2 Fo)/(Fo 2 f ) is a gooddescriptor of changes to functional RCII concentration perunit Chl a. However, the different regression slopesdemonstrate that the relationship between (Fm 2 Fo)/(Fo

2 f ) and the actual concentrations of RCIIs per unit Chla are not the same for phases 1 and 2 and thus cannot beassumed to be a constant for the entire sampling period.The appropriate linear relationship was applied to the insitu (Fm 2 Fo)/(Fo 2 f ) measured daily throughout each ofthe two phases to derive nO2.

Fig. 3. Comparison of (A) the reciprocal of the Emerson and Arnold number, nO2 (mol O2 (mol Chl a)21), with the blank correctedFRR fluorescence-based ‘‘functionality’’ factor, Fv/Fo 5 (Fm 2 Fo)/(Fo 2 f) (dimensionless), for all data collected throughout phases 1and 2. (B) The effective absorption coefficient (Eq. 3) where Eo (l) is calculated by the in situ (achl [in situ], m2 (mg Chl a)21) versus theFRR fluorometer excitation spectra (achl [FRR], m2 (mg Chl a)21). (C) Chl a concentration (mg Chl a m23) with the blank-correctedmaximum dark-adapted fluorescence yield (Fm 2 f, instrument units). Regression equations are given in Table 2.

Table 2. Regression equations and correlation coefficients for variables compared in Fig. 3. Variables are compared as independent(y) versus dependent (x). Regression slopes for nO2 versus the blank corrected functionality factor, (Fm 2 Fo)/(Fo 2 f ), were significantlydifferent between phases 1 and 2, tv56: 2.071.

Variables (y vs. x) Slope Intercept r2 n p

nO2 vs. (Fm 2 Fo)/(Fo 2 f ) (phase 1) 0.00025 20.00003 0.990 4 ,0.05nO2 vs. (Fm 2 Fo)/(Fo 2 f ) (phase 2) 0.00034 20.00001 0.629 6 ,0.05Chl a vs. Fm 2 f 22.46 24.92 0.905 14 ,0.001

achl (in situ) n achl (FRR) 0.467 0.000 0.916 14 ,0.001

Spring bloom photosynthesis 2069

Second, determinations of achl (FRR) and achl (in situ)

were compared and found to covary according to a single

linear relationship throughout the entire sampling period

(Fig. 3B, Table 2). The regression from this relationship

provided a single correction factor that was applied to each

daily measurement of s PSII9 throughout the sampling

period.Third, discrete values of Chl a were compared with blank

corrected dark-adapted maximum fluorescence yields, Fm

2 f, measured from the second bench top FRR fluorometerto develop a regression equation describing the relationshipbetween Chl a and the maximum fluorescence yield(Fig. 3C, Table 2). The regression was then applied toestimate Chl a from in situ measurements of Fm 2 f.Calculation of

PPGROSS

O2 requires the Chl a during the day.

However, a true dark-adapted Fm can be measured only in

darkness after nonphotochemical fluorescence quenching

has completely relaxed. Therefore, Chl a was estimated

from the mean Fm measured the night before and after each

diurnal phase.

In situ pH and estimation of CO2—Water temperatureand pH were measured every 15 min at a depth of 1 musing the system described in Maberly (1996) (Fig. 2E).These data, plus weekly measurements of alkalinity (A),were used to calculate the total concentration of inorganiccarbon (TCO2, mol CO2 m23, as the sum of free CO2,HCO 2

3 , and CO 223 ) using the first and second dissociation

coefficients corrected for temperature and ionic strength(pK 19 and pK 29), and the calculated concentration ofhydroxide ions ([OH2]) (Maberly 1996),

TCO2 ~ 1z10(pK01{pH)

�z10(pH{pK

02)�=

1z2 10(pH{pK02

� �� �A{ OH{½ �ð Þ

Therefore, TCO2 was determined at 15-min intervals in thelake. This approach provides a high temporal resolutionbut has an inherent assumption that changes in TCO2

result from carbon exchange with the biota rather than,for example, advection of water with high TCO2 fromdepth.

Calculation of net and gross community productivity fromdiel changes of TCO2—Daily integrated net CO2 uptake,P

PNETCO2 (mol CO2 m23), was determined from the net

consumption of the total inorganic carbon concentration,

TCO2, at 1 m throughout daylight hours (Fig. 2F),PPNET

CO2 ~ TCO2 (dusk)�TCO2 (dawn) ð7Þ

TheseP

PNETCO2 determinations can be affected by vertical

mixing events and changes in autotrophic biomass during

the day. Therefore, in some circumstances, a diurnal

decrease of TCO2 that is indicative of net CO2 consumption

is not observed (e.g., Fig. 2F).A community respiration rate (R, mol CO2 m23 h21)

was determined to account for the contribution of

autotrophic and heterotrophic respiration. This correctionis also affected by environmental fluxes (mixing). R wascalculated from the mean rate of increase in TCO2

throughout each night period (Fig. 2F),

R ~ TCO2 dawnð Þ�TCO2(dusk)½ �=NL ð8Þ

where NL is night length between dusk and dawn (h). Toaccount for day-to-day variability in the environment andplanktonic community, we used the mean R from the nightbefore and after each diurnal change in TCO2. Dailyintegrated gross CO2 uptake,

PPGROSS

CO2 (mol CO2 m23),

was determined asPPGROSS

CO2 ~P

PNETCO2 z(R:DL) ð9Þ

where DL is night length between dawn and dusk (h).

Results and discussion

Diel variability of FRR photophysiological parameters—Estimates of daily integrated gross oxygen evolution,designated

PPGROSS

O2 , are driven by the diel patterns of Eo

(PAR), QOXA : QTOTAL

A , and s PSII9, and thus governed by the

incident PAR in both marine (Gorbunov et al. 2001;Suggett et al. 2001; Moore et al. 2003) and lake (Vincent etal. 1984; Oliver et al. 2003) environments. In EsthwaiteWater, Eod (PAR) 2 m above water and 1 m below thewater were 966 6 46 mmol photons m22 s21 and 179 6 27.2(mean 6 standard error for all sampling days) at the solarnoon maximum, respectively (Fig. 2A). Decreases ofQOX

A : QTOTALA and s PSII9 were also centered on the solar

noon (Figs. 2B, C). Daily integrated Eod (PAR) above (not

shown) and below (Table 3) the water were highest

throughout phase 2.PSII photochemical efficiency under ambient light, (Fm9 2

F9)/(F m9 2 f ), is an estimate of the fraction of absorbedexcitation energy used for PSII photochemistry and islowered by an increase in dissipation (quenching) viaphotochemical and nonphotochemical pathways. Aftera brief period of dark acclimation, such as within theenclosed chamber of the FRR fluorometer, the PSIIphotochemical efficiency can remain lower than dark-adapted values because nonphotochemical but not photo-chemical dissipation pathways remain active (Kolber andFalkowski 1993; Falkowski and Kolber 1995). Here, the PSIIefficiency is strictly defined as the maximum photochemicalefficiency under ambient light, (F m9 2 F o9)/(F m9 2 f )(Kromkamp and Forster 2003; Suggett et al. 2003) and, aswith the calculation of photochemical quenching (Eq. 2),assumes that the minimum fluorescence measured in theFRR enclosed chamber, F o9 FRR, is equal to F o9. Therefore,variations of PSII photochemical efficiency that are mea-sured in the FRR enclosed chamber will be indicative ofchanges in the amount of nonphotochemical quenching ofexcitation energy. From dawn until noon, PSII photochem-ical efficiency measured in the enclosed chamber decreasedby 34 6 5% and 46 6 10% (mean 6 standard error) forphases 1 and 2, respectively (Figs. 2B, 4).

(6)

2070 Suggett et al.

The effective absorption cross section measured atsteady state (i.e., phytoplankton acclimated to ambientlight), s PSII9, reflects the size of the light harvestingantenna and is reduced with increased energy dissipationvia nonphotochemical quenching in the antenna bed.Therefore, we used a second approach to determine theextent of nonphotochemical quenching as the decreasein effective absorption between dawn and noon(5s PSII9 :sPSII, Fig. 2C). The main drawback of thissecond approach is that the effective absorption can alsochange as a result of photoacclimatory and inhibitorydriven alterations to the light-harvesting pigment beds andreaction center complexes (Suggett et al. 2004). Therefore,mean sPSII measurements from the night before and aftereach diurnal phase were used to account for this possiblesource of error from day to day (Fig. 2C). s PSII9 decreasedrelative to sPSII by 22 6 5% and 28 6 12% between dawnand solar noon for phases 1 and 2, respectively (Figs. 2C,4).

In contrast to the effective absorption cross section, thePSII photochemical efficiency is indicative of nonphoto-chemical dissipation that occurs within both the antennaebed and the PSII reaction centers (Gorbunov et al. 2001).Nonphotochemical dissipation estimated from decreases of(F m9 2 F o9 FRR)/(F m9 2 f ) between dawn and noon wasgreater than that determined from decreases of effectiveabsorption cross section by ,0 and 50% (Fig. 4) but onaverage by 21% and 25% for phases 1 and 2, respectively.Given the assumptions above, this percentage suggestsa relatively small amount of PSII reaction center-basedquenching occurred in these phytoplankton communitiesand thus that the majority of RCIIs remained functionalthroughout.

Photosynthetic unit size—Strong correlations betweennO2 and measured Fv/Fo, (Fm 2 Fo)/(Fo 2 f ) for phases 1and 2 (Table 2, Fig. 3A) suggest that changes in nO2 occurpartly from modifications to the proportion of RCIIs thatare active (Babin et al. 1996). Fv/Fo changed by ,20–30%throughout the investigation, a magnitude that wasconsistent with the average amount of PSII reactioncenter-based quenching observed throughout phases 1(21%) and 2 (25%) (above, Fig. 4).

Previous FRR fluorescence-based estimates of P chlO2 have

used ‘‘typical’’ nO2 values of 5.0 1024 and 1.0?1023 mol O2

(mol Chl a)21 for eukaryotic phytoplankton and cyano-bacteria, respectively, based on data from laboratoryexperiments of few isolates (Moore et al. 2003; Raateojaet al. 2004; Smyth et al. 2004). To estimate the actual O2

yield from active RCIIs per unit Chl a, the ‘‘typical’’ nO2

is then multiplied by Fv/Fo normalized to a theoreticalmaximum ( <an Fv/Fo of 1.8; Babin et al. 1996). Ourmeasured values of nO2 for natural lake communitieswere 3.2 and 3.4?1024 mol O2 (mol Chl a)21 for phases 1and 2 (Table 3). These values are ,20% lower and 10%higher (respectively) than using the product of the‘‘typical’’ value of 5.0 1024 mol O2 (mol Chl a)21 and thefactor [Fv/Fo]/1.8 (data not shown) when direct measure-ments are not available. Therefore, and in agreement withobservations by Suggett et al. (2004), this alternativeapproach can yield an estimate of O2 evolved by activeRCIIs (Chl a)21 with considerable error, most likely fromassumed values of the ‘‘typical’’ nO2 and the theoreticalmaximum for Fv/Fo. Our measurements of nO2 here areconsistent with laboratory data for eukaryotic phytoplank-ton species under nutrient replete conditions (see Suggett etal. 2004).

Table 3. Mean 6 standard error (minimum–maximum) of variables measured at 1 m throughout phases 1 (28 February–24 March)and 2 (25 March–14 April) of the spring bloom.

Variable Phase 1 Phase 2

Daily integrated Eod (PAR)(mol photons m22)

3.060.5 (1.0–5.8) 5.160.4 (2.1–7.2)

Chl a (mg m23) 20.164.0 (7.8–34.2) 9.061.7 (4.4–14.1)sPSII (A2 photon21) 360614 (303–449) 445610 (316–511)nO2 (mol O2 (mol Chl a)21) 3.2?102464.2?1025 (2.3–4.0?1024) 3.4?102462.0?1025 (2.8–4.4?1024)

P chlO2

MAX (mol O2 (g Chl a)21 h21) 0.18260.018 (0.121–0.239) 0.25860.019 (0.149–0.406)

aPO2 (mol O2 (g Chl a)21 h21)?(mmol photons m22 s21)21

2.2?102361.5?1024 (1.8–2.6?1023) 2.9?102362.0?1024 (2.0–3.4?1023)

Predominant species (% cells) Asterionella formosa (93%) Asterionella formosa (55%)Anabaena circinalis (4%) Anabaena circinalis (17%)Cyclotella spp. (2%) Rhodomonas spp. (13%) Chrysochromulina

parva (11%)

Variables shown are daily integrated Eod (PAR) (mol photons m22), Chl a concentration (mg m23), mean dark-adapted effective absorption (sPSII, A2

photon21), reciprocal of the Emerson and Arnold number (nO2, mol O2 (mol Chl a)21), and the maximum rate and initial slope of FRR-based Chl a-normalized O2 production, P chl

O2MAX (mol O2 (g Chl a)21 h21) and aPO2 ((mol O2 (g Chl a)21 h21)?(mmol photons m22 s21)21), respectively. P chl

O2MAX

and aPO2 were determined from each daily light-response of P chlO2 in situ by least-squares nonlinear regression to the equation of Jassby and Platt (1976),

P chlO2 5 P chl

O2MAX . (1-exp(-aPO2

. PPFD/ P chlO2

MAX)). Data were only used where P chlO2 was clearly saturated with respect to the Eod (PAR). This criterion

provided n 5 7 and 16 from the 13 and 21 d of daily P chlO2 versus Eod (PAR) at 1 m from throughout phases 1 and 2, respectively. Each variable was

significantly different between phases 1 and 2 (t test with unequal sample sizes; data not shown). Microscopy measurements shown for phases 1 and 2were determined from 18 March and 01 April, respectively.

Spring bloom photosynthesis 2071

Community respiration—Nocturnal respiration rates ofCO2 (R) were 37 6 28% (mean 6 standard deviation) and130 6 107% of the mean diurnal net CO2 uptake rates forphases 1 and 2, respectively. Daily integrated gross CO2

uptake,P

PGROSSCO2 , was determined as the sum ofP

PNETCO2 and R (Eq. 9, Fig. 2F). Respiration rates, and

hence the difference between net and gross CO2 uptake,was greater for phase 2 than phase 1 (Table 4). Phase 1 wascharacterized by higher phytoplankton biomass than phase2 (see Chl a, Table 3). As such, our results are consistentwith enhanced heterotrophy:autotrophy under conditionsof lower phytoplankton biomass (del Giorgio et al. 1999)and a possible increase in grazing pressure. The differencesin R may also partly reflect changes in the planktoniccommunity assemblage. However, in our calculation ofgross CO2 uptake we must assume that heterotrophic

activity and hence respiration (R) is equal during the dayand night (Cole et al. 2000). In addition, the respirationterm will also account for day-night differences in TCO2

from environmental fluxes, for example, enhanced noctur-nal mixing leading to diel patterns in stratification andfrom autotrophic respiration. Consequently, it is arguablethat the actual in situ TCO2 uptake lies somewhere betweenthe estimates with and without the inclusion of R.

Comparing FRR fluorescence- and TCO2-based produc-

tivity—Daily integrated net CO2 uptake,P

PNETCO2 , was

linearly correlated with daily integrated gross oxygenevolution,

PPGROSS

O2 , during both phases: 85% and 67%of the variance in

PPNET

CO2 could be attributed to a linear

dependence onP

PGROSSO2 in phases 1 and 2, respectively

(Table 4).P

PNETCO2 was 53 6 10% (mean 6 standard error)

and 26 6 9% ofP

PGROSSO2 for phases 1 and 2, respectively

(Table 4, Fig. 5A). Note that for some days,P

PNETCO2 was

negative (Fig. 5A), thus indicating a net flux of TCO2 fromthe water column. These days corresponded with high windspeeds during phase 1 and a breakdown in water columnstratification during phase 2 (data not shown). Henceaccounting for the daily respiration term, R, was essentialfor comparing O2 production and CO2 uptake for the entiredata set.

Daily gross CO2 uptake,P

PGROSSCO2 , was strongly

linearly correlated with, but systematically lower than,PPGROSS

O2 (Table 4). The mean (6 standard error) ofPPGROSS

O2 :P

PGROSSCO2 was 1.01 6 0.04 for phase 1 and 1.53

6 0.13 for phase 2 (Table 4, Fig. 5B). Thus, the photo-

synthetic quotient relating FRR-based estimates of O2

evolved with gross community carbon assimilated was

higher throughout phase 2.There was relatively little day-to-day variability in the

relationship between gross CO2 uptake and gross FRR-based O2 evolution; only ,10–25% of the variance inP

PGROSSCO2 could not be explained by the linear regression

onP

PGROSSO2 (Table 4, Fig. 5). This provides strong

evidence of a systematic coupling between photosyntheticelectron transfer and CO2 fixation for the phytoplanktoncommunities during both phases 1 and 2. However, two ofthe main determinants of

PPGROSS

O2 , Eod (PAR) and

phytoplankton biomass, also varied significantly through-

out phases 1 and 2 (Table 3), Therefore, it is possible that

variations of gross CO2 uptake,P

PGROSSCO2 , may be

accounted for by the daily integrated Eod (PAR), SEod

(PAR), and Chl a without recourse to calculateP

PGROSSO2 .

The product of SEod (PAR) and Chl a accounted for 27to 42% of the variability in

PPGROSS

CO2 (Fig. 5C, Table 4).

This is ,30–50% lower than the amount of variability inPPGROSS

CO2 that can be attributed toP

PGROSSO2 (Table 4). In

addition, the extent of variability of both SEod (PAR) and

Chl a (the standard error was approximately 625% of the

mean) exceeded that observed for parameters describing

light absorption (standard error approximately 65–10% of

the mean) (Table 3). However, greater than 50% of

sampling days demonstrated that P chlO2 was saturated with

Fig. 4. Extent of nonphotochemical quenching determinedas the ratio of maximum photochemical efficiency or effectiveabsorption at noon to that at dawn for each day of sampling,

D(F m9 2 F o9 FRR)/(F m9 2 f) and Ds PSII9, respectively. Here, (F m9

2 F o9 FRR)/(F m9 2 f) and s PSII9 at dawn is equal to the blankcorrected maximum PSII photochemical efficiency under darkadaptation, (Fm – Fo)/(Fm – f) and the maximum PSII effectiveabsorption cross section under dark adaptation, sPSII. However,for simplicity, we have termed the ratio of either parameterbetween noon and dawn as D. Day-to-day photoacclimatory andphotoinhibitory driven variances in sPSII are accounted for asdescribed in the main text. All fluorescence parameters were takenfrom the FRR enclosed chamber. Dashed lines indicate D(F m9-F o9 FRR)/(F m9 2 f): Ds PSII9 limits of 1:1 and 1:2. For points thatfall on the 1:1 line, all of the nonphotochemical quenching can beattributed to the change in the effective cross section and thus toquenching that takes place in the pigment bed. For points that fallabove the 1:1 line, we infer that a proportion of the nonphoto-chemical quenching occurs in the reaction centers.

2072 Suggett et al.

respect to Eod (PAR) (see Table 3). Consequently, evidence

suggests that biomass and light cannot exclusively account

for the variability of production that was observed. Also,

that variability of the light-response characteristics ofPPGROSS

O2 , as quantified by FRR, and of the coupling

between gross O2 evolution and CO2 assimilation must

significantly account for the variability of community

metabolism.The higher photosynthetic quotient for phase 2 was

associated with a systematic alteration in the phytoplank-ton community structure and the lake environment(Table 3). Phase 1 was characterized by a diatom domi-nated assemblage (Table 3) and relatively high nutrientconcentrations (data not shown). Phase 2 was characterizedby significantly higher numbers of flagellates, Rhodomonas

spp. and Chrysochromulina parva, and the cyanobacteriumAnabaena circinalis. This change in the phytoplanktonassemblage between phase 1 and 2 was consistent withprevious changes that have followed an exhaustion ofsilicate and rapid stabilization of the water column(Maberly et al. 1994). The differences in the PQ areconsistent with observations from unialgal cultures ofdiatoms and cryptophtes (MacIntyre et al. unpubl. data).Specifically, MacIntyre et al. (unpubl. data) found that thediatom Thalassiosira weissflogii displays lower photosyn-thetic quotients of (,1.5 mol O2 (mol CO2)21) than thecryptophyte Streatula major (,2.5 mol O2 (mol CO2)21)under nutrient replete growth conditions. Thus, the di-rection and magnitude of the change that we observed inEsthwaite Water is consistent with observations fromunialgal cultures. Specific reasons for these taxonomic

Fig. 5. Comparison of pH-based daily integrated (A) net CO2 uptake (P

PNETCO2 , mol CO2

m23) and (B) gross CO2 uptake (P

PGROSSCO2 , mol CO2 m23) with FRR fluorescence-based daily

integrated O2 production (P

PGROSSO2 , mol O2 m23). The solid diagonal line indicates

a photosynthetic quotient of P GROSSO2 per unit CO2 uptake of 1. (C)

PPGROSS

CO2 with the productof daily integrated Eod (PAR) (mol photons m22) and Chl a concentration (g Chl a m23).Regression equations are given in Table 4.

Table 4. Bartlett type II regression equations and correlation coefficients for data compared in Fig. 5. Variables are compared asindependent (y) versus dependent (x). Regressions for daily net CO2 uptake (

PPNET

CO2 ) versus daily gross O2 evolution (PGROSSO2 ) exclude

data whereP

PNETCO2 is negative (see Fig. 5A). Regression slopes for

PPNET

CO2 and daily gross CO2 uptake (P

PGROSSCO2 ) versusP

PGROSSO2 and for

PPGROSS

CO2 versusP

Eod (PAR)?Chl a were significantly different (p , 0.05) between phases 1 and 2, tv 5 25:2.345and tv 5 30:3.583, respectively.

Phase Variables (y vs. x) Slope Intercept r2 n p

1 Daily net CO2 uptake vs.P

PGROSSO2

0.828 20.0026 0.845 12 ,0.001

Daily gross CO2 uptake vs.P

PGROSSO2

0.981 0.0001 0.805 13 ,0.001

Daily gross CO2 uptake vs.P

Eod (PAR) Chl a 0.394 20.0020 0.267 13 ,0.001

2 Daily net CO2 uptake vs.P

PGROSSO2

0.481 0.0009 0.667 17 ,0.001

Daily gross CO2 uptake vs.P

PGROSSO2

0.644 0.0010 0.728 21 ,0.001

Daily gross CO2 uptake vs.P

Eod (PAR) Chl a 0.229 20.0001 0.419 21 ,0.001

Spring bloom photosynthesis 2073

differences in the photosynthetic quotient are not clear butpresumably represent distinct photosynthetic architecturesof different higher taxonomic groupings.

Three processes may explain the differences observedbetween

PPGROSS

O2 andP

PGROSSCO2 (Prasil et al. 1996; Geider

and MacIntyre 2002; Wilhelm et al. 2004): (1) electron sinks

within the photosynthetic electron transfer chain,

(2) photorespiratory O2 consumption, and (3) consumption

of NADPH by essential photochemical processes other

than CO2 fixation, such as nitrate reduction and assimila-

tion. These pathways will be briefly discussed.Two electron cycling pathways contribute to turnover of

PSII, and hence FRR estimates of gross oxygen evolution,without net O2 evolution and thus without net CO2

fixation. First, cycling of electrons around PSII may occurunder high light (Falkowski et al. 1988; Prasil et al. 1996).These authors suggested that cycling of electrons aroundPSII may account up to ,30% of the PSII linear electronflow under exposure to light-saturated irradiances forcultures of Chlorella sp. and Chaetoceros gracilis. Second,oxygen may be reduced at PSI by the Mehler reaction(Kana 1992). These processes increase the minimumquantum requirement for the evolution of 1 mol O2

(Falkowski et al. 1988; Prasil et al. 1996; Noctor andFoyer 1998). Lewitus and Kana (1995) estimated that theMehler reaction can account for up to ,50% of light-saturated gross O2 evolution for isolates of eukaryoticestuarine phytoplankton species Pavlova sp., Closteriumsp., Cryptoimponas sp., and Hemiselmis sp. Our measure-ments of P chl

O2 were saturated with respect to Eod (PAR),suggesting a possible contribution of these two electroncycling pathways to P chl

O2, for more than 50% of samplingdays (see Table 3).

Photorespiration may also consume a proportion of theO2 evolved during linear electron transfer (Badger et al.2000; Ort and Baker 2002) where O2 successfully out-competes CO2 for ribulose 1,5-biphosphate (RuBP) at theactive site of RUBISCO. In this case, gross CO2

assimilation but not linear electron transfer (or gross O2

evolution) will decrease to yield a photosynthetic quotientfor the ratio of oxygen evolution to net carbon dioxidefixation that is lower than we calculate fromP

PGROSSO2 :

PPGROSS

CO2 . Photorespiration is stimulated when

there is relatively low inorganic carbon availability (Geider

and MacIntyre 2002). However, it is unlikely that

photorespiration was significant because the lake pH

remained relatively low throughout the investigation (data

not shown). Furthermore, addition of NaHCO3 did not

alter nO2 measurements (data not shown).NADPH produced from linear electron flow (

PPGROSS

O2 )

will be allocated between CO2 fixation and other energy-

demanding photosynthetic processes, for example, nitrate

and sulphate reduction and reductive biosynthesis, such as

lipid biosynthesis. Consequently, this allocation is reflected

in the stoichiometry for phytoplankton growth (Falkowski

and Raven 1997; Geider and MacIntyre 2002). Nitrate

assimilation requires a higher ratio of gross oxygen

evolution to carbon fixation than ammonium assimilation

(Laws 1991). The ratio of ammonium to nitrate was higher

during phase 2 than phase 1 as a result of both lower nitrate

and higher ammonium concentrations (data not shown).

Therefore, we might expect the photosynthetic quotient to

be lower in phase 2. Contrary to this expectation, we found

a higher photosynthetic quotient during phase 2. Despite

the changes in inorganic nitrogen concentrations between

phases 1 and 2, we do not have evidence to suggest nitrate

concentrations became limiting for microalgal growth or

that ammonia became the main source of nitrogen during

phase 2.We have demonstrated a tight coupling between daily

photosynthetic electron transfer rate (e.g., potential O2

production) and daily gross CO2 uptake in situ. Our resultsstrongly indicate that the community metabolism inEsthwaite Water during the spring bloom was driven bygross phytoplankton photosynthesis. Furthermore, theratio of light absorption to inorganic carbon fixationdepends on the taxonomic makeup of the phytoplanktoncommunity.

Our investigation provides further confidence in the useof the FRR technique as a measure of primary productivityin situ. Several investigations have previously used FRRfluorometry to assess microalgal productivity in situ(Suggett et al. 2001; Raateoja et al. 2004; Smyth et al.2004) but have been limited by assuming nO2 and lack ofknowledge of the fluorescence blank relative to the sample(Cullen and Davis 2003). Furthermore, implementation ofFRR fluorescence as a tool for directly measuring ‘‘pro-ductivity’’ is fraught with difficulty where photosyntheticpathways uncouple absorbed light, linear electron flow,and reductant partitioning from assimilated carbon(Kromkamp and Forster 2003; Suggett et al. 2004;Wilhelm et al. 2004). We have demonstrated an FRRapproach that overcomes uncertainties inherent to theseprevious P chl

O2 determinations.P

PGROSSO2 and

PPGROSS

CO2

were tightly coupled for these spring bloom phytoplankton

communities exhibiting little RCII damage (20–30%) and

hence RCII-based fluorescence quenching. There will no

doubt be condit ions in which P GROSSO2 diverges

fromP

PGROSSCO2 , such as an unfavorable growth environ-

ment and/or a change in phytoplankton community.

However, experimental programs that combine both bio-

physical (FRR) and gas exchange (diel pH and diel O2)

measurements hold promise for gaining significant insights

into the role of phytoplankton photophysiology in phyto-

plankton ecology.

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Received: 6 February 2006Accepted: 9 March 2006

Amended: 8 May 2006

2076 Suggett et al.