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European Journal of PhycologyPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/tejp20
In-line recording of PAM fluorescence of phytoplanktoncultures as a new tool for studying effects offluctuating nutrient supply on photosynthesisSebastian Lippemeier , Ralf Hintze , Klaus Vanselow , Peter Hartig & Franciscus Colijna Research and Technology Centre Westcoast of Kiel University, Hafentörn 1, 25761 Büsum,GermanyPublished online: 03 Jun 2010.
To cite this article: Sebastian Lippemeier , Ralf Hintze , Klaus Vanselow , Peter Hartig & Franciscus Colijn (2001) In-linerecording of PAM fluorescence of phytoplankton cultures as a new tool for studying effects of fluctuating nutrient supply onphotosynthesis, European Journal of Phycology, 36:1, 89-100
To link to this article: http://dx.doi.org/10.1080/09670260110001735238
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Eur. J. Phycol. (2001), 36: 89–100. Printed in the United Kingdom 89
In-line recording of PAM fluorescence of phytoplankton
cultures as a new tool for studying effects of fluctuating
nutrient supply on photosynthesis
SEBASTIAN LIPPEMEIER, RALF HINTZE, KLAUS H. VANSELOW, PETER HARTIGAND FRANCISCUS COLIJN
Research and Technology Centre Westcoast of Kiel University, HafentoX rn 1, 25761 BuX sum, Germany
(Received 25 October 1999 ; accepted 20 November 2000)
Turbidostat cultures of Dunaliella salina (Chlorophyceae) and Thalassiosira weissflogii (Bacillariophyceae) were grown at fluctuating
concentrations of nitrate, phosphate and silicate. In-line measurements of PAM fluorescence were used to monitor the effects of fluctuating
nutrient supply on the photochemical efficiency of photosystem II reaction centres of light-adapted cells (∆F}F «m). Besides the maximal
photochemical efficiency of photosystem II reaction centres of dark-adapted cells (Fv}F
m), chlorophyll a, particulate organic carbon,
nitrogen and phosphorus, and the cell number were measured frequently during the experiments. Following nutrient-replete growth, the
cells were supplied with medium from which either nitrate, phosphate or silicate was omitted. When significant effects of nutrient
starvation were indicated by the fluorescence parameters, a pulse of the deficient nutrient was added to the cultures. Our experimental
set-up for in-line fluorescence measurements provided sensitive and reproducible detection of the various fluorescence signals, revealing
strong influences of nutrient supply on the photochemical efficiency of photosystem II. In general the fluorescence values changed
substantially within 1–30 min after re-addition of the deficient nutrient. Addition of phosphate and silicate induced an immediate
characteristic decrease in fluorescence, whereas nitrate addition was characterized by a strong, delayed increase in fluorescence. Complete
recovery to pre-starvation fluorescence values took about 48 h in all experiments. The physiological background of nutrient uptake is used
to explain the observed tight couplings between fluorescence responses and nutrient re-addition. Our study clearly demonstrates that
in-line fluorescence measurements provide a new tool for the investigation of phytoplankton reactions to fluctuating nutrients and offer
the possibility to detect nutrient starvation in the field.
Key words : Dunaliella salina, fluorescence, in-line PAM, nitrogen, nutrients, phosphorus, silicon, starvation, Thalassiosira weissflogii,
turbidostat
Introduction
Many studies report effects of nutrient supply on theproductivity of phytoplankton (e.g. Herzig & Falkowski,1989 ; Sosik & Mitchell, 1991 ; Geider et al., 1998). Theseeffects are commonly investigated under steady-stateconditions in continuous cultures (Rhee, 1980). This doesnot take into account that, in the aquatic environment, thecells are subject to variations in nutrient supply due towind-driven upwelling, currents and advection (MacIsaacet al., 1985 ; Dugdale & Wilkerson, 1989). Apart fromthese large-scale fluctuations in nutrient regimes, micro-scale nutrient patchiness in aquatic ecosystems has beenproposed by Rothschild et al. (1999) and may be generatedby, for example, zooplankton activity (McCarthy &Goldman, 1979 ; Lehman & Scavia, 1982). In general, theimportance of nutrient fluctuations for phytoplanktongrowth is accepted. The relevance of time scales, however,is still a matter of debate.
Little is known about the reactions of phytoplanktonproductivity to fluctuations in nutrient supply. A classical
Correspondence to : S. Lippemeier. e-mail : lippem!ftz-west.uni-kiel.de
approach to investigate this topic is the determination ofnutrient uptake kinetics. These experiments are basedeither on the changes in nutrient concentrations in themedium after perturbation (Harrison & Davis, 1977 ;Goldman & Glibert, 1982 ; Parslow et al., 1984 ; Martinez,1991) or on the uptake rate of nutrient isotopes (MacIsaac& Dugdale, 1969 ; Fuhs & Canelli, 1970 ; Goering et al.,1973 ; Harrison & Davis, 1977). According to Parslow et
al. (1984) and Malara & Sciandra (1991), nutrient uptakekinetics can be calculated from the automated in-linedetection of nutrient concentrations, which provides ahigh temporal resolution of nutrient uptake.
All these uptake studies revealed fast changes in uptakecharacteristics induced by nutrient pulses. Obviouslyphytoplankton cells are capable of using even short andsmall pulses of nutrients very effectively for growth. Thisobservation was used to explain high growth rates inwaters with relatively low but fluctuating overall nutrientconcentrations (McCarthy & Goldman, 1979). However,the allocation of nutrients in the cell following uptake ishighly variable. Therefore the detection of uptake ratesallows only a limited assessment of the relationships
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90S. Lippemeier et al.
between fluctuating nutrient supply and cell productivity(Flynn, 1998).
In many studies fluorescence characteristics of phyto-plankton have been shown to provide an assessment ofthe photochemical efficiency of the reaction centres ofphotosystem II (PSII) (Genty et al., 1989). Moreoverseveral authors (Kolber et al., 1988, 1990 ; Geider et al.,1993a, b, 1998 ; Falkowski & Kolber, 1995 ; Berges et al.,1996 ; Olaizola et al., 1996 ; Lippemeier et al., 1999) haveshown that the availability of nitrate, phosphate, silicateand iron affects phytoplankton fluorescence in the fieldand in phytoplankton cultures. All these studies revealedthat nutrient deficiency induces a decreased photochemicalefficiency of reaction centres of PSII.
Detailed investigations of the physiological responsesto variations in ammonia, nitrate and phosphate supplyhave been carried out by several authors (Turpin &Weger, 1988 ; Holmes et al., 1989 ; Gauthier & Turpin,1997). In these investigations parallel measurements ofgas exchange and variable fluorescence, and the use ofspecific physiological inhibitors, revealed strong in-fluences of fluctuations in nutrient supply on phyto-plankton physiology. However, due to the experimentalset-up, these investigations were always restricted toresponses within a time frame of several minutes. Theresponses of phytoplankton cells to nutrient additionswere so quick that variable fluorescence has already beenapplied as a tool to identify the limiting nutrient in naturalphytoplankton communities (Wood & Oliver, 1995).
Despite the highly valuable information on phyto-plankton responses to fluctuating nutrient supply, theseinvestigations have always been impaired by the need totransfer a sample from the culture suspension or the waterbody into a measuring cuvette, with the concomitantchanges in light, temperature, gas exchange and mech-anical forcing (e.g. bubbling or stirring). However, in-linefluorescence measurements of phytoplankton cultureswithout sampling directly at the culture vessel, comparableto those in the present study, have been used in a smallnumber of investigations for nutrient-replete cultures(Ibelings et al., 1994 ; Kroon, 1994). As yet no experimentalapproaches have been used to investigate both the short-term (several minutes) and long-term (several hours todays) responses of phytoplankton cultures to fluctuatingnutrients without the need for sampling.
Hence the objective of the present study was todevelop an experimental set-up to investigate the re-sponses of phytoplankton to fluctuating nutrient supply(nitrate, phosphate and silicate) based on in-line measure-ments of fluorescence. We use the term ‘ in-line ’ to indicatethe difference from previous studies, in which fluorescencewas measured on-line in a bypass (Lippemeier et al., 1999)and not directly at the culture vessel. A conventionalPAM fluorometer was applied to detect the fluorescencesignals in-line via glassfibre-optics directly inside theculture suspension in ambient light, allowing fast measure-ments with a high temporal resolution.
We were thus able to test the hypothesis that in-line
fluorescence-based photosynthesis measurements providemore information about phytoplankton responses tonutrient fluctuations than classical uptake studies.
Materials and methods
Organisms and cultivation system
Dunaliella salina (culture collection of the University ofGo$ ttingen, Germany) and Thalassiosira weissflogii (isolatedfrom the German Wadden Sea, culture collection of theResearch and Technology Centre Westcoast of theUniversity of Kiel at Bu$ sum, Germany) were grown in F}2medium (Guillard & Ryther, 1962) prepared from artificialseawater (Harrison et al., 1980) (supplied with 371 µMsilicate and 10 nM selenium (final concentrations) forThalassiosira weissflogii). All inorganic nutrient concen-trations were calculated from known concentrations ofstock solutions.
Starvation in nitrogen, phosphorus or silicon wasinduced by a shift of the medium supply to a mediumlacking one of these nutrients. Starvation was verified bychanges in fluorescence signals, C}N and C}P ratios orcell number. We decided to measure cellular C}N and C}Pratios instead of the ambient nutrient concentrations in themedium because the former provide a more sensitiveindication of nutrient status (Hecky & Kilham, 1988 ;Wood & Oliver, 1995). After starvation of the cultures,the deficient nutrient was added directly to the culturesuspension to establish nutrient-replete conditions.
In all experiments the cells were grown in a flatphotobioreactor (42¬29¬2±5 cm) with glass walls andturbidostatic regulation (Fig. 1). The effective culturevolume of 2500 ml was maintained by an overflow. Byilluminating the reactor with fluorescent tubes (FluoraL18}77, Osram, Germany) from one side, a light path ofonly 2±5 cm inside the culture was achieved. Togetherwith the turbidostatic regulation of optical density, thisillumination set-up was chosen to minimize changes inlight climate due to self-shading of the cells duringcultivation. All cultures were grown at a continuousirradiance of 100 µmol m−# s−". For temperature control,the complete cultivation system was built in a growthchamber (KBW 240, Binder, Germany) providing aconstant temperature of 20³0±5 °C during all experi-ments.
Aeration and mixing of the cultures was provided bypumping filtered air (0±1 µm, 10 l min−") to the bottom ofthe culture vessel. This prevented cell growth on the glasswalls in front of the optical detection units for fluorescenceand turbidity. By introducing the air at the edges of theculture vessel, the formation of bubbles in front of thefluorescence and turbidity sensors located in the centre ofthe sidewalls (Fig. 1) was prevented.
Determination of the growth rate based on apparent
absorption at 875 nm
For the detection of absorption, a light-emitting diode
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PAM fluorescence under fluctuating nutrient supply 91
Fig. 1. Schematic drawing of the experimental set-up used for turbidostatic cultivation of the cultures and for in-line fluorescence
measurements.
(LED) peaking at 875 nm (³55 nm) and a photodiodewere mounted on opposing walls of the vessel facing eachother, with a light path of 2±5 cm through the algalsuspension (Fig. 1). The intensity of the LED was square-wave-modulated with a frequency of 5 kHz. The signal ofthe photodiode was processed by a lock-in amplifier(model 7260, EG&G, UK) using the 5 kHz signal whichmodulated the LED as reference signal.
Turbidostatic regulation of the cultures was based onthese absorption measurements at 875 nm. The absorptionat 875 nm correlated with the particulate organic carbon(POC) concentration of the cultures (partly shown inTable 1). At 12 s intervals, the absorption signal recordedby a PC was used to maintain a preset turbidity byappropriate regulation of medium influx by a peristalticpump. The dilution rate based on turbidity measurementsat 875 nm (D
)(&) was calculated from the pump-rate of
fresh medium:
D)(&
[day−"]¯ Pump-rate [ml day−"]}culture volume [ml](1)
The turbidostatic regulation provided constant POCvalues during the experiments. Hence POC was in steadystate and the dilution rate equals the growth rate µ basedon POC. We emphasize that µ was correlated with POCand therefore not strictly coupled to the cell division rate.
Fluorescence measurements
The optical configuration of a PAM-101}103 fluorometer(Walz, Germany) was modified, as shown in Fig. 1, and thecommon end of a branched glassfibre-optic was attacheddirectly to the glass wall of the culture vessel. A measuringlight LED (Walz, Germany) peaking at 655 nm and ahalogen lamp (KL 1500, Walz, Germany) for the gen-eration of saturating light pulses were each connected toseparate branches of the optic cable. The measuring lightmodulation frequency was adjusted to 100 kHz, toincrease the signal-to-noise ratio. The spectra of both thesaturating light halogen lamp and the measuring light LEDwere modified by a short-pass filter (DT Cyan special,Balzer, Germany). The fluorescence was detected by aconventional photodiode detector (ED101US, Walz, Ger-many) that was also connected to a separate branch of theglassfibre-optic (Fig. 1). A long-pass filter (RG 9, Schott,Germany) was used to protect the fluorescence detectorfrom excitation by the saturating, measuring and cul-tivation lights.
The repetitive pulse option in the PAM-software DA-100 was used for continuous recording of fluorescence.The sampling period was set to 5 min except for thephases of nutrient re-addition, when the data collectioninterval was reduced to 1 min to improve the temporalresolution. All in-line fluorescence measurements were
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92S. Lippemeier et al.
Table 1. Cell number, biochemical and fluorescence parameters of Dunaliella salina and Thalassiosira weissflogii grown under fluctuating
nutrient supply
Parameter
D. salina : N starvation T. weissflogii : P starvation T. weissflogii : Si starvation
Start
(N-replete)
N
starvation
End
(N-replete)
Start
(P-replete)
P
starvation
End
(P-replete)
Start
(Si-replete)
Si
starvation
End
(Si-replete)
Cell number
(10& ml−")
® 5 12±7 4±2 2±9 4±5
Chla 1730 605 1404 838 753 953 826 654 970
(µg l−") (394) (21) (170) (5±2) (45) (96) (56) (79) (29)
POC 54±1 52±4 52±8 64±5 61±0 61±8 65±7 65±7 62±4(mg l−") (1±5) (1±8) (4±9) (0±2) (0±9) (3±1)Fv}F
m0±68 0±52 0±71 0±64 0±59 0±63 0±65 0±56 0±67
(r.u.) (0±011) (0±003) (0±005) (0±003) (0±007) (0±002) (0±006) (0±005) (0±004)
Data are based on single or duplicate measurements, and the values in parentheses are standard errors. Cell number for Thalassiosira weissflogii during
fluctuating silicate supply is shown in Fig. 5. (®), not measured.
conducted in the presence of actinic cultivation light. Thusaccording to the nomenclature of van Kooten & Snel(1990) the measurements provide determination of theactual (F ) and the maximal (F «
m) fluorescence under actinic
light.From these two parameters, the photochemical
efficiency of the PSII reaction centres of light-adapted cells(∆F}F «
m) was calculated according to Genty et al. (1989) :
∆F}F «m
¯ (F «m®F )}F «
m(2)
Several times during the experiments, 10 ml samples wereincubated in the dark at 20 °C for 1 h. Subsequently theminimal fluorescence (F
!) and the maximal fluorescence
after the application of a saturating light pulse (Fm) (van
Kooten & Snel, 1990) were determined with a dual-modulation fluorometer (PSI Instruments, Czech Republic)as described in Nedbal & Trtı! lek (1995). The maximalphotochemical efficiency of the PSII reaction centres(F
v}F
m) of dark-adapted cells was calculated as the mean of
two measurements of each sample :
Fv}F
m¯ (F
m®F
!)}F
m(3)
Other measurements
Photosynthetically active radiation (PAR) in the culturevessel was measured with a calibrated 4π quantum sensor(0±5 cm diameter, ZEMOKO, The Netherlands) in com-bination with a datalogger (LI 1000, Licor, USA).
Chlorophyll a (Chla) was analysed in duplicate for eachsample after filtration of 20 ml samples onto WhatmanGF}F filters, frozen at ®20 °C, by high-performanceliquid chromatography (HPLC) according to the methodof Mantoura & Llewellyn (1983).
Cell numbers were determined with a flow cytometer(FacsVantage, Becton-Dickinson, USA). Three millilitresamples were preserved with formalin (1%) and keptfrozen at ®20 °C directly after sampling until analysis.Data presented for cell numbers are based on counts of at
least 20¬10$ cells from each sample. Light microscopywas used to control the cell numbers determined with theflow cytometer.
Samples for particulate carbon (POC), particulate ni-trogen (PON) and particulate phosphorus (POP) werefiltered on precombusted Whatman GF}C filters andfrozen at ®20 °C. POC and PON were determined usinga CHN analyser (CHN-O-RAPID, Heraeus, Germany).POP was determined according to the method of Koroleff(1983). POC was determined in duplicate from samplestaken during nitrogen starvation in Dunaliella salina andsilicon starvation in Thalassiosira weissflogii. During phos-phorus starvation in Thalassiosira weissflogii, POC wasdetermined once from each sample.
Results
Nitrogen and phosphorus starvation were investigatedin both Dunaliella salina and Thalassiosira weissflogii. Ingeneral, the reactions of all investigated parametersdepended on the type of fluctuating nutrient (nitrogen orphosphorus) and not on the species investigated in thisstudy. To prevent redundancy, therefore, only the resultsof nitrogen starvation in Dunaliella salina and phosphorusstarvation in Thalassiosira weissflogii are presented. Theeffect of silicon starvation in Thalassiosira weissflogii ispresented also.
Nitrogen starvation in Dunaliella salina
Fig. 2 shows the time course of C}N ratio, growth rate andthe fluorescence parameters F, F «
mand ∆F}F «
mfor a
Dunaliella salina culture during growth under nitrogen-replete and nitrogen-depleted conditions.
The POC concentration calculated as the mean of 15samples taken at different times during the experimentremained constant at 53±1 mg l−" (³2±9 mg l−"). Chlaconcentration, cell number and F
v}F
mchanged substan-
tially in relation to the nitrate supply (Table 1). Prior to
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PAM fluorescence under fluctuating nutrient supply 93
Fig. 2. Variations with time in (A) C}N ratio, (B) growth rate
and (C ) in-line fluorescence parameters during cultivation of
Dunaliella salina under fluctuating nitrate concentrations. Nutrient
treatments are as indicated in the figure. The figure shows one
representative set of data for nitrogen limitation in Dunaliella
salina.
starvation, the C}N ratio was 8±5 and growth rate was0±43 day−" (Fig. 2A, B ).
Supplying the cells with nitrate-free medium for 65 hcaused the C}N ratio to increase to 19±1, and the growthrate to decrease rapidly (Fig. 2A, B ). With increasingnitrogen starvation, the fluorescence parameters F and F «
m
decreased from 0±37 to 0±24 and from 0±70 to 0±33,respectively. As a consequence ∆F}F «
mdecreased by 40%
from 0±47 to 0±28 (Fig. 2C ).After this 65 h starvation period, 2±5 ml of a nitrate
solution was added directly to the culture, resulting in afinal nitrate concentration of 883 µM. Immediately fol-lowing the nitrate pulse, F and F «
mbegan to increase (Fig.
3A) whereas the growth rate continued to decrease andremained zero for the next 8 h (Fig. 2B). Two hours afterthe nitrate pulse, F and F «
mhad reached values of 0±26 and
0±36 respectively. During the next hour, F and F «m
decreased slightly again. Three hours after nitrate re-addition both fluorescence parameters started to increaseagain. Due to a steeper increase in F «
mrelative to F, the
photochemical efficiency (∆F}F «m) also increased rapidly
and reached a maximum value of 0±52 approximately 24 hafter the nitrate pulse. Another 24 h later, ∆F}F «
mhad
stabilized at 0±47, which was similar to its value at the startof the experiment.
Fig. 3. Variations with time in in-line fluorescence parameters
within the first 6 h after nutrient re-addition during the
experiments shown in Figs 2C, 4C and 5C. Re-addition of (A)
nitrate to Dunaliella salina, (B) phosphate and (C ) silicate to
Thalassiosira weissflogii. Nutrient treatments are as indicated in the
figure.
Eight hours after the nitrate pulse, the growth ratestarted to increase steeply and reached a maximum valueof 0±8 day−" 18 h after the pulse (Fig. 2B). Twelve hourslater the growth rate stabilized at 0±36 day−", which wascomparable to that in nitrogen-replete conditions at thestart of the experiment. The C}N ratio decreased by 10%during the first 2±25 h after nitrate re-addition. A C}Nratio of 9±5 was established 11 h later, which was slightlyhigher than at the start of the experiment (Fig. 2A). Incontrast to the C}N ratio and the fluorescence values, cellnumbers increased only slightly during the first 12 h afternitrate re-addition (not shown) and reached a maximumvalue of 12±7¬10& cells ml−" after 36 h (Table 1).
Phosphorus starvation in Thalassiosira weissflogii
Fig. 4 shows the time course of C}P ratio, growth rate andthe fluorescence parameters F, F «
mand ∆F}F «
mfor a
Thalassiosira weissflogii culture during growth under phos-phorus-replete and phosphorus-depleted conditions. ThePOC concentration calculated as the mean of five samplestaken at different times during the experiment remainedconstant at 62±4 mg l−" (³1±9 mg l−"). Chla concentra-tion, cell number and F
v}F
mchanged substantially in
relation to the phosphate supply (Table 1). Prior to
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94S. Lippemeier et al.
Fig. 4. Variations with time in (A) C}P ratio, (B) growth rate and
(C ) in-line fluorescence parameters during cultivation of
Thalassiosira weissflogii under fluctuating phosphate
concentrations. Nutrient treatments are as indicated in the figure.
The figure shows one representative set of data for phosphorus
limitation in Thalassiosira weissflogii.
starvation, C}P ratio was 183 and growth rate was 0±49day−" (Fig. 4A, B).
After 65±25 h in phosphate-free medium, the cellularC}P ratio had increased to 453±8 and the growth rate haddecreased to zero 3 h before the re-addition of phosphate(Fig. 4A, B). The fluorescence parameters F and F «
m
decreased from 0±24 to 0±16 and from 0±52 to 0±24respectively with increasing phosphorus starvation. Thisresulted in a decrease in ∆F}F «
mby 31% from 0±54 to 0±37
(Fig. 4C ).After this 65±25 h starvation period, 2±5 ml of phos-
phate solution was added directly to the culture resultingin a final phosphate concentration of 35 µM. Immediatelyafter the phosphate pulse, F and F «
mdecreased markedly by
17% and 30% to 0±13 and 0±17 respectively (Fig. 3B) and∆F}F «
mdecreased by 35% to 0±24. After 15 min this
decline was followed by a steep increase in F (22% to0±16), F «
m(39% to 0±28) and ∆F}F «
m(80% to 0±43) for the
next 15 min. Subsequently F and F «m
continued to increaseat a lower rate for the next 21 h, yielding maximal valuesof 0±26 and 0±59, respectively (Fig. 4C ). ∆F}F «
mreached a
maximal value of 0±62 at 18 h after the phosphate pulse.Subsequently all fluorescence signals stabilized at valuesslightly lower than before phosphorus starvation.
Fig. 5. Variations with time in (A) cell number, (B) growth rate
and (C ) in-line fluorescence parameters during cultivation of
Thalassiosira weissflogii under fluctuating silicate concentrations.
Nutrient treatments are as indicated in the figure. The figure
shows one representative set of data for silicon limitation in
Thalassiosira weissflogii.
Immediately after the phosphate pulse, the growth rateincreased steeply from zero to 1±2 within 1 h (Fig. 4B).This was directly followed by a sudden decrease to 0±05and two subsequent maxima of 1±2 day−" within the next18 h. Finally the growth rate stabilized at 0±42 day−" at24 h after the phosphate pulse. As discussed below, it isunlikely that these variations in the absorption at 875 nmreflect true changes in growth rate. The C}P ratio was alsoaffected by the phosphate re-addition and decreased from453±8 to 185 in the first 30 h after the pulse (Fig. 4A). Cellnumbers had risen to 4±5¬10& cells ml−" at 30 h afterphosphate re-addition, which was slightly higher than atthe beginning of the experiment (Table 1).
Silicon starvation in Thalassiosira weissflogii
Fig. 5 shows the time course of cell number, growth rateand the fluorescence parameters F, F «
mand ∆F}F «
mfor a
Thalassiosira weissflogii culture during growth undersilicon-replete and silicon-depleted conditions. The POCconcentration (calculated as the mean of seven samplestaken at different times during the experiment) remainedconstant at 64±6 mg l−" (³2±2 mg l−"). Chla concentra-tion, cell number and F
v}F
mchanged substantially in
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PAM fluorescence under fluctuating nutrient supply 95
relation to the silicate supply (Table 1). Prior to starvation,4±9¬10& cells m l−" and a growth rate of 0±51 day−" weremeasured (Fig. 5A, B).
After 63 h in silicate-free medium, the cell number haddecreased to 2±8¬10& cells ml−" just before the silicate re-addition and the growth rate had decreased to zero 3 hbefore the silicate re-addition (Fig. 5A, B). The fluorescenceparameters F and F «
mdecreased from 0±24 to 0±14 and from
0±54 to 0±22, respectively, with increasing silicon star-vation. Due to a stronger decrease in F «
m(59%) than
F (42%), ∆F}F «m
decreased by 36% from 0±56 to 0±36(Fig. 5C ).
After this 63 h starvation period, 17±5 ml of silicatesolution was added directly to the culture resulting in afinal silicate concentration of 371 µM. This additioninduced an immediate strong decrease in F and F «
mby 7%
and 38% to 0±13 and 0±16, respectively, within the first10 min (Fig. 3C ). In the following 50 min, F increased to0±15 and F «
mincreased to 0±26. Subsequently F and F «
m
slightly decreased again for 30 min by 2% and 8%,respectively. ∆F}F «
mdecreased immediately after the
pulse, and recovered to 0±38 over the next 50 min. Afterthis, the steeper increase in F «
mrelative to F resulted in a
steep rise in ∆F}F «m
to a maximum value of 0±6 at 20 hafter the silicate pulse (Fig. 5C ). Subsequently all fluores-cence values stabilized at values comparable to those insilicon-replete conditions at the start of the experiment.
The C}N ratio was also affected by the silicate re-addition and had decreased by 34% to a value of 7±2 at25 h after the pulse (not shown). In the following 47 h, theC}N ratio increased to 9±8, which was comparable to theC}N ratio determined before the silicate starvation. Thegrowth rate started to increase steeply 30 min after silicatere-addition and reached a maximum value of 0±9 day−"
within the next hour (Fig. 5B). During the following 7±5 h,the growth rate first decreased and finally increased againto 1±1 day−". In the next 39 h, the values stabilized and afinal growth rate of 0±5 day−" was established, which wascomparable to that in the silicon-replete conditions at thebeginning of the experiment. The cell number increasedcontinuously from 2±8¬10& ml−" at the end of thestarvation phase to 4±6¬10& ml−"within 72 h after silicatere-addition (Fig. 5A).
Discussion
Turbidostatic regulation of cell growth
The turbidostatic regulation of the cultures provided aconstant concentration of particulate organic carbon(POC). In contrast, the cell numbers changed withoutaffecting the turbidity of the cultures, indicating thatchanges in cellular carbon content were induced bynutrient fluctuations. This is in agreement with the generalprinciples of turbidostatic systems (Richmond, 1986). Thestrong changes in the C}N and C}P ratios during nitrogenand phosphorus starvation, respectively, and the declinein the growth rate (µ ) to zero during nutrient starvation
in all experiments, clearly demonstrate that the physiologyof the cells was strongly affected.
The steep increases in growth rate following phosphateand silicate re-addition (Figs 4B, 5B) might be explained bythe fact that the nutrient-starved cells were arrested intheir cell cycle before cell division and showed asynchronized increase in growth rate following re-addition of the deficient nutrient. This was shown in detailby Brzezinski (1992) for the growth rate (based on cellnumbers) of silicon-deficient Thalassiosira weissflogii. In thecase of phosphorus-deficient cells, a surge in cell divisionwas possibly induced by phosphate re-addition due to avery fast build-up of previously lacking ATP and phos-pholipids. Alternatively, it might be speculated that theuptake of silicate and phosphate led to fast changes in theoptical properties of the cells (scattering, reflectance),causing the observed strong variations in the turbiditysignal.
In-line fluorescence measurements
In general, our experimental measuring set-up provided aconsistent and sensitive in-line detection of fluorescencerevealing immediate reactions to nutrient fluctuations (Fig.3A–C ) as well as long-term responses (Figs 2C, 4C, 5C ),which allows the determination of recovery times afternutrient re-addition. Measurements of fluorescence withand without agitation resulted in identical values for F andF «m
(not shown), so that the cultivation irradiance and themixing of the cultures did not interfere with reproduciblein-line fluorescence measurements.
Unlike an earlier study (Lippemeier et al., 1999), thefluorescence signals were detected directly in the culturesuspension with actinic light and not after a short darkadaptation. Consequently, the fluorescence measurementsin the present study can be compared directly withmeasurements of the photosynthetic efficiency of thereaction centres of PSII of light-adapted cells (∆F}F «
m, Eq.
2 ; Genty et al., 1989 ; Geel et al., 1997 ; Hartig et al., 1998).
Which mechanisms are responsible for the strong influences
of nutrient regime on the fluorescence signals ?
In this study, we used a new fluorescence-based approachto investigate the effects of varying nutrients on thefluorescence characteristics of the cells. We did not focuson the physiological background of these effects and willtherefore relate our findings to the results of other authorsin order to compare our results with the results obtainedwith other techniques and provide some physiologicalevidence to justify our findings. We will go through theeffects of different nutrient regimes on the fluorescenceparameters F
v}F
m(Table 1), F, F «
mand ∆F}F «
m.
Effects of nutrient fluctuations on Fv}F
m. A decrease in F
v}F
m
similar to our findings (Table 1) was reported for nitrogenstarvation in Phaeodactylum tricornutum (Geider et al.,1993a), nitrogen limitation in Dunaliella tertiolecta (Berges
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96S. Lippemeier et al.
et al., 1996 ; Geider et al., 1998) and Thalassiosira weissflogii
(Berges et al., 1996). The same was reported for phos-phorus starvation in Phaeodactylum tricornutum, phos-phorus limitation in Dunaliella tertiolecta (Geider et al.,1998) and silicon starvation in Thalassiosira weissflogii
(Lippemeier et al., 1999).A covariation of F
v}F
mwith a decrease in the PSII
reaction centre protein D1, which is proportional to thenumber of functional PSII centres, was observed fornitrogen, phosphorus and iron starvation in Phaeodactylum
tricornutum (Geider et al., 1993a), and for phosphorusdeprivation in Chlamydomonas reinhardtii (Geider et al.,1993a ; Wykoff et al., 1998). Berges et al. (1996) observeda decrease in F
v}F
mwhich corresponded to a decrease in
active PSII centres per cell (measured by oxygen flashyields) in nitrogen-limited Thalassiosira weissflogii. Ingeneral, however, a decrease in the number of functionalPSII centres (i.e. centres with D1) is not equivalent to areduction in F
v}F
m, which is a relative measure of the
maximal photochemical efficiency of PSII centres (Wykoffet al., 1998). A decline in F
v}F
msuggests damage to some
PSII centres, resulting in a reduced fraction of functionalPSII reaction centres with increased nutrient starvation.
As proposed by Geider et al. (1993a), the repair ofdamaged PSII centres in nitrogen-starved cells may beimpaired because the lack of nitrogen-rich amino acidsleads to an accumulation of non-functional PSII centres. Inphosphorus-starved cells, the repair of damaged PSIIcentres may be impaired by reduced ATP regeneration.Considering the strong influence of silicate supply onFv}F
mof Thalassiosira weissflogii (Fig. 5C and Lippemeier et
al., 1999), decreased protein synthesis in silicon-starvedcells (Werner, 1977) could lead to a reduced repair ofdamaged PSII centres resulting in a decrease in F
v}F
m.
These effects of nutrient supply on the photosyntheticapparatus provide a reasonable background to explain thestrong nutrient-regulated changes in F
v}F
mobserved in
our study. Even if the relative responses of Fv}F
mthat we
observed are comparable with those in the literature, thedegree of nutrient-induced variations in F
v}F
mis different.
However, this might be explained by the fact that theeffects of nutrient regime are dependent on the type ofcultivation (e.g. batch, chemostat) and the length of thestarvation period, as well as on the species investigated(Geider et al., 1998).
Effects of nutrient fluctuations on ∆F}F «m. Additional quench-
ing mechanisms besides the ones related to the damage toPSII centres have to be taken into account whenconsidering the stronger effects of the different nutrientfluctuations on the photochemical efficiency of light-adapted cells (∆F}F «
m) detected in-line compared with
effects on Fv}F
m. In particular, so-called energy-quenching
(qE) might be responsible for the observed variations in∆F}F «
m.
The decrease in the growth rate, which was observedduring all nutrient starvations investigated in this study, isstrong evidence that the reactions involved in the Calvin
cycle and any related biomass build-up are reduced duringnutrient starvation. As a result of this, the need for ATPwill decrease, finally leading to an increased transthyla-koidal pH gradient. This has been shown to result inelevated energy-quenching (qE), which enhances fluores-cence quenching (Weis & Berry, 1987) and influences∆F}F «
m. The reduced need for ATP is also responsible for
a downregulation of the linear electron transport. ∆F}F «m
therefore provides an indirect measure of the linearelectron transport activity.
A decrease in Calvin cycle activity in nitrogen-deficientgreen algae may be associated with the decrease inRubisco activity reported for nitrogen-limited Chlorella
emersonii (Beardall et al., 1991). In phosphorus-deficientSelenastrum minutum, a reduced need for protons due toreduced ATP synthesis during phosphate starvation couldbe responsible for an additional increase in pH gradientbesides reduced ATP consumption (Gauthier & Turpin,1997).
These physiological considerations provide a back-ground to partly explain the build-up of a pH-dependentfluorescence quenching in light-adapted cells duringnutrient starvation, which could account for the strongerdecrease in the photochemical efficiency of light-adaptedcells (∆F}F «
m) than dark-adapted cells (F
v}F
m) in nitrate-
and phosphate-starved cells in our study. The strongeffects of silicon starvation on ∆F}F «
m(Fig. 5C ) have not
been observed in previous studies, and the physiologicalbackground for the strong coupling of silicate supply andfluorescence has still to be investigated.
The observed recovery of all investigated parameterswithin 2 days of nutrient re-addition in this study showsthat the nutrient-related effects were reversible, and thatthe repair of damaged proteins and de novo synthesis weresufficient to re-establish the nutrient saturated operationalactivity.
Effects of nutrient re-addition on ∆F}F «m. In the first minutes
and hours after re-addition of the limiting nutrient, thefluorescence values are likely to be influenced in generalby the complex regulation of the need for energy, redox-power and carbon skeletons during nutrient uptake(Holmes et al., 1989 ; Turpin & Bruce, 1990 ; Gauthier &Turpin, 1997). Therefore, significant differences in thetemporal changes of the fluorescence parameters after re-addition of the limiting nutrient are to be expected.
After the re-addition of nitrate, the photochemicalefficiency of light-adapted cells (∆F}F «
m) remained un-
changed (Fig. 3A), possibly because electrons are neededfor the uptake of nitrate, primarily for the nitrate}nitritereductase reaction. This is in accordance with the obser-vations of Holmes et al. (1989) that oxygen evolutionremains constant during nitrate uptake. Because theATP}electron ratios during carbon fixation and nitrateuptake are similar (Turpin & Bruce, 1990), quenchingremains constant, and this may explain why nitrate re-addition did not affect the fluorescence values within thefirst 30 min (Fig. 3A). In contrast, the uptake of ammonium
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PAM fluorescence under fluctuating nutrient supply 97
does not require electrons provided by photosynthesis, sothat the addition of ammonium to nitrogen-limited cellsinduced a strong pH-related fluorescence quenchingwithin a few seconds (Turpin & Weger, 1988). However,we observed a slow increase in F «
mand F 30 min after
nitrate re-addition. The exact mechanism behind thisobservation is unknown; possible explanations could be aslight decrease in qE or a state transition towards PSIIinduced by an increased PSI acceptor pool during thenitrate uptake process (Vanselow, 1993).
The third hour after nitrate re-addition might begoverned by the conversion from nitrogen assimilation tocarbon assimilation. This would include an initial decreasein ATP requirement because of replete internal nitrogenstores, which results in a period of enhanced pH-based qE(Holmes et al., 1989). This might be reflected in thereduction in F and F «
min the third hour after nitrate re-
addition (Fig. 3A). Three hours after re-addition of nitrate,fluorescence levels started to increase again. This could becaused by a recovery of Calvin cycle activity, leading toan increased ATP demand and a reduction in qE.
In contrast to nitrate re-addition, the re-addition ofphosphate and silicate resulted in an immediate strongdecline in the fluorescence-parameters F «
mand F (Fig. 3B,
C ). This could be caused by the build-up of a strong pH-dependent quenching related to the uptake of thesenutrients, as was reported by Gauthier & Turpin (1997) forphosphate re-addition in Selenastrum minutum. This in-crease in pH gradient may be related to a reduced need forphotosynthetically produced ATP, since the requirementfor ATP during phosphate uptake could be providedeither by dark respiration (Gauthier & Turpin, 1997) or byan enhanced cyclic electron flow around PSI, which couldbe connected to a state transition towards PSI. As has beenproposed by Wood & Oliver (1995), a further contributionto the build-up of the pH gradient during phosphateuptake could also be related to decreased carbon fixationvia substrate limitation of Rubisco induced by an increasedATP:ADP ratio.
The fact that the photochemical efficiency of light-adapted cells (∆F}F «
m) is reduced, along with F and F «
m,
indicates a reduced electron transport rate during theinitial part of phosphate and silicate uptake. Even thoughthe correlation of ∆F}F «
mwith photosynthetic oxygen
evolution is dependent on the cultivation conditions (Geelet al., 1997), this is in agreement with the observation thatgross oxygen evolution is immediately reduced byphosphate re-addition to phosphorus-deficient Scenedes-mus (Healey, 1979) and Selenastrum minutum (Gauthier &Turpin, 1997). These findings demonstrate a limitedrequirement for redox power during the initial phase ofphosphate uptake, which is in contrast to the fluorescenceresponse observed during the uptake of nitrate.
Despite the strong influence of silicon supply on thephysiology of diatoms, the effects of silicate re-additionhave received little study (Lippemeier et al., 1999).However, as after phosphate re-addition, there seems tobe a strong build-up of qE (Lippemeier et al., 1999) due to
a decreased need for redox power during the initialsilicate-uptake.
The strong depression of fluorescence in the first30 min after silicate re-addition (Fig. 3C ) was not observedin an earlier study (Lippemeier et al., 1999) with on-linefluorescence detection in a darkened bypass system. In-line detection of fluorescence in ambient light obviouslyreveals other fluorescence responses to silicate re-additioncompared with fluorescence detection in the dark after ashort dark adaptation in a bypass. This is in agreementwith the above assumption of an enhanced pH gradient inthe light-adapted cells during silicate starvation.
The temporal resolution of in-line measurements offluorescence and growth revealed similar recovery timesfor the three investigated nutrients (nitrogen, phosphorus,silicon) in our study in the range of 48 h. However, asmentioned above, the degree of starvation and the amountof nutrient added will strongly influence the absolutevalues, so that a comparison of recovery times even withinexperiments with the same limiting nutrient is notstraightforward.
Generally, we conclude that the changes in fluorescencereflect complex changes in the balance between the light-harvesting processes and the processes which generateand utilize energy sources (ATP and NADPH). All thephysiological evidence provides a background that en-ables only a limited explanation of several aspects of theobserved modulations of fluorescence signals in relationto nutrient regime (Figs 2C, 3A–C, 4C, 5C ). Furtherinvestigations of the exact mechanisms involved in thesefluorescence reactions are clearly needed.
Are the fluorescence responses suited to the detection of
nutrient starvation in the field ?
Because of the close connections between nutrient supplyand the fluorescence signals of the cells, the potential of in-line fluorescence measurements as a fast and convenienttool for the identification of the deficient nutrient innatural phytoplankton communities has been investigatedby Wood & Oliver (1995). These authors reported thatthe fluorescence responses after nutrient enrichment canbe related to the deficient nutrient in cultures and coulddetect nutrient limitation in natural phytoplankton com-munities.
The fluorescence responses observed in the presentstudy were nutrient-specific for nitrate and, at least forDunaliella salina and Thalassiosira weissflogii, there seemedto be no species-specific fluorescence responses to nitrateand phosphate re-addition. However, Wood & Oliver(1995) observed a strong fluorescence increase in Micro-cystis aeruginosa following phosphate re-addition. This iscontradictory to the strong immediate decrease in fluores-cence that was reported by Gauthier & Turpin (1997) forthe re-addition of phosphate in Selenastrum minutum andthat we observed in Dunaliella salina (not shown) andThalassiosira weissflogii (Fig. 4C ). Additional evidence forspecies-specific fluorescence responses has been reported
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98S. Lippemeier et al.
for ammonium addition in nitrogen-limited Chlorella
pyrenoidosa and Selenastrum minutum (Elrifi et al., 1988).In addition to species-specific responses, there is strong
evidence that the characteristics of the signals (i.e. responsetime and rate of increase or decrease) are stronglydependent on the nutrient status of the cells, as has beenshown for nitrogen-starved Dunaliella tertiolecta (Beardallet al., 1995). Hence it might be possible to identify thedeficient nutrient but it will be very difficult to assess thedegree of starvation based on fluorescence responsesalone.
In our study, the fluorescence parameters remainedunchanged in the first 30 min after nitrate re-addition (Fig.3A), but a strong decrease in the fluorescence parameterswas recorded immediately after phosphate and silicate re-addition (Fig. 3B, C ). Because of the physiological back-ground of nitrate uptake, this is likely to be a characteristicof nitrate re-addition in general (Gauthier & Turpin, 1997).Hence it might be difficult to obtain a significantfluorescence reaction to addition of nitrate during a testwithin a few minutes.
However, nitrate-limited cells have an increased uptakecapacity for ammonia and show quick fluorescenceresponses upon addition of ammonia (Turpin & Weger,1988). Thus addition of ammonia could be used for afluorescence-based detection of nitrogen starvation.Finally, non-specific changes in fluorescence after nutrientspiking, as reported by Wood & Oliver (1995) afterammonium addition to nitrogen- and phosphate-starvedMicrocystis aeruginosa, may present a general problem thatshould be considered in fluorescence-based nutrient tests.
Obviously all the different factors that affect thefluorescence responses have yet to be investigated to beable to fully characterize the nutrient status of naturalphytoplankton communities. However, we conclude thatthe results obtained so far indicate that fluorescence is apromising tool for identifying the nutrient actuallylimiting phytoplankton growth.
Fluorescence in comparison with classical nutrient uptake
studies
Classical nutrient uptake studies revealed changes inuptake rates within seconds, and indicated strong in-fluences of nutrient supply on phytoplankton productivity(MacIsaac & Dugdale, 1969 ; Fuhs & Canelli, 1970 ;Goering et al., 1973 ; Harrison & Davis, 1977 ; Goldman &Glibert, 1982 ; Parslow et al., 1984 ; Martinez, 1991).However, Flynn (1998) has pointed out that the use ofnutrient uptake kinetics to study the effects of fluctuatingnutrients on phytoplankton productivity is problematic.Apart from methodological problems, this is mainly dueto post-uptake processes, which do not necessarily haveto keep pace with the nutrient uptake itself. To evaluatethe competitiveness of a species, therefore, it is notsufficient to know about nutrient uptake rates : one mustalso obtain information about the variations in thesubsequent physiological reactions of the cells.
Even though the fluorescence measurements do notprovide direct assessment of nutrient uptake, we concludethat the observed changes in fluorescence are a conse-quence of nutrient uptake. Since the fluorescence measure-ments provide an estimation of the photochemicalefficiency of the reaction centres of PSII (∆F}F «
m, Eq. 2), we
further conclude that they provide more informationabout the reactions of cells to fluctuating nutrients thanexperiments based on the determination of nutrient uptakekinetics alone.
However, it is still not trivial to differentiate betweenthe use of photosynthetically produced energy assessedby ∆F}F «
mfor basic cellular turnover metabolism or for
actual growth (Flameling & Kromkamp, 1998 ; Hartig et
al., 1998). Hence the observed changes in fluorescencealone are most suited to investigate the short-termenergetic responses to nutrient fluctuations. For anassessment of the influences of nutrient regime on overallphytoplankton primary production, a combination offluorescence-based photosynthesis measurements andfinal parameters of cell productivity (e.g. growth rate)must be favoured.
Applications and benefits of in-line fluorescence in future
nutrient studies
Recent studies have shown the importance of nutrients inthe production of toxic substances during harmful algalblooms (Anderson et al., 1990 ; Pan et al., 1996). Accordingto the present study, in-line fluorescence measurementsare a promising method to investigate the role of nutrientsin the temporal development of toxic algal blooms. Thismay include the detection of the limiting nutrient and thedetermination of the response times of the photosyntheticefficiency during fluctuating nutrient regimes. Based onour findings, we conclude that our set-up for in-linefluorescence measurements is a useful alternative toclassical nutrient uptake experiments, providing newinsights into nutrient regulated physiology on a broadtime scale.
Acknowledgements
Technical assistance by B. Egge, C. Reineke, G. Bojens, B.Hein and W. Voigt is gratefully acknowledged. Dr M.Reckermann is thanked for determination of cell numberswith the flow cytometer. Comments by two anonymousreviewers and discussion with Dr E. Young and ProfessorJ. Beardall provided substantial improvement of themanuscript. This work was supported by the DeutscheBundesstiftung Umwelt under grant 07255.
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