AQUATIC BIOLOGYAquat Biol

Vol. 22: 95–110, 2014doi: 10.3354/ab00586

Published November 20

INTRODUCTION

Scenescence and mass culture

Microalgae mass culture generally aims for highbiomass yields, often associated with intended nutri-ent starvation to increase lipid contents (Jacobsen etal. 2010, Liu et al. 2013). Nitrogen starvation lowersprotein contents, RuBisCO levels, and activates bio-

chemical responses that lead to an elevation of lipidbodies by a process that is not entirely understood.Algae mass-culture conditions are frequently lesscontrolled, the exponential growth phase might havepassed during the time of cell harvest and cells haveexperienced senescence. Under these conditionscells can exhibit acclimation properties which arecomparable to nutrient starvation conditions. Lowerphotosynthetic performance is one result of senes-

© The authors 2014. Open Access under Creative Commons byAttribution Licence. Use, distribution and reproduction are un -restricted. Authors and original publication must be credited.

Publisher: Inter-Research · www.int-res.com

*Corresponding author: s.ihnken@gmail.com

Light acclimation and pH perturbations affect photosynthetic performance in Chlorella mass culture

Sven Ihnken1,*, John Beardall2, Jacco C. Kromkamp1, Cintia Gómez Serrano3, Moacir A. Torres4, Jirí Masojídek5, Irene Malpartida3, Robert Abdala3,Celia Gil Jerez3, Jose R. Malapascua5, Enrique Navarro6, Rosa M. Rico3,

Eduardo Peralta3, João P. Ferreira Ezequil6,7, Félix Lopez Figueroa3

1Royal Netherlands Institute for Sea Research (NIOZ), PO Box 140, 4400 AC Yerseke, The Netherlands2School of Biological Sciences, Monash University, Clayton, Victoria 3800, Australia

3Departamento de Ecología, Facultad de Ciencias, Universidad de Málaga, Campus Universitario de Teatinos s/n, 29071 Málaga, Spain

4Departamento de Bioquímica, Institute de Química, Unversidade de São Paulo, SP, Brazil5Institute of Microbiology, Academy of Sciences, 37981 Trebon, Czech Republic

6Pyrenean Institute of Ecology (CSIC), Av. Montañana 1005, Zaragoza 50059, Spainl7Departamento de Biologia, Universidade de Aveiro, Aveiro 3810-193, Portugal

ABSTRACT: Chlorella spp. are robust chlorophyte microalgal species frequently used in mass cul-ture. The pH optimum for growth is close to neutrality; at this pH, theoretically little energy is re-quired to maintain homeostasis. In the present study, we grew Chlorella fusca cells in an open, out-door, thin-layer cascade photobioreactor (TLC), under ambient photon flux at the theoreticallypreferred pH (7.2), and let the culture pass the exponential growth phase. Using pH drift experi-ments, we show that an alkalization to pH 9 supported photosynthesis in the TLC. The increasedphotosynthetic activity under alkaline conditions was a pH-dependent effect, and not a dissolvedinorganic carbon (DIC) concentration- or light intensity-dependent effect. Re-acidification (in onestep or in increments) lowered gross oxygen production and increased non-photochemical quench-ing in short-term experiments. Gross oxygen production and electron transport rates in PSII wereuncoupled during the pH perturbation experiments. Electron transport rates were only marginallyaffected by pH, whereas oxygen production rates decreased with acidification. Alternative electronpathways, electron donation at the plastid terminal oxidase and state-transitions are discussed as apotential explanation. Because cell material from the TLC was not operating at maximal capacity,we propose that alkalization can support photosynthesis in challenged TLC systems.

KEY WORDS: Chlorella · Mass culture · pH perturbance · Chlorophyll fluorescence · Oxygen production · pH drift · Photosynthesis · Photoprotection

Contribution to the Theme Section ‘Environmental forcing of aquatic primary productivity’ OPENPEN ACCESSCCESS

Aquat Biol 22: 95–110, 2014

cence (Humby et al. 2013). When cells enter the sta-tionary growth phase, chloroplast structures becomedisorganized, LHCII, D1, PsaA, Cyt f, and RuBisCOlevels decline, and photosynthesis is down-regulatedwhile non-photochemical quenching (NPQ) remainsstable compared to the exponential growth phase(Humby et al. 2013). These substantial changeswithin the chloroplast might affect the cell’s responseto external factors such as pH and light intensity.Gardner et al. (2011) showed that a Chlorella straindemonstrated elevated lipid contents and displayedmorphological changes at high pH, which indicatesthat the elevated pH induced stress to the cell.

Homeostasis: effect of external pH manipulation

The external pH under which the cell is capable ofoperating at maximal capacity is species-specific butusually range between pH 6 and 8.5 (Taraldsvik &Mykleastad 2000, Hinga 2002, Lundholm et al. 2004,Liu et al. 2007, Middelboe & Hansen 2007). AlthoughChlorella spp. prefer a pH between 6 and 6.5 (Myers1953), Chlorella vulgaris can grow effectively at analkaline pH of 10.5 (Goldman et al. 1982). Chlorellasaccharophila grows optimally at pH 7.0 but canmaintain homeostasis when external pH is as lowas 5.0 (internal pH 7.3) (Gehl & Colman 1985). Thisstrain can successfully grow at pH 2.5 (Beardall1981), which shows its immense internal pH regula-tion capacity. Internal pH is generally maintainedwithin narrow limits above ~pH 7 in Chlorella (Smith& Raven 1979, Beardall & Raven 1981, Sianoudis etal. 1987) and other taxa (Dixon et al. 1989, Kurkdjian& Guern 1989). Homeostasis is maintained by 2 mainprocesses: proton binding and H+ translocation be -tween cell organelles and the external medium byactive, energy-demanding, or passive ion exchangemechanisms (Smith & Raven 1979). The energydemand for active proton translocation is estimatedto be 1 ATP per H+ transported (Briskin & Hanson1992). The abundance of H+ ATPase complexes canbe controlled by the cell via pH sensing (Weiss & Pick1996). However, Nielsen et al. (2007) showed thatlight intensity (and hence energy supply) did not cor-relate with cell growth under extreme pH conditions,which indicates that elevated light input does notnecessarily enhance the cell’s capacity to regulateinternal pH. In short-term pH manipulations, H+

pumping is important (Bethmann & Schönknecht2009); however, under longer-term (growth) condi-tions, anion exchange might be the predominant pro-ton translocation process (Lew 2010). It is not clear

how internal pH is regulated in cells that have passedtheir exponential growth phase.

Carbon acquisition and pH

Changes in the media’s pH or supplementation ofCO2 affects the bicarbonate equilibrium. IntroducingCO2 into the medium lowers the pH and shifts the bi-carbonate equilibrium towards higher concentrationsof CO2 and bicarbonate at the expense of carbonate. Ifthe pH is increased at a constant pCO2, the dissolvedinorganic carbon (DIC) concentration increases, whilethe CO2(aq):HCO3

− ratio decreases. The form of DIC isrelevant for the cellular DIC acquisition system, whilethe concentration of DIC matters when substantialrates of photosynthesis must be sustained. Mostaquatic primary producers possess means of elevatingthe CO2 concentration at RuBisCO, which suppressesRuBisCO’s oxygenase function and provides sufficientCO2 even when high photon flux (PF) allows high CO2

fixation rates in the Calvin-Benson-Bassham-cycle.Most algae can regulate these CO2-concentratingmechanisms (CCMs) effectively (Giordano et al.2005). Chlorella spp. possess effective CCMs and ac-quire both CO2 and/or HCO3

− actively (Shelp & Can-vin 1980, Beardall 1981, Beardall & Raven 1981), andare able to raise the pH in a closed vessel to pH 11(Myers 1953), which shows that cells are capable ofcarrying out effective photosynthesis. The interlinkedpH and CO2 concentration behavior makes it difficultto separate both effects.

In the present study, we tested Chlorella fusca cellsgrown in an open, outdoor, thin-layer cascade photo-bioreactor (TLC) under post-exponential growthphase conditions. We investigated if cells are suscep-tible to high PF, are limited by external DIC concen-trations or affected by changes in the medium pH.We perturbed pH in short-term (i.e. minutes) andlong-term (i.e. hours: pH drift) experiments to test thecells’ capacity to regulate pH-related photosynthesis.The results show that cells performed better in alka-line conditions and were stressed by acidification ofthe media.

MATERIALS AND METHODS

Organism, growth conditions and growth history

The chlorophyte microalga Chlorella fusca (Cul-ture Collection of Marine Microalgae, ICMAN-CSIC,Cádiz, Spain) was grown in an outdoor thin-layer

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cas cade photobioreactor (TLC3; TLC number isgiven to enable comparison with to other studiesusing the same experi mental setup) in September2012 in southern Spain (Málaga), using Bold’s BasalMedium modified with 3-fold nitrate content plus theaddition of vitamins (3N-BBM-V) (Andersen et al.2005). The cell suspension was held in an open tank(~120 l), pumped to the top of a flat panel slide (4 m2,2% inclination), and allowed to flow back to the tankby gravity (surface to volume ratio 27 m−3, 145 l work-ing volume). The cells received full sunlight on thecascade (for a duration of ~11 s) and were exposed tolow light conditions in the tank. Completion of a single cycle took approximately 70 s. Macronutrients(NaNO3, MgSO4 and KH2PO4) were added once aday using common farming fertilisers (WelgroHydroponic, Comercial Química Massó); nu trientconcentrations were ≥540 mg l−1 (PO4

− and SO4), and≥650 mg l−1 (NO3

−; R. Abdala pers. obs.). The TLCwas aerated; pure CO2 gas was injected into the airstream (final 1%) and the pH was maintained atbetween 7.2 and 7.6. pH drift experiments showedvery limited capacity of the cells to increase the pHby means of photosynthesis (see Fig. 1A), even whenDIC was added in form of NaHCO3 (see Fig. A1A inthe Appendix), or when diluted with fresh medium(Fig. A1C). We therefore tested the photophysiologi-cal fitness in a light acclimation ex periment (see Figs.2 & 3), which showed that photosynthetic capacitywas not severely im paired. However, increasing thepH by substantial addition of NaHCO3 (20 mM) inthe TLC increased pH drift (see Fig. 1). pH pertur-bance experiments were carried out after pH adjust-ments had been performed in the TLC. Cell numbersde creased for 2 d prior to the experiments (seeFigs. 4–7) (from ~1 × 106 to ~4 × 105 cells ml−1), but re -mained stable thereafter (counted using a Neubauerchamber; R. Abdala pers. obs.). At that point, the TLChad been maintained for 12 d, at a temperature of~25°C. The TLC used in the present study was part ofa nutrient-concentration effect study. A TLC withreplete nutrients concentrations (TLC presented inthe present study), was maintained parallel to a TLCwith low nitrogen (TLC–N) and another TLC withlowered sulphate concentrations (TLC–S). We ex -pected the TLC with replete nutrients to show high-est cell numbers. However, maximal cell concentra-tions were lower than expected (TLC of the presentstudy, i.e. replete nutrients = 2 × 107 cells ml–1,TLC–S = 2.3 × 107 cells ml–1, and TLC–N = 1 ×106 cells m–1l; R. Abdala pers. comm.). The experi-ments in the present study were carried out after themaximal TLC capacity had passed, and cell suspen-

sions experienced grazing, grazer treatment andwere contaminated with Nannochloris sp. and Chla -my domonas sp. cells in variable concentrations.

More information about the hydrodynamics of theTLC used and light exposure effects in the TLC onphoto synthetic performance is given in Jerez (2014,this Theme Section). Further information on photoac-climation in the TLC will be published elsewhere (J.C. Kromkamp et al. unpubl.).

pH drift experiments

Samples were withdrawn from the TLC in the morn-ing, transferred to 20 ml glass scintillation vials, andeither continuously exposed to ambient sunlight orshaded by neutral density filters. pH was measuredseveral times a day (Basic 20, Crison Instruments),calibrated with standard laboratory solutions. Measure -ment duration was standardized to 1 min vial−1 toavoid erratic gas exchange amongst replications. Tem -peratures were in the range of 25 to 28°C and main-tained by placing vials in a non-controlled tub waterbath and regularly exchanging the cooling fluid.

Light acclimation experiment

Cell suspensions for continuous light exposure ex -periments were withdrawn from the TLC in theevening. 250 ml were filled into 500 ml conical glassflasks, which were kept in the dark overnight andthen exposed to ambient light conditions (HL, highlight) the following day while aerated with ambientair (~1 l min−1). Shading was achieved by neutral den-sity filters to 70% (ML, medium light) or 30% (LL, lowlight) of the ambient PF. Temperature conditions weremaintained at approximately 25 ± 3°C by placingflasks in a styrofoam tub filled to one-third with water(which was replaced when needed). At given times,2 ml samples were withdrawn, immediately placedin an AquaPen fluorometer (AquaPen, P.S.I.) and fastfluorescence induction curves measured. Maximumand effective quantum yield of PSII (Fv/Fm and Fv’/Fm’)and Vj (accumulation of the ‘primary’ acceptor of PSII,QA

−) were computed by the software provided by theinstrument. Initial samples were dark-incubated over -night. Other samples were measured in the absenceof actinic light, which allows QA

− to oxidize. However,because samples were not dark-acclimated prior tomeasurements, nor exposed to ambient actinic light,residual NPQ can lower the fluorescence signal anddecrease variable fluorescence. This results in F0’ dur-

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ing the O phase of the fast fluorescence inductioncurve, rather than F’ or F0 (i.e. quantum yields areFv’/Fm’ rather than ΔF/Fm’) (van Kooten & Snel 1990,Kromkamp & Forster 2003).

Fast fluorescence induction curves

Fast fluorescence induction curves represent theconsecutive reduction of electron carriers in PSII andthe plastoquinone (PQ) pool within about 1 s of illu-mination with saturating light. After the O phase(first data point at t = 50 µs), QA gets reduced until theJ phase is reached (100% QA

–, 0% QA, 2 ms), whilethe consecutive reduction of QB and formation ofplastoquinol (PQ pool reduction) is associated withthe inflection (I phase) and the maximal fluorescencesignal (P phase at t = 1 s) by a fully reduced electrontransport chain from the PSII reaction centre to thePQ pool (Tomek et al. 2001, Zhu et al. 2005). Thisconcept is commonly accepted, however, alternativeinterpretation of fast fluorescence induction curveshave been frequently presented due to the highnumber of fluorescence quenchers in PSII and thephotosynthetic unit (Strasser et al. 1995, Bukhov etal. 2004, Stirbet & Govindjee 2012). The reductionstate of the PQ pool, for instance, was not a determi-nant factor for maximal fluorescence signals duringP, but the reduced PQ pool has been shown to affectthe J phase (Tóth et al. 2005, 2007). Nevertheless, wewill interpret data of this study following Zhu et al.(2005), where signals show the accumulation of thefollowing electron carrier: J phase = QA

−, P phase =QA

− QB2−, PQ is fully reduced. Vj was calculated as

(J − O) / (P − O), with J phase = F2ms (where F = fluo-rescence), O = F50µs, P = F1s. 1 − Vj is indicative forthe electron transport capacity past QA. Low valuesrepresent a lower probability that electrons are efficiently transported. Note that P = Fm (or Fm’) inmultiple-turnover saturation pulse fluorometers (e.g.pulse-amplitude modulation, PAM), while J is equi -valent to maximal fluorescence in a single-turnoverfluorometer (e.g. fast repetition rate fluorometer, FRRf).

pH perturbations: simultaneous PAM, O2 and pHmeasurements

Cell suspensions were withdrawn from the TLCand placed in a 20 ml glass scintillation vial. The lidwas modified to allow submersion of a standard pHelectrode (Basic 20, Crison Instruments), and an oxy-gen optode (Microx TX3 PreSens) into the cell sus-

pension, while minimizing the contact surface of thecell suspension with ambient air. The optical fibre ofa Mini-PAM (Walz) was placed against the top one-third of the vial and orientated perpendicular to theactinic (white LED) light source, which provided aconstant PF of 140 µmol photons m−2 s−1. Higheractinic PF was achieved by usage of the internal lightsource of the fluorometer and exposed cells to 400µmol photons m−2 s−1 when switched on. However,cells moved into and out of the additional light fieldsince only a part of the scintillation vial was exposedto the additional PF. This can limit comparison withthe FRRf experiment, where the entire volume of thecell suspension was ex posed to PF conditions. Cellsuspensions were stirred con tinuously. After a ~4 minacclimation phase, additions of known amounts ofHCl perturbed the pH, which was recorded manu-ally every 30 s. To test for DIC limitation, 300 µl ofNaHCO3 (1 mM final concentration) were injected atthe times indicated. Experiments were conductedfrom morning until noon, before high ambient lightcould cause inhibition.

pH perturbations: fast repetition rate fluorescencemeasurements

FRRf fluorometers use a different excitation andmeasurement protocol compared to PAM instru-ments. While PAM measures Fm or Fm’, during a multiple turnover (of PSII) saturation pulse (approx.800 ms duration), FRRf uses a sequence of sub-saturating flashlets to consecutively reduce PSII to asingle turnover. The FRR fluorometer used (Fast-Tracka Mark II, Chelsea Technology Group) wasequipped with a bench-top illumination extension(FastAct, Chelsea Technology Group) and was set toapply a sequence of 100 flashlets, each 1 µs long andspaced 50 µs apart. Data from 12 such sequenceswere averaged by the standard FRRf software (Fast-Pro) and produced quantum yields (Fv/Fm, ΔF/Fm’) ofa single turnover in PSII, i.e. all QA reduced, PQ-poolnot affected. An iterative curve fit of a single excita-tion flashlet sequence provides measurements of thefunctional absorption cross-section of PSII (in nm2). Asequence of single measuring flashlets after the sin-gle turnover excitation protocol shows QA

− re-oxida-tion kinetics (τPSII), i.e. the electron transport capacityfrom a fully reduced QA. Further information aboutFRRf fluorometry can be found in Kolber & Falkowski(1992, 1993) and Kolber et al. (1998).

A total of 3 ml of microalgae culture was with-drawn from the TLC on the same day as PAM pH

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Ihnken et al.: Light and pH effects on photosynthesis in Chlorella 99

perturbation experiments were carried out, and im -mediately placed in the FRR fluorometer. An internalLED provided actinic PF of 3, 140, 400, 140 and0 µmol photons m−2 s−1 in this sequence for the timesindicated. Acidification 5 min after the start of theprotocol was achieved by addition of 8.6 µl of 0.5 MHCl. After 7 min, the pH was brought back up byinjection of 51 µl 0.1 M NaOH. Samples were homo -genized by aeration between each saturation flashletapplication, which enables gas exchange between thecell suspension and air, and complicates data inter-pretation regarding the bicarbonate system and pH.

RESULTS

pH drift experiments

pH drift was monitored daily. Until the pH wasincreased in the TLC, incubations could elevate thepH by a maximum of 0.5 pH units (examples aregiven in Fig. A1 in the Appendix). To test if cellswere DIC limited, we added small amounts of DIC(400 and 800 mM), which increased the initial value(pH 7.11) marginally (pH 0.06 and 0.23 units respec-tively). Incubations with increased DIC concentra-tions showed similar pH rise kinetics compared to thecontrol (Fig. A1A,B). pH drift experiments wherefresh media was added to cell suspensions from the

TLC (1:1 by volume) did not stimulate pH drift com-pared to the control, neither did CO2 bubbling per-formed before the experiment (Fig. A1C). This indi-cates that cells were not nutrient starved or depleted.When the pH in the TLC was adjusted (from pH 7 to9), pH drift increased (initial pH 8.47, final pH 9.57;Fig. 1). No pH increase was recorded in suspensionscollected before the DIC addition. A stronger pHdrift was observed on the following day (initial pH8.65, final pH 11.5 ± 0.06; Fig. 1B). A similar effectwas pre viously observed in an other TLC (TLC2)(S. Ihnken pers.obs., J. C. Kromkamp et al. unpubl.),where the pH was increased from pH 7 to 8.5 and driftexperiments showed increased pH rise (initial pH 8.5,final pH 10.5).

Fig. 1B shows that shielding the vials at 70% of am-bient PF (i.e. ML) resulted in the highest final pH.Higher and lower PF levels showed a similar pH driftcapacity, with some variation due to photoinhibition(HL), or light limitation (LL). Control flasks, which ex-perienced the same treatment but remained closed forthe entire period, showed similar final pH values com-pared to vials that were used for pH measurements,indicating that the measurements and the ac -companying brief exposure to air did not influence theresults. In samples which experienced full ambient PF(HL), however, a lower final pH was found in the con-trol vials (pH 11.09 ± 0.25 and pH 10.02 ± 0.18 foropened and continuously closed vials respectively).

Light acclimation status

Fig. 2 shows the light acclimationcapacities in a separate experimentwhere cells were withdrawn from theTLC and continuously exposed to am-bient PF. Fv’/Fm’ decreased with in-creasing ambient PF in all treatments.The lowest quantum yields werefound in full light exposed samples af-ter the highest ambient PF, while MLcells had already started to recover atthis point (t = 15:00 h). HL cells recov-ered from low midday yields in theevening. LL cells lowered values onlymarginally around noon. Fv’/Fm’ de-pressions in HL and ML samples werepartly caused by impairment of elec-tron transport capacities past QA. 1 −Vj decreased by 1 unit at the highestPF, but recovered later during the day(Fig. 2B). Low re-oxidation turnover

8 10 12 14 16 18 20

Local time (h)

pH

A B

8 10 12 14 16 18 20

HLMLLLvials not opened

After pH adjustmentBefore pH adjustment

12

11

10

9

8

7

Fig. 1. pH drift experiments with Chlorella fusca cells collected (A) before andafter the pH was increased by addition of NaHCO3

− in the thin-layer cascadephotobioreactor (TLC) (refer to ‘Results’ section for DIC-effects versus pH-ef-fects). (B) Light intensity effect on pH-adjusted suspensions to full-strengthambient photon flux (HL), medium light (ML: 70% of ambient photon flux),and low light (LL: 30%). Cells were collected from the TLC in the morning,and exposed to natural sunlight throughout the day. Glass scintillation vialsshielded cells from a high fraction of UV light. Vials in (A) were shaded to ML levels. Temperature was kept below 28°C. Data show means ± SD when plot

symbol is extended (n = 3). Times are given in 24 h format

Aquat Biol 22: 95–110, 2014

for electron carriers past QA, as shown by 1 − Vj, werealso visible in low P phase values during the fast fluo-rescence induction in HL samples (Fig. 3). The lowestP phase values were found in the afternoon (15:00 h,HL), but samples re covered thereafter. J phasesignals were only re pressed in HL samples but re-mained low in the evening. In LL samples, the Jphase was hardly affected; however, P phase valuesincreased over the course of the day, with higher val-ues in the evening than in the morning.

To test if cells suffered from DIC limitation, sam-ples were withdrawn from the experiment (Fig. 3)at 3 times (morning, noon, afternoon), exposed toactinic PF of approximately 140 µmol photons m−2 s−1

for 2 min, spiked with DIC to a final concentrationof 300 µM, and ΔF/Fm’ followed for 4 min by a Mini-PAM. ΔF/Fm’ was not enhanced by DIC additions(see Fig. A2 in the Appendix).

pH perturbation: decreasing pH

Perturbation experiments were carried out with sub-samples of the TLC, 2 d after the pH was in creasedfrom ~7.2 to ~9. Acidification of the medium loweredthe fluorescence signal rapidly (Fig. 4A). Both F ’ (i.e.steady-state fluorescence under actinic light condi-tions) and Fm’ (maximal fluorescence mea sured duringa saturation pulse by the fluorometer) decreasedwithin approximately 1 s to a lower state when the pHwas dropped. Continuously recorded fluorescencesignals indicate a recovery from acidification, as can beseen by a slow increase in F ’ and Fm’ until the pH wasraised. The decrease in Fm’ was caused by a rapid in-crease in NPQ. However, lower pH did not affect rela-tive electron transport rates (rETR) effectively; valuesremained stable after HCl additions.

Acidification decreased gross oxygen evolution from11.2 ± 1.74 to 3.7 ± 2.61 µM l−1 min−1 (data averagedfrom Figs. 4 & 5). This is surprising as fluorescencemeasurements suggest a steady continuation of rETR.Acidification therefore led to an uncoupling of oxy-gen and variable fluorescence based measurementsof photosynthesis.

Supplementing suspensions with DIC (1 mM) didnot increase rETR. Photosynthetic O2 production,how ever, appeared to be stimulated in 1 samplewhen the DIC amendment was performed underacidic conditions, but not in another (Fig. 4B but notC). Cells of the different TLCs were tested regularlyfor DIC limitation where the photosynthetic perform-ance remained unaffected by DIC additions at low orhigh growth pH (data not shown).

100

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B

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um y

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’)

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Vj

HL

MLLL

0.6

0.4

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Fig. 2. Chlorella fusca. Photosynthetic parameters calcu-lated from fast fluorescence induction curves shown inFig. 3. Samples were withdrawn from the TLC and con-tinuously exposed to HL, ML or LL (see Fig. 1). Datashow (A) effective quantum yields and hourly averagedphoton flux and (B) QB redox parameter 1 − Vj. Sampleswere dark-acclimated for ≤2 s before measurements wereperformed. Effective quantum yields are therefore denotedas Fv’/Fm’ ([Fm’ − Fo’] / Fm’ and not ΔF /Fm’, which requiresapplication of a saturation pulse under actinic irradiance).Efficient quantum yields are indicative of the efficiency ofphoton usage for photosynthetic electron transport. 1 − Vjrepresents the probability that an electron in QA

− movesfurther to electron carriers including the plastoquinone(PQ) pool. The higher the value (efficient Fv’/Fm’, or 1 −Vj), the higher the photosynthetic competence of thesample. Data show mean ± SD (n = 3; n = 2 for fluores-cence induction curves shown in HL for t > 15:00 h;

where n = 2 due to failure of aeration in 1 replicate)

Ihnken et al.: Light and pH effects on photosynthesis in Chlorella 101

pH perturbation: increasing pH

Increasing the pH rapidly increased F ’ and Fm’ toinitial values or higher (Fig. 4). As a result, NPQ decreased to initial levels or lower. Negative NPQvalues occurred when initial Fm’ was lower than Fm’after the pH treatment. Because NPQ was calcu-lated using the first Fm’ value instead of Fm,negative NPQ values can occur, and indicate alower NPQ state compared to values at the start ofthe experimental protocol. Photosynthesis measuredby rETR was not affected by the single-step pHincrease. Photosynthetic oxygen production, how-ever, was stimulated by addition of NaOH. Initialvalues were not quite restored, which could havebeen be due to high oxygen concentrations in thevials at the end of the measurements due to abuildup of O2 by photosynthesis.

Photon flux effect at low and high pH

When the actinic PF was in creased4-fold at low pH, rETR slowlyincreased during the entire period ofelevated PF (Fig. 5). Decreasing Fm’values caused NPQ to increase untilthe pH was raised after 5 min. pH ele-vation caused a similar responseunder high PF conditions compared tolow PF conditions (Fig. 4). PF elevationafter cells were exposed to pH shiftsdid not induce a different responsecompared to PF elevation at low pH(Figs. 4 & 5). No pattern was found foroxygen production rates regarding PF.

Gradual pH amendments

When the pH was incrementallylowered, continuously recorded fluo-rescence and NPQ acclimation wasvisible (Fig. 6) in contrast to single-steppH perturbations, where very little ac-climation was visible. Accli ma tion topH increments was visible at pH >7.0,where alkalization increased the fluo-rescence signal, decreased NPQ andelevated oxygen production with high-est values between pH 7 and 8. Grad-ual increase of pH mirrored responsesto decreasing pH. Clearly, F ’ de-creased with acidification, and in-creased with alkalization.

High time resolution measurements by FRRf

Fig. 7 shows variable fluorescence responses topH perturbations in different PF levels. After a 3 minrelaxation phase in very low light, the low actiniclight (140 µmol photons m−2 s−1) was switched on andled to a temporary increase in F ’ and Fm’ followed bya drop and a quasi-steady state just before HCl wasadded (Fig. 7, dashed line), which decreased F ’ andFm’ further. NPQ was activated when the actiniclight was switched on and reached maximum valuesbriefly after acidification. rETR, however, was onlymarginally disturbed, which corroborates PAM mea -sure ments. The cells started to recover from acidaddition within approximately 40 s, as shown byNPQ down-regulation. Increasing the actinic light

O

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P

HLinitial–dark

9:00 HL

0.001 0.100Time (s) Time (s)

0.001 0.100

09:00

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LL

Fig. 3. Fast fluorescence induction curves of Chlorella fusca cell suspensionscontinuously exposed to HL, ML and LL (see Fig. 1) at different times of theday. A 250 ml cell suspension was withdrawn from the TLC, filled in 500 mlconical glass flasks, bubbled over night and exposed to measurement condi-tions on the following day. Samples were withdrawn from experimental condi-tions and dark-acclimated for ≤2 s before the measurement was performed.Data were normalized to O (initial fluorescence at t = 50 μs); vertical barsrange over 0.5 units. Data show mean ± SD (n = 3; n = 2 for fluorescence induc-tion curves shown in HL for t > 15:00 h where n = 2 due to failure of aeration in

one replicate)

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intensity to 400 µmol photons m−2 s−1 (Fig. 7, lightgrey bars) perturbed the NPQ down-regulation onlybriefly (~1 min). rETR clearly increased under addi-tional light, but was down-regulated upon addition ofNaOH and consequent rise of the pH. This is in con-trast to PAM measurements, where rETR remainedstable upon an alkalization at high PF (Fig. 5). Acidi-fication and alkalization appears to induce 2 phases,one imme diately after the pH adjustment for a dura-tion of approximately 1 min. In the secondary phase,the parameters F’, Fm’ and NPQ developed in the op -posite direction until the conditions were altered following the measurement protocol.

When cells were transferred to lower actinic PF, theeffective quantum yields increased, mainly due to arelaxation of NPQ as F ’ increased only slightly. In abrief dark period, F ’ decreased to initial values andFm’ responded marginally, suggesting that photopro-tective mechanisms were not active at the last lightphase of the experimental protocol. Photoinhibitionwas absent, as shown by Fv/Fm values which werenot repressed by the experimental treatment (0.37 ±0.08 and 0.37 ± 0.07 for the first and last quantumyield respectively). While acidification lowered QA

−

re-oxidation kinetics (τPSII) only slightly, addition ofNaOH increased τPSII almost 2-fold (Fig. 7 C), whichsuggests a QA

− re-oxidation acceleration. The PSIIfunctional cross section (σPSII) decreased when theactinic PF of 140 µmol photons m−2 s−1 was switchedon due to a NPQ decrease. Acidification caused aslow rise in σ’PSII until initial values were restored.The increase of the PF from 140 to 400 µmol photons

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(0.93)

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(0.90)

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(0.93)

6.3 (0.79)

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6.5 (0.95)

4.5 (0.94)

Fig. 4. Chlorella fusca. Photosynthesis and photoprotectionin response to rapid pH perturbations. From to top to bottomin each panel, data show gross O2 evolution and correlationcoefficient (in brackets), pH, continuous fluorescencerecording, non-photochemical quenching (NPQ), relativeelectron transport rates (rETR) and actinic photon flux (greyshaded area; 140 µmol photons m−2 s−1 in the low photonflux, LL, phase; 450 µmol photons m−2 s−1 in the high PF, HL,phase) measured simultaneously. rETR and photoprotectionNPQ were determined using variable fluorescence withrETR = ΔF/Fm’ × photon flux, and NPQ = (Fm’* − Fm’) / Fm’,where Fm’* = the first Fm’ value of the experimental protocol.Fm was not measured as cells were not transferred to thedark prior to measurements. Data show responses to pHamendment at (A) constant photon flux, while in (B) and (C)the light intensity was increased at the end of the protocol.(C) shows a repeat measurement from (B) for error estima-tion purpose. pH perturbations were achieved by addition ofsmall volumes (≤300 µl) of acid (HCl) or base (NaOH). DIC(NaHCO3

− solution) was added as indicated by arrows toyield final 1 mM. Note that continuous fluorescence lines,pH and oxygen concentrations do not read on any y-axis. pH

was measured every 30 s

Ihnken et al.: Light and pH effects on photosynthesis in Chlorella

m−2 s−1 only caused a minor dip in this ‘recovery pro-cess’. Interestingly, addition of NaOH caused a slowdecrease in σ’PSII. Note that the changes in σ’PSII aregenerally in the opposite direction to NPQ, as mightbe expected, but that the kinetics differ.

DISCUSSION

Light intensity stress

Chlorella spp. are known to grow well, even underchallenging conditions. In the TLC used in the pres-ent study, the cells were exposed to full sunlight forsome tens of seconds, then remained for some time inlow light (Jerez et al. 2014). This light treatment chal-lenges the photosynthetic apparatus of the cells andrequires effective regulation of photosynthesis andphotoprotection. Although diatoms perform betterthan green algae under fluctuating light (Wagner etal. 2006), chlorophyta convert photon energy effi-ciently to biomass (Wilhelm & Jakob 2011) and aresuccessfully used for mass culture. To test the strainused for high light resistance, we exposed cells con-tinuously to full strength sunlight for an entire day(≤1800 µmol photons m−2 s−1, daily irradiance dose~150 kW m−2). Cells were able to cope with continu-ous full strength ambient light conditions as shownby fast fluorescence induction curves. Quantum effi-ciencies and QB reduction kinetics were lower inhigh light, but could recover in the afternoon. Fre-quently, P phase values were lower than J, whichcould be caused by a selective operation of the activefluorescence quenchers, or indicate photodamage inPSII. Photodamage impairs QA

− reduction kineticsand might lower the degree of QB and PQ pool reduc-tion capacities, potentially due to the proximity of QB

to the susceptible D1 protein (Aro et al. 1993, Jansenet al. 1999). However, the full photosynthetic poten-

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(0.88)

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NP

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Gross oxygen evolution11.3 µM l–1 min–1

(0.98)

Gross oxygen evolution11.7 µM l–1 min–1

(0.95)

Gross oxygen evolution12.3 µM l–1 min–1

(0.95)

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Fig. 5. Combined effects of pH perturbance and photon fluxon photosynthesis and photoprotection in Chlorella fusca.Photon flux was (A) elevated when cells were exposed toacidic conditions and maintained while pH was broughtback up again in a single mode, or (B) by 2 consecutive baseadditions to initial values. The low light relaxation phasewas intermitted in (C). Relative electron transport rates(rETR) and photoprotection non-photochemical quenching(NPQ) were determined using variable fluorescence withrETR = ΔF/Fm’* photon flux, and NPQ = (Fm’* − Fm’) / Fm’,where Fm’* = the first Fm’ value of the experimental proto-col. Fm was not measured as cells were not transferred tothe dark prior to measurements. For legends and protocol

explanations refer to Fig. 4

Aquat Biol 22: 95–110, 2014

tial could be restored in lower PF in the late after-noon and early evening.

Samples were dark-acclimated only very briefly(≤2 s), which will oxidize primary electron acceptorsin PSII (QA

−), but not secondary electron acceptors orthe PQ pool (Strasser et al. 1995), or relax photo -protective NPQ. Although the rapid phase of energydependent quenching (qE) might relax due to a dissi-pation of the ΔpH-gradient within seconds, xantho-

phyll cycle-mediated qE requires minutes, as do state-transitions (Müller et al. 2001, Ihnken et al. 2011a).Residual NPQ in illuminated samples can explain theobvious difference between initial samples, whichwere dark-acclimated overnight, and samples takenat low PF. Unexpected, however, was that even LLsamples showed similar Fv’/Fm’ values compared toML and HL samples in the morning, where the PF atthe vessel surface in LL treatments was ≥200 µmolphotons m−2 s−1. In situ rapid light curves showedlight saturation at approximately 500 µmol photonsm−2 s−1 (J. C. Kromkamp pers. comm.), which indi-cates that LL cells were light-limited except for thehighest PF at noon. Interesting, even LL samples ap-pear to be photoinhibited around noon and needed torepair in the afternoon, as shown by a lag of Fv’/Fm’ up-regulation after the highest light values in the di-urnal cycle. Theoretically, chlorophyta perform betterunder continuous PF (as provided in the PF experi-ment) compared to fluctuating light (in the TLC)(Wagner et al. 2006), but Chlorella cells can acclimateto various, and fluctuating, PF regimes without com-promising growth (Kroon et al. 1992a,b), or be stimu-lated by fluctuating light compared to continuouslight exposure (Wijanarko et al. 2007). However, it ispossible that a change in the light exposure treatmentaf fected the photosynthetic performance in the pres-ent study. Cells experienced fluctuating light undergrowth conditions in the TLC and were then exposedto continuous PF for 1 d in the PF experiment. Pho-toacclimation may take hours to days, and is an en-ergy-dependent process (Post et al. 1984, Wilhelm &Wild 1984, Havel kova-Dousova et al. 2004). It is pos-sible that LL samples needed longer to adjust to thechanged PF regime due to lower energy capturecompared to ML and HL. However, LL conditions inpH drift experiments did not restrain pH drift, whichshows that cells were still able to perform well, atleast when the initial pH was high (Fig. 1B).

Generally, cells were resistant to severe photo-dam-age due to effective repair, or efficient and progres-sive photoprotection. These results show that cellswere able to cope with in situ, and partly very high,PF without major damage. The very restricted pHdrift at pH ~7 was therefore not due to impairment ofthe photosynthetic machinery.

pH or DIC effects?

pH drift experiments showed very low activity atapproximately pH 7.2. The low photosynthetic activ-ity at this pH is surprising, as Chlorella sp. showed

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Fig. 6. Chlorella fusca. Effects on photosynthesis and photo-protection by consecutive pH adjustments at constant pho-ton flux. (A) and (B) show separate experiments performedon the same day. A DIC addition was performed at the end

of (B) (arrow) with a final 0.7 µM

Ihnken et al.: Light and pH effects on photosynthesis in Chlorella

highest growth rates at pH 7 or lower (Myers1953, Goldman et al. 1982), and can raise the pHto values well over pH 10 (Myers 1953) due to thepresence of efficient CCMs and bicarbonateuptake capacity (Shelp & Canvin 1980, Beardall1981, Beardall & Raven 1981). We were not ableto measure the DIC concentration, but suspecteda DIC limitation when the pH was low. At anacidic pH, the total DIC concentration is muchlower than at an alkaline pH. At pH 7.0, forinstance, [DIC] = 74 µM, while at pH 9.0 [DIC] =6300 µM when solutions are in air equilibrium(temperature = 25°C, salinity = 0), calculatedusing R; (R Development Core Team 2013) pack-age seacarb 3.0 (Lavigne & Gattuso 2011). The insitu DIC concentrations would have been muchhigher because cell suspensions were main-tained at pH ~7.2 by aeration with CO2 gas. DICaddition experiments did not stimulate pH drift,nor rETR. The DIC additions were sufficientlysubstantial to enable photosynthesis for severalhours if DIC would have been limiting (gross O2

production ~10 µM l−1 min−1 ≈ CO2 consumption;DIC additions 400, 800 mM). We therefore con-cluded that the DIC concentration was notrestricting photosynthesis in the TLC, or in pHdrift experiments. After the pH was raised bybicarbonate addition in the TLC, pH drift wasstimulated. Final pH values were high andshowed substantial DIC acquisition and photo-synthetic capacity compared to other studies andspecies (Merrett et al. 1996, Choo et al. 2002,Ihnken et al. 2011b). An in crease in pH also ele-vated growth in a Chlorella mass-culture (Cas-trillo et al. 2013). Similar conclusions were madeusing marine algae in situ. The H+ concentration,and not the DIC concentration, was the primaryfactor for cell regulation, growth and photosyn-thesis in moderate pH manipulation experiments(Lundholm et al. 2005, Hansen et al. 2007, Mid-delboe & Hansen 2007). Unfortunately, it is notclear what drives these effects due to the com-plexity of pH and inter-relationships with differ-ent cell functions (Smith & Raven 1979, Felle2001). However, it can be concluded thatimpaired pH drift was neither due to DIC limita-tion, nor to PF effects in the present study.

pH-dependent acclimation patterns

O2 evolution was repressed when the pH wasdecreased from alkaline conditions to pH 6.5.

105R

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II’

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II

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Fig. 7. Chlorella fusca. Fluorescence parameters measured byFRRf in response to manipulated pH and actinic light conditions.High initial pH (9.3) was lowered by addition of HCL (dashedline) to a theoretical pH 6.5. After 5 min, the cell solution was al-kalized by injection of NaOH (dotted line) to a theoretical pH 8.5.Grey scale bars indicate actinic photon flux: 3, 140, 400 and 0µmol photons m−2 s−1. Data in (A) show minimal fluorescence indarkness or actinic light (F’), maximal fluorescence during a sin-gle turnover flashlet application (Fm’) and effective (or maximal)quantum yields; (B) shows relative electron transport rates (rETR)and non-photochemical quenching (NPQ). The iterative curve fitduring the single turnover fluorescence emission allows determi-nation of the functional absorption cross-section of PSII (σPSII); (C)Re-oxidation kinetics of QA

− after the single turnover are shown by τPSII. Data show mean ± SD for n = 3

Aquat Biol 22: 95–110, 2014106

Cells grown at various pH levels show species-dependent pH effects (Schneider et al. 2013). Cyano-bacteria growth was depressed by acidification(Wang et al. 2011), while seagrass photosynthesisdecreased with increasing pH (Invers et al. 1997).Unfavorable pH conditions increased dark respira-tion and repressed photosynthetic activity in Euglena(Danilov & Eke lund 2001) and cyanobacteria, whileFv/Fm was not affected by pH (Liao et al. 2006).

It was expected that a pH increase would be lessfavored by Chlorella. Alkaline conditions increasethe likelihood of cell damage due to increased con-centrations of reactive oxygen species at high pH(Liu et al. 2007). At pH 7, internal pH values are similar to the external medium (Smith & Raven 1979,Beardall & Raven 1981, Gehl & Colman 1985, Sia -noudis et al. 1987), which suggests an energeticadvantage at this pH due to low costs for homeo -stasis. Proton regulation can be energetically expen-sive (Smith & Raven 1979, Briskin & Hanson 1992)and involve regulative ATPase activity (Weiss &Pick 1996). Active proton pumping might only beem ployed by the cell during short-term regulation(Beth mann & Schönknecht 2009), while steady-statehomeostasis might be facilitated mainly by anionexchange (Lew 2010) in Chlorella fusca by a K+/H+

antiport system (Tromballa 1987).However, the substantial pH perturbations in the

present study were carried out in a shock-mode,which is likely to require active H+ pumping andentail ATP consumption (Bethmann & Schönknecht2009). Because the intra-cellular pH could not be de -termined, it is not clear if (and how fast) homeostasiswas reached. Photoautotrophic cells can regulate in-ternal pH in time scales of minutes (Bethmann &Schönknecht 2009), but no information of rapid inter-nal pH regulations (in seconds) was found in the literature for Chlorella. Limitations of internal pHmaintenance requires extreme external pH (Gehl &Colman 1985), or osmotic stress (Goyal & Gimmler1989) and induced internal pH changes of approxi-mately 0.9 and 0.15 units respectively. F ’, and Fm’ val-ues related positively with pH in the present study:the higher the pH, the higher the fluorescence signal.NPQ was activated by acidification. Similar resultshave been found for thylakoid fragments (Rees et al.1992, Heinze & Dau 1996). In these studies, fluores-cence responded in seconds, and a steady-state wasreached within 1 min. These results can be explainedby the quenching of the NPQ component qE, whichwas directly triggered by pH, irrespective of photo-synthetic ETR. A lumen pH of 6 clearly activates qE

(Kramer et al. 1999). Similarly, Jajoo et al. (2012)

showed that pH changes can induce a thylakoidmembrane re-organisation in pea thylakoid mem-branes. However, in the present study intact cellswere used, and cell compartment membranes as wellas pH regulatory mechanisms are expected to regu-late internal pH effectively. If external pH perturba-tion could affect qE directly, the cells’ pH regulationwould be severely impaired. The exponential growthphase was passed in the TLC, which is very likely tohave lowered the cell fitness. Cells in the stationaryphase show distinctively different genetic activity,photophysiological characteristics (Humby et al. 2013)and are more susceptible to external stress factors(Randhawa et al. 2013). However, senescence effectsare under-investigated (Humby et al. 2013). It appearslikely that cells exploit alternative electron transportroutes to increase energy dissipation to compensatefor decreased photosynthetic energy quenching ca-pacity. During ionic stress in higher plants, electronacceptance at the plastid terminal oxidase site(PTOX) accounted for up to 30% of the electron trans-port in PSII (Stepien & Johnson 2009). Because PTOXactivity consumes oxygen while electron transport isfacilitated, this mechanism could explain the discrep-ancy between rETR and gross oxygen evolution in thepresent study. Electrons are being transported anddonated to molecular oxygen at the PTOX, with wateras an end product (Kuntz 2004, Cardol et al. 2011).However, increasing, not decreasing, pH supportedPTOX-mediated O2 consumption (Josse et al. 2003),i.e. reaction kinetics operate in the opposite directionand are therefore not likely to explain low oxygenevolution rates in acidified samples in the presentstudy. It is still un known if PTOX activity cangenerally act as a safety valve under stress conditionsin higher plants, partly due to open questions regard-ing reduction kinetics in this pathway (Trouillard et al.2012, Laureau et al. 2013). Nevertheless, it is possiblethat this mechanism is employed under pH shock con-ditions in Chlo rella as electron redox systems can par-ticipate in homeostasis (Houyoux et al. 2011).

A segregation between ETR and oxygenetic photo -synthesis can be explained by state-transitions(Ihnken et al. 2014). Further studies must evaluate ifstate-transitions occur in Chlorella under conditionsof pH stress and test the hypothesis that qT is the rea-son for changes in variable fluorescence readingsand the seeming segregation of rETR and O2 evolu-tion. A deviation from ETR/O2 photosynthesis linear-ity can also be explained by other mechanisms, suchas cyclic electron flow around PSII. Cyclic electrontransport in PSII suggests photosynthetic electrontransport, but electrons are cycled within PSII and do

Ihnken et al.: Light and pH effects on photosynthesis in Chlorella

not contribute to photosynthesis. Al ternatively, nitratereduction, elevated oxygen consuming processes suchas the Mehler reaction (Asada 2000, Heber 2002),photorespiraton/ chloro respiration (Beardall et al. 2003,Peterhansel & Maurino 2011) or mitochon drial respi-ration can cause deviation between fluorescence andoxygen based measurements.

A pH effect on the inter-PS-system redox chain wasclearly shown in the present study. QA

− re-oxidationkinetics (τPSII; Fig. 7C) increased with alkalization.Faster QA

− re-oxidation, most likely due to a higheroxidation state of the PQ pool, was not facilitated byelevated electron transport, as rETR decreased whenthe base was added. This clearly shows that alterna-tive electron routes are employed by the cells whenthe pH of the external medium is perturbed. How-ever, rapid alkalization causes cells to aggregatetemporarily (Castrillo et al. 2013), which can causeself-shading similar to the package-effect (Berner etal. 1989). It is therefore possible that high τPSII valuesare artifacts caused by cell aggregation after additionof the alkaline solution.

DIC acquisition

Costs for DIC acquisition are expected to be lowerat pH 6.5 due to the higher CO2:HCO3

− ratio com -pared to pH 9.5. Chlorella possesses effective CCMsand can actively acquire both CO2 and HCO3

− (Shelp& Canvin 1980, Beardall 1981, Beardall & Raven 1981,Matsuda & Colman 1995a,b). Predominantly, bicar-bonate CCMs are anion transporters which can oper-ate as symport or antiport systems using Na+ (Badgeret al. 2002) or Cl− (Young et al. 2001). Because anionregulation is also involved in homeostasis, HCO3

− ac-quisition and consumption might be related tointernal pH regulation. Cells acclimated to alkalinepH might combine bicarbonate acquisition and pHregulation, although uptake of HCO3

− does not appearpreferable as bicarbonate must dissociate to CO2

(which is fixed in the Calvin-Benson-Bassham-cycle)and OH− (which hydrolyses to water under ‘consump-tion’ of H+). Nevertheless, cells ex hibited higher pho-tosynthetic rates at alkaline pH compared to measure-ments taken at pH 6.5. Compared to optimal growthconditions, RuBisCO contents are down-regulatedwhen cells enter the stationary growth phase (Humbyet al. 2013). A CCM up-regulation can account for lowRuBisCO levels (Beardall et al. 1991). If CCM induc-tion is controlled by pH in Chlorella, an alkalizationmight allow higher photosynthetic rates due to CCMup-regulation, or activation.

CONCLUSIONS

The present study shows 2 surprising pheno mena.Firstly, cells showed higher performance at elevatedpH, a DIC concentration- and light in tensity-independent response. As this is in conflict with themajority of the literature values (where Chlorellaspp. preferred pH ≤ 7), the precondi tioning of thecells (having passed the maximal growth phase) islikely to have influenced the outcome of the experi-ments. The second surprising finding is related to thedeviation of oxygen and fluorescence based meas-urements of photosynthesis. Acidification loweredgross oxygen production, but not electron transportrates in PSII. The reason for this might be related tostate-transitions which could not be detected duetechnical limitations in the present study. Neverthe-less, alkalinisation clearly increased the physiologi-cal performance of cultures that are not operating atmaximum efficiency.

Acknowledgements. The authors express their special grat-itude to the local coordinators of the bio-technology work-group, Irene Malpartida and Roberto Abdala. Specialthanks to the Spanish GAP coordinators Félix L. Figueroaand Jesús Mercado, and the Spanish GAP committee MaríaSegovia, Nathalie Korbee, Roberto Abdala, Rafael Conde,Francisca de la Coba, Andreas Reul and Irene Malpartida.Experiments were carried out under support from theDepartment of Ecology of the Universidad de Málaga. TheNetherlands Institute for Sea Research (NIOZ) providedacknowledged travel funds for J.C.K. and S.I. Three anony-mous reviewers contributed through constructive criticism.

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Submitted: December 2, 2013; Accepted: June 20, 2014 Proofs received from author(s): August 22, 2014

Appendix.

10 12 14 16 18

8.0

7.8

7.6

7.4

7.2

7.0

8.0

7.8

7.6

7.4

7.2

7.0

8.0

7.8

7.6

7.4

7.2

7.0

Local time (h) Local time (h)

pH

no DIC addition+ 400 mM+ 800 mM

7.0 7.2 7.4 7.6 7.8 8.0+800 mM (pH)

No

DIC

ad

diti

on (p

H)

10 12 14 16 18p

H

TLC 2TLC 3 + CO250:50 diluted with replete medium

A B C

Fig. A1. pH drift experiments. (A) Cells were supplemented with NaHCO3−; (B) shows a scatter plot of (A) for samples without

DIC amendment and samples where an 800 mM bicarbonate supplement was performed. (C) Shows additional pH drift ex -periments from TLC2 and TLC3, where cell suspensions were bubbled with CO2 prior to pH drift experiments, and samples

that were diluted with fresh, nutrient replete medium (50:50 by volume). Data are mean ± SD (n = 3)

–2 –20 2 4

Time before DIC addition (min)

morningnoon HLnoon MLnoon LL

0 2 4

morningevening HLevening MLevening LL

A B1.3

1.2

1.1

1.0

0.9

Incr

ease

of ∆

F/F m

’ (re

lativ

e)

Fig. A2. Samples of the photon flux (PF) experiment (see Figs. 2 & 3) were withdrawn, exposed to a constant PF of 140 μmolphotons m−2 s−1 and supplemented with 300 µM dissolved inorganic carbon (DIC) at t = 0 min, while ∅F/Fm’ values wererecorded every 30 s. Data were normalized to the first value. Data show mean (n = 3), SD were omitted for readability but

rarely exceeded 5% with the exception of evening light limitation (LL) samples, where 20% SD were common