Influence of different degrees of N limitation on photosystem II performance and heterogeneity of Chlorella vulgaris

Algal Research 26 (2017) 84–92

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Influence of different degrees of N limitation on photosystem II performance and heterogeneity of Chlorella vulgaris

MARK

Giorgos Markoua,b,⁎, Ly H.T. Daoc, Koenraad Muylaerta, John Beardalld a

School of Agricultural Production, Infrastructure and Environment, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece Laboratory Aquatic Biology, KU Leuven Kulak, E. Sabbelaan 53, 8500 Kortrijk, Belgium c Faculty of Biology, Hanoi National University of Education, Hanoi, Vietnam d School of Biological Sciences, Monash University, Melbourne, VIC 3800, Australia b

A R T I C L E I N F O

A B S T R A C T

Keywords: Microalgae N limitation Chl fluorescence Antenna heterogeneity

Nitrogen (N) limitation is considered as the most efficient strategy to induce the accumulation of lipids, carbohydrates or other target compounds in microalgal biomass. However, along with biomass biochemical composition, alterations in N limitation affect the photosynthetic apparatus and result in decreased growth. In this study, Chlorella vulgaris was cultivated in semi-continuous mode with different degrees of N limitation and chlorophyll (Chl) fluorescence analyses were used to investigate the effect of N limitation on photosystem II (PSII) performance, in terms of structural and functional heterogeneity. As expected, N limitation resulted in the decrease of quantum yield and calculated OJIP parameters related to PSII performance. N limitation resulted in a significant increase of trapped energy per reaction center (RC) and subsequently to higher dissipation of excess energy. However, despite the negative effect of N limitation on the number of RCs, the electron transport beyond QA as well the capacity of reducing/re-oxidation of plastiquinone were not negatively affected, implying that performance of RCs was not affected by N limitation. Photochemical quenching (qp) increased of as N limitation increased while the curve of non-photochemical quenching (NPQ) was unimodal, i.e. increased up to a level of N limitation and then decreased as N limitation degree increased further. The overall results of the present study suggest that the decrease of PSII performance was due to a reduction of the number of RCs accompanied with higher energy dissipation a probable outcome of the decreased need for reductant by cells due to lower metabolic activity under N limitation.

1. Introduction Microalgae are a highly promising feedstock for the production of various bioproducts, such as feed and food supplements, biofuels, natural colorants, antioxidants etc. The microalgal biomass content of given target compounds such as lipids, carbohydrates, pigments etc. is variable and can be modulated by altering cultivation conditions. Nutrient limitation, particularly N, is often an effective strategy to increase specific target compounds in the biomass. Numerous studies have demonstrated that N stress is an efficient strategy for accumulation of lipids, carbohydrates, and carotenoids (such as astaxanthin, lutein etc.) [1–3]. When microalgae are deprived of N, the synthesis of N-rich biomolecules (proteins, chlorophylls) is reduced and C-rich biomolecules (carbohydrates and/or lipids) are accumulated [2,4–7]. Even though N limitation results in an accumulation of target compounds, biomass growth rate declines significantly, resulting in an overall decrease of the productivity of the target compound [7]. In

⁎

order to optimize the N limitation cultivation strategy and to increase productivity, it is of great importance to better understand the trade-off between the accumulation of C-rich compounds and the decrease of growth potential. It is well documented that N limitation results in a decrease in protein content of microalgal biomass [6] and in particular in proteins associated with the photosynthetic apparatus [8]. It has been suggested that N limitation selectively affects the photosynthetic machinery, impacting mainly PSII [9]. N limitation reduces the rate of synthesis of the D1 protein and the chlorophyll-binding protein CP47, which are both found in the core of PSII, resulting in the loss of PSII reaction centers (RCs). Likewise, N limitation results in the decrease of cell Chl content [10] having an overall negative effect on photosynthetic performance. Photosynthetic performance can be assessed through Chl fluorescence analysis. Chl fluorescence is a widely used technique which provides fundamental information about PSII. Chl fluorescence analysis has been employed successfully to asses PSII performance of higher

Corresponding author at: School of Agricultural Production, Infrastructure and Environment, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece. E-mail address: [email protected] (G. Markou).

http://dx.doi.org/10.1016/j.algal.2017.07.005 Received 20 March 2017; Received in revised form 15 June 2017; Accepted 5 July 2017 2211-9264/ © 2017 Elsevier B.V. All rights reserved.

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every day. The experiments were conducted with six different N concentrations (added as NaNO3), namely 7.5, 15, 22.5, 30, 37.5 and 150 mg-N/L. The latter concentration was chosen so that the associated cultures would act as a control treatment with excess N. The experiments were conducted in 250 mL cultures using 380 mL clear glass vessels with a diameter of 76 mm placed in a temperature controlled room (22 °C) aerated with filtered air for agitation and to avoid carbon limitation. The illumination of the cultures was performed on the one side of the photobioreactors using 28 W cool fluorescence tubes (Osram, Germany) with light intensity of 75 ( ± 2.5) μmol photons/ (m2 s) measured in the middle of the empty photobioreactor. The intensity of 75 μmol m− 2 s− 1 is close to growth saturating for this species and was chosen to avoid N availability becoming a limiting factor at higher N levels. The semi-continuous cultures were kept for at least 15 days (3 × 1/dilution rate) to reach a steady state and then measurements were performed in samples withdrawn for at least three sequential days. The cultures were diluted so that all Chl fluorescence measurements were done on samples with the same biomass concentration (optical density; OD750nm of 0.2 ( ± 0.02)). Cells were adapted to darkness (at least 15 min) and Chl fluorescence measurements were recorded using the portable pulse-amplitude modulation (PAM) fluorometer, AquaPen AC-C 100 for OJIP and NPQ protocols, or a dual modulation kinetic fluorometer FL 3500 (Photon System Instruments, Czech Republic) for all other measurements, employing their dedicated FluorPen 1.0 and FluorWin 3.7 software, respectively. All measurements were performed using two independent culture replicates and at least three measurements per replicate (total six measurements per treatment) and their average along with the relative standard deviation (SD% = 100 ∗ SD / Average) is given.

plants, eukaryotic microalgae and cyanobacteria under various environmental conditions [11,12] and especially under stress conditions, such as high temperature [13], high salinity [14], high light intensity [15], heavy metal toxicity [16] or nutrient limitation [17]. Chl fluorescence originates in the re-emission of light energy that was absorbed by the light harvesting complex and that could not be transformed by photochemistry. Generally, the absorbed light energy displays three pathways: (i) it is either utilized for photochemical processes (photochemistry) or emitted as (ii) fluorescence or (iii) heat. These three pathways are in competition and any increase of the yield of one will result in a decrease of the other two. By measuring the yield of Chl fluorescence, information about changes in the efficiency of photochemistry can be gained [18,19]. When dark adapted cells are exposed to a strong saturating light pulse, the Chl fluorescence increases from a minimum value (F0) to a maximum (Fm) and displays a polyphasic transient (referred to as the OJIP curve). The most widely accepted interpretation of the underlying mechanism for the Chl fluorescence rise is the QA model, which considers that the Chl fluorescence rise reflects the gradual reduction of QA, a quinone that is an early electron acceptor in the electron transport chain between PSII and PSI [20,21]. PSII displays heterogeneity in terms of light-harvesting antenna composition and in its capacity to reduce the plastoquinone (PQ) pool. Antenna heterogeneity is related to differences in the antenna size and the energy transfer between the various components. Antenna heterogeneity arises from two major types of RCs, i.e. the PSIIα and PSIIβ RCs, while the heterogeneity in the PQ pool reducing capacity is due to the presence of a number of different RCs that are either able or unable to transfer electrons from QA− to QB [22]. PSIIα RCs are the most abundant and are localized on the appressed membrane region of the thylakoid and contain 210–250 Chl molecules, while PSIIβ RCs are localized in the non-appressed membrane region of the thylakoid and are 2–3 times smaller, in terms of their bound Chl, than PSIIα RCs [23]. It has been reported that environmental stress can negatively affect PSII heterogeneity, by increasing PSIIβ RCs as well by increasing the QB non-reducing centers [16,24,25]. Antenna heterogeneity analysis could thus be used to characterize whether there is a structural and/or functional change during stress conditions that may have a negative impact on PSII performance and biomass growth. Detailed studies on effect of nutrient limitation on the PSII in terms of active/inactive RCs, QA re-oxidation kinetics and heterogeneity (PSIIβ and QB non-reducing centers) are rare [26]. To have a better understanding of the effects of N limitation on microalgae, in the present study, C. vulgaris was cultivated in semi-continuous mode reaching steady-state cell growth under different degrees of N limitation. Biomass growth, biochemical composition and Chl fluorescence measurements and analysis were performed in order to examine in more detail the effect of N limitation on microalgal growth. Given that there is an increasing interest in producing microalgal biomass with higher lipid content that can be obtained under N limitation, as well the fact that in algal-based wastewater treatment systems N can be a limiting nutrient, the information from the present work may enhance our understanding of microalgal response under N limitation for a more efficient operation of mass culturing facilities.

2.2. OJIP fluorescence transients The fluorescence transients of the OJIP test reflect the kinetics and heterogeneity involved in the reduction of the plastoquinone (PQ) pool [27]. The model (model of QA) on which the Chl fluorescence analysis is based can be described briefly as follows: photons are absorbed by the antennae pigments (ABS, absorption flux) which are excited. A part of the excitation energy, the trapping flux (TR), is transferred to the reaction center while the remaining part is dissipated either as fluorescence or as heat. The TR is converted in the RCs to redox energy by reducing the electron acceptor QA to QA−, which is then re-oxidized to QA leading to electron transport (ET) and consequently to photochemistry [20]. Dark adapted oxygenic microorganisms, after illumination with high actinic light intensity, show a polyphasic rise of fluorescence. The typical fluorescence rise displays four steps, i.e. the O-, J-, I-, and Psteps. The different steps are obtained typically at 50 μs, 2 ms, and 60 ms, for O, J and I step, respectively, while P is the maximum fluorescence recorded (at 250–400 ms). Fluorescence transients were recorded up to 2 s with data acquisition every 10 μs for the first 2 ms and at 1 ms intervals thereafter. The terms and formulae for the various parameters of the OJIP test are shown in Table 1 and were based on Strasser et al. [20] unless otherwise stated in the text. OJIP curves were normalized to chl using the ratio Chl/B as the normalization factor.

2. Materials and methods 2.1. Microorganism and cultivation conditions

2.3. QA− re-oxidation kinetics The microalga Chlorella vulgaris (SAG 211-11b) was cultivated in a modified Bold's Basal Medium (BBM) containing 1 g/L NaHCO3, 175 mg/L KH2PO4, 25 mg/L CaCl2, 10 mg/L Na2EDTA, 75 mg/L MgSO4·7H2O, 5 mg/L FeSO4·7H2O and 1.0 mL of trace elements stock solutions: 2.86 g/L H3BO3, 20 mg/L (NH4)6Mo7O24, 1.8 g/L MnCl2·4H2O, 80 mg/L CuSO4 and 220 mg/L ZnSO4·7H2O. C. vulgaris was cultivated in semi-continuous mode. Dilution rate was set at 0.2/day and the feeding of the fresh medium was performed

The QA− re-oxidation kinetics were recorded after a single turnover flash, with actinic flash duration of 50 μs and intensity of 2500 μmol photons / (m2 s). The total duration of the test was 50 s. The kinetic data were recorded with six points per decade. The QA− re-oxidation process consists of three phases (fast, medium, slow). In order to calculate their half-times of decay, the kinetics were fitted to the following exponential function [28]: 85

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Table 1 Parameters, formulate and terms used in the OJIP test. (Adapted from [20]). Parameters

Formulae

Terms

VJ VI M0 φPo Ψo φEo φDo φPAV

(F2 ms − F0) / (Fm − F0) (F60 ms − F0) / (Fm − F0) 4 ∗ (F300 μs − F0) / (Fm − F0) TRo/ABS = 1 − (F0 / Fm) = Fv / Fm ETo/TRo = 1 − VJ ETo/ABS = [1 − (F0 / Fm)] ∗ Ψo = = 1 − ΦPo-(F0 / Fm) φPo ∗ (1 − Vav) = φPo ∗ (Sm / tFm)

ABS/RC TRo/RC ETo/RC DIo/RC PIAbs

Mo ∗ (1/VJ) ∗ (1/φPo) Mo ∗ (1/VJ) Mo ∗ (1/VJ) ∗ Ψo (ABS/RC) − (TRo/RC) (1 / (ABS/RC)) ∗ (φPo / (1 − φPo)) ∗ (Ψo / (1 − Ψo)) log(PIAbs)

Variable fluorescence at the J step Variable fluorescence at the I step Approximated initial slope of the fluorescence transients Maximum quantum yield for primary photochemistry (at t = 0) Probability that a trapped exciton moves an electron into the electron chain beyond QA (at t = 0) Quantum yield for electron transport (at t = 0) Quantum yield of energy dissipation (at t = 0) Average quantum yield of primary photochemistry (from t = 0 to tFm), Sm is the normalized complementary area over the OJIP curve, and tFm is the time in which Fm is recorded Absorption flux per RC Trapped energy flux per RC (at t = 0) Electron transport flux per RC (at t = 0) Dissipated energy flux per RC (at t = 0) Performance index (absorption basis)

DFAbs

∗

∗

Driving force

∗

F(t ) = Fr + A1∗e−K1 t + A2∗e−K2 t + A3∗e−K3 t

2.6. Photochemical and non-photochemical quenching

where F(t) is the fluorescence at time t, K1, K2, and K3 are the decay rate constants, A1, A2, and A3 are the amplitudes of the fluorescence associated relaxation phases (fast, medium and slow, respectively), while Fr is the remaining fluorescence at the end of the decay. DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] was also used (50 μM) as an electron transport inhibitor.

Photochemical and non-photochemical quenching measurements and analysis were routinely performed using an AquaPen PAM fluorometer (PSI Instruments). The NPQ protocol begins by measuring the minimal level of fluorescence Fo in dark adapted cells. After recording Fo, a short saturating flash reduces the PQ pool and Fm is recorded. A short dark relaxation period is followed, and then actinic light is supplied for tens to hundreds of seconds to provoke the Kautsky effect and fluorescence signals (Ft) are recorded. After that, a sequence of additional saturating flashes is applied on top of the actinic light to measure the NPQ and the effective quantum yield measuring Fm′ in light adapted state. NPQ was calculated as (Fm − Fm′)/Fm′ [34]. Two parameters that F′ − F can be calculated using the data of the NPQ protocol are: qp = F ′M − Ft′ M 0 which is a quantification of the photochemical quenching and ′ FM − FM NPQ = F ′ , which reflects the non-photochemical quenching. qp M reflects the level of open PSII centers and it gives higher values when the photochemistry potential is high. In contrast, NPQ increases as the fluorescence is quenched due to processes other than photochemistry [19,35].

2.4. S-states test for the determination of the inactive PSII reaction centers The Oxygen Evolving Complex (OEC) generates oxygen after a series of oxidations of four intermediate states (S0 → S4). The PSII RCs of dark adapted cells are in the S0 and S1 states (S0/S1; slash indicates a mixture of Sx/Sy). In the S-states test, the five S-states are advanced stepwise by short actinic light flashes, i.e. first flash advances S1/S2, second flash S2/S3, third flash S3/S4 → S0 and the fourth flash S4 → S0/ S1 [29,30]. S-states were advanced by 50 μs long flashes at 200 ms periods. S-states tests were employed for the determination of the contribution of inactive PSII RCs, since the fluorescence decay after the fourth flash (S4 → S0/S1) is controlled by inactive RCs (Kaftan et al., 1999). Inactive PSII were calculated according to [24]:

Inactive PSII RCs (%) = 100∗ΔF4 [(F3d F0) − 1]

2.7. Chlorophyll to biomass content

where F3d is the first fluorescence signal after the third flash, ΔF4 = 4th (F4th 99ms / F0) − 1, and F99ms is the fluorescence 99 ms after the fourth flash.

To obtain a proxy for the ratio of Chl to biomass (Chl/B), the optical density (OD) of the light absorption of 680 nm relative to that at 750 nm was calculated [36]. Biomass concentration (dry weight) was measured indirectly by optical intensity (OD) at 750 nm. The selected wavelengths are ascribed to the maximum Chl absorption and to biomass turbidity, respectively reflecting possible variations of Chl content in biomass.

2.5. Antenna, and reducing site heterogeneity To determine the antenna heterogeneity, a flash fluorescence induction (FFI) analysis was performed after applying a 50 μs saturating actinic light flash. FFI causes a transient closure of PSII RCs and can be used for the determination of antenna heterogeneity [31]. The semi-log plot of the double normalized (0 and 1) complementary area over the fluorescence induction curve gives a curve that consists of a first fast sigmoidal component ascribed to PSIIα and a slow linear component ascribed to PSIIβ. The intercept of the slow and linear phases reflects the proportion of PSIIβ RCs [24,32,33]. For the determination of reducing site heterogeneity, a double pulse OJIP was performed in order to determine the contribution of QB non-reducing centers. More specifically two fluorescence transients were induced by two subsequent light pulses (each of 1 s duration). The QB non-reducing centers were determined as:

2.8. Lipid analysis Lipids were measured by the sulfo-vanillin method [37]. Briefly, 50 μL of cell suspension containing 200–500 mg/L of lipids (to achieve this biomass, samples were concentrated by centrifugation) was added to 0.5 mL of 96% sulfuric acid and then samples were placed in boiling water for 10 min. They were left at room temperature to cool down, followed by the addition of 1.25 mL of phosphoric-acid/vanillin solution which was prepared by dissolving 0.12 g of vanillin in 20 mL deionised water and 80 mL of 85% phosphoric acid. The samples were incubated at 37 °C for 15 min and the OD was measured at 530 nm. For the construction of a standard curve, canola oil (Sigma Ardrich) was used [37].

NQB = [(Fv Fm ) first pulse − (Fv Fm )second pulse] (Fv Fm ) first pulse 86

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P

2.9. Intracellular N content

150 mg-N/L

I

For the determination of intracellular N, the residual nitrate concentration was measured in the cultivation medium (before the addition of the fresh medium) using the salicylic- sulfuric acid method [38]. Briefly, in 125 μL samples containing 0–60 mg-N/L, 250 μL of 5% salicylic acid in 96% sulfuric acid (w/v) solution was added followed by the addition of 2.5 mL of 4 N NaOH. After 30 min, the optical density at 410 nm was measured. The difference between the concentration of added and the remaining N was considered as the amount of N that was taken up by cells.

37.5 mg-N/L 30 mg-N/L

Fluorescence

J

22.5 mg-N/L 15 mg-N/L

7.5 mg-N/L

O

2.10. Statistical analysis

TF

The number of samples that have been measured was at least six (two culture replicates with at least one measurement per replicate for three sequential days). Performing semi-continuous cultures it is considered that each day represented a different population of cells, therefore the replicates from a culture on different days is considered as independent replicates. Statistical analyses were performed using analysis of variance (ANOVA) to compare the mean differences between the N limitation treatment and the control. Data were tested for their normality (Shapiro-Wilk test) and for homogeneity (Equal Variance Test). All compared data groups passed the tests. Statistical significance (p) was set at 0.05. The statistical analysis was performed using the data analysis software SigmaPlot 12.5.

0.01

0.1

1

10

100

m

1000

10000

Time (ms) Fig. 1. Log scale OJIP Chl fluorescence transients of C. vulgaris cultivated in semi-continuous mode with different degrees of N limitation. Chl fluorescence was normalized to chlorophyll using the ratio Chl/B as the normalization factor.

characterization of PSII performance and heterogeneity. 3.2. PSII performance and PSII RCs activity Fig. 1 illustrates the OJIP Chl fluorescence transients of C vulgaris cultivated under different degrees of N limitation. As shown there, N limitation clearly affected the Chl fluorescence yields. In general, the level of fluorescence at the different time-points in the OJIP curve (F0, FJ, FI, and Fm) was lower as the N supply was lower. Because the biomass concentration (OD750nm = 0.2) was standardized across the different N supply levels, this indicates that the various cellular Chl content affected the fluorescence transients. Even if the signals were normalized to the ratio Chl/B (Ft/(C/B)), the Chl fluorescence yields (F0, FJ, FI, and Fm) were still significantly different (data not shown), reflecting that N limitation did not only affect Chl fluorescence due to the decrease of biomass Chl content, but probably also due to potential changes in the PSII performance or heterogeneity. As shown in Fig. 1, N limitation affected the shape of the OJIP transients, but unlike other stress factors such as ammonia toxicity [42], high temperature [13] or high salinity [43] in which the OJIP curve tends to become almost a straight line, in N limited cultures the Chl fluorescence curves preserved the typical OJIP shape. When the OJIP shape is a straight line as it has been reported in ref. [13,43], then it indicates that electron transport after QA is inhibited and that OEC cannot provide electrons to PSII and to reduce PQ [13,43]. Probably this indicates that, even at the highest degree of N limitation where cells do not have the potential to grow further, the growth inhibition is not similar to other environmental stresses. In Table 3, some selected parameters, which were calculated based on the OJIP Chl fluorescence signals, are listed. These parameters are related to specific energy fluxes, flux ratios, performance indexes, and

3. Results and discussion 3.1. Biomass growth and Chl content It is well documented that N limitation of microalgae decreases microalgal growth and biomass production along with the alteration of the biomass biochemical composition [7]. The most notable change in biochemical composition is the degradation of proteins and photosynthetic pigments (chlorophylls) and the accumulation of carbonaceous storage molecules such as lipids [6,10,39]. The steady-state biomass concentration in relation to the provided N concentration is shown in Table 2. Biomass concentration increased proportionally with increasing N concentration, (up to a maximum that was obtained with N concentrations of 37.5 and 150 mg-N/L). Moreover, intracellular N content decreased with decreasing N concentration in the medium, suggesting that N gradually limited growth [40] of C. vulgaris cultivated under the specific experimental conditions. Clearly there is a negative relationship between Chl/B (Chl to biomass) and N concentration, reflecting a decreased Chl content with increased degree of N limitation. Lipid analysis showed that N limitation triggered the accumulation of lipid. These observations are generally in line with previously published studies, which report that the degree of nutrient limitation proportionally affects the microalgal biochemical composition and growth [7,41]. To give an insight into the effect of N limitation on growth, cells of C. vulgaris cultivated under different degrees of N limitation were subjected to Chl fluorescence measurement and analysis for the

Table 2 Relationship between N concentration and steady-state biomass concentration, Chl to biomass ratio (Chl/B) and lipids content in C. vulgaris cultivated in semi-continuous mode under different N limitation degrees. Values represent the average ± SD/average (%). N concentration (mg-N/L) 7.5 Biomass concentration (g/L) Intracellular N (mg-N/g) Chl/Biomass (OD680/OD750) Lipids (%) a

0.258 ± 0.02 29 ± 2 1.16 ± 0.04 48 ± 2

15

22.5 a

0.415 ± 0.01 36a ± 1 1.22a ± 0.03 34a ± 1

30 a

0.551 ± 0.03 41a ± 3 1.28a ± 0.03 28a ± 2

Denotes statistical significant differences between two sequential pairs of treatment.

87

37.5 a

0.646 ± 0.03 46a ± 2 1.32 ± 0.03 24a ± 2

150 a

0.721 ± 0.03 52a ± 2 1.38 ± 0.05 15a ± 0.8

0.706 ± 0.07 76a ± 3 1.39 ± 0.03 11a ± 0.4

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Table 3 Specific fluxes and phenomenological fluxes of C. vulgaris cultivated in semi-continuous mode with different N concentrations. Values represent the average ( ± SD/average, %). Parameter

M0 Ψo ΦEo ΦDo ΦPAV ABS/RC TRo/RC ETo/RC DIo/RC PIAbs DFAbs ΦPo (or Fv/Fm) a

N concentration N (mg-N/L) 7.5

15

22.5

30

37.5

150

0.618a ± 0.07 0.497a ± 0.07 0.322a ± 0.04 0.352 ± 0.004 921 ± 9 1.898 ± 0.08 1.230 ± 0.05 0.612 ± 0.09 0.668 ± 0.03 0.976 ± 0.22 0.010 ± 0.002 0.648 ± 0.006

0.449a ± 0.02 0.596a ± 0.02 0.404a ± 0.02 0.324a ± 0.02 913 ± 9 1.644a ± 0.05 1.112a ± 0.01 0.663 ± 0.03 0.533a ± 0.04 1.903a ± 0.3 0.279a ± 0.05 0.676a ± 0.02

0.399a ± 0.04 0.629a ± 0.023 0.441a ± 0.02 0.299 ± 0.006 912 ± 9 1.529a ± 0.09 1.072a ± 0.05 0.673 ± 0.01 0.457a ± 0.04 2.642a ± 0.5 0.422a ± 0.08 0.702a ± 0.007

0.332a ± 0.03 0.654a ± 0.02 0.468a ± 0.02 0.285 ± 0.01 917 ± 8 1.341a ± 0.05 0.959a ± 0.04 0.627 ± 0.03 0.382a ± 0.05 3.568a ± 0.5 0.552a ± 0.01 0.715a ± 0.01

0.271a ± 0.02 0.692a ± 0.01 0.505a ± 0.01 0.270 ± 0.01 917 ± 9 1.205a ± 0.02 0.880a ± 0.02 0.608 ± 0.01 0.326a ± 0.01 5.059a ± 0.6 0.704a ± 0.08 0.730a ± 0.01

0.213a ± 0.03 0.738a ± 0.03 0.542a ± 0.02 0.266 ± 0.01 915 ± 9 1.101a ± 0.07 0.808a ± 0.05 0.596 ± 0.04 0.292 ± 0.02 7.179a ± 1.3 0.856a ± 0.15 0.735 ± 0.01

Denotes statistical significant differences between two sequential pairs of treatment.

driving forces for photochemistry [20]. As mentioned before, the Chl fluorescence rise of the OJIP test reflects the kinetics and heterogeneity involved in the reduction of the plastoquinone pool, QA. Among the parameters, M0 expresses the net closing rate of RCs during illumination. As shown in the Table 3, M0 increased as N limitation degree increased, suggesting that the rate of RCs closure increased due to N limitation, i.e. electron transport chain (ETC) was faster reduced under N limitation. However, since M0 is also expressed as M0 = TRo/ RC − ETo/RC, and given that ETo/RC (electron transport beyond QA) didn't change significantly between the different degrees of N limitation (Table 3), it could be concluded that the closure rate of the RCs was increased due to the increase of trapping flux per RC (TRo/RC). Since the closure of RCs is equivalent to the reduction of QA, it implies that N limitation results in a more rapid reduction of QA. Moreover, given that the ETo/RC was not affected by N limitation, the probability and the quantum yields for electron transport beyond QA (Ψ0 and φΕΟ, respectively) were affected only by the higher TRo/RC. This suggests that N limitation may result in a decrease of active RCs, i.e. synthesis of less RCs due to the N limitation. The higher trapped energy from fewer RCs, given that the transfer flux (ETo/RC) was not affected, resulted in higher dissipation of excess energy (as it is expressed from the increased values of ΦDO and DI0/RC). On the other hand, ABS/RC, which can be used as a good indicator of the apparent antenna size, increased as N limitation increased (Table 3). The increase of ABS/RC is considered to reflect inactivation of some RCs [20]. The calculation of the percentage of the RCs that became inactive due to N limitation (=ratio of the ABS/RC of the control to ABS/RC of each treatment) gave the following values: 42%, 33%, 28%, and 9%, for 7.5, 15, 22.5, 30 and 37.5 mg-N/L, respectively. Based on this, N limitation has a negative effect on the active RCs. However, as is shown in Fig. 2 the contribution of inactive PSII RCs, as calculated from the S-states measurements, was not changed significantly between the different degrees of N limitation. These results suggest that the increase of ABS/RC was not due to the inactivation of existing RCs, as it is frequently observed when cells are subjected to various stress conditions, such as high light intensity or temperature [21], but rather from the decrease of the absolute amount of active RCs because under N limitation fewer RCs are synthesized [10]. It is known that under N limitation, chloroplast division is inhibited, leading to a decrease in the amount of thylakoid membrane per chloroplast [26] while the synthesis of protein D1 also decreases, resulting in synthesis of fewer functional RCs [44,45]. Under N limitation the optical absorption cross section also increases [45]. However, it is not clear whether the decrease of Chl content is proportional to the decrease of the synthesis of RCs or whether RC and Chl are decreased to different degrees. ABS/RC could be considered also as a ratio of the RC chlorophyll to Chl of the antenna (Chlant/ChlRC) [46]. Given that total Chl is

Fig. 2. Proportion of the inactive PSII RCs calculated from the S-states measurements (illustrated in the inset panel) of C. vulgaris cultivated in semi-continuous mode with different degrees of N limitation.

the sum of Chlant and ChlRC, our results suggest that under N limitation there is a possible increase of Chl/RC. A probable outcome of the increase of Chlant over ChlRC, along with the decrease of the number of RCs is thus the increase of TRo/RC. Remarkably, the average quantum yield of primary photochemistry (φPAV) was not significantly affected by the variation of N limitation degree. Since the complementary area (from t = 0 to t = TFm) was significantly smaller as N limitation increased (data not shown) the similarity in values of φPAV originated in the shorter time required to reach Fm (Fig. 1). This shorting of TFm reflects that the RCs were closed in a shorter time under N limiting conditions, probable due to the increased TRo/RC. However, the parameters related to PSII performance (PIAbs, DFAbs, and φPo (or Fv/Fm)) were gradually decreased as N limitation degree increased, reflecting the decrease of the potential for biomass growth. These results are in line with the lower biomass concentrations that were observed in the cultures. The decrease of Fv/Fm is frequently obtained in microalgae cultivated under N and/or P limitation [10,17,47]. A change in Fv/Fm however, could be due to a change in the efficiency of photochemical and non-photochemical quenching. As shown in Fig. 3, the qp parameter increased as the degree of N limitation increased. The value of qp is an indicator of the fraction of RCs that are open, i.e. QA in the oxidized state. In Table 4, the half-time (t1/2) and the corresponding amplitude (A) of the three phases of the QA re-oxidation kinetics are shown. The fast phase (related to re-oxidation of QA− by QB) was not affected by N limitation (statistically not significant; p > 0.05), while the medium and slow phases displayed lower t1/2 as N limitation increased. The medium and slow phases reflect the binding of quinone to the QB site and the recombination of 88

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Fig. 3. NPQ and qp of C. vulgaris cultivated in semi-continuous mode with different degrees of N limitation as it was obtained by applying different N concentrations.

QA− with the S2 state of OEC, respectively [26,48]. These faster kinetics of QA re-oxidation may have resulted in a faster re-opening of RCs and thus in higher qp values. In the presence of DCMU, the QA− re-oxidation kinetics are an indicator of the condition of the donor side of PSII [48]. Although there was a trend of QA− re-oxidation kinetics to be slower with increasing N limitation degree (Table 4; Fig. 4), the results were not statistical significant and didn't show any difference between the N limitation degrees. Probably this reflects that the donor side condition (activity or performance) was slightly affected, or even not at all, by N limitation. The lifetime of QA− depends mainly on the rate of forward electron transport and consequently on the redox state of the ETC. As ETC becomes reduced fewer QA− will be re-oxidized [21]. Given that TFm of the OJIP curve (Fig. 1) was shorter it would be expected that under N limitation the ETC would be reduced faster, affecting also the QA− re-oxidation kinetics. Since QA re-oxidation kinetics are probed using a single turnover flash of 50 μs, which is not adequate to fully reduce the ETC, the results suggest that the faster QA− re-oxidation kinetics should be either due to stronger binding of QA− to the QB site or stronger recombination of QA− with the S2 state of OEC. Herrig and Falkowski [45] reported that under N limitation of Isochrysis galbana the quantum requirement for oxygen evolution was increased, which suggest that the faster QA− re-oxidation kinetics was due to a stronger recombination of QA− with the S2 state of OEC, i.e. OEC consumed more energy under N limitation. In overall, given that ETo/RC (Table 3) was not affected significantly from N limitation, the results for QA− reoxidation suggest that N limitation does not negatively affect the capacity for reducing/re-oxidation of PQ. The higher oxidized state of the PSII acceptor and higher qp values under N limitation has been observed also in maize leaves [49]. On the other hand, non-photochemical quenching analysis, revealed that NPQ had a unimodal shape. This suggests that NPQ mechanism(s) were not able to be activated under a strong degree of N limitation,

Fig. 4. QA- re-oxidation kinetics of C. vulgaris cultivated in semi-continuous mode with different degrees of N limitation as it was obtained by applying different N concentrations.

while with increasing N availability and increasing Chl content the NPQ mechanism(s) were able to function and quench (non-photochemically) a significant part of the absorbed energy (given that the ABS/RC still was high; Table 3) while at higher N availability the NPQ values were decreased again because there was no need for quenching (since the cells had the typical biochemical composition under non-stress conditions; Table 2). These results, especially under the highest N limitation degree, might explain the observation of Kolber, et al. [44] that under N starvation the microalga Thalassiosira was susceptible to photoinhibition, because, as it is well known, NPQ provides photoprotection [19].

3.3. Antenna and reducing site heterogeneity As shown in Fig. 5, N limitation did not result in an increase of the proportion of PSIIβ RCs and of QB non-reducing centers (p > 0.05). These results may indicate that under N limitation the antenna heterogeneity was not altered. A probable conclusion that could be drawn is that the decrease of the observed Chl fluorescence yields (see Section 3.2) is not caused by altering the heterogeneity of the RCs, but rather by the decrease of the number of active RCs. The results for heterogeneity are in line with the observation that N limitation did not increase the inactive RCs, but resulted in a decrease in the absolute number of the RCs. Similar results were also observed in Mg/S deficient Spinacia oleracea L. plants, where QB non-reducing centers were not altered due to nutrient starvation [50].

Table 4 QA re-oxidation kinetics parameters. Values represent the average ( ± SD/average, %). N concentration N (mg-N/L) 7.5

15

22.5

30

37.5

150

Without DCMU Fast phase (t1/2, μs) Medium phase (t1/2, ms) Slow phase (t1/2, s) A1/A2/A3 (%)

785 ± 79 5.2 ± 0.4 58 ± 10 41/32/27

656a ± 26 5.8 ± 0.2 76a ± 4 48/30/23

642 ± 6 6.0 ± 0.1 79 ± 1 49/29/22

612a ± 6 6.3 ± 0.4 79 ± 4 51/28/22

640 ± 71 6.9 ± 0.6 89a ± 6 51/28/22

641 ± 77 7.7 ± 0.7 94 ± 8 54/26/20

With 50 μM DCMU Fast phase (t1/2, ms) Medium phase (t1/2, s) Slow phase (t1/2, s) A1/A2/A3 (%)

409 ± 78 2.5 ± 0.2 94 ± 24 11/49/40

364 ± 22 2.1a ± 1 87 ± 17 11/51/38

365 ± 62 2.3 ± 0.1 91 ± 12 11/49/40

336 ± 40 2.1 ± 0.1 82 ± 2 12/49/39

326 ± 29 1.6a ± 0.2 71 ± 8 11/52/37

322 ± 32 1.7 ± 0.4 71 ± 8 11/52/37

a

Denotes statistical significant differences between two sequential pairs of treatment.

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Fig. 5. (a) Proportion of PSIIβ RCs and (b) QB non-reducing centers of C. vulgaris cultivated in semi-continuous mode with different degrees of N limitation as it was obtained by applying different N concentrations.

dissipating the excess energy that cannot be utilized. In scaled-up microalgal cultures, especially in open ponds, the most significant growth limiting factor is light, resulting in a relative low biomass concentration (for open ponds typically 0.3–0.5 g/L). Light limitation occurs due to the low light penetration into the deeper regions of the culture because light is absorbed by the cells in the shallower levels the cultures, or, in the case of algal wastewater treatment systems, because of the turbidity of the wastewater [55–57]. A suggestion to cope with the low light penetration inside cultures is to develop microalgal species with smaller antenna size. i.e. biomass with lower Chl content [58,59]. In typical cells, a normal Chl content can result in higher package effect, decreasing the efficiency of light harvesting per unit of Chl [60]. It has been shown that mutant microalgal strains with smaller antenna sizes have improved light harvesting and higher photosynthetic efficiency because cells with lower Chl content display a lower Chl package effect than in cells with higher Chl content [58,59,61]. It has been hypothesized that cultures with smaller antenna sizes due to nutrient limitation will have a lower shading effect and deeper light penetration and hence will be favored by the higher light availability [17]. However, regarding cells grown under nutrient limitation, although lower Chl content is achieved, it comes along with higher apparent antenna size, lower growth rates, higher energy dissipation or higher respiration and lower Rubisco and C fixation rates [10,60], so the higher light penetration will not advantage higher biomass productivities.

3.4. Overall effect of N limitation on microalgal growth and biomass production Recently, there has been an increasing trend of studying microalgal cultivation under N limitation for the accumulation of target compounds, such as lipids or carbohydrates as feedstock for the production of biofuels or other products. Most of the related studies focus on biomass growth and biochemical composition issues and only few investigate the effect of N limitation on the PSII performance [17,51]. Since changes in the PSII performance reflect changes in the photosynthetic activity, and thus in the biomass growth, probing PSII performance could serve as a tool to understand better why growth is reduced under N limitation. The changes observed in the photosynthetic machinery and in metabolic activity under N limited conditions, may reflect the adjustment of the whole cell organization to maintain balance between reductant supply and demand [10]. Schematically, photosynthetic organisms possess two main process sides, i.e. (i) the photosynthetic machinery which generates and provides electrons (reductants) and (ii) the various metabolic processes that use the produced electrons. The energy absorbed and transported through the light harvesting complex is utilized to drive various processes involved in photochemistry. These two sides under given conditions function in balance [10]. Despite the decrease of Chl content, the results of the present study suggest that there was no heterogeneity alteration under N limitation. Typically, stress conditions that negatively affect PSII result in an increase of inactive RCs and higher contribution of PSIIβ and QB non-reducing centers [16,25,28]. The unvaried properties found here suggest that although the number of RCs decreased, their performance was comparable to that of control cultures. Even so, a decrease in quantum yields (Table 3) along with the decrease of Chl content and biomass growth was observed. It is concluded that N limitation did not affect the overall ability of the RCs to capture light and to transport energy for biomass synthesis (photochemistry) but rather the decrease of the overall PSII activity was due to a higher energy dissipation, a probable outcome of the decreased need for reductant by cells with low N availability and lower metabolic. These conclusions are supported by previous works. Sauer et al. [52] reported that control and N limited cells of the cyanobacterium Synechococcus PCC 7942 displayed the same responses, indicating that the remaining PSII units exhibited nearly normal properties. Moreover, in another work it was concluded that growth of nutrient starved (P and S) plants was not limited by the photosynthetic reactions [53]. It seems that growth is controlled at another level of regulation than photosynthesis [53]. Thus, the effect of N stress is very different from that of other stressors. Under N limitation the functioning of the RCs seems to not be compromised, but that there is mainly a decreased number of synthesized photosynthetic units, instead of inactivation of existing ones, and a probably decrease of demand for reductants due to a decrease of terminal energy acceptors [54]. The results suggest that the energy demand by processes downstream from light harvesting dictates the overall PSII activity by

4. Conclusions N limitation degree resulted, as expected, in a gradual decrease of biomass growth, in accumulation of lipids, and in a decrease of chlorophyll content. Chl fluorescence analysis show that increasing N limitation degree the quantum yields and some of the calculated parameters related with PSII performance decreased mainly due to higher dissipation of excess energy. QA re-oxidation was faster under N limitation which explains the higher qp. QA re-oxidation kinetics in the presence of DCMU revealed that the donor side condition (activity or performance) was slightly/barely affected, or even not at all by N limitation. Moreover, antenna and reducing site heterogeneity was not affected by N limitation, reflecting that even though the number of RCs was decreased, their performance was not affected/compromised. These results suggest that the decrease of PSII performance was due to a decrease in the number of RCs in proportion to N availability with a higher energy dissipation, a probable outcome of the decreased need for reductant by cells due to lower metabolic activity.

Conflict of interest Authors declare that there is no conflict of interest.

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