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Journal of Applied Phycology ISSN 0921-8971Volume 28Number 3 J Appl Phycol (2016) 28:1509-1520DOI 10.1007/s10811-015-0726-y

Impacts of nitrogen and phosphorusstarvation on the physiology ofChlamydomonas reinhardtii

Manoj Kamalanathan, MattiaPierangelini, Lauren Ann Shearman,Roslyn Gleadow & John Beardall

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Impacts of nitrogen and phosphorus starvationon the physiology of Chlamydomonas reinhardtii

Manoj Kamalanathan1& Mattia Pierangelini1 & Lauren Ann Shearman1

&

Roslyn Gleadow1& John Beardall1

Abstract The importance of algae-derived biofuels has beenhighlighted by the current problems associated with fossilfuels. Considerable past research has shown that limiting nu-trients such as nitrogen and phosphorus increases the cellularlipid content in microalgae. However, limiting the supply ofnutrients results in decreased biomass, which in turn decreasesthe overall lipid productivity of cultures. Therefore, nutrientlimitation has been a subject of dispute as to whether it willbenefit biofuel production on an industrial scale. Our researchexplores the physiological changes a cell undergoes whenexposed to nitrogen and phosphorus limitations, both individ-ually and in combination, and also examines the biotechno-logical aspects of manipulating N and P in order to increasecellular lipids, by analyzing the lipid production. We showthat nitrogen starvation and also nitrogen plus phosphorusstarvation combined have a more profound effect on the phys-iology and macromolecular pools of Chlamydomonasreinhardtii than does phosphorus starvation alone. The photo-synthetic performance of C. reinhardtii underwent drasticchanges under nitrogen starvation, but remained relatively un-affected under phosphorus starvation. The neutral lipid con-centration per cell was at least 2.4-fold higher in all thenutrient-starved groups than the nutrient-replete controls, butthe protein level per cell was lower in the nitrogen-starved

groups. Overall, nitrogen starvation has a more dramatic effecton the physiology and neutral lipids and protein levels ofC. reinhardtii than phosphorus starvation. However, the levelof total lipids per volume of culture obtained was similaramong nutrient-replete and all of the nutrient-starved groups.We conclude that combined nitrogen and phosphorus starva-tion does not likely benefit biofuel production in terms ofenhanced lipid or biomass production.

Keywords Nitrogen . Phosphorus . Microalgae . Biodiesel .

Photosynthesis

Introduction

The production of biofuels from algal biomass represents apotential alternative source to the currently used fossil fuels,stocks of which are rapidly becoming depleted (Chisti 2007;Brennan and Owende 2010). In the past few years, severalstudies and reports have been published on the optimizationof biofuel production by algae. In some microalgae, lipids area major macromolecular pool, which can be converted intobiofuels (Hu et al. 2008; Benemann 2008). Many studies fo-cusing on biofuel production have shown that inducing nutri-ent limitation (nitrogen and phosphorus limitation, hereafterreferred to as N and P limitation) can havemajor influences onoleogenesis and cell lipid content (El-Sheek and Rady 1995;Gardner et al 2011; Yeh and Chang 2011; Zhang et al. 2013).Even though these studies show that the lipid content per cellincreases on nitrogen and phosphorus limitation, the maxi-mum biomass achievable was lower under these conditions.Inducing N or P limitation may interfere with the synthesis ofimportant cellular components, thereby limiting the ability ofcells to synthesize sufficient resources for cell multiplication.The negative effects of nutrient limitation, especially nitrogen,

Electronic supplementary material The online version of this article(doi:10.1007/s10811-015-0726-y) contains supplementary material,which is available to authorized users.

* Manoj Kamalanathanmanoj.kamalanathan@monash.edu

1 School of Biological Sciences, Monash University,Clayton, Victoria 3800, Australia

J Appl Phycol (2016) 28:1509–1520DOI 10.1007/s10811-015-0726-y

Received: 15 June 2015 /Revised and accepted: 28 September 2015 /Published online: 10 October 2015# Springer Science+Business Media Dordrecht 2015

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on the photosynthetic apparatus and other cellular macromol-ecules are thus believed to affect the overall growth of thealgae, directing any carbon assimilated to increase the cellularlipid content.

Most of the published reports focusing on the manipulationof nutrient limitation to obtain high lipid production have fo-cused on either nitrogen or phosphorus limitation alone, andhave not considered the combined effects of applying both Nand P limitation together (Terry et al. 1985; Kilham et al.1997; Praveenkumar et al. 2012; Chu et al. 2014). AlthoughLiebig’s law of the minimum suggests that only one (the mostlimiting) nutrient will be growth-limiting at any one time(Browne 1942), recent work suggests that co-limitation bytwo or more nutrients occurs naturally, especially betweennitrogen and phosphorus (Elser et al. 2007; Harpole et al.2011). Moreover, the effects of the combined limitation bythese nutrients on the photosynthetic apparatus and perfor-mance have been virtually ignored, mostly due to the assertionof Liebig’s law of the minimum. However, there is some ev-idence indicating the occurrence of co-limitation in algae,which in turn affects primary productivity (Shaked et al.2006; Wyatt et al. 2010; Harpole et al. 2011). Therefore, thepossibility of synergistic effects of combined nitrogen andphosphorus starvation on lipid synthesis has become an areaof interest. It is important to understand the physiological im-pacts of individual and combinedN and P starvation to be ableto better exploit its use for biofuel production. Therefore, thisstudy focuses on the effects on macromolecular pool produc-tion and the photosynthetic apparatus of Chlamydomonasreinhardtii under both individual, N or P, and combined (Nand P) limitation. The study will help in understanding thephysiological reasons behind lower biomass and macromole-cule productivity caused by inducing both individual andcombined N and P limitation.

Materials and methods

Chlamydomonas reinhardtiiCS-51 cultures obtained from theAustralian National Algae Culture Collection (ANACC),CSIRO,were grown under continuous illumination at 60μmolphotons m−2 s−1 at a temperature of 18 °C in the MLA medi-um (Bolch and Blackburn 1996). Axenic cultures (500 mL in1000-mL flasks; n=3) were inoculated in control MLA medi-um to obtain an initial cell density of 2.25×104 cells mL−1 andallowed to grow until exponential phase (day 4). The cultureswere then split into four parts. The first part was kept in thecontrol conditions and the other three were resuspended inMLAmedia with either no source of phosphorus (dipotassiumphosphate), or no source of nitrogen (sodium nitrate), or nosource of either nitrogen or phosphorus. Cultures were thenallowed to grow until they reached stationary phase (day 8).

Analysis of growth parameters Cell numbers were mea-sured by microscopy using an improved Neubauer hemocy-tometer. Growth rates (μ) were calculated using the averageslope of the plot of logn of cell numbers per milliliter versustime, during the exponential phase. The total dry biomass perliter was determined by filtering a known volume of algalculture through a 25-mm-diameter pre-weighed GF/C filter(GE Healthcare-1822-025), followed by 60-min incubationat 100 °C and re-weighing of the filter.

Analysis of growth medium components N and P concen-trations and pH in the culture mediumwere measured through-out the course of the experiment. A bench top pH meter(Jenway-3510) was used to measure the pH of the medium.N concentrations were determined by measuring the nitratelevels in the media as described by Cataldo et al. (1975)).Culture was centrifuged at 3000×g for 10 min and a sample(5 μL) was incubated with 20 μL 5 % salicyclic acid for20 min. The absorbance readings were taken at 410 nm afterthe addition of 300 μL 3 M NaOH to the solution. P concen-trations were determined by the methods described by Chenet al. (1956) and Christianson and Dunham (2005). Then 10 %ascorbic acid, 2.5 % ammonium molybdate, and 6 N sulfuricacid were added to the supernatant and the solution incubatedfor 90 min at 37 °C. The absorbance was then read at 820 nm.

Analysis of photosynthetic performance Chlorophyll a andb, and carotenoid levels were determined using the same ace-tone extract derived from treating the pellet of 5 mL culturecentrifuged at 3000×g for 10 min with 5 mL of 90 % acetone.This was followed by overnight extraction at 4 °C in completedarkness and centrifugation of the suspension at 3000×g for10 min at 4 °C. The pellet obtained in this procedure was usedfor protein analysis. The absorbance of the supernatant wasread at 630, 647, 664, and 750 nm to measure chlorophyll aand b levels and at 480 nm to measure carotenoids. Chloro-phyll and carotenoid concentrations were then calculatedusing the equations described by Jeffrey and Humphrey(1975) and Jensen (1978), respectively.

The photosynthetic parameters like maximum PSII quan-tum yield (Fv/Fm), relative electron transport rates (rETRmax),light harvesting efficiency (α), and light saturation parameter(Ek) were measured using a pulse amplitude modulated fluo-rescence system (PHYTOPAM,Walz, Germany), operated bya Phyto-Win software (version 2.13). The samples were darkadapted for 15 min prior to measurement. The built-in fittingfunction was used to deriveFv/Fm, rETRmax,α, and Ek values.

Chlorophyll polyphasic fluorescence induction (FI) curveswere generated using a double-modulation fluorometer (Pho-ton Systems Instruments, Czech Republic). For the purpose,the wizard PEA built-in software FluorWin was used (Hill andRalph 2008). OJIP fluorescence transients (Strasser andGovindjee 1992) were measured by applying a 6-s flash at

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the 80 % light intensity setting (>15×106 μmol photons(PAR) m−2 s−1) to a sample (cells taken from exponentialphase of growth on day 6), dark-acclimated for at least5 min (Perreault et al. 2009). In order to standardize the re-sults, the fluorescence values from the OJIP transients werenormalized to the minimal fluorescence at the O step (50 μs)as described in Petrou et al. (2011). OJIP transients were an-alyzed on the basis of FO=fluorescence at 50 μs, FJ=fluores-cence at 2 ms, FI=fluorescence at 3–40 ms, and FP=fluores-cence at 100–350 ms, using the equations reported in Zhanget al. (2010) and Strasser et al. (2000). The fluorescence am-plitude of the IP step (ΔFIP) was calculated according toCeppi et al. (2012). Non-photochemical quenching (NPQ)was calculated from the decline of fluorescence after the Pstep up to 6 s, according to the equation reported in Antalet al. (2009). A full list of the fluorescence parameters exam-ined is given in Table 2.

Analysis of neutral lipids and proteins The Nile Red fluo-rescent dye (Sigma-Aldrich) was used to determine the rela-tive neutral lipid levels in cells throughout the course of theexperiment as described by Cooksey et al. (1987). A DC Pro-tein Assay kit (BIO-RAD) was used to determine the totalprotein content for each treatment group.

Statistical analysis The results were statistically analyzed byusing one-way ANOVA with multiple comparisons of themean of each group with the mean of every other group usinga Tukey test. These statistical analyses were performed usingGraphPad Prism software (version 6.0f).

Results

Growth analysis

The effects of N and P limitation on the growth ofC. reinhardtii are shown in Fig. 1, Table 1, and SupplementaryFig. 1. The final cell numbers in the nutrient-replete cultureswere significantly higher than in the N (p<0.0001), P(p<0.0001), and NP (p<0.0001) starved cultures (Fig. 1;Table 1), with the N- and NP-starved groups yielding lowercell numbers than the P-starved and control group. Althoughthe P-starved cells had similar growth rates to the controlgroup, the growth rates were considerably lower for both N(p=0.0066) and NP (p=0.0025) starved cultures than for con-trol group cells (Table 1). The maximal dry biomass values(Supplementary Fig. 1) were significantly lower for P (p=0.0497), N (p=0.0497), and NP (p=0.0236) starved culturesthan they were for the control group. To understand how Nand P starvation independently affected growth, we measuredboth extracellular conditions such as pH, nitrate, and phos-phate levels of the medium during growth and physiological

aspects including photosynthetic parameters and the macro-molecular pools of the cells.

Analysis of growth medium components

The pH of both N- and NP-starved cultures fluctuated aroundneutral values with an average pH of 7.4 (SupplementaryFig. 2). However, the pH of the P-starved cultures becamemore alkaline as growth progressed and followed the trendobserved for the control treatment (p=0.9956). The nitrateand phosphate measurements indicated that the concentrationsof nitrate in the N- and NP-starved cultures (Fig. 2) and ofphosphate in the P- and NP-starved cultures (Fig. 3) werevirtually zero from day 4 onwards. This confirms that all threegroups were truly starved of extracellular nutrients. The rate ofdecline of nitrate and phosphate levels is an indication of therate of nitrate and phosphate uptake by the cells. Therefore, anindirect measure of the rate of nitrate and phosphate uptakecan be obtained by calculating the slope of decline in thenitrate and phosphate levels in the media. The nitrate uptakerates calculated in this way showed that the uptake rates weresimilar between the control (−0.09±0.01 day−1) and P-starved(−0.09±0.02 day−1) groups. On the other hand, the rate ofphosphate uptake was significantly different (p=0.0248) be-tween the control (−128.32±27.29 day−1) and N-starvedgroups (−49.91±27.51 day−1).

Analysis of photosynthetic parameters

The levels of chlorophyll a per cell (Fig. 4a) in control and P-starved treatments were significantly higher than under both N(p<0.0001) and NP starvation (p<0.0001). In contrast, the P-

Fig. 1 Average cell number±standard deviation for C. reinhardtii undercontrol, with no phosphorus, with no nitrogen, and no nitrogen andphosphorus groups (n=3)

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starved cells had a similar chlorophyll a content to controlcells. The cellular levels of carotenoids showed a similar trendto that for chlorophyll a (Fig. 4b). Both N- and NP-starvedgroups had significantly lower carotenoids than the control(p<0.0001) and P-starved groups (p<0.0001), which had sim-ilar carotenoid levels to one another. On the other hand, thecarotenoids/chlorophyll a ratio is significantly higher underN-starved (p=0.00001) and NP-starved (p=0.00002) cells rel-ative to controls, due to the much reduced chlorophyll a con-centration. P-starved cells also had significantly lowercarotenoids/chlorophyll a in comparison with N-starved (p=0.0003) and NP-starved (p=0.0005) cells (Fig. 4c).

Multiple photosynthetic parameters, such as Fv/Fm whichis the maximum quantum yield of PSII, also widely consid-ered as stress indicator, rETRmax which indicates a relativemeasure of electron transport rates through PS II, α, whichis a measure of the light harvesting efficiency, and Ek, whichindicates the light saturation parameter, were derived with aWalz PhytoPAM fluorometer (Cosgrove and Borowitzka2011, Masojídek et al. 2011). The Fv/Fm values were signifi-cantly lower in cells starved with N (p=0.0012) and NP (p=0.0302) compared to the control (Fig. 5a), whereas the P-starved group had similar Fv/Fm values to the control. The αvalues were significantly lower in the N-starved (p=0.0003)and NP-starved (p=0.0059) cells as compared to the control.The P-starved group had significantly higher α values relativeto N-starved (p=0.0002) and NP-starved groups (p=0.0049).

On the other hand, the α values of P-starved cells were verysimilar to the control group (Fig. 5b). The Ek values of thecontrol group cells were significantly lower compared to bothN-starved (p<0.0001) and the NP-starved group (p=0.0002)(Fig. 5c). A similar phenomenon was observed with the P-starved group, with lower Ek values as compared to N-starved (p<0.0001) and NP-starved (p<0.0001) cells. Alsothe Ek values of P-starved cells were slightly lower than thecontrol group. However, the rapid light curves revealed thatthere were no significant differences in the rETRmax valuesamong all the treatments (Fig. 5d).

The analysis of the polyphasic fluorescence intensity (FI)measured for the four experimental conditions (Fig. 6) is con-sistent with the modifications of the photosynthetic apparatusdescribed above and shows that the differences in the O-J-I-Psteps are mainly driven by the N availability in the culturemedium. This is also represented by the extrapolated parame-ters describing the physiological characteristics of PSII andwhich are reported in Table 2. The Vj parameter, describingthe rise of initial slope of the polyphasic FI, was higher for theN- and NP-starved groups with respect to the control and P-starved groups (p<0.0001). TheMO parameter, describing thenet rate of reaction center’s closure, was higher for the N- andNP-starved groups with respect to the control and P-starvedgroups (p<0.0001). The ABS/RC parameter, describing the

Table 1 Average final cell numbers and growth rates (μ) from day 5 to 8 (±standard deviation) for C. reinhardtii under control, with no phosphorus,with no nitrogen, and no nitrogen and phosphorus groups (S=3) [significant differences are indicated by different letters (a, b, and c)]

Control (±SD) No phosphorus (±SD) No nitrogen (±SD) No nitrogen and phosphorus (±SD)

Final cell numbers×105.mL−1 5.72 (±0.43)a 3.75 (±0.28)a 2.7 (±0.102)a 2.6 (±0.108)a

μ (day−1) 0.5 (±0.04) 0.45 (±0.01) 0.35 (±0.02)b 0.33 (±0.04)b

Fig. 2 Average nitrate levels in the media±standard deviation forC. reinhardtii under control, with no phosphorus, with no nitrogen, andno nitrogen and phosphorus groups (n=3)

Fig. 3 Average phosphate levels in the media±standard deviation forC. reinhardtii under control, with no phosphorus, with no nitrogen, andno nitrogen and phosphorus groups (n=3)

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absorption flux per reaction center, was also higher for the N-and NP-starved groups with respect to the control and P-starved groups (p=0.0001). Other parameters like TRO/RC,ETO/RC, and DIO/RC were also higher in N- and NP-starvedgroups relative to the control and P-starved groups (p<0.0001,p=0.0005 and 0.0006). However the Fv/Fm values obtainedfrom the OJIP curve were lower for N- and NP-starved groupsrelative to the control and P-starved groups (p=0.0003), sim-ilar to the Fv/Fm values obtained by the PAM fluorometer.

Analysis of proteins and neutral lipids

Protein measurements showed that at the end of the experi-ment, both the N- and NP-starved groups had significantlylower protein/cell levels as compared to the control(p<0.0001 and <0.0001) and the P-starved group (p<0.0001and 0.0001) (Fig. 7). Nile Red analysis of neutral contentshowed that all the N- and P-starved groups had significantlyhigher neutral lipid/cell content as compared to the controlgroup (p<0.0001) (Fig. 8). A gradual decrease in both pro-teins and neutral lipid/cell through time was observed; thiscould be possibly due to the dilution effect caused by cellularmultiplication.

To understand the biotechnological aspect of N and P star-vation, we have also expressed the Nile Red analysis resultson a per milliliter culture basis instead of per cell, and thisindicated that the total neutral lipid per milliliter was similaracross all the groups (Fig. 9). This suggests that although thenutrient limitation increases the neutral lipid content of thecell, it also decreases growth, resulting in lower cell number,each with higher neutral lipid content. On the other hand,growing cultures in nutrient-replete conditions leads to highercell number each with lower neutral lipid content, resulting insimilar overall neutral lipid levels as the nutrient-limitedgroup. Therefore, N and P starvation would not benefit biofuelproduction in terms of lipid productivity. However, it is ex-pected that NP starvation will lead to a lower use of totalnitrate and phosphate, which in turn should reduce the costof media production.

Discussion

Nutrient limitation has been a widely used strategy for increas-ing the lipid content of microalgae; however, its effect on thephotosynthetic physiology of the organism is virtually ig-nored. Here, we studied the effect of nitrogen and phosphorus

�Fig. 4 a Average chlorophyll a cell−1±standard deviation; b averagecarotenoids per cell±standard deviation; c average carotenoidschlorophyll a−1±standard deviation for C. reinhardtii under control,with no phosphorus, with no nitrogen, and no nitrogen and phosphorusgroups (n=3)

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starvation, both individually and in combination, on the phys-iology of C. reinhardtii. This microalga has been extensivelystudied and is considered as the model organism for photo-synthetic studies. Although the oleaginicity ofC. reinhardtii islimited, testing conditions that stimulate lipid production inthis alga only increases the robustness of the experiment. Wealso carried out a cost analysis to see whether such strategiesof nutrient starvation are economically viable on an industrialscale. The growth was reduced in the nutrient-starved groups,similar to our previous work studying the role of phosphorusand the use of the N-assimilation inhibitor MSX (unpublisheddata), where both phosphorus and nitrogen starvation affectedthe growth of C. reinhardtii, and many other reports (Singhand Kumar 1992; El-Sheek and Rady 1995; Illman et al. 2000;Zhila et al. 2005; Solovchenko et al. 2008; Ördög et al. 2011).A similar growth pattern was found between the N- and NP-starved treatments, with both groups showing the lowestgrowth rate, suggesting that nitrogen starvation had more se-vere effects on cell multiplication than phosphorus starvation.

The growth medium components and the photosyntheticparameters were analyzed to understand the effects of nutrientstarvation. The unchanged pH in N- and NP-starved condi-tions could be reflective of the limited photosynthesis in thosecultures while the high pH under P starvation could be relatedto higher photosynthetic activity, which led to CO2 depletionin the culture medium (Hofslagare et al. 1983, 1985; Axelsson1988). The external phosphate levels in the medium suggestedthat P starvation does not affect nitrate uptake by the P-starved

cells. However, the absence of nitrogen had a considerableeffect on the phosphate uptake rate of the N-starved cells. Thismay contribute to the slower growth rate of the N-starvedcells. Overall, the extracellular measurements indicated thatnitrogen starvation had a more severe effect than phosphorusstarvation on growth, by affecting the phosphate uptake abilityand also possibly by lowered photosynthetic activity asreflected in the pH measurements.

Fig. 5 a Average maximumquantum yield of PSII values±standard deviation; b averagelight harvesting capacity alpha(α)±standard deviation; c averagelight saturation parameter (Ek)±standard deviation; d averagerelative electron transport ratevalues±standard deviation forC. reinhardtii under control, withno phosphorus, with no nitrogen,and no nitrogen and phosphorusgroups (n=3)

Fig. 6 O-J-I-P transients analyzed on cells of C. reinhardtii undercontrol, with no phosphorus, with no nitrogen, and no nitrogen andphosphorus groups. Results are the average of measurements performedon three independent culture replicates

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The chlorophyll levels were lower in N- and NP-starvedgroups. This is commonly observed in studies with N starva-tion in algae (Terry et al. 1985; Kolber et al. 1988; Singh andKumar 1992; Latasa and Berdalet 1994; Berges et al. 1996b).5-Aminolevulinic acid is a precursor of chlorophyll which issynthesized either by condensation of succinate and glycine ordirectly from glutamate (Beale and Castelfranco 1973; Oh-hama et al. 1982; Fufsler et al. 1984). The limited supply ofnitrogen in N- and NP-starved cells might affect the cells’ability to synthesize amino acids like glycine and glutamate,limiting the synthesis of 5-aminolevulinic acid that in turnleads to lower chlorophyll levels in the cell. Similar to chlo-rophyll, carotenoid levels were lower in the N- and NP-starved group; this suggests that it is the availability of nitro-gen that affects the level of carotenoids in the cell. As ob-served here, many other studies have shown lower carotenoidlevels under nitrogen limitation (Li et al. 2012; Kim et al.2013). However, the chlorophyll/carotenoids ratio was higherin the N- and NP-starved groups; this is consistent with mostof the nitrogen limitation studies with algae (Berges et al.

1996; Masojídek et al. 2000; Pirastru et al. 2012). The de-crease in carotenoid level may be due to the limiting step ofdimerization of geranyl-pyrophosphate as suggested byRichmond (1986). Also, the light intensity used in our exper-iment (60 μmol photons m−2 s−1) was not high enough toinduce higher photoprotective carotenoid production (Sagarand Briggs 1990; Vidhyavathi et al. 2008; Couso et al.2012). Kim et al. (2013) clearly showed that higher light in-tensities accompanied by N limitation usually lead to highercarotenoid levels, whereas lower light intensities with N lim-itation caused a decrease in total carotenoid levels. The ob-served increase in carotenoids/chlorophyll a ratio may be dueto the large decrease in chlorophyll a levels. The observeddecrease in pigments specifically in N- and NP-starved cells,and not in P-starved cells, clearly indicates that nitrogen isessential for chlorophyll and carotenoid synthesis.

Fv/Fm (PhytoPAM) values clearly show that N starvationand not P starvation was the main stressor in our experiments.As discussed above, nitrogen and not phosphorus is needed

Table 2 Parameters extrapolated from the O-J-I-P transients (±standard deviation) and calculated according to the equations of *Zhang et al. (2010)),+Ceppi et al. (2012) and †Antal et al. (2009)) [significant differences are indicated by different letters (a, b, and c)]

Control No phosphorus No nitrogen No nitrogen and phosphorus

*Fv/Fm Maximum quantum yield 0.68 (0.01)a 0.72 (0.01)a 0.57 (0.05)b 0.56 (0.03)b

*MO Initial slope of the fluorescence transient 0.58 (0.051)a 0.49 (0.015)a 1.17 (0.075)b 1.09 (0.12)b

*Vj fluorescence at the J-step 0.52 (0.01)a 0.49 (0.01)a 0.62 (0.03)b 0.57 (0.02)c+ΔFIP Fluorescence amplitude of the I-P step 0.45 (0.02)a 0.54 (0.04)b 0.27 (0.03)c 0.3 (0.02)c, d†NPQ Non-photochemical quenching 1.7 (0.05)a 2 (0.3) 2.2 (0.3) 2.3 (0.2)b

*ABS/RC Absorbed flux per PSII RC 1.6 (0.2)a 1.4 (0.04)a 3.3 (0.5)b 3.4 (0.4)b

*TRO/RC Trapped flux per PSII RC 1.1 (0.1)a 1 (0.01)a 1.9 (0.1)b 1.9 (0.2)b

*ETO/RC Electron transport flux per PSII RC 0.5(0.04)a 0.5(0.01)a 0.7(0.1)b 0.8(0.03)b

*DIO/RC Dissipated flux per PSII RC 0.5 (0.06)a 0.4 (0.03)a 1.5 (0.4)b 1.5 (0.3)b

Fig. 7 Average protein cell−1±standard deviation for C. reinhardtiiunder control, with no phosphorus, with no nitrogen, and no nitrogenand phosphorus groups (n=3)

Fig. 8 Average relative neutral lipid cell−1±standard deviation forC. reinhardtii under control, with no phosphorus, with no nitrogen, andno nitrogen and phosphorus groups (n=3)

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for the synthesis of chlorophyll and other components of thephotosynthetic apparatus; therefore, N starvation has a severeand immediate effect on the Fv/Fm values. Lower Fv/Fm

values for N-starved cells are commonly observed in manystudies (Richardson et al. 1969; Kolber et al. 1988; Bergeset al. 1996; Gordillo et al. 1998; Berden-Zrimec et al. 2008).Our previous data has shown that phosphorus starvation doesnot affect the photosynthetic apparatus (Kamalanathan et al.2015). Also, Chlamydomonas has been shown to store a largeinternal storage pool of phosphorus in the form ofpolyphosphates (Siderius et al. 1996; Eggink et al. 2012;Aksoy et al. 2014), which might be sustaining the phosphorusdemand when availability of P in the environment is low.These factors might account for the absence of any effect seenon Fv/Fm values in the P-starved cells.

The light harvesting complex of chlorophytes mostly com-prises chlorophyll a and b (Durnford et al. 1999). The lower αvalues in N- and NP-limited groups might thus be related tothe lower chlorophyll a/cell content in these cells. This sug-gests that the ability of cells starved with nitrogen to harvestlight in low light conditions might be poor compared to that ofcontrol or P-starved cells. The Ek value represents the onset oflight saturated photosynthesis (Sakshaug et al. 1997;Pierangelini et al. 2014) and is derived from the ratio ofrETRmax to alpha. The higher Ek value of both N- and NP-starved cultures seems to be influenced by lower alpha values.Similarly, rETRmax values and differences in cellular pig-ments, Fv/Fm, α, and Ek values between the N-starved groups(N- and NP-starved) and the non-N-starved groups (controland P-starved) suggest that N starvation causes changes in thelight harvesting apparatus that maintain a reduced influx ofenergy through light capture despite the restricted capacity tosynthesize the pigments and proteins necessary for the photo-synthetic machinery.

A slightly higher Vj was also found for the N-starved cellswith respect to NP-starved cells. The initial rise of the FI (O-J)depends on the reduction of QA, the primary acceptor of PSII(Boisvert et al. 2006). A rapid non-sigmoidal increase of theO-J step has been related to physiological characteristics suchas a low energetic connectivity between PSII units and a highabsorption cross-section of PSII antennae (Antal et al. 2009).Thus, similarly in C. reinhardtii, the more rapid and less sig-moidal O-J fluorescence rise for cells grown in the N- and NP-starved condition (higher Vj andMO) could suggest that underN limitation, the PSII units are energetically isolated and witha large absorption cross-section of their antenna (Lavergneand Trissl 1995; Trissl 2003; Antal et al. 2009; Solovchenkoet al. 2013). However, the increase of absorption cross-sectionof PSII antenna indicated by the O-J step is contradicted by thelower Chl a cell content and the expectation therefore ofsmaller PSII antennae. Moreover, cells exposed to N stresscan mobilize molecules of Chl a and other pigments as aninternal source of N, resulting in a decrease of photosyntheticunit size (Perry et al. 1981). Thus, as suggested by the highervalues of ABS/RC, the O-J steps could reflect only an appar-ent increase of PSII antenna size, attributed to eitherinactivated PSII reaction centers which transfer their antennato the remaining active PSII or changes in the PSIIα/β-centersheterogeneity (Falkowski and Kolber 1995; Strasser et al.2000; Zhang et al. 2010; Stirbet 2011; de Marchin et al.2014). In our case, the shape of the O-J step is not indicativeof a defined change of PSIIα/PSIIβ heterogeneity. However,the increase of the apparent PSII antenna size can be associ-ated with the inactivation of PSII centers, which can be in-ferred from the lower Fv/Fm (measured both with PhytoPAMand FI) inC. reinhardtiiN-starved cells with respect to controland P-starved conditions (p=0.0003). The remaining activePSII seems to have higher ability to trap excitation (TRO/RC), transferring electrons from QA to QB (ETO/RC) and dis-sipating excitation (DIO/RC) (Zhang et al. 2010; Stirbet andGovindjee 2011). In keeping with the changes in the O-J rise,C. reinhardtii cells exposed to N and NP starvation also dif-fered in the J-I-P steps. Compared to other experimental con-ditions, fluorescence intensity for the J-I-P steps declines inmicroalgal cells exposed to higher irradiances (Petrou et al.2011). A decline of the P step was also registered for N-depleted microalgal cells (Petrou et al. 2012). In addition,Solovchenko et al. (2013) reported that changes in O-J-I-Punder different light treatments and N-deprived cells couldbe related to changes in pigment (Chl a and carotenoids) com-position. Thus, the lowering of the J-I-P portion of FI curves inC. reinhardtii cells under N- and NP-starved conditions, be-sides the inactivation of PSII, could be attributed to the de-crease of Chl a inside the cells. Overall, the photosyntheticmeasurements indicated that the N- and NP-starved cells wereseverely affected in photosynthetic apparatus and perfor-mance, whereas the P-starved cells behaved more like control

Fig. 9 Average relative neutral lipid mL−1±standard deviation forC. reinhardtii under control, with no phosphorus, with no nitrogen, andno nitrogen and phosphorus groups (n=3)

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group cells. This further supports the notion that nitrogen star-vation has a more severe effect than phosphorus starvation onthe photosynthetic physiology of C. reinhardtii.

The protein levels were lower in N- and NP-starved groups,which is consistent with many other N-limitation studies(Berdalet et al. 1994; Kilham et al. 1997) and was not surpris-ing as (a) nitrogen is essential for protein synthesis and (b)cells might start degrading proteins in order to satisfy othercellular nitrogen requirements. On the other hand, the P-starved group had similar protein levels to the control groupcells. A similar observation was seen in a study carried out byBerdalet et al. (1994), which suggested a reduced number ofribosomes could sustain the observed protein synthesis underP starvation provided nitrogen is still available.

The neutral lipid per cell of all the nutrient-starved groupswere higher; this is consistent with most of the nutrient limi-tation studies carried out to date (Tornabene et al. 1983; El-Sheek and Rady 1995; Kilham et al. 1997; Zhila et al. 2005; Liet al. 2008; Converti et al. 2009; Stephenson et al. 2010; Yehand Chang 2011; Uslu et al. 2011; Ördög et al. 2011;Praveenkumar et al. 2012; Zhang et al. 2013; Ito et al. 2013;Chu et al. 2014). Except for the last day of the study, all thenutrient-limited treatments had similar cellular neutral lipidcontent. Nutrient limitation usually leads to slower growth rateand eventually growth arrest or death of the cells. At the sametime, cells continue to photosynthesize and energy and fixedcarbon are available for neutral lipid synthesis. Therefore, wesee an increase in neutral lipids on a cellular level. On the lastday of the experiment, we observed a higher cellular neutrallipid content in the N- and NP-starved groups in comparisonto both P-starved and control group. There are two possibleexplanations for this; firstly, the P-starved cells were still mul-tiplying whereas the N- and NP-starved groups had nearlystopped. Secondly, nitrogen limitation could have led to rear-rangement of intracellular macromolecular pools, leading tomobilization of the carbon from proteins to neutral lipid syn-thesis under N and NP starvation as clearly observed in aprevious infrared spectroscopic study (Giordano et al. 2001).

Both measurements of extracellular parameters (pH, nutri-ent levels) and the physiological analysis showed that N- andNP-starved cells behaved in a similar fashion and P-starvedcells behaved more closely to control cells. This clearly sug-gests that the effects of nitrogen starvation on C. reinhardtiiare more severe and dominant than the effects of phosphorusstarvation and that there was no synergistic effect of N and Pco-limitation. However, even though P-starved cells behavedlike the nutrient-replete group, the biomass yield was stillsignificantly lower. Though there was no difference observedin the measured parameters between control and P-starvedcells, phosphorus is needed for nucleic acid synthesis, cellularenergy currency ATP, reducing equivalents like NADPH andmembrane phospholipids (Holm-Hansen 1970; Berdalet et al.1994). These processes may have been affected under

phosphorus starvation, which may have affected the cells’ability to multiply and grow at the same rate as the controlgroup, thereby resulting in relatively lower biomass.

In conclusion, nutrient limitation has been a widely usedand accepted way of increasing the lipid content of the algalcells for biofuel production. Here, we studied the effects ofnutrient limitation (both nitrogen and phosphorus individuallyand in combination) on the physiology of the cells. We foundthat nitrogen limitation has a severe effect on the photosyn-thetic apparatus of the cells. This limits the growth of cells andhence the total amount of neutral lipids that can bemade underN starvation.

Phosphorus starvation resulted in similar neutral lipid levels tothe N-starved cells and no significant improvement on the max-imal biomass of the P-starved cultures over N-starved cultures.The biomass yield was clearly limited by phosphorus, but theneutral lipid production and photosynthetic ability was not asthese parameters remained high and comparable to those ofnutrient-replete cultures. Although relatively higher neutral lipidcontent was expected under the combination of both NP starva-tion, there was no significant difference in the cellular neutrallipid levels in relation to the individual N and P starvation effects.The effect of N starvation on all the photosynthetic parametersmeasuredwasmore dominant than P starvation in theNP-starvedcells. As a result, the NP-starved cells behaved more like N-starved cells in all respects.

The total neutral lipid produced was, however, similar be-tween the nutrient-limited and the nutrient-replete group; thisis mainly due to the difference in biomass and cellular neutrallipid content. The nutrient-limited group resulted in highercellular neutral lipid content but lower biomass, whereas thenutrient-replete cells resulted in lower cellular neutral lipid buthigher biomass, therefore producing similar total neutral lipidlevels among the different treatments. Therefore, using nutri-ent limitation is not necessarily beneficial in terms of totallipid production but does allow the use of lower levels ofnitrogen and phosphorus, and that might decrease the cost ofthe production for nitrogen and phosphate for growth media.Also, nutrient limitation results in lower biomass with higherneutral lipid content, hence decreasing the amount of biomassto be treated in downstream processing, saving the cost, time,and labor involved. At the same time, if the biomass is to beused for other purposes like animal and fish feed, and fertilizerproduction, then nutrient limitation may not be the best way ofgrowing cells as it leads to lower cellular protein levels.

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