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- Environmental Advances 4 (2021) 100070
Contents lists available at ScienceDirect
Environmental Advances
journal homepage: www.elsevier.com/locate/envadv
Copper affects photosynthetic parameters of N- or P-limited
Ankistrodesmus densus
Giseli Swerts Rocha∗, Evaldo L.G. Espíndola
NEEA/CRHEA, São Carlos School of Engineering, University of São Paulo (USP), Avenida Trabalhador Sãocarlense, 400, Parque Arnold Schmidt, CEP 13566-590, São
Carlos, SP, Brazil
a r t i c l e i n f o a b s t r a c t
Keywords: Algae require micro- and macronutrients for optimal growth and metabolism. Under limitation or excess of the
Metal nutrients in the environment, they can adapt their photosynthetic machinery to cope with the new concentrations
Nitrogen available to decrease damage to their performance. In the present study, to evaluate a microalga’s responses to
Phosphorus
a macronutrient’s limitation and the excess to a micronutrient, we acclimated the well-distributed freshwater
Photosynthesis
microalga Ankistrodesmus densus to N- or P-limited medium before exposing it to sublethal copper (Cu) concen-
Quenching
trations. Our results indicate that Cu affected the chlorophyll a concentration in N- and P-replete conditions,
while the N- or P-limitation affected chlorophyll a concentration, maximum and effective quantum yield of pho-
tosystem II (PS II). Within the time frame of 72 h, and the maximum Cu concentration used (1.26 μM Cu2+ ), the
addition of Cu to N- or P-limited algae resulted in synergism in all of these parameters, except in chlorophyll
concentration under P limitation. In addition, the combination of Cu with N- or P-limited algae decreased the
photochemical quenching (qP) and increased the non-photochemical quenching (qN and NPQ). The values ob-
tained in Y (NPQ) – i.e., the quenching of regulated energy loss in PS II – indicate that the combination of Cu and
N- or P-limited algae induced the activation of photoprotective mechanisms. Under the highest Cu exposure, the
changes obtained from N- or P-limited algae were similar, indicating that under low metal concentrations, the
concentration of macronutrient is responsible for changing the chlorophyll concentration, qN, NPQ, and Y (NPQ);
however, at higher concentrations of metal, Cu apparently drives these changes. All of the parameters evaluated
were affected under N or P limitation and Cu combination, indicating a synergism. Based on the present study
results, we suggest using Ankistrodesmus densus in ecotoxicological studies due to its sensitivity and adaptation
to adverse scenarios.
1. Introduction Nitrogen and P affect the functioning of algae (Lai et al., 2011), and
they can limit the algal metabolism and aquatic primary productivity
Algal physiology and biochemistry can be affected by the availabil- (Beardall et al., 2001). The importance of these nutrients is related to
ity of nutrients, such as the macro- (e.g., phosphorus – P, and nitrogen their role in several metabolic events, e.g., N is part of nucleic acids, pro-
– N; required in concentrations of 10−5 to 10−3 M) and micronutrients teins, and chlorophylls (Geider and La Roche, 2002), while P is a con-
(e.g., trace metals: zinc, copper, manganese, required in concentrations stituent of membrane lipids (phospholipids), nucleic acids and ATP. Phy-
of 10−10 to 10−8 M). The lack or limitation of macronutrients can lead to toplankton can optimize their uptake, and photosynthetic machinery
changes in chlorophyll a fluorescence (Petrou et al., 2008), and down- can be adjusted under nutrient limitation (Smith and Yamanako, 2007;
regulation of photosynthesis under N- (Malapascua et al., 2014) or P Rocha et al., 2018). Nitrogen limitation can decrease photosynthetic ac-
limitation (Rocha et al., 2018). Apparently, P and N metabolisms are tivity (Geider et al. 1993; Berges et al. 1996; Benvenuti et al. 2015),
mutually dependent, i.e., P availability can facilitate N2 fixation in an and some authors report more serious damage in photosynthetic perfor-
N-limited environment. At the same time, N enrichment in a P-limited mance of C. reinhardtii under N- than P starvation (Kamanalathan et al.,
scenario can increase the expression of genes involved in P uptake, 2016), probably due to the lower production of photosystem II (PSII)
polyphosphate formation in Cyanophyceae (Wang et al., 2018), and an proteins under N limitation.
increase in P uptake under N-limitation was also observed in green mi- Copper is important in plastoquinone pool oxidation (Peers and
croalgae Chlorella vulgaris (Chu et al., 2013) and Chlamydomonas rein- Price, 2006), iron assimilation (Merchant et al., 2006), and other pro-
hardtii (Kamanalathan et al. 2016). cesses. The lack of copper in culture media can affect the growth
∗
Corresponding author.
E-mail addresses: swertsbio@gmail.com (G.S. Rocha), elgaeta@sc.usp.br (E.L.G. Espíndola).
https://doi.org/10.1016/j.envadv.2021.100070
Received 17 October 2020; Received in revised form 29 April 2021; Accepted 25 May 2021
2666-7657/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
- G.S. Rocha and E.L.G. Espíndola Environmental Advances 4 (2021) 100070
rate (Lombardi and Maldonado, 2011). In higher concentrations than Serial dilutions were made from the copper solution of CuCl2 Titrisol
required by algae, it can result in the formation of Cu-chlorophyll, 1000 mg L−1 (Merck) in ultra-pure water (Barnstead Easy Pure II,
which does not have fluorescence and can decrease the antenna size Thermo Scientific, Dubuque, IA, USA). Samples were acidified with 1%
(Küpper et al., 1998); cause nutrient limitation (Serra et al., 2010); affect HNO3 and measured by atomic absorption spectrophotometry with a
the phosphate uptake (Nalewajko and Paul, 1985) and induce phosphate graphite furnace (Analytic Jena; Zeenit 60, graphite tube wall, 324.8
starvation (Verma et al., 1993). The concentration of Cu that can affect nm absorption line, 0.8 nm slit). The determined values in samples dif-
the algal responses can vary according to the species. Some character- fered less than 10% of the nominal concentrations.
istics, such as the extracellular ligands released by algae, can decrease Three experimental replicates were used for each treatment in the
the copper’s toxicity (Lombardi and Vieira, 2000). The concentration of toxicity tests, using 500 mL polycarbonate Erlenmeyer flasks containing
Cu can vary according to the environment; while the Cu concentration 200 mL of sterile culture medium. Cells were inoculated, providing an
in the Mediterranean coastal wetland varies from 4.8 × 10−3 to 0.31 μM initial cell density of approximately 5 × 104 cells mL−1 . Exponentially
(Andreu et al., 2016); it can reach values from 0.14 to 1.55 μM in UK growing Ankistrodesmus densus cells N- (1 mM N – control and 0.01 mM
rivers (Neal and Robson, 2000) and 0.75 to 3.66 μM in Egyptian rivers N – 1% N) or P-acclimated (50μM P – control and 5 μM P - 10% P)
(Mansour and Sindky, 2002). The toxicity of Cu is related to the con- were exposed for 72 h to 4 copper concentrations: 3.6 × 10−4 (con-
centration of free ions Cu2+ (Lombardi et al., 2007) because of cellular trol); 0.6; 1.2 and 1.8 μM, which were defined after preliminary tests.
uptake availability (Sunda and Huntsman, 1998). Although the effects Since the free ions are the bioavailable fraction of the metal, we calcu-
of the major nutrients, such as P and N, affecting the metal uptake are lated the Cu2+ ions through the chemical equilibrium model MINEQL+
still not totally understood (Wang and Dei, 2001; Ji and Sherrel, 2008); 4.62.3 (Environmental Research Software, Hallowell, ME, USA), which
in the presence of more nutrients, there are more ligand sites to copper, estimated that 70% of added copper was in the free form in the WC
reducing its bioavailable fraction (Riedel and Sanders, 2003). Phospho- culture medium; thus 0.00025 (control); 0.42; 0.84 and 1.26 μM Cu2+
rus can bind metals as a means of detoxification (Paulsson et al., 2002), were available to the algae in the medium. The concentration of N or P
and P limitation is linked with higher Cu toxicity (Guasch et al., 2004; in the medium did not affect the speciation of Cu in the medium signifi-
Rocha et al., 2016). cantly. In all of the treatments, the mean values of the copper speciation
Microalgae are in the base of aquatic food webs, and are very sen- in WC medium is mainly Cu2+ (≈70-72%); CuOH+ (≈17.5%); CuCO3 aq
sitive to changes in the environment, adapting their physiology to new (≈7.7%) and CuSO4 aq (≈1.5%).
scenarios, and transferring contaminants to the higher trophic levels. For chlorophyll determination, samples were well-mixed, 10 mL
To obtain more information on the concomitant stress of the lack of were filtered onto cellulose ester membranes (0.45 μm pore size - Mil-
macronutrients and excess of a micronutrient, the present study evalu- lipore), and extracted with dimethylsulfoxide (Shoaf and Lium 1976).
ated the effects of copper exposure to the widely distributed freshwater Blanks were performed using a filter without algal culture. Absorp-
microalga Ankistrodesmus densus. The alga was acclimated to N- or P- tion was measured at 664 and 647 nm wavelengths, and the concen-
limited medium to check if the algal physiology responses are related to tration of chlorophyll a was calculated, as described by Jeffrey and
the limitation of a macronutrient or with the excess of a micronutrient, Humphrey (1975).
or if there is a synergism between macronutrient limitation and excess
of micronutrient. Thus, we assessed the impacts of the stressors on pho-
tosynthetic parameters, such as the maximum and effective quantum 2.2. PAM fluorescence measurements
yield of PS II, photochemical and non-photochemical quenching.
Using a Phyto - PAM I (Walz, Germany) equipped with an ED-
101US/MP optical unit, we were able to assess the chlorophyll fluores-
2. Material and methods cence in 4 different excitation wavelengths (470 nm, 520 nm, 645 nm,
and 665 nm), using automatic Gain adjustment, i.e., Gain was automat-
2.1. Algal cultures ically adjusted according to the concentration of the sample. Samples
(3 mL) were dark-adapted for 20 min before measuring the initial (F0 ,
The freshwater microalga Ankistrodesmus densus (Chlorophyceae) light intensity 1 μmol photons m−2 s−1 ) and the maximum fluorescence
was kept in WC medium (Guillard and Lorenzen, 1972), without adding (FM , saturating light pulse 0.2 s, 2000 μmol photons m−2 s−1 ) to ob-
EDTA, and cultured at pH 7.0, under a light intensity of 130 μmol pho- tain the variable fluorescence (FV = FM – F0 ). With the values of F0 ,
tons m−2 s−1 , photoperiod of 12:12 h light: dark cycle and temperature Fv , and FM , we obtained the maximum photosynthetic efficiency of PSII
of 22 ± 2°C. The algae were acclimated to this medium for 5 months (Φ = Fv /FM ). The quantification of these parameters occurred at 2, 24,
before using them for toxicity tests. The cultures were gently shaken 48, and 72 h after metal exposure.
manually twice per day. Culture media were sterilized through auto- At 72 h, the samples were exposed to continuous actinic light (128
claving for 20 min at 121°C before inoculation. Sterile conditions, using μmol photons m−2 s−1 ). New saturating light pulses were applied ev-
a UV chamber, were used in culture manipulation. The experiment’s ery 20 s - for 10 min - to obtain light-adapted sample parameters. The
materials were washed in neutral detergent and kept for 7 days in 1 M steady-state chlorophyll fluorescence (Fs ) and maximum fluorescence in
HNO3 for metal cleaning. light (F’m ) allowed the calculation of the operational quantum yield of
Algal cells were acclimated to 2 nitrogen (N): 1 mM N (control) and PS II (((Y(II) = (F’m - Fs )/F’m ))), photochemical quenching ((qP = (F’m
0.01 mM N (1 % N), and 2 phosphorus (P) concentrations: 50μM P (con- - Fs ) / (F’m - F0 )), non-photochemical quenching ((qN = 1- [(F’m - F’0 )
trol) and 5 μM P (10% P), as described by Rocha et al. (2016; 2018). / (Fm - F0 )]) (Juneau et al. 2002), and Stern-Volmer non-photochemical
The nutrient concentration present in the control is the original from the quenching ((NPQ = (Fm - F’m )/F’m )) (Maxwell and Johnson 2000). The
WC medium. The other treatments were defined after preliminary tests fractions of energy used for photosynthesis, and dissipated as fluores-
with different N and P. The conditions of illumination, temperature, pH, cence and heat, via photoprotective mechanisms ((Y (NPQ)) or not ((Y
and photoperiod were the same as those described for maintaining the (NO)), were calculated according to equations 1 and 2, respectively
stock culture. Exponentially growing cells were transferred every 72 h (Klughammer and Schreiber, 2008):
to a fresh medium containing the different concentrations of N or P. The
cells were checked, and measurements in PhytoPAM were taken daily to ( ) ( )
Y(NPQ) = Fs ∕F’ m − Fs ∕Fm (1)
confirm acclimation before starting the toxicity tests (Rocha et al. 2018;
2021a). Phosphate (PO3− 4
) and nitrate (NO−
3 ) concentrations were deter-
mined in the culture media as described in APHA (1995). Y(NO) = Fs ∕Fm (2)
2
- G.S. Rocha and E.L.G. Espíndola Environmental Advances 4 (2021) 100070
Fig. 1. Chlorophyll a concentration (μg mL−1 ) of Ankistrodesmus densus at 72 h
Fig. 2. Maximum quantum yield of photosystem II of Ankistrodesmus densus
exposed to different Cu, N and P concentrations. Bars with different superscript
at 72 h exposed to different Cu, N and P concentrations. Bars with different
letters are significantly different (P < 0.05).
superscript letters are significantly different (P < 0.05).
2.3. Statistical analyses
The maximum quantum yield of PS II (Φ = Fv/Fm) at 72 h is shown
The data were tested for normality and homogeneity of variance.
in Figure 2 and Suppl. Tables 1 and 2. The addition of Cu or the lower
Statistically significant differences among treatments and controls were
amounts of N and P decreased Φ values from 3%-9% compared to con-
determined using one-way ANOVA and Tukey’s post hoc at p < 0.05
trol. In the combination of two stressors (N- or P-limitation + Cu), P-
(Minitab 16; SigmaPlot 11, Systat). The data were obtained from three
limited cultures were the most affected, i.e., P limitation combined with
experimental replicate cultures and are presented as the mean ± SD of
higher Cu resulted in a 22% decrease in Φ values. In comparison, N lim-
the replicates.
itation and Cu decreased Φ values by 15%. The not remarkable decrease
The calculation of synergism and antagonism was performed when
in Φ values under N (≈ 9%) or P (≈6%) limitation indicates the accli-
the endpoints evaluated were significantly lower in the treatments than
mation of the algae to the nutrient stress (Kromkamp and Forster, 2003)
the control, assuming independent action of macronutrients (N or P) and
without a relationship between the maximum quantum yield and nutri-
Cu. The predicted and observed effects, and synergy ratio was calculated
ent status (Harrison and Smith, 2013), and not compromising the phys-
according to equations 3, 4, and 5, respectively (Gottardi et al., 2017):
iological state of algae (Kromkamp et al., 2008). This decrease in Φ
𝑀𝑎𝑐 𝐶𝑢 values was much lower than that observed by White et al. (2011) in the
𝑃 𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑 = 𝑥 (3)
𝐶 𝐶 freshwater microalga Chlorella sp, where the maximum quantum yield
where Mac refers to the value of the endpoint obtained under N or P values were 36% and 28% lower than the control under N- and P limita-
limitation; Cu, under copper exposure, and C refers to control. tion, respectively. In a study by Kamanalathan et al. (2016) with the mi-
𝐶𝑜𝑚𝑏 croalga Chlamydomonas reinhardtii, N limitation affected the maximum
𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑 = (4) quantum yield of PS II more than P limitation, while in the present study
𝐶
where the comb is the endpoint’s value under the combination of N or the Φ values under N- and P-limitation were similar.
P limitation and Cu exposure. The data from photochemical (qP; Fig. 3A) and non-photochemical
(qN – Fig. 3B; NPQ – Fig. 3C) quenching show that the energy distribu-
𝑃 𝑟𝑒𝑑𝑖𝑐𝑡𝑒𝑑
𝑆𝑦𝑛𝑒𝑟𝑔𝑦 𝑟𝑎𝑡𝑖𝑜 = (5) tion in PS II was not affected by the increase of Cu in nutrient replete
𝑂𝑏𝑠𝑒𝑟𝑣𝑒𝑑
media or by the decrease of N or P; however, the addition of Cu in
3. Results and discussion nutrient-limited media affected the quenching. The decrease in qP and
increase in qN and NPQ under N or P limitation combined with Cu expo-
Chlorophyll a concentration from A. densus was affected by the lower sure suggest synergism and indicate the damage in the light use and the
concentration of N or P and higher Cu in the medium. Under N or P limi- activation of the photoinhibition mechanisms (Suppl. Table 1; Suppl. Ta-
tation, the chlorophyll concentration decreased compared with control; ble 2). The decrease in qP, more pronounced in the combination under
however, the addition of Cu had a higher impact in control cultures, P-limitation and highest Cu, indicates a loss or a decrease in the percent-
while in N- and P-limited cultures, the macronutrient seems to be the age of active reaction centers (Juneau et al., 2002; Kalaji et al., 2014),
main source of changes (Fig. 1; Suppl. Table 1; Suppl. Table 2). The which can result in lower carbon assimilation (Krause and Jahns, 2003),
decrease in chlorophyll a concentration under copper exposure (in con- affecting all the photosynthetic process. However, the values of qN and
trol cells) can result from the formation of the non-functional and non- NPQ in the present study suggest that the nutrient limitation and addi-
fluorescent Cu-chl (Küpper et al., 1998). This decrease in pigment is tion of Cu did not result in damage to the photoprotection mechanisms,
expected under nutrient limitation, and the higher decrease obtained and the increase in these parameters was an active defense of algae
in N-limited cultures can be related to the direct role of nitrogen in the to avoid photoinhibition. Juneau et al. (2002) did not detect qN in C.
synthesis of chlorophylls (Turpin, 1991; Geider et al., 1993; Ivorra et al., reinhardtii exposed during 96 h to 0.38, 0.76, and 1.52 μM Cu, while
2002). However, our data differ from White et al. (2011), where chloro- Lombardi and Maldonado (2011) observed a decrease in NPQ of batch
phyll concentration was more affected in Chlorella sp under N- than P- cultures of Phaeocystis cordata exposed to 7.9 × 10−5 and 3.1 × 10−4 μM
limitation. While P-limitation caused higher decrease in chlorophyll a Cu2+ , which suggests impairment of photoprotective mechanisms in
concentration than N-limitation, the addition of metal was not synergis- their studies. In a study by Rocha et al. (2021b) with the microalga Sele-
tic. On the other hand, in N-limited cultures, synergism was observed in nastrum gracile, the authors did not observe a linear change in the values
the highest copper concentration. of qN and NPQ with the increase of Cu in the medium as observed in the
3
- G.S. Rocha and E.L.G. Espíndola Environmental Advances 4 (2021) 100070
Fig. 3. Photochemical (qP; A) and non-photochemical (qN, B; NPQ, C) quench- Fig. 4. Y (II) (A), Y (NO) (B) and Y (NPQ) (C) of Ankistrodesmus densus at 72 h
ing of Ankistrodesmus densus at 72 h exposed to different Cu, N and P concen- exposed to different Cu, N and P concentrations. Bars with different superscript
trations. Bars with different superscript letters are significantly different (P < letters are significantly different (P < 0.05).
0.05).
addition of Cu in the control treatment, while Y (II) values decreased
present study, i.e., the qN and NPQ values of S. gracile increased in con- with the lower concentration of N or P in the medium. In addition, the
centrations of 2.4 and 4.8 × 10− 2 μM Cu2+ , while at 9.6 × 10−2 μM Y (II) was more affected with the increase of metal in nutrient limited
Cu2+ these values decreased and they were not different from the treatments, especially under P limitation (Fig. 4A; Suppl. Table 1; Suppl.
control. Table 2). The parameter Y (NO), which quantifies the fraction of energy
In the present study, we observed that effective quantum yield of dissipated passively in fluorescence and heat, was affected by the P con-
photosystem II ((Y (II)) values were not significantly affected by the centration in the medium. Copper addition did not change the values of
4
- G.S. Rocha and E.L.G. Espíndola Environmental Advances 4 (2021) 100070
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Declaration of Competing Interest ical composition of Prorocentrum donghaiense to different nitrogen and phosphorus
concentration. J. Exp. Mar. Biol. Ecol. 405, 6–17.
Lombardi, A.T., Hidalgo, T.M.R., Vieira, A.A.H., Sartori, A.L., 2007. Toxicity of ionic cop-
The authors declare that they have no known competing financial per to the freshwater microalga Scenedesmus acuminatus (Chlorophyceae, Chlorococ-
interests or personal relationships that could have appeared to influence cales). Phycologia 46, 74–78.
the work reported in this paper. Lombardi, A.T., Maldonado, M.T., 2011. The effects of copper on the photosynthetic re-
sponse of Phaeocystis cordata. Photosynth. Res. 108, 77–87.
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