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Cadmium Exposure and Phosphorus Limitation Increases Metal Content in the Freshwater Alga Chlamydomonas reinhardtii Rachel E. Webster,† Andrew P. Dean,† and Jon K. Pittman*,† †
Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, U.K.
bS Supporting Information ABSTRACT: The characteristics of metal accumulation in freshwater microalgae are important to elucidate for a full understanding of metal cycling and toxicity in a freshwater system. This study has utilized an elemental profiling approach to investigate the impacts of Cd exposure and phosphorus (P) availability on metal accumulation after 7 days in batch culture-grown Chlamydomonas reinhardtii. Multivariate statistical analysis of the elemental data demonstrated distinct responses between both stresses. Sublethal concentrations of Cd (up to 15 μM) caused increased accumulation of Co. Cu, Fe, and Zn content also increased in response to enhanced Cd concentrations but only when P availability was low. While Cd exposure effected the accumulation of a few specific metals, P limitation increased the accumulation of all essential trace metals and macronutrients analyzed including Co, Fe, K, Na, and Zn but not Mn. The accumulation of Cd also markedly increased in response to P limitation. The impact of P availability on essential metal accumulation was the same when either inorganic P or an organic P source (glycerophosphate) was used. These results highlight the potential risks of metal toxicity for freshwater microalgae and aquatic food chains when P availability is limiting and which can be exacerbated by Cd pollution.
’ INTRODUCTION The contamination of the environment by toxic concentrations of metal pollutants, largely through industrial practices such as mining,1 is damaging to ecosystems and extremely hazardous to human health. The increasing worldwide contamination of freshwater with metals is a major environmental problem due to the impact on aquatic organisms and on human health through unsafe drinking water.2 Cd is a particular concern as its industrial use is increasing and significant amounts of Cd waste continue to be produced and discharged into water.3 In most organisms, Cd has no biological function and accumulation of this metal, even at low concentration, can be extremely toxic to plants and animals and can cause many illnesses in humans.3 Furthermore, it is a persistent environmental pollutant that can easily accumulate throughout the food chain.4 Although other transition metals such as Zn, Cu, Fe, and Mn are essential to most organisms and deficiency symptoms can be observed if their availability is reduced, these essential metals are toxic if they accumulate to high concentrations. Indeed elevated concentrations of essential and nonessential trace elements can be highly toxic in lower organisms, particularly freshwater microorganisms, such as microalgae, and in crustaceans and fish which feed on microalgae.5 7 Microalgae are the key point of entry of a metal pollutant into the food chain; therefore, the characteristics of algal metal uptake will partly determine the impact of the pollutant on the freshwater ecosystem. The toxicity and effect of a metal on an algal cell in an aquatic environment will depend on the metal speciation and hence the external concentration of free metal, the concentration of metal r 2011 American Chemical Society
bound to the cell (biotic ligand interaction) and the degree of metal accumulation and internalization.8,9 These properties are determined by a variety of chemical and biological parameters such as pH, complexation with external ligands, competition by other ions, the abundance and kinetics of metal uptake pathways, and subcellular sequestration.9 11 Many of these parameters have been quantified and modeled in microalgae with regard to Cd accumulation. However, it is less clear what impact Cd exposure has on the accumulation of other metals into microalgae, in particular essential trace metals and macronutrients. The eukaryotic unicellular chlorophyte Chlamydomonas reinhardtii is an established and well-used experimental model in which to study metal homeostasis and metal toxicity in microalgae.9,12 Moreover, it is a representative aquatic phytoplankton species which is ubiquitous throughout freshwater environments worldwide. Recent studies have begun to take a systematic approach to understand the affects of Cd on C. reinhardtii by quantifying transcriptomic, proteomic, and metabolomic changes in response to Cd exposure.13 16 Here we have utilized an elemental profiling approach to quantify ionomic changes in C. reinhardtii in response to long-term (7 day) Cd exposure. The ionome can be regarded as the mineral nutrient and trace element composition of an organism and has been quantified for a variety of species including yeast and plants.17 Received: March 10, 2011 Accepted: August 2, 2011 Revised: June 27, 2011 Published: August 02, 2011 7489
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Figure 1. (A) Predicted concentrations of free Cd2+ calculated using Visual MINTEQ in media containing increasing concentrations of glycerophosphate (TAgP) and PO43- (TAP) with the addition of 15 and 40 μM CdCl2. (B) Chlamydomonas reinhardtii growth (as a percentage of growth in the absence of Cd) after 7 days in 1 mM TAgP and TAP medium in the presence of 20 and 40 μM Cd. * indicates significant difference (p e 0.05) between 20 and 40 μM Cd treatments within TAgP grown cells and # indicates significant difference (p e 0.05) between TAgP and TAP treatments. Biomass (C) and intracellular Cd content (D) after 7 days, of cells grown in TAgP medium containing 0.01, 0.1, and 1 mM P and in the presence of 0, 5, 10, and 15 μM Cd. * indicates significant difference (p e 0.05) between no Cd and Cd treatments. For each data set error bars correspond to the standard error of the mean of 3 replicate culture flasks, and data are representative of 2 4 independent experiments.
Moreover ionomic analysis will be a powerful means to determine an organism’s physiological response to environmental stress, as recently demonstrated by a study of plant ionomic response to Fe deficiency stress.18 This study has quantified the C. reinhardtii ionome in response to Cd alongside varying concentrations of external phosphorus (P), given that P is present in variable amounts in the environment. Anthropogenic activities have caused pollution of P in some rivers and lakes;19 however, most freshwaters are low in P.20 We have examined Cd exposure in conditions of reduced P availability (10 μM) and compared them to conditions of higher P concentrations (0.1 mM and 1 mM P). This has allowed the identification of specific trace metals and macronutrients whose accumulation into the cell is affected by Cd treatment and/or by P availability. Collectively, this study provides insight into the interactions between Cd pollution, P nutrition, and metal homeostasis and highlights the potential of increased metal toxicity in organisms in environments with limiting P and exposure to Cd.
’ MATERIALS AND METHODS Strains and Growth Conditions. C. reinhardtii wild type strain 137+ (CCAP 11/32C or CC-125) and the cell wall
deficient strain cw15 (CCAP 11/32CW15+ or CC-425) were grown photoheterotrophically as sterile axenic batch cultures in Tris-acetate-phosphate (TAP) liquid medium21 and in TAgP liquid medium, in which inorganic phosphate was replaced with the organic P source β-glycerophosphate (disodium salt: C3H7Na2O6P). Cells were grown in three versions of TAP or TAgP medium containing 1 mM P, 0.1 mM P, or 0.01 mM P. All media was buffered to pH 7.0 with Tris. The concentrations of metals and nutrients present in each medium are shown in Table S1. For Cd exposure treatments, various concentrations of CdCl2 from 5 to 40 μM were added to media. For low-N treatments, cells were grown in modified TAP medium containing 0.7 mM NH4Cl instead of 7 mM NH4Cl. To start the cultures, 0.5 mL of early stationary phase cells grown in high-P media was centrifuged and washed twice in Milli-Q water to remove residual P and then added to 200 mL of high-P, intermediate-P, low-P, or low-N medium in 250 mL flasks, giving an initial cell count of ∼65 103 cells ml 1. For each experiment cultures were grown in replicates of 3 4 in a growth room on an orbital shaker at 120 rpm at 22 °C with a 16-h light:8-h dark light regime and a photon flux of approximately 150 μmol m 2 s 1. Cellular Elemental Analysis. After 7 days growth 10 mL of cells were harvested for determination of metal content. Metal 7490
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content was quantified on a per cell basis or a per biovolume basis so cell density and cell biovolume was determined by cell counting and microscopy imaging as described.22 The harvested cells were EDTA washed to remove externally bound metals by centrifugation at 3000 g for 10 min, resuspension, and mixing of the cell pellet with a 10 mL volume of 1 mM EDTA for 5 min and then recentrifugation and washing with a 15 mL volume of MilliQ water. Cells were centrifuged again at 3000 g for 10 min, and cell pellets were oven-dried at 60 °C for 24 h and then digested in 0.5 mL of ultrapure concentrated nitric acid (67%) at 100 °C for 3 h. Samples were diluted in Milli-Q water and analyzed by inductively coupled plasma atomic emission spectroscopy (ICPAES) (Perkin-Elmer Optima 5300) using internal standards. Speciation Modeling and Statistical Analysis. Chemical speciation modeling of the growth medium was performed using Visual MINTEQ version 3.023 with a fixed pH of 7.0 and with the same components present in TAgP and TAP media without or with varying concentrations of CdCl2. Differences between treatments were assessed using one-way and multivariate ANOVA. When significant differences were detected at a 95% level of confidence, the multirange Tukey’s test was applied. ANOVA and discriminant function analysis was performed using SPSS version 16.
’ RESULTS AND DISCUSSION Cd Exposure under Different P Availabilities. Cd exposure treatments were performed by growing C. reinhardtii in TAgP medium which contains the organic P source glycerophosphate instead of inorganic PO43- which was predicted to bind Cd2+ leading to the formation of insoluble Cd-PO43- precipitates and reduced concentration of free Cd2+ (Figure 1A). Metal speciation prediction indicated that the concentration of free Cd2+ would be higher in TAgP medium compared to TAP medium containing PO43- (Figure 1A and Table S1). An increased concentration of bioavailable Cd2+ would be expected to inhibit C. reinhardtii growth and this was confirmed experimentally as shown by a significant reduction in cell density in TAgP medium at high Cd exposure (>20 μM), whereas the density of cells grown in TAP medium at 20 and 40 μM CdCl2 was not markedly reduced (Figure 1B). The observed differences in cell density were not due to the type of P source as C. reinhardtii could grow to equivalent cell densities in TAgP and TAP media and the P content in cells grown in each media was similar (Table S2), indicating that C. reinhardtii can use both forms of P, as suggested previously.24 For further analysis with Cd we chose a concentration range up to 15 μM in TAgP medium which did not induce toxicity to the cells so that the effects of sublethal Cd exposure on cellular metal quota could be examined. Three Cd treatments of 5, 10, and 15 μM were tested, which were predicted to give free Cd2+ concentrations of ∼2, 10, and 25 nM Cd2+ (Table S1). Cd exposure was assessed under three P conditions: 1 mM P, 100 μM P, and 10 μM P. Previous studies have demonstrated that external P limitation (to 1.6 μM) or complete P starvation substantially impacts on C. reinhardtii growth and physiology.25,26 The P limitation imposed in this study also caused a significant reduction in intracellular P concentration compared to high-P cells (Table S2). Likewise cell density, as an indicator of biomass yield, was substantially reduced with approximately 4-fold less cells in the low-P treatment compared to 1 mM P and 0.1 mM P treatments (Figure 1C) indicating that only the lowest P concentration resulted in P limited cells. Cd exposure led to a concentration-dependent increase in cellular Cd content measured
Figure 2. Discriminant function analysis based on intracellular metal content and calculated on the basis of Cd treatment (A) and P treatment (B). Intracellular metal content was determined at day 7 in cells grown in TAgP medium containing 0.01, 0.1, and 1 mM P and in the presence of 0, 5, 10, and 15 μM Cd. The individual elements that determine each function are identified in Table S3. Groups or multiple groups are clustered on the basis of significant discrimination. Each plot represents data from 15 replicate culture flasks from 5 independent experiments for each Cd treatment/P treatment group.
after 7 days (Figure 1D). Cells were EDTA-washed to remove cell wall-bound metal so that only internalized Cd was measured. Comparative analysis of a cell wall-deficient strain confirmed that this washing procedure was sufficient for determination of intracellular metal content only and not cell wall-bound metal (Figure S1). Cd accumulation was substantially enhanced in the low-P cells compared to the higher-P cells (Figure 1D). At a Cd concentration of 5 or 10 μM, there was no difference between the level of Cd accumulation in the high-P and intermediate-P cells, but in cultures containing 15 μM Cd there was a difference in the magnitude of Cd accumulation between the intermediate-P and high-P cells as well as significant accumulation in the low-P cells. Despite an increased cellular Cd quota, Cd exposure up to 15 μM did not further reduce cell growth under any of the P conditions (Figure 1C). Multivariate Analysis of the Algal Ionome in Response to Cd or P Treatment. The impact of the Cd treatments on 7491
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Figure 3. Effect of Cd treatment on intracellular Ca, Co, Cu, Fe, Mg, and Zn content after 7 days, in Chlamydomonas reinhardtii grown in TAgP medium containing 0.01, 0.1, and 1 mM P and in the presence 0, 5, 10, and 15 μM Cd. * indicates significant difference (p e 0.05) between no Cd and Cd treatments. nd = not detected. Error bars correspond to the standard error of the mean of 15 replicate culture flasks from 5 independent experiments.
essential metal accumulation in combination with the P treatments was examined by ICP-AES analysis of EDTA-washed cells. Thirteen metal/metalloid elements were quantified: B, Ca, Co, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, Se, and Zn. The pH was buffered to pH 7.0 in all treatments so that the influence of pH on metal uptake10 was not a variable parameter in this study. The intracellular elemental content data sets were analyzed by discriminant function analysis (DFA) to examine whether it was possible to classify the cells on the basis of Cd treatment and/or on the basis of P treatment and identify specific ionomic signatures for the respective stresses. DFA is a multivariate statistical analysis which examines how dependent variables discriminate the groups. Combinations of dependent variables are identified as functions which may separate the groups. The analysis found that on the basis of Cd treatment, there was overlap between all treatments, but there was significant
discrimination provided by function 1 between the 0 and 5 μM Cd treatments and the 10 and 15 μM Cd treatments (Figure 2A, Table S3). Function 1 was significant (Wilk’s Lambda = 0.642, p = 0.042) and correlated with the Cd, Cu, Co, Se, and Zn profiles (Table S3). For DFA on the basis of P treatment there was highly significant discrimination of the 0.01 mM P treatment from the higher P treatments by function 1 (Wilk’s Lambda = 0.054, p < 0.01) (Figure 2B), which is correlated with 10 of the elements including Zn, Na, and Fe (Table S3), but there was also significant discrimination between 1 mM P and 0.1 mM P treatments on the basis of function 2 (Wilk’s Lambda = 0.541, p < 0.01), which is correlated with the Ca, Cd, Cu, and Mg profiles. This suggests that the effect of P limitation on the cell’s ionome overrules the effect of Cd and the effect of P on the ionome appears to be global. Multivariate ANOVA confirmed this as each element was a significant dependent variable 7492
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Figure 4. Effect of P treatment on intracellular content of B, Ca, Co, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, Se, and Zn after 7 days, in Chlamydomonas reinhardtii grown in TAgP medium containing 0.01, 0.1, and 1 mM P and in the presence of 15 μM Cd. * indicates significant difference (p e 0.05) between 0.01 mM and 0.1 or 1 mM P treatments. Error bars correspond to the standard error of the mean of 15 replicate culture flasks from 5 independent experiments.
(p < 0.05) in response to P treatment except for Mn (p = 0.545). Furthermore, this analysis determined that there was no interaction between P treatment and Cd treatment. Multivariate analysis can therefore be used to visualize ionomic variation in response to specific stresses in C. reinhardtii, and we can use such ionomic variation to discriminate cells on the basis of individual stresses, similar to previous ionomic analysis in plants exposed to Fe or P stress.18 Metal Accumulation Changes in Response to Cd Exposure. DFA indicated that the impact of Cd exposure on the C. reinhardtii ionome was not as substantial as the impact of P stress. This was quantified further by performing multivariate ANOVA. The multivariate test of significance for the Cd treatment indicated that the effect of Cd on the ionome was specific, effecting just six metals: Ca (p = 0.045), Co (p = 0.001), Cu (p = 0.040), Fe (p = 0.048), Mg (p = 0.017), and Zn (p = 0.001). The cellular concentrations of these metals were quantified (Figure 3). Intracellular accumulation of four of the trace metals, Co, Cu, Fe, and Zn was enhanced by 15 μM Cd exposure. Cd-induced accumulation of Cu, Fe, and Zn was only significant under P limiting (0.01 mM P) conditions, while Cd-induced Co accumulation was significantly enhanced under all P conditions and in response to 10 μM Cd (Figure 3B). Cd treatment (from 5 to 15 μM) led to a significant reduction in content of the macronutrients Ca and Mg but only when the cells were grown in 1 mM P conditions (Figure 3A and 3E). A reduction in metal content following addition of a second metal may be due to competitive inhibition and might indicate metal interaction at a single uptake site.9,27 However, additive or synergistic effects are often observed when multiple metal mixtures are examined,27 as have been found here for Co, Cu, Fe, and Zn interactions with Cd. Analogous effects have been described previously in C. reinhardtii including increased Ni content in the presence of Pb28 and increased Pb content in the presence of Cu12 or Cd.13 The mechanisms by which Cd exerts toxicity are not fully understood. An increase in oxidative stress appears to be a consequence of Cd exposure, and Cd-dependent induction of oxidative stress response proteins have been observed in C. reinhardtii.14 Unlike Cu, Fe, or Zn, Cd is not a redox active metal and does not generate reactive oxygen species by the Fenton reaction. The
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data here suggest that one additional mechanism by which Cd may exert toxicity is via the induction of increased metal burden within the cell, including the accumulation of redox active metals, and perturbation of normal metal nutrient homeostasis. Although this study examined elemental accumulation following sublethal Cd exposure, we have observed that accumulation of these metals continues to be enhanced at higher Cd treatments (>20 μM) that induce cell toxicity (data not shown). Metal Accumulation in Response to P Availability. The concentrations of metals in P replete cells (Figure 4) were equivalent to those previously determined in C. reinhardtii grown in TAP medium.29,30 Comparisons with the previously determined ionomes of yeast, bacteria, and the plant Arabidopsis17,31,32 found that the relative profiles of these elements were very similar between the species but with a clear difference being the greater accumulation of Mn in C. reinhardtii and Arabidopsis compared to yeast and bacteria, probably due to the increased essential requirement of Mn by photosynthetic organisms.33 Multivariate analyses indicated that P limitation results in significant global elemental changes within C. reinhardtii. All essential trace metals and nutrients quantified exhibited significant changes in accumulation in response to 0.01 mM P treatment compared to 1 mM P with the exception of Mn where only the change in accumulation between 0.01 mM and 0.1 mM P cells was significant (Figure 4). Most metals accumulated within the cells in response to limiting P, but the trend was reversed for Mg which was reduced in the limiting P treatments compared to the 1 mM P cells. These P limitation responses were observed in cells treated with 15 μM Cd (Figure 4) and were generally equivalent in cells grown in the absence of Cd apart from Co accumulation being poorly detectable in the absence of Cd (Figure S2). Ionomic changes in response to P limitation have also been observed in Arabidopsis. In leaves of this plant an increase in Fe, Zn, and As and a decrease in Co and Cu were observed following P limitation.18 Metal changes in response to altered P status therefore appear to be common in higher plants and algae, yet there appear to be specific differences between C. reinhardtii and Arabidopsis which may be due in part to the differences in metal abundance and speciation in soil compared to water as well as potential difference in metal nutrition requirements between the species. In response to P limitation C. reinhardtii cells increase in biovolume, principally due to an increased abundance of starch and lipid storage granules.22 We also observed that in response to 0.01 mM P treatment, cell biovolume significantly increased, but, interestingly, the P limitation-induced biovolume increase was inhibited by the presence of Cd (Figure S3A). The size increase of P limited cells did not account for the observed increases in metal content. When calculated on the basis of cell biovolume rather than per cell, cellular Zn content (Figure S3B) and Cd content (Figure S3C) was still higher in 0.01 mM P cells compared to 1 mM P cells, while Mn showed the opposite pattern (Figure S3D). The reduction in Zn content in the high-P cells compared to the low-P cells might also be argued to be a result of cellular dilution of Zn due to cell division of the rapidly growing high-P cells. However, Zn content in these cells at day 7 (0.17 ( 0.006 109 atoms cell 1) was equivalent to the Zn content at the start of the growth period (0.09 ( 0.001 109 atoms cell 1), indicating that this was not the case. The impact of P availability on metal accumulation in microalgae has been examined for a few metals in a few previous studies, but some of the results are conflicting. Supporting the 7493
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Environmental Science & Technology data described here, a previous study of the freshwater alga Chlorella compared the effects of various low PO43- concentrations (25 nM-10 μM) on short-term metal accumulation and found evidence for an increase in accumulation of Cd, Co, and Zn in response to P limitation after 1 or 2 days.34 However, contrasting results were observed in a study of metal uptake in C. reinhardtii over a 4 h period which indicated that P limitation (0.1 μM P compared to 10 μM P) reduced Zn and Cd accumulation.35 Here, we have determined metal accumulation over an extended growth period (at 7 days). This might suggest that the short-term kinetics of Zn and Cd accumulation are very different than those observed over the full growth period of the population. However, the results in our study cannot be directly compared with these previous experiments due to a number of differences in the parameters used. In particular, both previous studies used semicontinuous cultures for microalgae growth instead of batch cultures in which P limitation will not be induced immediately.25 To examine whether the lower P concentrations (
