Scenedesnus rotundus isolated from the petroleum effluent employs alternate mechanisms of tolerance to elevated levels of Cadmium and Zinc

Scenedesmus rotundus was isolated from metal contaminated petroleum industry effluent and its tolerance to Cadmium and Zinc was tested using different concentrations of CdCl2 and ZnCl2 ranging from 0.001 mM to 1.0 mM of Cd and 0.03 mM to 1.21 mM of Zn amended in Bolds Basal medium. The changes in cell count recorded at regular intervals upto a period of 24 days revealed a concentration dependent inhibition in growth. Concentration of the metal, at which 50% of the cells are live and metabolically active referred to as EC50 was calculated as 0.04 mM for Cd and 0.2 mM for Zn. Further, the effect of EC50 of the metals on the protein content, uptake of metals at varying pH, oxidative stress markers including lipid peroxidation, protein oxidation andnd oxygen uptake, levels of enzymatic antioxidants such as catalase and superoxide dismutase and non-enzymatic antioxidants namely, GSH and PC4 were determined. Though a direct correlation could not be drawn between pH and metal uptake, the compartmentalization of the metal during the lag phase and exponential phase was evident, most of the metal was present in extracellular fractions in the former, while in the later it was internalized. Our study shows a clear correlation between toxicity of Cd and the ability of the algae to synthesize PC4 from GSH and chelate it leading to detoxification, while Zn treatment led to an increase in the activity of catalase and superoxide dismutase and replete GSH pools. Further the changes in the cell wall structure at EC50 of Cd and Zn were studied. This is the first report on effect of heavy metals on the structural modifications of the cell wall of Scenedesmus in general and Scenedesmus rotundus in particular, indicating appearance of granules on the entire cell surface in both Cd and Zn treatments, with the degree of granulation increasing in the order of pH 12 > 10 > 8 in Cd treatment. Further structures of higher order resembling minute wheels are observed in Cd treated cells are also reported.


Effect of pH on growth and uptake of cadmium and zinc. The algal cells were grown in Bold's Basal
Medium amended at EC 50 concentrations of Cd and Zn at different pH ranging from 5-12, (5, 8, 10 1nd 12) with native pH of the medium 6.8 serving as control. Cells were harvested on Day 4 and Day 12. A modified method of Franklin et al. 44 was used to determine intracellular and extracellular concentrations of Cd and Zn. Algal cells were harvested by centrifugation and the supernatant representing the soluble metal fraction was referred to as fraction F1. The pellet was rinsed thoroughly with 2 mL Bold's Basal media and centrifuged. The supernatant representing the concentration of metal absorbed to the cells was referred to as fraction F2. The pellet was washed with 2 mL of EDTA solution (0.01 M EDTA, 0.1 M K 2 H 2 PO 4 pH 6) and centrifuged, the resulting supernatant referred to as F3 represented the concentration of the metal adsorbed on to the surface of the alga. The algal cell pellet was digested in 2 mL concentrated Nitric acid for 10 hours and the fraction representing intracellular metal concentration was referred to as Fraction F4. The culture flasks in which the algal cells were grown were rinsed with 10% HNO 3 representing the concentration of the metal absorbed to the glass was referred to as fraction F5. All the fractions were analysed using atomic absorption spectrometer (Element AS AAS4141, (Electronic Corporation of India) and the metal concentrations calculated against standard CdCl 2 and ZnCl 2 solutions.

Effect of Cd and Zn on the levels of oxidative stress markers. Lipid peroxidation. Algal extract
(1 mL) was taken in a 10 mL test tube and mixed with 1 ml of TBA. The mixture was heated in a boiling water bath at 95 °C for 60 minutes. The test tubes were cooled at room temperature and absorbance was measured at 532 nm using UV-visible spectrophotometer.
Protein oxidation. Carbonyl derivatization reaction with DNPH was performedaccording to Levine et al. 45 with some modifications. To 5 mL of algal cell extract, 18% SDS was added and mixed. Equal volume of 20 mM DNPH in 20%trifluoroacetic acid (TFA) was added followed by 20 mL of 2 M Tris in 30% glycerolwith 0.74 M β-mercaptoethanol. The samples were incubated at 37 °C for 30 min. Samples without DNPH served as control. The absorbance of thesupernatant was measured at 375 nm Oxygen uptake. Oxygen uptake was measured by oxygen electrodes withcomputerized data acquisition described byBaker et al. 46 . The algal cells were shaken inbeakers to ensure continuous aeration of the media. Changes in the steady state concentration of oxygen inbeakers containing algal cells was compared to beakers with medium alone (100%) to estimate the basal rate of respiration. www.nature.com/scientificreports www.nature.com/scientificreports/ Effect of Cd and Zn on the activity of enzymatic antioxidants. The algal cells were centrifuged and homogenized with silica beads in 2-ml vials by application of two times 60-s cycle. The homogenised sample was resuspended in the Bolds Basal Medium used for enzyme assays.
Superoxide dismutase (SOD). SOD was estimated as per the procedure described by Kakkar et al. 47 . Algal homogenate (0.5 ml) was diluted to 1 mL with 0.5 mL of distilled water. To the suspension 0.25 mL of ice coldethanol and 0.15 mL chloroform were added. The mixture was shaken for 1 minute and centrifuged at 2000 rpm. To the supernatant, 1.5 ml of carbonate buffer was added. The reaction was initiated by the addition of 0.4 ml epinephrine and change in optical density per minute was measured at 480 nm in a double beam UV-VIS spectrophotometer (UV 1700, Szhimadzu). SOD activity was expressed as U/mg of protein. Change in optical density per minute at 50% inhibition to adrenochrome transition by the enzyme is taken as one enzyme unit.
Catalase. Catalase activity was assayed by the method of Sinha et al. 48 . To 0.1 ml algal homogenate, 1.0 ml eachof phosphate buffer and hydrogen peroxide were added. The reaction was arrested by the addition of 0.2 ml dichromate acetic acid reagent. Standard hydrogen peroxide in the range of 4 to 20 µl was taken and treated similarly. The tubes were heated in a boiling water bath for 10 min and read at 570 nm in a double beam UV-VIS spectrophotometer (UV 1700, Szhimadzu). Catalase activity was expressed as U/mg protein.
Ascorbate peroxidase. Ascorbate peroxidase (APX) activity was assayed following Nakano and Asada 49 . The reaction mixture consisted of 100 μL ascorbate (7.5 mM), 100 μL algal extract, 100 μL H 2 O 2 (300 mM) and 2.7 mL of 25 mM potassium phosphate buffer with 2mMEDTA (pH7.0). Change in absorbance at 290 nm (ε = 2.8 mMcm −1 ) was considered as a measure of oxidation of ascorbate. Ascorbate peroxidase activity was expressed as U/mg protein.  www.nature.com/scientificreports www.nature.com/scientificreports/ Effect of Cd and Zn on algal thiol content (GSH, and PCn). Algal growth was followed for 12 days, corresponding to the time for the algae to reach the end of the exponential phase. One mL aliquots were taken at regular time intervals and cell counts were determined by haemocytometer. The algal cells were harvested at the density of 6 × 10 6 cells/ml and centrifuged (3000 × g for 10 min at 20 °C). The preparation of the sample and derivatization steps were followed from 50 .
Extraction of GSH and PCs. The algae were filtered onto 0.8 µm Membrane and placed in 2 ml of 50 mM HEPES and 50 mM NaCl (pH 7.5) containing 1 mM TCEP. The cells were homogenised, centrifuged and supernatant filtered through 0.2 µm membrane.  www.nature.com/scientificreports www.nature.com/scientificreports/ Derivatization of GSH and PCs with mBBr. The extracted sample was dissolved in 300 µl of 6.3 mM DTPA with 0.1% (v/v) TFA. The derivatization protocol was followed from F. Else C. Sneller 2000 51 . 450 µl of 200 mM HEPES buffer, pH 8.2, containing 6.3 mM DTPA was mixed with 10 ml of 25 mM mBBr. Derivatization was carried out for 30 mins at 45 °C in the dark. The reaction was stopped by adding 300 µl of 1 M MSA. The samples were stored in the dark at 4 °C until HPLC analysis. Standard GSH PC3 and PC4 were also derivatized following the same procedure.
HPLC analysis. PCs were separated on Agilent 1260 infinity analytical column. Before injection, the column was equilibrated in methanol and water, the derivatized sample was injected and run in a 12-25% (v/v) methanol gradient for15min and then with a linear gradient ethanol from 15 to 29 min. Next, a linear gradient was used www.nature.com/scientificreports www.nature.com/scientificreports/ from 35% to 50% (v/v) methanol, from 29 to 50 min after injection. Fluorescence was monitored with an excitation wavelength of 380 nm and emission wavelength of 470.
Effect of Cd and Zn on algal cell wall morphology. Scenedesmus rotundus was grown in Bolds Basal medium at varying pH amended with EC 50 concentrations of Cd and Zn. During the exponential growth phase the algal cells were centrifuged, the pellet was washed with phosphate buffer saline for 3 times. 2.5% glutaraldehyde was added and incubated at room temperature for 30 minutes. The pellet was washed with Na-phosphate buffer for 3 times and the pellets were collected. The sample was dehydrated with 30%, 50%, 70%, 80%, 90% ethanol for 10 minutes each and at 100% ethanol for 1 h. The structural changes were examined using Scanning Electron Microscope (Quanta 200 FEG) Statistical analysis. All the experiments were performed in three biological and three technical replicates.
Results were expressed as mean ± SD for six replicates. All the data were statistically evaluated with SPSS/19.0 software. www.nature.com/scientificreports www.nature.com/scientificreports/

Results and Discussion
Isolation and growth of Scenedesmus rotundus. Based on the morphology and 18S rDNA sequence data, the species was identified as Scenedesmus rotundus. The effects of different concentrations of Cadmium and Zinc on the growth of Scenedesmusrotundus are presented in Fig. 1. Algal growth rate was affected by exposure to Cadmium and Zinc at higher concentrations. Among the different concentrations of Cadmium studied, growth of the alga was comparable to control, up to a concentration of 0.05 mM, at higher concentrations a rapid decline in growth was observed. Zn treated cultures of Scenedesmusrotundus tolerated the heavy metal upto the highest concentration of 1.21 mM studied, however a dose dependent reduction in growth was observed.
Based on the results obtained from the growth of Scenedesmus rotundus at varying concentrations of Cadmium and Zinc, EC 50, values, i, e concentration of the metals at which 50% of the cells are live and metabolically active was calculated. EC 50   www.nature.com/scientificreports www.nature.com/scientificreports/ Effect of Cd and Zn on total chlorophyll. Alterations in the pigment content are generally considered as a measure of physiological competence. Exposure of algal cells to metals above their tolerance limits results in reduction in the pigment content indicating bulk metabolic perturbations. In the present study the total chlorophyll of Scenedesmus rotundus showed significant variations when compared to the control in both Cd and Zn treatments (Fig. 2). Treatment with Cd caused a marked reduction in the total chlorophyll content across all the treatments studied, these results are in line with the findings of 55 .

Effect of EC 50 concentrations of Cd and Zn on the total protein content. Protein content in algal
cells is influenced by metal toxicity. Scenedesmusrotundusthe total protein content was less in Zinc and Cadmium during the lag phasewhen compared to the control gradually increased in the exponential phase (Fig. 3). Among the two metals studied, Zinc treatment shows the higher protein contentwhen compared to the Cadmium. The increase of soluble protein is attributedto a variety of metabolic processes in cells, heavy metal stress can induce related stress protein gene expression, which is a defense mechanism of plants to environment stress 56 . Some reports indicate increased protein content in algae under metal stress, a decrease of soluble protein content was detected in Cdtreated C. sorokiniana cells (REF). Probably, chlorophylls and proteins, and even the chloroplast proteins, represented an emergency source of nitrogen and sulphur to ensure cell growth.
Effect of pH on the uptake and internalization of cadmium and zinc. pH may affect the uptake of Zn and Cd by phytoplankton in water bodies by three possible mechanisms (1) the kinetic parameters of the Zn and Cd uptake systems may be directly affected; (2) the uptake systems for Zn and Cd may be up-or down-regulated; and (3) the bioavailability of the metals may change the localization of Cd and Zn in the intracellular compartments during exponential phase is attributed to the regulatory mechanisms operating within the cells.
Concentration of Cd and Zn were estimated in different fractions on Day 4 and Day 12 at EC 50 concentrations of Cd and Zn at varying pH, pH 5, pH 6.8, (Control), pH 8, pH 10 and pH 12. Figures 4 and 5 depict the concentration of Cd and Zn in different fractions. During the lag phase of growth (Day 4) most of Cd was distributed between the soluble, adsorbed and absorbed fractions, while Zn was distributed between soluble and absorbed fractions. On the contrary, during exponential phase (Day 12) a large fraction of Cd was present in the intracellular fraction, while Zn was distributed between the absorbed and intracellular fractions. A direct correlation between the effect of pH and metal uptake by the alga was not observed in the present study. Similar observations were also reported by 57,58 . This could probably result due to buffering activity of numerous acid base groups, organic moieties and cell surface hydroxyls. Variation in pH in the bulk medium should be greatly attenuated at the surface of the cells as it is at particle surfaces with acid-base functionalities 59 .

Effect of Cd and Zn on the activity of enzymatic antioxidants. Synthesis of enzymatic and
non-enzymatic anti-oxidants is a common response encountered in algae upon exposure to heavy metals. Enzymatic antioxidants such as such as superoxide dismutase, catalase, and ascorbate peroxidase and low molecular weight compounds non-enzymatic antioxidants such as carotenoids and glutathione often are involved in scavenging ROS. Damage to the cells often results Catalase and Super oxide dismutase activities are frequently reported to increase due to heavy metal stress 55 . Oxidative stress markers, lipid peroxidation, and oxygen uptake showed significant increase in Cd treated cells, while protein oxidation was significant in Zn treated cells when compared to the control. In the present study activity of catalase and super oxide dismutase increased with respect to control, a significant increase was recorded in Zn treated cultures when compared to Cd treated cultures, while ascorbate peroxidise activity showed significant increase in Cd treatment as compared to Zn treated cultures (Fig. 6).

Effect of Cd and Zn on algal thiol content (GSH, and PCn).
As a protective measure, cells also induce the synthesis of protective proteins. These can be either metal chelators or enzymatic or non-enzymatic antioxidants or both. Metal chelators such as PCs 60-64 are cysteine-rich peptides with an ability to coordinate metals to sulfhydryl groups on the protein. Phytochelatin synthesis appears to be the central point in the stress response to heavy metals in almost all the species studied. Most reports indicate induction of the phytochelatin synthase or an increase in the levels of PCs in the cells when exposed to heavy metals in a concentration-dependent and dose-dependent manner. Several types of PCsare reported ranging from PC 2 to PC 4 with respect to the metal studied, the concentration and the duration of exposure. The response to heavy metal stress in Scenedesmus critically influenced by the thiol GSH, and phytochelatins. Several independent studies corroborate the fact that these components are involved in the cells' antioxidant metabolism to confer resistance to the heavy metal by efficient sequestration of the metal ions. Different metals tend to induce different patterns of regulation of the above said components. The most extensively studied is www.nature.com/scientificreports www.nature.com/scientificreports/ Cadmium, which has been shown by several independent studies to increase the GSH and phytochelatin content in the cell 65,66 . Figures 7 and 8 represents the variations in the levels of PC 4 in Scenedesmus rotundus exposed to EC 50 concentration of Cadmium and Zinc. While PC4 levels increased in both the treatments, induction of PC4 was strong in Cadmium treatment when compared to Zinc treated cells. The elevated levels of PC under Cd stress is also reported by Tukaj et al. 67 . Cd stress induces the synthesis of PC [2][3][4] . In the present study induction of PC 4 was very strong in Cd treated Scenedesmus rotundus and the finding is in line with many other reports indicating strong induction of PCs in algal cells subjected to heavy metal stress.
Being in a highly reduced state under optimal conditions, shifting towards a more oxidized state in response to increased intracellular ROS availability and mechanisms that link such shifts to altered redox state, and therefore biological activity, of target proteins mark glutathione as a candidate transmitter of intracellular ROS signals. GSH content of the cell is crucial in determining the cells' viability under heavy metal stress. Kov´aˇcik et al. 34 , reported that, old cultures that inherently had a lower GSH were not as viable as the new cultures that had a higher levels of the same. In the present study GSH levels showed different response to Cd and Zn treatments. While GSH pools were depleted upon Cd treatment, Zn treatment improved the levels of intracellular GSH, by nearly 5 fold when compared to the control. A correlation between falls in the GSH levels with simultaneous rise in PCs levels has been reported in many papers. Increased rates of PC synthesis require the formation of a complex between GSH and a heavy metal. Therefore it can be inferred that while Scenedesmus rotundus alleviates Cd toxicity via www.nature.com/scientificreports www.nature.com/scientificreports/ complexation to PC 4 leading to sequestration and detoxification, Zn toxicity is handled via other detoxifying mechanisms involving the GSH Ascorbate cycle, as high levels of GSH are persistent in cells exposed to Zn. Thus it can be reiterated that the optimal levels of GSH are crucial given its role in ROS scavenging and antioxidant activity and also confirming its role as a precursor to PC synthesis.
Wang et al. 23 , reported decrease in the GSH content and increased PC synthesis upon exposure of cells to As (II) and As(V), under phosphate limitation, while Kov´aˇcik et al. 28 , reported sharp increase in PC content induced by Cd accompanied by a significant decrease in the GSH content in the new cultures under short term exposure. On the contrary, Jacinto et al. 68 , reported increased peaks of Zn-GSH and Hg-PCs complexes supported that increased PC synthesis might is favored by the increased GSH levels. While many studies report an inverse correlation between GSH and PC content in cells exposed to different heavy metals, few studies report direct correlation. Exposure to Pb increased the levels of GSH and cysteine and hence increased activity of phytochelatin synthase and induction of PC 5 in addition to PC 2-4 69 . In the present study, while Scenedesmus rotundus responded  www.nature.com/scientificreports www.nature.com/scientificreports/ to Cd stress by using up the GSH pool towards synthesis of PC4 leading to sequestration for detoxification, the response to Zn was skewed towards retention of GSH pools and increase in activity of antioxidant enzymes.

Effect of Cd and Zn on algal cell wall morphology.
The cell wall is a complex structure composed of cellulose and polysaccharides, cross-linked with structural proteins. The cell wall represents a physical barrier against the entry of heavy metals into the symplastic compartment. Cell wall polysaccharides play a crucial role in heavy metal binding and accumulation in the cell wall 70,71 . One of the advantages of precipitating metals within the cell wall is their strong metabolic inactivation 72 . Figures 9 and 10 depict the scanning electron micrographs of Scenedesmus rotundus grown in different pH at EC 50 concentrations of Cd and Zn. Marked changes in the architecture of the cell wall were observed in both the treatments. This is the first report on effect of heavy metals on the structural modifications of the cell wall in Scenedesmus sp. in general and Scenedesmus rotundus in particular. One of the major changes observed is the appearance of granules on the entire cell surface of the Cd and Zn treated samples. Such granules were sparsely and uniformly distributed in the control cells and cells grown at pH 5, while the cells grown at pH of 8, 10 and 12, an increase in the granulation is reported. Further the degree of granulation increased in the order of 12 ˃ 10 ˃ 8 with in Cd treatments. Samadani et al. 73 , report an increase in cell granularity was observed in Cd exposed cells which the authours attributed to Cd-S containing molecule complexes. Scenedesmus treated with EC 50 of Zinc at different pH showed marginal reduction in cell size, however did not show variation in granulation as compared to the control. Zinc being an essential trace metal, is probably internally sequestered, while the toxic non -essential Cd may be precipitated in the cell wall and a portion of it rendered metabolically inactive. Andosch et al. 74 , reported the link between compartmentalization of heavy metals and the toxicity of these metals. Based on ultrastructural details of cellular organelles they reported that Cd and Cr, which were less compartmentalized into intracellular organelles, were more toxic than Al, Zn, and Cu.
Further structures of higher order resembling minute wheels are observed in Cd treated cells across all the pH, with the degree of order increasing with increased pH. The higher order structures as observed in Cd treated cells were completely absent in cells exposed to EC 50 concentration of Zinc. A comparison of these structures could be drawn to cellulose synthase complexes in the plasma membrane of Arabidopsis 75,76 (Figs 11 and 12). Do these represent synthetic machinery for formation of new wall material in Scenedesmus rotundus exposed to Cd stress or are these structures associated with the efflux of the metal has to be investigated to understand the functional significance of these structures with respect to alleviating metal toxicity in general and Cd tolerance in particular.