Abstract
In this study, Scenedesmus sp. and Chlorella sp. were exposed to 100, 150, 200 mg/L of lead and 10, 50, 100 mg/L of cobalt for 10 days. The chlorophyll content at Pb (200 mg/L) was found to be 2.35 ± 0.15 μg/mL in Scenedesmus sp. and 2.58 ± 0.02 μg/mL in Chlorella sp. on the 10th day. Scenedesmus sp. and Chlorella sp. exposed to Co (100 mg/L) showed a decline in chlorophyll content (0.83 ± 0.09 μg/mL and 0.74 ± 0.08 μg/mL) respectively. Furthermore, Scenedesmus sp. and Chlorella sp. exposed to 100 mg/L of lead showed the highest lipid peroxidation measured using malonaldehyde (MDA) (10.60 μmol/g and 6.24 μmol/g), superoxide dismutase (SOD) (49.04 U/mL and 49.32 U/mL) and catalase (CAT) (237.74 nmol/min/mL and 373.48 nmol/min/mL) activity, respectively. Scenedesmus sp. and Chlorella sp. exposed to 200 mg/L of lead showed elevated MDA (4.89 μmol/g and 5.14 μmol/g), SOD (32.05 U/mL and 37.80 U/mL) and CAT (121.78 nmol/min/mL and 160.46 nmol/min/mL) activity, respectively. Scenedesmus sp. and Chlorella sp. showed a high tolerance for 100 mg/L of lead and 10 mg/L of cobalt. As the concentration of lead and cobalt was increased, cell growth declined and elevated levels of stress biomarkers were observed. This study helps to understand plant tolerance levels and presents their candidature for treating wastewater with high lead and cobalt content.
HIGHLIGHTS
Scenedesmus sp. and Chlorella sp. were explored for expression of stress biomarkers when exposed to various concentrations of Pb and Co.
Chlorophyll content at higher concentrations of Pb and Co declined in both species.
SOD and catalase activity were upregulated at higher concentrations of Pb and Co in both species.
MDA, SOD and catalase were identified as physio-chemical bioindicators.
Graphical Abstract
INTRODUCTION
Increase in metal toxicity has severely deteriorated the environment in recent years. Metal contamination is one of the major causes of toxicity around the world and its presence in the environment can be natural or man-made (Stankovic et al. 2014). These metals are hazardous for phytoplanktons, zooplanktons and even for human lives, especially with their chronic effects (Stankovic et al. 2014). Heavy metals slowly accumulate in microalgae and plants near water bodies (Rai et al. 1981). Occurrence of heavy metals, either naturally or due to human activities, may cause toxicity to the surrounding water bodies. Lead is one of the important metals to be studied for its toxicity and various studies have been done using microalgae. Stichococcus bacillaris has been used to check the toxicity of lead and production of thiol after 24 hours of exposure (Pawlik 2002). Lead is known to have adverse effects on members of Chlorophyceae at high doses, causing changes in morphology of cells, growth and chlorophyll content affecting photosynthesis (Rosko & Rachlin 1977; Christensen et al. 1979; Rai & Chandra 1992; Pawlik 2002). Cobalt also affects the photosynthesis process and is involved in Fenton-like copper reactions that result in the formation of hydroxyl radicals (Moorhouse et al. 1985).
Lower concentrations of lead below 1 mg/L have been found to accelerate growth in Cladophora and concentrations up to 7.5 mg/L had no inhibitory effect on its growth (Cao et al. 2015). Another study reported that lead concentrations of up to 50 mg/L did not affect the photosynthesis in Chlorococcum sp. and concentrations higher than 100 mg/L produced a decline in photosynthesis of cells (Chang-En & Zheng-Yu 2007). The stimulatory effect of Pb on algal cells can be attributed to the hormetic effect of metals. In a reported study, the authors found that low concentrations of lead (6 mg/L) stimulated growth by 3.92% due to the hormetic response of algae, i.e., stimulation of growth at low concentrations and inhibition at higher concentrations of heavy metals (Sacan et al. 2007). Similarly, in another study, it was found that Botryococcus braunii treated with 4.5 mg/L of cobalt favored rapid growth in 5% CO2. A cobalt-enriched environment can enhance photon utilization, giving better light conversion efficiency and thus help in rapid growth (Cheng et al. 2020).
In the current study, the tolerance levels of Scenedesmus and Chlorella sp. were tested against high concentrations of cobalt and lead. It was evident from previous studies that, in some cases, the lower levels of cobalt and lead showed no marked differences such as growth inhibition of cells. Microalgae are the primary producers of aquatic ecosystems and exposure to acute or chronic concentrations of heavy metals can disturb the redox balance of the cells. The exposure of metal pollutants to microalgae induces oxidative stress because they are involved in reactive oxygen species (ROS) generation through different mechanisms (Stohs & Bagchi 1995). Several peroxidases, such as superoxide dismutase (SOD), catalase (CAT), and APX, respond to generated ROS. Here, SOD is the first line of defense against the superoxide molecules, converting them to hydrogen peroxide (Hassan & Scandalios 1990), which is then available for CAT to act upon. So, the response molecule produced against generated ROS is dependent on the ROS generation mechanism too.
Heavy metals can interfere with a wide range of biological activities in microalgae. Tolerance of the microalgae towards the high concentrations of heavy metals is one of those biological activities in which the microalgae manage metals by resistance and deposition in tissues (Kaplan 2013). Absorption of heavy metals involves surface interactions, and the functional groups present on the cell surface play a vital role in this. The process of biosorption is biphasic (Roy et al. 1993; Das et al. 2008) including two major steps of adsorption and absorption. The process of adsorption is not dependent on metabolites and is comparatively faster than absorption (Hassett et al. 1981; Gadd 2009). However, the process of absorption is slower and involves the active transport of metals inside cells and their binding to specific metabolites for detoxification. This whole process of resistance shown by microalgae as ‘tolerance’ results in expression and inhibition of certain compounds which can be considered as the biochemical markers of tolerance. Whenever microalgae are exposed to heavy metals like Pb or Co in various concentration ranges (ppb to ppm), biochemical features like pigment concentration decline, indicated by hampering of photosynthesis and decline in chlorophyll content. Similarly, other biochemical molecules of microalgae both enzymatic and non-enzymatic may be expressed to sustain tolerance towards ROS generated by heavy metals during their interaction with the cells. Therefore, an upregulated expression of antioxidant enzymes in microalgae towards heavy metal exposure can express the tolerance limit of specific heavy metals by different species. In addition to photosynthetic activity, morphological changes, and growth rate, which are usually represented through chlorophyll content to understand the effect of xenobiotic elements, there is a need to collect information on antioxidant enzymes. Xenobiotic elements are not an essential nutrient requirement of organisms. So, presence of these elements does not only hamper cells growth, but it also initiates the release of stress biomarkers. Thus, it is also important to measure the activity of these antioxidant enzymes released as stress biomarkers, along with accessing changes in chlorophyll and cellular morphology. Therefore, biomarkers associated with oxidative stress seem to be more accurate in terms of toxicity studies (Pikula et al. 2019). Cystoseria indica was used to study biomarkers after being exposed to Cd, Cu, Zn, Pb, Hg, Ni, and Cr (Sinaei et al. 2018). However, there is need to gather more information to identify and establish pollution biomarkers using microalgae to measure as changes in the environment (light, pH, temperature, metal toxicity and pesticides etc.). The species experiencing the change should have moderate range of tolerance. Antioxidant enzymes from microalgae are important for assessment of toxicity imposed on cells. As microalgae are well known for high metal accumulation efficiency, they can be potential candidates to be used for phycoremediation.
In our previous research, we have explored the potential of Scenedesmus sp. as a candidate for biofuel production (Anand et al. 2019) and Chlorella sp. for nanoparticle biosynthesis (Kashyap et al. 2019). In the current study, these species have been explored for their potential to indicate changes, with a broad tolerance limit for heavy metals in terms of antioxidant enzymes. These species were found to be more robust and ready to be used in the open environment as per our previous research (Anand et al. 2019; Kashyap et al. 2019). These two species have been explored widely for lipids and carotenoids etc. in the past, governed by stress induction. Thus, making them a candidate to handle metal toxicity with a high tolerance range. Both metals Pb and Co cannot be degraded naturally and therefore making them available for the process of biomagnification leads to a major problem of food chain. Chlorophyll a content and antioxidant enzymes like SOD, CAT, and malonaldehyde (MDA) have been used to assess the stress imposed on cells at different concentrations of Pb and Co. There are very few studies reported for determining the tolerance capacity of Chlorella and Scenedesmus sp. at such high concentrations of Pb and Co. Therefore, this study can add to the ongoing research of utilizing microalgae for treating wastewater from battery, radiation and lithium based industries. The wastewater from these industries has very high levels of lead and cobalt. Furthermore, research areas which can be explored based on the current study are biorefinery approaches using the wastewater as growth medium and utilizing algal biomass for production of high value-added compounds.
MATERIALS AND METHODS
Cultivation conditions
Two freshwater microalgae, Scenedesmus sp. and Chlorella sp. were grown in BG-11 medium (Stanier et al. 1971). The growth conditions were 12:12 hours of light and dark photoperiods with white LED light intensity of 3000 lux and temperature of 28 ± 5 °C. pH of the medium was 7.2. The microalgae species used were indigenously isolated from wastewater in the Indore region, India and therefore had a robust nature.
Experimental set up
Scenedesmus sp. and Chlorella sp. were exposed to various concentrations of cobalt (Co) and lead (Pb) spiked in BG-11 medium. The concentration range was 10, 50 and 100 mg/L (represented by Co10, Co50, Co100, respectively) for Co and 100, 150 and 200 mg/L (represented by Pb100, Pb150 and Pb200, respectively) for Pb. Algae cells were exposed to these concentrations with an initial cell density of 0.3 (OD680 nm). Chlorophyll a estimation was carried out at varying time periods and metal concentrations. On the 10th day, cells were harvested to estimate the levels of antioxidant enzymes (SOD and CAT) and lipid peroxidation through MDA in the presence of Pb and Co. The 10th day was chosen as the cells are in mid log phase and multiplying quickly. At this stage they are supposed to respond quickly to any stress imposed. The concentration ranges were chosen based on other publications where researchers have exposed blue-green algae to lead from 0 to 200 ppm (Pinchasov et al. 2006). Similarly, for cobalt also the concentration was decided after going through the literature, where it was found that microalgae exposed to cobalt (0 to 200 μmol/L-23.06 ppm) was used for tolerance and bioremediation studies (Mei et al. 2007).
Chlorophyll estimation
Estimation of lipid peroxidation of cells via malondialdehyde (MDA)
Estimation of catalase (CAT) and superoxide dismutase (SOD) activity
Estimation of antioxidant enzymes for assessment of toxic effects of heavy metals was performed after extraction of protein from the cells (Liu et al. 2018). The crude protein extract was then used to estimate CAT and SOD activity under the effect of various concentrations of Co and Pb in monocomponent exposure.
Catalase activity of cells was measured using a Colorimetric kit (Item No. 707002 Cayman 1180 E. Ellsworth Rd-Ann Arbor, MI, USA) as per the instructions given in the manual by the manufacture. Similarly, SOD activity was performed on cells treated with heavy metals using an enzyme kit (Item No. 706002).
RESULTS AND DISCUSSION
Algal growth and chlorophyll accumulation
Chlorophyll a estimation of Scenedesmus sp. and Chlorella sp. was carried out using the methodology described previously. The result obtained showed that the chlorophyll a content of cells under the effects of Pb and Co in a monocomponent stress environment declined in comparison to control cells, which were not exposed to heavy metals. The chlorophyll a content of Scenedesmus sp. cells treated with Pb100, Pb150 and Pb200 was found to be decreased, i.e., 9.98 ± 0.72 μg/mL, 3.61 ± 0.16 μg/mL and 2.35 ± 0.14 μg/mL, respectively on 10th day of exposure when compared to control (10.65 ± 0.37 μg/mL). Similarly, chlorophyll a content of Chlorella sp. treated with Pb100, Pb150 and Pb200 were also found to be less, i.e., 9.88 ± 0.97 μg/mL, 3.03 ± 0.26 μg/mL and 2.58 ± 0.03 μg/mL, respectively, on the 10th day in comparison to the control (12.90 ± 0.64 μg/mL), which can be observed from Figure 1(a) and 1(b). The cells of Scenedesmus sp. treated with Co also showed a decrease in chlorophyll a content at higher concentrations. The chlorophyll a content of Scenedesmus sp. cells treated with Co10, Co50 and Co100 were found to be 4.65 ± 0.30 μg/mL, 0.90 ± 0.11 μg/mL and 0.83 ± 0.09 μg/mL, respectively on the 10th day (Figure 2(a)). Chlorella sp. treated with Co also showed a decrease in chlorophyll a content as shown in Figure 2(b) and was found to be 9.04 ± 0.31 μg/mL, 0.89 ± 0.06 μg/mL and 0.74 ± 0.08 μg/mL for Co10, Co50 and Co100, respectively on the 10th day. The chlorophyll a content in Scenedesmus sp. cells treated with Pb100 (6.41 ± 0.56 μg/mL) and Co10 (4.47 ± 0.33 μg/mL) did not show much decline on the 5th day when compared with 5th day untreated cells (5.80 ± 0.24 mg/L). Similarly, the cells of Chlorella sp. treated with Pb100 (4.04 ± 0.14 μg/mL) and Co10 (3.29 ± 0.18 μg/mL) showed almost same amount of chlorophyll a as in the control (4.07 ± 0.14 μg/mL) on the 5th day. However, on the 10th day all samples treated with either Pb or Co showed significant decline in chlorophyll a content as compared to the control as stated above. Our results showed that the overall chlorophyll a content decreases in comparison to control and falls in line with the earlier reported studies.
Chlorophyll a content of (a) Scenedesmus sp. (b) Chlorella sp. treated with Pb100, Pb150 and Pb200.
Chlorophyll a content of (a) Scenedesmus sp. (b) Chlorella sp. treated with Pb100, Pb150 and Pb200.
Chlorophyll ‘a’ content of (a) Scenedesmus sp. (b) Chlorella sp. cells treated with Co10, Co50 and Co100.
Chlorophyll ‘a’ content of (a) Scenedesmus sp. (b) Chlorella sp. cells treated with Co10, Co50 and Co100.
A study done on Scenedesmus YaA6 and Chlorella sp. FIeB1 to assess the toxicity of Pb showed that both in long-term and short-term exposure to Pb, the chlorophyll content of the cells declines in both species, which is also evident from our results (Dao & Beardall 2016). In another study, after exposure of Chlorella sorokiniana to Pb the chlorophyll a content decreased up to four-fold in comparison to the control (Carfagna et al. 2013). Effect of Zn, Co and Cd on Chlorella pyrenoidosa Chick S-39 decreased the release of photo-induced oxygen by the cells. This led to a suppressed production of chlorophyll fluorescence due to exposure to these metals and contributed towards inactivation of photosystem II (PSII) (Plekhanov & Chemeris 2003). They reported a decrease in photosynthetic oxygen release, which affected the electron transport chain by 60% in cells exposed to Co, 45% in cells exposed to Cd and 85% for Zn. So, evidently the chlorophyll fluorescence was suppressed.
MDA analysis as stress marker
Lipid peroxidation of cells is an indicator that the cells are under stress. MDA is the well known product of lipid peroxidation, which has been estimated in the current study in which two freshwater microalgae were exposed to varying concentrations of heavy metals (Pb and Co). As evident from the results of the chlorophyll analysis, cells of Scenedesmus sp. and Chlorella sp. had low chlorophyll content due to stress imposed by Pb and Co as represented in Figures 1 and 2. MDA levels from both species under the effect of different concentrations of Pb and Co in a monocomponent exposure environment showed that the lipid peroxidation of the cells increased with increasing concentrations of these metal ions. This decrease in chlorophyll and increase in MDA level (Tables 1 and 2) indicates that the cells were under stress as the concentration of Pb and Co increased.
MDA, SOD and catalase activity under varying concentrations of Pb and Co in Scenedesmus sp.
Metal levels . | MDA (μmol/g) . | SOD (U/mL) . | CAT (nmol/min/mL) . |
---|---|---|---|
Pb100 | 3.08 | 10.14 | 88.12 |
Pb150 | 4.73 | 22.74 | 111.71 |
Pb200 | 4.89 | 32.05 | 121.78 |
Co10 | 3.45 | 13.67 | 75.63 |
Co50 | 9.86 | 29.60 | 123.34 |
Co100 | 10.60 | 49.04 | 237.74 |
Metal levels . | MDA (μmol/g) . | SOD (U/mL) . | CAT (nmol/min/mL) . |
---|---|---|---|
Pb100 | 3.08 | 10.14 | 88.12 |
Pb150 | 4.73 | 22.74 | 111.71 |
Pb200 | 4.89 | 32.05 | 121.78 |
Co10 | 3.45 | 13.67 | 75.63 |
Co50 | 9.86 | 29.60 | 123.34 |
Co100 | 10.60 | 49.04 | 237.74 |
MDA, SOD and CAT activity under varying concentrations of Pb and Co in Chlorella sp.
Metal levels . | MDA (μmol/g) . | SOD (U/mL) . | CAT (nmol/min/mL) . |
---|---|---|---|
Pb100 | 3.93 | 7.65 | 59.33 |
Pb150 | 4.12 | 19.32 | 139.82 |
Pb200 | 5.14 | 37.80 | 160.46 |
Co10 | 4.65 | 16.25 | 90.72 |
Co50 | 5.67 | 21.80 | 234.18 |
Co100 | 6.24 | 49.32 | 373.48 |
Metal levels . | MDA (μmol/g) . | SOD (U/mL) . | CAT (nmol/min/mL) . |
---|---|---|---|
Pb100 | 3.93 | 7.65 | 59.33 |
Pb150 | 4.12 | 19.32 | 139.82 |
Pb200 | 5.14 | 37.80 | 160.46 |
Co10 | 4.65 | 16.25 | 90.72 |
Co50 | 5.67 | 21.80 | 234.18 |
Co100 | 6.24 | 49.32 | 373.48 |
Maximum MDA level was observed in both Scenedesmus sp. and Chlorella sp. at Pb200, i.e., 4.89 μmol/g and 5.14 μmol/g, respectively. MDA concentration in Scenedesmus sp. and Chlorella sp. exposed to the highest concentration of cobalt (Co100) was found to be 10.6 μmol/g and 6.24 μmol/g, respectively (Tables 1 and 2). MDA level showed an increase by 1.6-fold in Scenedesmus sp. treated with 200 mg/L of Pb as compared to cells treated with the lowest concentration i.e., Pb100 (3.08 μmol/g). However, there was no significant difference observed in MDA levels at Pb150 (4.73 μmol/g) and Pb200 (4.89 μmol/g). This suggested that MDA production reached saturation level after exposure to Pb150 and did not increase further significantly. Similarly, Scenedesmus sp. cells treated with Co100 showed a three-fold increase in MDA level when compared to the lowest concentration. i.e., Co10. It was found that a similar pattern was followed by cells of Chlorella sp. and that the MDA level increased up to 1.3-fold at Pb200 and by 1.4-fold at Co100 when compared to Pb100 and Co10, respectively. The generation of ROS is one of the major responses of algae in adverse growth conditions, which ultimately results in cell death. The results obtained agreed with those obtained by other researchers, showing increase in MDA level after exposure to higher concentrations of heavy metals e.g., MDA levels increased significantly when cells of Cladophora were exposed to Pb (Cao et al. 2015). A study reported enhanced MDA levels in microalgal cells up to 0.6 μmol/106 cells at 10 μmol/L of cobalt, which fell in line with our study, suggesting the expression of MDA under heavy metal stress (Mei et al. 2007). Fenton-type reaction of cobalt with hydrogen peroxide to produce hydroxyl radicals make it an oxidative stress inducing factor, which results in expression of MDA when exposed to heavy metals (Moorhouse et al. 1985).
CAT and SOD activity as biomarkers for heavy metal toxicity
As observed from the MDA results the cells experienced stress in the form of ROS generated due to stress caused by heavy metals (Pb and Co). CAT and SOD are stress biomarker molecules, which can counter this stress by breaking down the ROS molecules (H2O2, hydroxyl radical, singlet oxygen and superoxide radicals) (Lesser 2006) and neutralizing its effect (Liu & Pang 2010). Therefore, it was necessary to study the impact of high concentrations of these metals on the expression of Pb and Co. Scenedesmus sp. and Chlorella sp. treated with various concentrations of Pb and Co were tested for CAT and SOD activity. SOD is the first line of defense against stress and its expression helps to defend the cell against stress encountered by exposure to high concentrations of heavy metals. There was a significant increase in SOD activity as the concentration of Pb and Co increased in both Scenedesmus sp. and Chlorella sp. SOD activity in Scenedesmus sp. treated with Pb200 (32 U/mL) was maximum, i.e., 3.1-fold higher than Pb100 (10.1 U/mL). Similarly, cells treated with Co100 (49 U/mL) also showed an increase by 3.6-fold when compared to Co10 (13.7 U/mL). Chlorella sp. showed significant increase in SOD activity of cells treated with Pb200 (37.8 U/mL), i.e., five-fold higher as compared to Pb100 (7.6 U/mL). Co-treated Chlorella sp. showed an increase in SOD levels at Co100 (49.3 U/mL) by three-fold when compared to Co10 (16.3 U/mL). The above results observed in the study indicated that SOD activity was high in cells with higher concentrations of Pb and Co. Such high levels of SOD activity also indicated that the cells are under tremendous stress at high concentrations of Pb and Co on the 10th day. SOD activity of Scenedesmus sp. and Chlorella sp. under various concentrations of Pb and Co is shown in Tables 1 and 2, respectively. A similar study has been done on Chlorella lessleri and Scenedesmus sp. in which the cells of these two microalgae were exposed to various concentrations of copper for one week. They reported an increment in the SOD activity along with CAT activity at higher metal concentrations (Sabatini et al. 2009).
Catalase activity was also estimated in cells of Scenedesmus sp. and Chlorella sp. Catalase breaks down the hydrogen peroxide (H2O2) produced from various metabolic activities of the cells. CAT is considered as one of the major enzymes along with SOD, which comes into action whenever cells encounter any kind of stress. In the present study, the results obtained revealed that the cells of Scenedesmus sp. treated with Pb100 (88.12 nmol/min/mL), Pb150 (111.71 nmol/min/mL) and Pb200 (121.77 nmol/min/mL), respectively, showed considerable CAT activity (Table 1). However, there was no significant difference between the CAT activity of cells at Pb150 and Pb200. This could be due to involvement of other enzymes like ascorbate peroxide (AP) as observed in a study done on Ulva rigida where it was found that increase in H2O2 did not increase the CAT activity, but the activity of AP was increased (Collén & Pedersén 1996).
There was a significant increase in CAT activity of Scenedesmus sp. treated with Co50 (123.33 nmol/min/mL) and Co100 (235.74 nmol/min/mL) when compared to cells treated with Co10 (75.63 nmol/min/mL) (Table 1). High CAT activity suggests that the cells are under stress and the identification of these stress biomarkers proves that heavy metals can induce stress in cells, leading to increased activity of antioxidant enzymes. Chlorella sp. treated with Pb150 (139.81 nmol/min/mL) and Pb200 (160.45 nmol/min/mL) did not show much difference in the CAT activity, but in comparison to cells treated with Pb100 (59.32 nmol/min/mL), there was a significant increase in CAT activity. Similarly, Chlorella sp. cells treated with Co10 (90.72 nmol/min/mL), Co50 (234.18 nmol/min/mL) and Co100 (373.48 nmol/min/mL) showed an increase in the CAT activity, demonstrating that the increase in concentration of cobalt led to increase in stress imposed on cells, as shown in Table 2. SOD and other metabolic enzymes led to the production of H2O2, which is then split by CAT activity. As observed and discussed, heavy metal exposure to Scenedesmus sp. and Chlorella sp. results in enhancement of CAT activity in our experiments. Similar study was performed on Chlorella sp. MM3 and cells exposed to polyaromatic compounds and heavy metals resulted in an increase in CAT activity and lipid peroxidation (Subashchandrabose et al. 2017).
Correlation studies to understand the effect of spiked BG-11 medium with Pb and Co on activity of SOD, CAT and MDA
To understand the relationship between different concentrations of Pb and Co on activity of antioxidant enzymes, a fitting curve was plotted. As observed from the linear regression curve (Figure 3) for MDA production at different concentrations of Pb ad Co, there was positive correlation between the concentration of heavy metals used and MDA production. A fitting curve was plotted for varying concentrations of Pb and Co in relation to SOD and CAT activity in both species (Figures 4 and 5). The positive correlation of MDA, SOD and CAT with varying concentrations of Pb and Co in both Scenedesmus sp. and Chlorella sp. suggested that the species were responding well to the stress imposed by heavy metals. Positive correlation also indicated the efficiency of using Scenedesmus sp. and Chlorella sp. as bioindicators and their metal tolerance capacity. However, a positive correlation does not indicate the level of significance, therefore a t-test was performed to find out the p-value at level of confidence (α 0.05). The correlation between MDA, SOD, and CAT activity was calculated and it was found that the correlation of changes in Scenedesmus sp. under cobalt exposure were non-significant with MDA and SOD, whereas it was significant for CAT with level of confidence at 0.05, as observed from the t-test (one tailed). Scenedesmus sp. exposed to lead showed significant correlation for MDA with SOD and a non-significant correlation for CAT. Chlorella sp. treated with different concentrations of Pb and Co were also tested for the correlation of MDA values obtained with SOD and CAT at 0.05 level of confidence to perform a t-test. The correlation coefficient values for Chlorella sp. under influence of Pb showed MDA with SOD, and CAT as 0.94 and 0.87, respectively, and when exposed to Co the correlation coefficient values were found to be 0.88 and 0.92, respectively. Chlorella sp. showed a significant positive correlation for MDA, SOD and CAT under the influence of lead and cobalt.
Positive correlation of MDA production with cobalt in (a) Scenedesmus sp., (b) Chlorella sp.; and with lead in (c) Scenedesmus sp., (d) Chlorella sp.
Positive correlation of MDA production with cobalt in (a) Scenedesmus sp., (b) Chlorella sp.; and with lead in (c) Scenedesmus sp., (d) Chlorella sp.
Positive correlation of SOD activity with cobalt in (a) Scenedesmus sp., (b) Chlorella sp.; and with lead in (c) Scenedesmus sp., (d) Chlorella sp.
Positive correlation of SOD activity with cobalt in (a) Scenedesmus sp., (b) Chlorella sp.; and with lead in (c) Scenedesmus sp., (d) Chlorella sp.
Positive correlation of CAT activity with cobalt in (a) Scenedesmus sp., (b) Chlorella sp.; and with lead in (c) Scenedesmus sp., (d) Chlorella sp.
Positive correlation of CAT activity with cobalt in (a) Scenedesmus sp., (b) Chlorella sp.; and with lead in (c) Scenedesmus sp., (d) Chlorella sp.
The change in metal concentration led change in the activity of these antioxidant enzymes in different species, making them an efficient biological indicator of heavy metal pollution caused by Pb and Co. Chlorella sp. seems to be a better candidate in comparison to Scenedesmus sp. in terms of correlation studies. SOD and CAT showed a much better correlation in both species as compared to MDA. Thus, making SOD and CAT better biomarker molecules in comparison to MDA. Some researchers have used correlation studies to show a correlation between ammonia and dissolved oxygen (DO) levels, whereas few have used it to describe the antioxidant capacity of microalgae against phenolic compounds (Chaudhuri et al. 2014; Nandigam et al. 2016). In the present work we have used it to show a correlation between heavy metal concentrations and activity of antioxidant enzymes under stress.
R2 values can be converted to ‘r’ which is known as the determent of correlation and the correlation values usually match with this ‘r’. Determinant of correlation can be calculated by square rooting the values of R2. In Table 3 the values of ‘r’ for MDA, SOD and CAT have been calculated, which recommends that the levels of expression of these enzymes have positive correlation with the independent variable of metal concentration used. The level of confidence or alpha was set at 0.05 to test the significance of correlation with the above-mentioned sample size. T-test also supported the level of confidence (α = 0.05) for Chlorella sp. exposed to lead where the p-value as 0.01 for one tailed t-test was found for correlation of MDA, 0.04 for CAT and 0.01 for SOD. This suggests a correlation with the increasing concentrations of Pb and that expression of MDA, CAT and SOD is significant. Scenedesmus sp. exposed to lead showed significant correlation with a p-value value of 0.01 for MDA and 0.01 for SOD but for CAT the p-value was 0.07, which shows that the correlation between Pb concentration and CAT was not significant. This shows that the CAT activity increased due to lead toxicity in Scenedesmus sp., but the increase was not found to be statistically significant. Therefore, MDA and SOD were found to be better indicators of metal toxicity in Scenedesmus sp. compared to CAT. Scenedesmus and Chlorella sp. exposed to cobalt showed a significant correlation in terms of expression of CAT with a p-value of 0.02 and 0.05, respectively.
Correlation values of MDA, SOD and CAT under influence of Pb and Co
Variables . | Correlation (r) with MDA (μmol/g) . | Correlation (r) with SOD (U/mL) . | Correlation (r) with CAT (nmol/min/mL) . |
---|---|---|---|
Scenedesmus sp. | |||
Lead (Pb) | 0.81 | 0.90 | 0.99 |
Cobalt (Co) | 0.92 | 0.88 | 0.99 |
Chlorella sp. | |||
Lead (Pb) | 0.96 | 0.99 | 0.92 |
Cobalt (Co) | 0.89 | 0.96 | 0.99 |
Variables . | Correlation (r) with MDA (μmol/g) . | Correlation (r) with SOD (U/mL) . | Correlation (r) with CAT (nmol/min/mL) . |
---|---|---|---|
Scenedesmus sp. | |||
Lead (Pb) | 0.81 | 0.90 | 0.99 |
Cobalt (Co) | 0.92 | 0.88 | 0.99 |
Chlorella sp. | |||
Lead (Pb) | 0.96 | 0.99 | 0.92 |
Cobalt (Co) | 0.89 | 0.96 | 0.99 |
CONCLUSIONS
The current study shows the induction of peroxidases such as enzymes in algal cells treated with high concentrations of Pb and Co. The chlorophyll content of Scenedesmus sp. treated with Pb200 and Co100 significantly declined on the 10th day. Similar results were obtained for Chlorella sp. treated with Pb200 and Co100 in terms of chlorophyll content analyzed on the 10th day of the experiment. There are relatively very few studies using with such high concentrations of Pb and Co to understand the behavior of algal cells. This work successfully demonstrated the increase in levels of antioxidant enzymes (SOD and CAT), lipid peroxidation (via MDA) and inhibitory effects of Pb and Co on cells through chlorophyll measurements. However, Chlorella sp. seems to be a better indicator, as it showed better correlation with changing concentrations of metals and antioxidant enzymes including MDA. Chlorella sp. was particularly more sensitive to lead and had better tolerance capacity for higher concentrations of lead. The significant level of correlation for MDA, SOD and CAT with changing metal concentrations of Pb further proves this analysis. This study showed that the change in metal concentration correlates with the expression of stress biomarkers even at acute concentrations. Further research is needed to understand the exact mechanisms responsible for ROS generation and expression of stress biomarkers. Transcriptomics can be used to understand the relationship between ROS generation and transcription factors specific for stress biomarkers expressed in the presence of heavy metals.
CONFLICTS OF INTEREST
There are no conflicts to declare.
ACKNOWLEDGEMENTS
Authors are thankful to SERB – ECR project (ECR/2017/001567) for providing financial assistance. Mrinal Kashyap is thankful to University Grants Commission (UGC) India for a predoctoral research fellowship (SRF). The funding agency has not played any role in experiment design or decisions regarding publication of manuscript. Authors would like to acknowledge Sophisticated Instrumentation Centre (SIC), IIT Indore for instrumentation facilities.
DATA AVAILABILITY STATEMENT
All relevant data are included in the paper or its Supplementary Information.