Response of Antioxidant Enzyme Activities of the Green Microalga Chlorococcum sp. AZHB to Cu²⁺ and Cd²⁺ Stress

Abstract

The green microalga Chlorococcum sp. AZHB, isolated from a wastewater treatment plant, can endure extremely environments contaminated by heavy metals, but little information is available about the physiological changes of microalgal cells after exposure to heavy metals. In this study, the response of antioxidant enzyme activities of this microalgal species were examined in batch cultures exposed to different concentrations of Cu²⁺ or Cd²⁺ for 10 days. The malondialdehyde (MDA) content and activities of peroxidase (POD) and superoxide dismutase (SOD) increased with the increasing concentration of Cu²⁺ and Cd²⁺ from 0 to 200 mg/L. The activity of catalase (CAT) increased with the increase in concentrations of Cu²⁺ and Cd²⁺ from 0–0.1 mg/L and 0–5 mg/L, respectively, and decreased from 0.1 mg/L Cu²⁺ and 5 mg/L Cd²⁺, respectively. Our results suggest that the defense mechanisms of Chlorococcum sp. AZHB to heavy metals should be involved in the improvement of the antioxidant enzyme activity in microalgal cells.

1. Introduction

Industrial wastewater contains cocktails of heavy metals including cadmium and copper, which can adversely affect aquatic ecosystems and human health [1,2]. The conventional techniques used for removing heavy metals from wastewater include lime precipitation, electrochemical treatment, ion exchange, and active carbon adsorption [3,4]. However, these physicochemical techniques have obvious shortcomings, for instance, incomplete metal removal and the production of toxic sludge [5,6]. Thus, new technologies are needed to decrease the levels of heavy metals to environmentally safe levels in a cost-effective and environmentally friendly manner.

Algae are at the base of aquatic food chains and they play a key role in algae-based sewage treatment systems such as algal oxidation ponds, algal biofilm treatments, algal bioreactors, and algal biosorption and immobilization [6,7,8]. In addition, algae can be widely used to treat wastewater where other organisms have difficulty surviving due to the contamination of heavy metals. In previous studies, several algal species have been used as biosorbents for removing toxic metals from wastewaters [5,9,10,11]. We isolated a green microalgal species, Chlorococcum sp. AZHB, from a wastewater treatment plant in Arizona, USA, and found that this species could endure extreme environments contaminated by heavy metals. Our previous study has reported on the removal of Cu²⁺ and Cd²⁺ by this microalgal species and the effects of algal growth, cell structure, and physiological characteristics after exposure to these two metals [12]. However, little information is available on the enzymatic changes of this microalgal species when it touches heavy metals.

Heavy metals are toxic to algal cells at elevated concentrations, and they can induce enzymatic defense systems to protect algal cells from oxidative stress [13]. Superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) are enzymatic reactive oxygen species (ROS) scavengers, participating in cell protection under stress conditions [14,15,16]. SOD, the first line of defense of algal cells against ROS, catalyzes the reaction of the disproportionation of the superoxide anion to oxygen and H₂O₂, which is further catalyzed the production of H₂O by CAT and POD [17,18]. In addition, malondialdehyde (MDA), a secondary lipid peroxidation product, serves as an indicator of cell damage produced by reactive oxidative intermediates (ROIs) [14,19]. Thus, determining the activities of the aforementioned enzymes is an important step in demonstrating the defense mechanisms of algal cells to heavy metals.

Therefore, the present study was specifically designed to investigate the changes of MDA, SOD, CAT, and POD in Chlorococcum sp. AZHB after exposure to Cu²⁺ and Cd²⁺, which could help improve the understanding regarding the microalgal physiological mechanisms involved in the tolerance of heavy metals.

2. Materials and Methods

2.1. Test Microalgal Species

The green microalgal species, Chlorococcum sp. AZHB, was collected and isolated from a wastewater treatment plant in Arizona, USA, and then cultured with a BG11 agar medium [12]. It was then transferred to a BG11 liquid medium under unialgal conditions for fast growth in order to obtain enough biomass to conduct the following exposure experiments. The microalgal species was maintained in Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences (FACHB).

2.2. Experimental Setup

The stock solutions of copper (i.e., 200 and 20 mg/L) were prepared with copper sulfate (CuSO₄·5H₂O), and were added to the BG11 medium to obtain working concentrations of 0, 0.01, 0.1, 1, 10, 50, 100, and 200 mg/L, respectively. The stock solutions of cadmium (i.e., 2000 and 50 mg/L) were prepared with cadmium chloride (CdCl₂·2.5H₂O), and were added to the BG11 medium to obtain working concentrations of 0, 0.1, 1, 5, 10, 50, 100, and 200 mg/L, respectively.

The precultivated microalgal cells were centrifuged at 3000 r/min for 10 min, and the pelleted microalgal cells were washed twice with 15 mg/L NaHCO₃ and re-suspended in sterile Milli-Q water for inoculating into the BG11 medium with Cu²⁺ or Cd²⁺. Culturing was carried out in 250 mL flasks containing 150 mL culture volume at 25 ± 1 °C in the LRH-250-G illuminating chamber (Taihong, Guangdong, China). Light was provided by continuous cool white fluorescent lamps at 35–40 μmol·photons m⁻²·s⁻¹ with a dark/light cycle of 14/10. The flasks were shaken four times every day, and the experiments lasted for 10 days. All of the treatments were performed in triplicate.

2.3. Extraction and Detection of MDA, SOD, CAT and POD

After 10 days of culturing, cultures of 150 mL were filtered using pre-weighed 0.45 μm pore-size filters, and the filters with microalgal cells were then weighed with an analytical balance. The difference in the final weight and the weight before filtration was regarded as the wet weight of microalgal cells. After adding five-fold PBS buffer (50 mmol/L, pH = 7.8) than the microalgal weight in a mortar, the microalgal cells were ground three times with a pestle under liquid nitrogen. After 15 min of centrifuging at 10,000 r/min at 4 °C, the supernatant was finally diluted to 25 mL with the PBS buffer (50 mmol/L, pH = 7.8). The resulting mixture of enzymes was used for determination of MDA, SOD, CAT, and POD. The detection of MDA and SOD followed the method described by Tang [20], and the detection of POD and CAT followed the method described by Li [21]. A SOD enzyme activity unit was defined as the amount of SOD enzyme that was required to inhibit 50% of NBT photo-reduction. A POD enzyme activity unit was defined as 0.01 change in A470 per minute. A CAT enzyme activity unit was the amount of enzyme required to decompose 1 mg H₂O₂ per minute.

2.4. Statistical Analyses

Differences in the MDA content and activities of SOD, CAT, and POD of among concentrations were examined using one-way analysis of variance (ANOVA), followed by the SNK test. Data were shown as mean +1 S.D. (n = 3). Pearson correlations among the enzymatic activities and the concentrations of Cu²⁺ or Cd²⁺ were performed. Analyses were performed through the use of the SPSS 19.0 package (Armonk, NY, USA the procedures). A value of p < 0.05 was considered to be statistically significant.

3. Results and Discussion

Copper is a trace element essential for living organisms because of its essential function as a cofactor in enzymes, including SOD, where one of the metal prosthetic groups consists of Cu. However, it could also cause damage to organisms at elevated concentrations [6,22]. For example, lower concentrations (i.e., 0.01–0.1 mg/L) of Cu²⁺ had no obvious effect on the growth of Chlorococcum sp. AZHB, but Cu²⁺ concentrations of 1–10 mg/L decreased the growth rate of this species. The growth was completely inhibited when the concentrations of Cu²⁺ were greater than 50 mg/L [12]. Cadmium is a non-essential element for organisms, but it is toxic when its concentration accumulates to a certain degree in organisms [23,24]. Concentrations of Cd²⁺ of lower than 1 mg/L had no obvious effect on the growth of Chlorococcum sp. AZHB. However, the microalgal growth was reduced at 5–10 mg/L Cd²⁺, and was completely inhibited when the concentrations of Cd²⁺ were higher than 50 mg/L [12].

In this study, we found that Cu²⁺ and Cd²⁺ also produced dramatic effects on the MDA concentration and SOD, POD, and CAT activities (Figure 1, Figure 2, Figure 3 and Figure 4). The MDA concentration increased with the increasing concentration of Cu²⁺ or Cd²⁺, but no significant difference occurred between the control and treatments at lower concentrations of Cu²⁺ or Cd²⁺ (Figure 1). MDA production is an indicator of cell peroxidation [14,19], and therefore higher concentrations of MDA in the treatments of elevated concentrations of Cu²⁺ or Cd²⁺ indicated the production of high oxidative damage caused by heavy metals. When stress appeared, the metabolic balance of free radicals within the cells of the plant was destroyed, which further promoted the generation of free radicals [14,25]. One of the toxic consequences of excessive free radicals was that membrane lipid peroxidation was initiated or enhanced, and the membrane system of cells was then damaged. Plant cells would die in extreme cases [25]. With the increasing contents of MDA, the oxidative damage caused by Cu²⁺ and Cd²⁺ stress increased with the increasing concentration of Cu²⁺ and Cd²⁺ in the medium. Soto et al. [26] also reported lipid damage in the cell membrane for the microalga Pseudokirchneriella subcapitata after exposure to 0.025 to 0.1 mg/L copper, expressed as a significant increase in MDA, which was consistent with the present study.

The peroxidation protective enzyme systems (i.e., activity of POD and SOD) in the microalgal cells increased with the increasing concentration of Cu²⁺ and Cd²⁺ (Figure 2 and Figure 3), showing the increasing activity of the antioxidant enzymes in this species when exposed to the two heavy metals. However, the activity of CAT showed a different change pattern (i.e., inverse-V shape) (Figure 4). The enzyme activity of CAT increased with the increasing concentration of Cu²⁺ from 0–0.1 mg/L and Cd²⁺ from 0–5 mg/L, and then decreased when the concentrations of Cu²⁺ and Cd²⁺ were greater than 0.1 and 5 mg/L, respectively (Figure 4). The inverse-V shape in the CAT activity had also been found in a previous study on mercury-induced CAT change in the green microalga Chlamydomonas reinhardtii [27], which may be attributable to the inhibition of CAT enzyme synthesis or the change in assembly of enzyme subunits at an extremely high concentration of heavy metals [28]. The enzymatic defense systems including SOD and CAT could transform O₂ and H₂O₂ into less active substances [18], and reduce or eliminate the attack to the membrane lipids, which thus could be protected without peroxidation [25]. This should be one of the physiological mechanisms that Chlorococcum sp. AZHB tolerated Cu²⁺ and Cd²⁺ stress. These results indicate that Cu²⁺ and Cd²⁺ could activate SOD, POD, and CAT, which was consistent with previous studies on heavy metal-induced toxicity to antioxidant enzymes in the algae Chlamydomonas reinhardtii [27], Ectocarpus siliculosus [29], Pseudokirchneriella subcapitata [26], and Scenedesmus sp. [18].

Significant correlations occurred between the concentration of Cu²⁺ or Cd²⁺ and the MDA concentration and activity of SOD or POD (

Table 1. Pearson correlations among the enzymatic activities and Cu²⁺ concentrations.

	Cu2+ Concentrations	MDA	SOD	POD	CAT
Cu2+ concentrations	1.000	0.923 **	0.780 *	0.821 *	−0.576
MDA		1.000	0.949 **	0.962 **	−0.431
SOD			1.000	0.992 **	−0.396
POD				1.000	−0.436
CAT					1.000

* p < 0.05, ** p < 0.01. MDA, malondialdehyde concentration, μmol/g ww; SOD, activity of superoxide dismutase, U/g ww; POD, activity of peroxidase, U/g ww·min; CAT, activity of catalase, U. ww, wet weight.

and

Table 2. Pearson correlations among the enzymatic activities and Cd²⁺ concentrations.

	Cd2+ Concentrations	MDA	SOD	POD	CAT
Cd2+ concentrations	1.000	0.789 *	0.763 *	0.879 **	0.154
MDA		1.000	0.960 **	0.974 **	0.640
SOD			1.000	0.950 **	0.652
POD				1.000	0.523
CAT					1.000

* p < 0.05, ** p < 0.01. MDA, malondialdehyde concentration, μmol/g ww; SOD, activity of superoxide dismutase, U/g ww; POD, activity of peroxidase, U/g ww·min; CAT, activity of catalase, U. ww, wet weight.

), which indicated that oxidative stress increased linearly with the increasing concentration of heavy metals. However, no correlation occurred between the CAT concentration and the concentration of Cu²⁺ or Cd²⁺ (Table 1 and Table 2), which suggests the complexity of changes of CAT activity induced by heavy metals. In fact, heavy-metal-induced CAT activity may remain unchangeable [30], or increase [26], decrease [28], or increase first then decrease with the increasing concentration of heavy metals [27]. Thus, the CAT activity may be affected by several factors, including the exposure concentration of heavy metals, microalgal species, exposure time, or other the unfavorable environmental conditions.

The main factors leading to differences in the toxicity of heavy metals to microalgae include different microalgal species, physiological and environmental conditions, and the form of heavy metals [30]. Compared with other microalgae, including Chlorella and Scenedesmus [6,22,23], Chlorococcum sp. AZHB could endure higher concentrations of Cu²⁺ and Cd²⁺, with the highest removal efficiency being about 88% [12]. The microalgal species that are applied to wastewater treatment should have a high tolerance to heavy metals and a strong accumulation capacity of heavy metals [6]. Thus, the microalgal species Chlorococcum sp. AZHB could be harnessed to treat wastewaters contaminated with heavy metals including copper and cadmium.

Microalgae have several mechanisms (Figure 5) to defend against the toxicity of heavy metals, including the exclusion of metal ions by the cell wall [31] and intracellular detoxification by binding to polyphosphate or phytochelatins [6]. The cell wall of Chlorococcum sp. AZHB had no obvious changes at exposures of 0–1 mg/L Cu²⁺ or 0–5 mg/L Cd²⁺, but became thicker at higher concentrations of Cu²⁺ or Cd²⁺ [12]. However, the number of pyrenoids increased largely upon exposure to lower concentrations of Cu²⁺ or Cd²⁺, but were reduced to one or disappeared after exposure to higher concentrations of these two heavy metals [12]. This suggests that Cu²⁺/Cd²⁺-induced toxicity defending mechanisms to this species should be involved in both microalgal extracellular and intracellular detoxification when exposed to heavy metals. This study further demonstrated that antioxidant enzyme systems should also be involved in toxicity defending against Cu²⁺ and Cd²⁺ stress.

4. Conclusions

The MDA content in the green microalga Chlorococcum sp. AZHB increased with the increasing concentrations of Cu²⁺ or Cd²⁺, suggesting that oxidative damage occurred after exposure to the two heavy metals. The response mechanism of this microalgal species might involve the linear improvement of the antioxidant enzyme activity predominantly POD and SOD, with an increase in the tested metal concentrations. However, the response of CAT activity increased first and then decreased with the increasing concentrations of heavy metals. Further study is warranted about the changes in the isozyme zymograms of SOD, POD, and CAT under Cu²⁺ and Cd²⁺ stress in order to comprehensively explain the defense and response mechanisms of microalgal cells to heavy metals.