Effects of Cd and MPs/NPs on the growth of M. aeruginosa
Effects of Cd and MPs/NPs on algal growth withprolonged time
Algal density as a useful indicator to reflect the growth effect under environmental stress was analyzed in Fig. 1. The density of algal cells cultured in different treatment groups was lower than that in the control group, indicating that both MPs/NPs and Cd ions had inhibition effects on the growth of algae. The increased Ir (Fig. S1) of algal cells with the prolonged exposure time showed that the effects of each treatment group used to study the growth of algae had a cumulative effect. Both MPs and NPs had a certain inhibition effect on the growth of algae. This result was similar to that of our previous report showing that MPs had a weak effect on M. aeruginosa due to the adsorption of nutrients onto MPs (Wang et al. 2021). The impact of NPs alone was greater than that of MPs, which proved that the smaller the size of the particles are, the stronger the identifiable presence of toxicity is (Anbumani and Kakkar 2018; Sjollema et al. 2016; Zhang et al. 2017). It can be determined that NPs may attach to the surface of phytoplankton and reduce the activity of microalgae by disrupting their cell morphology and organelle function (Liu et al. 2020; Wei et al. 2020). In the presence of Cd, the inhibition of the algae growth was greatly enhanced. Similar studies have shown that Cd with a concentration of 2 mg/L can significantly inhibit the growth of mixotrophic Ochromonas gloeopara, which may be caused by nucleic acid decomposition in alga cells by preventing the formation of phosphate (Wu et al. 2021). In addition, high concentrations of metal ions can induce the production of ROS, which can easily interact with nucleic acids and cause DNA damage in algal cells eventually (Deng et al. 2020). This DNA damage was expected since heavy metals have a reputation for destroying the permeability of cell membranes and hindering the exchange of substances and the uptake of nutrients, which affects the normal growth of algae (Wu et al. 2021).
In the combined system, a similar trend was found when looking at the activity of Cd alone. But the Ir was decreased slightly in the existence of PS due to the adsorption ability of heavy metal ions. Moreover, the presence of NPs could decrease the Ir more than MPs even though there was no significant difference between them. Due to the smaller size and higher specific surface area, NPs had the higher adsorption ability of metal ions compared to MPs which could decrease the effluence of Cd ions (Bhagat et al. 2021).
Effects of different concentrations of MPs/NPs on algal growth
A various concentration of MPs/NPs was studied to explore the effect of MPs and NPs on algae growth in both stand-alone MPs and NPs as well as the combined MP/NP system. After 96 h of exposure time, the Ir gradually improved with the increasing concentration of polystyrene (PS) (Fig. 2a) in the single pollution system, due to the shielding effect and enhanced interaction between plastics and alga (Wang et al. 2020). In the joint system of Cd and PS, even more inhibitory effects to algal growth became evident compared to PS alone. The results indicated that the addition of heavy metals (particularly Cd) may greatly increase their toxic effect on algae and play a key anti-algae role in the combined system. At the same time, it was also found that the Ir of algal cells declined as the PS dose increased in the combined system. This may be related to the variances in the accumulation of Cd in cells along with the PS concentrations. Moreover, the Ir of alga treated with NPs was slightly lower in the system where the alga was also treated with MPs, even though statistically, no significant difference was found in the performance of NPs compared with MPs.
To further understand the toxicity effect of MPs/NPs in the combined system, we also investigated the accumulation of Cd in algal cells under different concentrations of PS. After 96 h of exposure time, with the increasing concentrations of MPs/NPs, the accumulation of the Cd in cells decreased obviously (Fig. 2b). Meanwhile, in the presence of NPs, Cd concentration decreased more significantly than it did when in the presence of MPs. This could be explained by the higher adsorption capacities of metal ions onto NPs than MPs (Fig. S2). Although the accumulation of Cd inside the cells after the MPs and NPs treatments was distinct, the difference of the toxic effect on algae between them was not significant. This may be caused by the promote of higher number of EPS generated by algae under the stress of NPs, which could combine with more metal ion Cd in the solution (Shen et al. 2018). Meanwhile, the complex effect of the EPS with heavy metal ions could reduce the toxic effect of Cd in the combined system.
Effects of different concentrations of Cd on algal growth
With the increase of Cd concentration, the Ir of alga increased continuously after 96 h of exposure (Fig. 3a). The inhibition effect on alga increased gradually with the highest inhibition rate reaching up to 92.8 % under the stress of Cd (2.0 mg/L) alone. The presence of Cd may have a remarkable influence on the normal metabolic process of algae (Horcsik et al. 2007). Heavy metals have some effect on the survival of algae by disturbing their physiological processes including endocytosis and food absorption (Wu et al. 2021). Naveed et al. determined that Cd could competitively replace Ca2+ and Mg2+ by entering into cells and inhibiting the activity of enzymes that require Ca2+ and Mg2+, thereby affecting the metabolic activity and photosynthesis of organisms (Naveed et al. 2019).
The combined system of MPs/NPs and Cd also had harmful effects to the algae. With the increasing of Cd concentration, the Ir was enhanced from 52.8% to 89% under the stress of MPs and Cd. The Ir was increased from 48.9% to 86% under the combined system of NPs with Cd after the addition of Cd. In the presence of NPs, the inhibition effect was suppressed when compared to MPs in the joint system. The main reasons could be the higher adsorption ability of NPs compared to MPs Meanwhile, the NPs could promote the algae to produce more amount of EPS, which may have complexing with metal ions (Shiu et al. 2020; Naveed et al. 2019). EPS may involve in the extracellular defense mechanism that tried to prevent the metal ions from entering into cells (Yan et al. 2021).
The accumulation of Cd in algal cells under different concentrations of Cd was also investigated (Fig. 3b). As the concentration of Cd increased, the accumulation of Cd in cells was enhanced gradually. The results indicated that Cd could enter into the cell through the cell membrane, causing a physiological disorder and toxic effects. Meanwhile, the accumulation of Cd in algal cells was lower in the presence of MPs and NPs compared to Cd alone, which indicated that the presence of MPs and NPs can inhibit Cd from entering into cells by adsorption behavior. In the combined system of NPs and Cd, the accumulation of Cd in algal cells was lower than that of MPs, which may be explained by the enhanced adsorption capacity of Cd onto NPs, which decreased the entry of Cd ions into the cell at the same time. Although the accumulation of Cd was varied greatly according to the amounts of Cd inside the cells, the difference of the toxicity between MPs and NPs in the joint system was not significant. The possible reasons were the toxic effects of cadmium which play a key role in the compound system (Liao et al. 2020), the agglomeration of nanoparticles may have some effect on the toxicity (Si et al. 2015), and the complexation of extracellular polymers with heavy metal ions (Sendra et al. 2017).
Effects of Cd and MPs/NPs on Chl-a with time
Chl-a is used as a standard index to reflect the growth and proliferation of algae as shown in Fig. 4. In the presence of Cd, the Chl-a content of algae was obviously reduced more than in the contact with MPs/NPs alone. The existence of Cd has a significant inhibitory effect on the Chl-a content of algae. The addition of Cd will reduce the biosynthesis and content of Chl-a by destroying the chloroplast structure, inhibiting the expression of photosynthesis-related genes, thereby thwarting the photosynthesis and growth of algae. The effect of NPs on the Chl-a content of algae was slightly greater than the effect of MPs, which is consistent with the results of Wu et al. (2019). This can be explained by the accumulation of intracellular ROS in the presence of NPs which can damage the cellular structure and hinder chlorophyll synthesis. The production of ROS can affect the function of chloroplasts, resulting in the disturbance of photosynthesis and metabolism (Zhang et al. 2020).
The combined system also had a high inhibition effect on the content of Chl-a which was lower than the inhibition effect of Cd alone but higher than that of PS alone. But the difference between MPs and NPs was not significant. The results demonstrated that in the presence of MPs/NPs, the combined system had a weakened effect on the photosynthesis of alga compared to Cd alone.
SOD activity and MDA content
To further investigate the toxic effects of MPs, NPs and Cd on algae, we studied the enzymatic activities of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) under different treatment groups. The antioxidant enzyme SOD, which is responsible for scavenging superoxide free radicals in cells, is considered a protective characteristic to the antioxidant system (Lu et al. 2018). MDA, which is formed during the degradation of aliphatic acids, is a sign of lipid peroxidation. The higher the content of MDA is, the deeper the degree is of cell membrane lipid peroxidation (Ni et al. 2018). Both the SOD activity and MDA content expressed the highest value under the stress of a single Cd group, indicating that Cd had the strongest toxic effect on algal cells (Fig. 5). The presence of MPs and NPs alone showed a certain effect on the toxicity of algae based on the SOD and MDA values due to the physical adsorption ability of algae onto MPs/NPs which would inhibit the photosynthesis of algae cells (Wang et al. 2020). Because of the large specific surface area of NPs, it is easier to accumulate and adsorb on the surface of microalgae and cause more damage to the cell membrane compared to MPs. In addition, NPs may block the transport of external substances from entering into microalgae, thus impede the exchange of matter and energy inside and outside the cells. Moreover, NPs may also induce the production of superoxide radical (O2-) by microalgae that enhance its inhibitory effect on algae (Yan et al. 2021). Meanwhile the MPs had less effect on the antioxidant system of algae than the NPs.
The toxicity of the combined system was lower than a single Cd system but higher than the MPs and NPs alone, which indicated that the presence of MPs and NPs could reduce the bioavailability of Cd in the composite system. The levels of antioxidant enzymes and growth inhibition rate were significantly increased in both Cd and combined treatment groups. The disruption of photosynthesis and the increase of the level of antioxidant enzyme may lead to membrane lipid peroxidation which cause the cell damage and growth inhibition. The addition of NPs in the joint system could decrease the activity of SOD and the content of MDA more obviously when compared to MPs, because NPs have a certain adsorption effect on Cd (Fu et al. 2019). It can also stimulate the algae to produce more EPS to complex heavy metals, thus reducing the toxic effect of Cd in the composite system (Mao et al. 2018).
Due to the detoxification mechanism of algae under the stress of pollutants, the algae may produce antioxidant enzymes to eliminate the damage caused by ROS (Wu et al. 2021). Both MPs, NPs and Cd could induce oxidative stress to cells which relate to the rise of ROS, leading to cell membrane damage and even death ultimately (Thiagarajan et al. 2019). The organism would develop an effective antioxidant defense system to maintain normal intracellular oxidative stress in response to excessive production of ROS. Therefore, the level of ROS was considered as a typical biomarker and has traditionally been used to evaluate the toxicity effect under stress (Miao et al. 2019).
As shown in Fig. 6, there was more ROS produced under the stress of Cd and MPs/NPs than the control group. The order of ROS percentages is listed as follows: Cd alone > MPs +Cd > NPs +Cd > NPs alone > MPs alone. The results confirmed that the more obvious toxicity was found in the presence of Cd compared to MPs/NPs alone, which is consistent with the SOD and MDA results. Thiagarajan et al. (2019) found that MPs could stimulate algal cells to produce ROS by restricting light irradiation and nutrient transfer. In addition, harmful metabolites are kept from entering the algal cells, which affects the normal activities of algae. NPs can hinder photosynthesis by attaching to the surface of algae, leading to the increase of ROS in algal cells (Bhattacharya et al. 2010). Moreover, NPs may interact with the enzymes in algae and affect its ecosystem function (Miao et al. 2019). In the presence of Cd, NPs can interact with algae through electrostatic adsorption and complexation, which results in the inhibition of enzymes needed for the growth and metabolism of algae, occurrence of the oxidative stress reactions and the generation of ROS—all of which can eventually produce toxic effects (Naveed et al. 2019). Therefore, in order to relieve the harmful effects of ROS, organisms will develop an effective antioxidant defense system to maintain normal levels of oxidative stress in cells and eliminate ROS by activating microbial antioxidant mechanisms. As shown in Fig. 4, the production of ROS may reflect the response to the stress of pollutants and affect algal photosynthesis and metabolism (Middepogu et al. 2018; Zhang et al. 2020). In our research, the antioxidant system of M. aeruginosa was seriously damaged under the stress of Cd both in single and combined system, leading to a more obvious oxidative stress response of algal cells when compared to MPs and NPs alone.
EPS make up the layer of biomacromolecular matrix covering the surface of cells produced by algae. The main components of EPS include polysaccharides and proteins, which are usually presented as a dynamic bi-layer structure composed of S-EPS and B-EPS (Naveed et al. 2019). The secretion of EPS by algae used to regulate the metabolic activities in the cells seems more like a self-protection mechanism under external stress such as heavy metals. EPS could react with metal ions by ion exchange, coordination interaction and complexation with its rich active functional groups in order to change the migration behavior and bioavailability of metals by organisms. The adsorption capacity of heavy metals is related to the EPS content of protein and polysaccharide. Meanwhile, S-EPS and B-EPS have different antitoxic abilities under external attack. Chen et al. found that more EPSs were produced by Chlorella vulgaris(C. vulgaris) when the algae was treated with Cd, while B-EPS was more effective than S-EPS in reducing the toxicity of Cd(Chen et al. 2015).
This study presents an analysis of both S-EPS and B-EPS contents of algae in different treatment groups. As shown in Fig. 7(a), less effect on the polysaccharide content is noted at different treatment groups compared to the production of protein in S-EPS. The content of protein was significantly increased in S-EPS in the presence of Cd due to the self-protection mechanism of algae. Compared to MPs, NPs could accelerate the alga to produce more EPS due to the smaller particle size, easy agglomeration, enhanced interaction and higher toxicity (Cunha et al. 2019; Nava et al. 2021). Because of the high content of amino, carboxyl, hydroxyl and other complex functional protein groups in EPS, the EPS in algae can potentially bind with heavy metals, thereby alleviating the negative effects of Cd on algal cells (Zhou et al. 2016).
Polysaccharide and protein of B-EPS in each treatment group were also promoted when compared with the control group (as seen in Fig. 7b). The highest content of B-EPS was found in the presence of Cd alone compared to the other treatments. The binding capacity of B-EPS with Cd could prevent metal ions from entering into cells thereby reducing the toxicity of metal ions. Meanwhile, Cd can also hinder the nutrient absorption of cells and the excretion of metabolites from intracellular activities that cause certain damage to algal cells. With the coexistence of NPs, both polysaccharide and protein in B-EPS were noticed to be higher than that found in MPs. The results can be further verified by the smaller accumulation of Cd in cells in the presence of NPs than in the presence of MPs in the combined system.
Aggregation of the algae and MPs/NPs
As shown in Fig. 8(a), M. aeruginosa in the control group has integrity of cellular structure and easily aggregates. After contact with MPs, algae were adsorbing and surrounding the surface of MPs in order to obstruct the normal growth of algae (Fig. 8b). In the presence of NPs, lots of sticky substances had generated around the algae cells which was speculated as EPS (Wang et al. 2021; He et al. 2019) (Fig. 8c). The EPS was increased significantly by the self-protection mechanism of algae under the stress of external pollutants (Wang et al. 2021; Li et al. 2019). Under the stress of Cd alone, the most abundant EPS was noted as shown in Fig. 8f. We also deduced that the presence of Cd not only promotes the algal cells but can also produce more EPS by self-protection, which also leads to the morphological damage of cells and the reduction of the algal cell density, resulting in the greater toxic effects (Wang et al. 2021).
In the composite system (Fig. 8d, e), more obvious EPS was generated by algae after the treatment of NPs and then MPs in the presence of Cd, which was in accordance with the former results of the EPS production. Meanwhile, the morphology of algae cells was damaged in the combined system shown in the SEM images, which revealed that the combined system of MPs/NPs with Cd had more of a negative effect on the algal cells than MPs or NPs alone. The existence of Cd with high toxicity in this system may contribution to this effect.
Extracellular and Intracellular MC-LR contents
M. aeruginosa is a representative species of cyanobacteria in freshwater, which is a main cause of the algal blooms. M. aeruginosa will release MCs that threaten the health of aquatic organisms. MC-LR, as one of the most typical types of MCs, contains both intracellular and extracellular MC-LR, which was considered in this research. The amount of extracellular MC-LR may be related to membrane lipid peroxidation and protein membrane synthesis (Zhang et al. 2020). Meanwhile, the dead algal cells also release intracellular MC-LR outside the cell, resulting in a higher extracellular MC-LR content.
As shown in Fig. 9, the maximum MC-LR both inside and outside the cells was produced under the stress of Cd, which was related to the stress reaction of cells. In addition, the osmosis of the metal ions through the membrane had the same effect on the algal metabolic activity that increased the permeability of the cell, thereby stimulating the release of the MC-LR (Xu et al. 2019). In comparison, NPs could promote more production and release of MC-LR than MPs due to the greater influence on lipid peroxidation of a cell membrane, which in turn affects the fluidity and permeability of membrane (Feng et al. 2020).
In the composite system, the generation of MC-LR was higher than in the single MPs/NPs system but lower than in the single Cd system. There was no significant difference between MPs and NPs in the combined system based on the comprehensive influencing mechanism such as the membrane lipid peroxidation caused by cell membrane damage, the size of the algal density, cell lysis and oxidative stress (Wu et al. 2020). In a word, the presence of MPs/NPs and Cd can induce MC-LR by M. aeruginosa, which gives rise to the cyanobacteria blooms and eutrophication of water especially in the existence with Cd.