3.1. Cd Removal Efficiency
The capacity of free-living Oscillatoria
sp. to remove Cd from artificially-contaminated aqueous samples is shown in Figure 1
. As can be appraised, Cd concentration from the medium (PBS, pH 7.4) decreased over time, and most of this reduction took place during the first 12–24 h, at both tested metal concentrations (i.e., 5.0 and 25.0 mg/mL). In addition, overall Cd removal performance by Oscillatoria
sp. was negatively affected by the metal concentration increase. A maximal removal rate of 66.0% was reached when the cyanobacterial cells were exposed to 5.0 mg/L of Cd for 24 h. However, at a metal concentration 5 times higher (i.e., 25.0 mg/L), a slight decrease of around 7% (58.8%) was recorded in Cd removal from the aqueous system at the same period of time. These results suggest that Oscillatoria
sp. might pose a series of biological mechanisms to deal with Cd intracellular toxicity.
Additionally, Cd removal from water fits a second-order reaction model (Table 1
), suggesting that the entire bioprocess depends not only on the metal concentration, but also on the number/type of metal-binding sites in the microalgal biomass, among other potential metabolic features.
In fact, a biphasic nature of Cd elimination can be clearly observed in Figure 1
, with an initial rapid phase that occurs in the first 4 h, and a slower one from 6 h onwards that reaches a plateau between 12–24 h of metal exposure. This non-linear Cd removal kinetics could be explained by the fact that living Oscillatoria
sp. utilizes two principal mechanisms for metal elimination. The fastest component of metal uptake is Cd adsorption on the cyanobacterial cell outer membrane, while the second most important mechanism for Cd removal is metal accumulation within the cells (Figure 1
). Moreover, the amount of accumulated Cd increased with time and was higher as the initial concentration of metal in the sample increased, suggesting the existence of some metabolic or physiological responses in Oscillatoria
sp. that depends on the metal concentration, and to a lesser extent, on the time of exposure to the toxic metal.
In general, metal accumulation in living organisms involves a series of complex biologically-regulated processes, such as metal translocation by transmembrane proteins (e.g., porins) and ionic channels, and the posterior interaction with cysteine- and glutamic acid-rich peptides, such as glutathione, phytochelatins, and metallothioneins [19
]. These peptides are able to form stable metal–protein complexes, thus reducing the deleterious effect of toxic metal ions on living cells [37
]. The occurrence of metallothioneins and their importance for heavy metal detoxification were reported in several organisms including plants, yeast, bacteria, mollusks, cyanobacteria, and mammals [38
]. In cyanobacteria, metallothioneins were first identified in cells adapted to grow in elevated levels of Cd or Zn [19
]. Moreover, [39
] reported the Cd-induced expression of a novel metallothionein (BmtA) in Oscillatoria brevis
and its importance for metal detoxification. In addition, these authors suggested that O. brevis
utilizes metallothionein as a primary heavy metal resistance mechanism to confer Cd tolerance to cells. A similar response might be used by other Oscillatoria
species to deal with heavy metal toxicity; however, more experimental evidence is needed to support this hypothesis.
Our results showed that an increase in Cd accumulation was detected when metal concentration increased from 5.0 to 25.0 mg/L, suggesting the induction of some response mechanisms (e.g., synthesis of metallothioneins, increased metal-storage/retention in vacuoles, etc.) involved in Cd bioaccumulation by Oscillatoria sp. However, the proportion of metal adsorbed was always higher than the amount of metal accumulated.
It was described that metal adsorption to cyanobacterial biomass occurs by a number of different physico-chemical interactions involving ionic bonds, cationic exchange, and chemical chelation. The presence of polysaccharides, proteins, and lipids on the surface of their cell walls containing different functional groups such as amino, hydroxyl, carboxyl, sulfate, and phosphate might act as binding sites for cationic metals [30
]. In addition, the presence of negative charges (e.g., uronic acids) in cyanobacterial exopolysaccharides was proposed to play an important role in the sequestration of cations, a phenomenon of major interest for metallurgical industry wastewater treatment [19
]. Therefore, a change in the functional groups of Oscillatoria
sp. surface is a key point to understanding the metal binding mechanism in these photoautotrophic microorganisms.
In this study, functional groups on the surface of the cyanobacterial biomass responsible for Cd adsorption were determined by using FTIR-ATR spectroscopy, with and without Cd exposure. As can be appraised in Figure 2
, several major bands at 3260, 2928, 1730, 1628, 1510, 1450, 1387, 1231, 1146, and 1026 cm−1
were observed in the spectrum of non-treated Oscillatoria
The broad band at 3500–3050 cm−1
was characteristic of the O–H and N–H groups [30
], whereas the bands at 2928 and 2853 cm−1
refer to the asymmetric and symmetric vibration of methylene (CH2
), respectively [28
]. The peaks at 1730 cm−1
correspond to the C=O bond. The bands within the region of 1500–1000 cm−1
were characteristic of the ester group. Several peaks were obtained in this region, indicating the stretching vibrations. Other bands of the spectrum could be attributed as follows: 1628 cm−1
group or –C–N of amide), 1510 cm−1
(–N–H bending), 1387 cm−1
stretching), 1231 cm−1
antisymmetric stretch), 1146 cm−1
(–C–O stretching of carboxylic acids), and 1026 cm−1
(–C–N and –C–C stretching vibrations) [40
]. The bands <930 cm−1
are the fingerprint zone and include phosphate and sulfur functional groups [28
Remarkable differences were observed in the spectra of Oscillatoria
biomass obtained with and without Cd treatment (Figure 2
). Many peaks in the spectrum of untreated cyanobacterial biomass shifted due to the presence of Cd in the medium. The shifting of peaks indicates that Oscillatoria
sp. possess a metal binding ability, indicating the usefulness of this microalgae for treating Cd-containing effluents. In addition, some other bands showed a lower intensity because of the presence of the metal. These changes could be due to the Cd removal by the filamentous microalgae. Therefore, our results proved that different functional groups (mainly anionic ones) present at Oscillatoria
sp. biomass such as carboxyl, phosphate, amide, amino, hydroxyl, sulfur, and others play a significant role in Cd adsorption. These results were consistent with related studies on the exposure of different microalgae to toxic metals [30
] characterized the functional groups potentially involved in divalent copper ions (Cu2+
) adsorption on dried algae biomass of Oscillatoria splendida
, by using FTIR spectroscopy. These authors described the presence of carbonyl, hydroxyl, and aliphatic chains acting as ligands for Cu binding. In the present study, the FTIR–ATR spectra obtained for Oscillatoria
sp. biomass (Figure 2
) with and without Cd-exposure were closely related to that reported by [41
], suggesting that the high content of carbohydrates and lipids with negatively charged groups (mainly carboxyl and phosphate) present in the cyanobacterial biomass was the most relevant factor in determining Cd binding.
Related to this, [15
] reported that Cd biosorption was maximal at a pH near neutrality, due to an increase in the number of negatively-charged ligands, which favored the binding of cationic metals, such as carboxyl and phosphate groups of lipopolysaccharides and phospholipids that were present in the outer membrane of Oscillatoria
. Moreover, at more alkaline pH values (e.g., pH > 8), Cd solubility could be lowered due to the formation of metal hydroxides. On the other hand, in acidic conditions (pH < 5), carboxyl and phosphate groups were closely linked to H+
ions, thereby making these sites unavailable for Cd cations. Therefore, in order to ensure an active biological uptake by the free-living microalgae, media pH regulation is another important parameter to be considered since it not only affects cell viability and acid–base behavior of the metal binding sites, but also metal chemistry and bioavailability.
As mentioned before, the efficiency of Cd elimination by Oscillatoria
sp. was negatively affected by the increase in Cd concentration from 5.0 to 25.0 mg/L. However, it is worth noting that at higher Cd levels of 50 mg/L, a greater metal removal efficiency was evidenced (>99%), probably due to an increasing cell damage (data not shown
). This fact could be associated with the exposure of additional metal-binding sites that enhanced Cd biosorption properties of the dead biomass, since non-living cells proved to be more efficient than living ones for metal elimination [15
]. However, when microalgae were used as biosorbents (i.e., dead or inactive biomass) for removing metal ions, their low growth rates and cell densities made the biomass production very costly to be further used as inactive biomass for wastewater treatment. In contrast, the removal of metal ions by live microalgae cultures could overcome this disadvantage. In addition, a bioremediation of some nutritional components that frequently contributed to the eutrophication of aquatic ecosystems always occurred simultaneously during microalgae cultures [42
Finally, one of the major hurdles in the removal of heavy metals by microalgae cultures is the high cost associated with the harvest of tiny cells from diluted cultures. A cost-effective strategy for removing microbial cells from diluted broth is gravity sedimentation facilitated by flocculation, particularly by the self-flocculation properties of some microbial species. Thus, compared to other regular cyanobacteria species, flocculating Oscillatoria sp. presents as an excellent candidate to be further explored for improved heavy metal tolerance, with the aim of applying it in metal-polluted wastewater management, due to the cost-effective sedimentation of these self-flocculating microorganisms.
3.2. Evaluation of Cd Toxicity in Oscillatoria sp.
Cyanobacteria are frequently challenged by toxic metals that have no function as nutrients. Due to their photoautotrophic lifestyle, cyanobacteria evolved to deal with toxic reactive oxygen species (ROS) produced by their metal-rich photosynthetic apparatus [19
]. In the present study, Oscillatoria
sp. showed different metabolic and physiological responses to Cd challenge. As can be seen in Table 2
, Cd toxicity in Oscillatoria
sp. live cells involves the generation of ROS, as shown by the increment in the levels of malondialdehyde (MDA). After 24-h of metal exposure, the content of MDA significantly increased by about 15% (p
< 0.05) when the cells were treated with 5.0 mg/mL of Cd, in comparison to the control untreated group, and up to 28% (p
< 0.05) with the highest tested metal concentration of 25.0 mg/mL. These results might be explained by the increase in Cd bioaccumulation by microalgae, showed in Figure 1
Metal toxicity is based on its chemical properties, and in general, promotes the production of ROS, the inactivation of enzymes, or the displacement of the normal metal cofactors in some cyanobacterial metalloproteins [19
]. It was described that Cd ions are able to induce oxidative stress at similar doses to the ones used in the present study in related microorganisms, such as Chlorella vulgaris
, Chlamydomonas reinhardtii
, and Spirogyra setiformis
]. Our results agreed with these observations; however, to the best of our knowledge, this was the first description of the Cd-induced oxidative stress in free-living Oscillatoria
The MDA content is considered an index of lipid peroxidation and a high level of MDA accumulation from peroxidation of unsaturated fatty acids can cause serious damage on nucleic acids and proteins [32
]. In addition, ROS reaction with lipids and proteins might cause membrane damage, biomolecule degradation, and enzyme inactivation [19
]. In fact, a decrease (~18%, p
< 0.05) in dehydrogenase (DHase) activity was detected in Oscillatoria
sp. cells exposed to Cd, with respect to the untreated control cells, after a 24-h metal exposure (Table 2
). The reduction of tetrazolium salts, such as TTC, to red-colored formazan through degradative dehydrogenases, is a widely accepted method for detecting the physiological state of several organisms, including microalgae cultures [33
]. Further, as TTC is a metabolic dye, suboptimal growth or stressful conditions (e.g., nutrient limitation, toxicant presence, etc.) could lead to inefficient TTC reduction by cells [46
]. Therefore, the quantity of intracellular formazan product, estimated by its absorbance at 485 nm, could be used as a surrogate marker of pollutant cytotoxicity [47
]. Hence, the results shown in Table 2
suggest that Oscillatoria
sp. is partially affected by Cd exposure and this might be related to metal-induced oxidative damage.
However, it is noteworthy that cyanobacterial DHase activity decreased to a similar extent when Oscillatoria
sp. was treated with 5.0 mg/L of Cd or with a metal concentration 5 times higher (i.e., 25.0 mg/L). In addition, no changes in cellular density (O.D.560nm
), protein content, and chlorophyll a
) concentration were observed after 24-h metal exposure between control cells and Cd-treated cyanobacteria (Table 2
). These results strongly suggest that Oscillatoria
sp. might develop different enzymatic and non-enzymatic intracellular antioxidant responses to depress the oxidative stress induced by the metal. Additionally, it is important to mention that cellular reduction of tetrazolium compounds might proceed independent of ATP production [45
], thus the observed decrease in the DHAse activity in Oscillatoria
sp. is not necessarily related to an ATP depletion in the cells. However, as DHase activity is also related to cell viability, it should be appropriate to further evaluate cyanobacteria growth after long-term Cd exposure.
As mentioned before, Oscillatoria
sp. seems to trigger some intracellular strategies to deal with Cd-induced oxidative damage. As can be appraised in Table 2
, Cd exposure in Oscillatoria
sp. interferes with the normal metabolism of carotenoids, since a significant reduction (~15%, p
< 0.05) in the total carotenoid content was observed in culture cyanobacteria exposed to 5.0 mg/L or 25.0 mg/L of Cd for 24 h. Carotenoids are known to play important roles as antioxidants and accessory light-harvesting pigments. These compounds are essential to photosynthesis, acting as secondary pigments. They are also pro-vitamin factors, and are involved in free radical elimination [48
]. Reactive oxygen species (ROS) can oxidize carotenoids, leading to a variety of oxidized products, including aldehydes, ketones, endoperoxides, and lactones. Some of these are reactive electrophile species that are bioactive and can induce changes in the gene expression, leading to acclimation of cells to stress conditions [49
]. Furthermore, carotenoids quench excess excitation energy to protect chlorophyll molecules (the main pigments responsible for collecting solar radiation during the photosynthetic process) from oxidative damage preserving the ATP levels [50
]. Therefore, our results suggest that Oscillatoria
sp. might improve its antioxidative defense system under the metal stressful conditions through a carotenoid-mediated ROS quenching. Related to this, it is noteworthy that the chl-a
content between the control and Cd-treated Oscillatoria
sp. cells did not statistically change, probably due to a faster hydrolysis of carotenoids, compared to chl-a
in the cells under metallic stress (Table 2
Moreover, the cyanobacteria culture did not show a marked reduction of their cell density (as a surrogate marker of biomass growth) and total protein content, as compared to the control group, after a 24-h metal exposure (Table 2
). In this context, [42
] recently proposed that the formation of photosynthetic pigments, in particular chl-a
, synchronized with the growth of the microalgal cells, could be used as an indicator for evaluating the removal efficiency of metal ions from polluted water.
Besides its impact on cyanobacterial oxidative stress, DHAse activity, and carotenoid content, Cd exposure also affected carbohydrate production by the filamentous microalgae. As shown in Table 2
, the content of soluble carbohydrates in Oscillatoria
sp. significantly increased (p
< 0.05) at both tested metal concentrations (i.e., 5.0 and 25.0 mg/L). These results might be explained by a reduction in carbohydrate utilization as a consequence of a lower rate in carbon assimilation caused by the reduction in the total carotenoid content. Further, the environmental stressful conditions caused by Cd exposure could induce the mobilization of soluble sugars where required, in order to preserve the osmotic homeostasis of the cyanobacterial cells, since these compounds not only act as structural cellular constituents, but also as intracellular signaling molecules involved in the regulation of metabolic processes associated with growth, ATP production, and survival of cells. Therefore, it is possible that the stress caused in Oscillatoria
sp. due to the intracellular accumulation of Cd prompts an increase in the amount of soluble carbohydrates as a protective mechanism, in order to counteract the oxidative damage, since these biomolecules play a very active role in cell energy management. A similar behavior was reported in plants exposed to heavy metal stress [51
], thus, it is possible that a similar mechanism could be used by photosynthetic microalgae.
Collectively, these data suggest that Cd-challenged Oscillatoria sp. might trigger an integrated reprogramming of their carotenoid and carbohydrate metabolism to protect, on one hand, the photosynthesis (i.e., preserve chl-a content), which normally provides ATP for cell growth and viability, and on the other hand, the protein synthesis, limiting in this way the poisoning incorporation of Cd into cells. Therefore, the tolerance of a cyanobacterium against heavy metal stress seems to be controlled by a complex and highly interrelated network of molecular and physiological approaches that help to counteract metal phytotoxicity. However, more evidence in Oscillatoria sp. is still needed at the molecular level (e.g., up- and down-regulation of genes), in order to understand these fascinating mechanisms. Additionally, large-scale performance assays and long-term metal removal studies using free-living cells are still needed to further design microalgae-based systems for industrial-scale wastewater management.