Physiological and biochemical parameters in macroalgae Chara sp. in response to oxidative stress following remediation of malachite green

Physiological and biochemical parameters in macroalgae Chara sp. in response to oxidative stress following remediation of malachite green
Introduction
The effluents of wastewater in some industries such as dyestuff, textiles, leather, paper, plastics, etc., contain various kinds of synthetic dyestuffs. The effluents of these industries are highly colored and the evacuation of these wastes into receiving waters causes intense damages to the environment and biological systems. In recent years a number of studies have focused on some micro/macro-organisms that are able to biodegrade and absorb dyes in wastewaters. Phytoremediation is a newly evolving field of science and technology that uses plants and algae to clean up polluted sites. This technology has been received attention lately as an innovative, cost-effective alternative to the more conventional water treatment methods.
In this study, macroalgae Chara sp. was used in order to decolorize a dye solution containing Malachite Green (MG). MG, a triarylmethane dye, is most widely used for coloring purposes such as dyeing silk, leather, wool and paper in textile industries. In addition, it is extensively used in the aquaculture industries as a biocide worldwide. Induction of oxidative stress and the related formation of reactive oxygen species (ROS) are frequent results of environmental stressors. The main purpose of the present investigation was to evaluate the potential of Chara sp. in remediation of MG, the study of changes in some physiological and biochemical parameters, including photosynthetic pigments content, phenolic compounds and the activity of some major antioxidant enzymes including peroxidase (POD), catalase (CAT) and superoxide dismutase (SOD) that can be involved in algae resistance to dye and/or its metabolism.

Materials and Methods
2.1. Algal biomass and dye removal
The algal species was acquired from Azna-lake (Khalkhal) in North of Iran. The algal species was washed with distilled water to remove macro/microscopic contaminations. According to its morphology and macro/microscopic observations, it was identified as Chara species belongs to Charophyta.
The decolorization experiments were carried out with different initial dye concentrations (7.5, 15 mg/L), pH values; (5.5–8.5), temperature; 25 °C and experiment time; 1-8 h. Treatments were carried out at 25 °C and pH = 8, and concentrations of 0, 7.5 and 15 ppm. Analysis of metabolites and enzyme assays were performed following two hours of treatment of algae with MG.

2.2. Enzyme activity assays
The algae was subjected to 7.5 and 10 mg/L MG in the nutrient solution for 2 h to investigate the effect
of the dye on antioxidant enzyme activity compared with a control. The algae biomass was homogenized in 0.1 M phosphate buffer solution (pH 7) containing 1% polyvinylpyrrolidone. The homogenate was centrifuged at 2000g at 4 °C for 20 min. The supernatant was used as the crude extract for enzyme activity and protein content assays.
Superoxide dismutase (SOD, EC 1.15.1.1) activity was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT). Peroxidase (POD, EC 1.11.1.7) activity was measured by spectrophotometry. Catalase (CAT, E.C. 1.11.1.6) activity was measured spectrophotometrically by following the dismutation of H2O2 at 240 nm for 3 min and calculated using extinction coefficient 39.4 M-1.cm-1.

2.3. Non-enzymatic assays
Plant photosynthetic pigments (chlorophyll a and b and total carotenoids) were measured spectrometrically at 470, 662, and 645 nm, respectively, using equations described by Lichtenthaler (1987) after extraction from leaves with 100% acetone. Malondialdehyd (MDA), as a marker of lipid peroxidation and oxidative stress, was estimated by measuring the thiobarbituric acid-reactive substances (TBARS). TBARS were determined from the solution absorbance at 532 nm.
Total phenolic compounds were determined using Folin-Ciocalteau reagent. The absorbance was measured at 720 nm by spectrophotometer. Flavonoid contents were measured by the aluminum chloride. Colorimetric assay as described previously. After 5 minutes absorbance of the solution read at 507 nm.

Results and discussion
After 2 h exposure of algae with 7.5 and 15 mg/L of MG, the content of photosynthetic pigments was determined. According to the results, the observed chlorophyll a (Chl a), chlorophyll b (Chl b) and the total chlorophyll content was decreased to 18.3% after 2 h exposure (P>0.05). Carotenoid content was signif‌icantly (P<0.05) increased (48%) compared with control group after exposure with 7.5 and 15 ppm of MG. These results showed that the increased level of carotenoids characterized by the antioxidant properties in response to dyestuffs is probably the part of the strategy adopted by Chara sp. to counteract the toxic effect of free radicals generated under oxidative stress. Chlorophyll content decreased may be due to the formation of proteolytic enzymes such as chlorophyllase which is responsible for the chlorophyll degradation and damaging the photosynthetic apparatus.

During bioremediation of MG, the levels of MDA increased in the presence nonsignificantly. Oxidative stress initiates lipid peroxidation of cell membrane polyunsaturated fatty acids.
Total phenol compounds were increased in algae by increasing the concentration of dye matter (P <0.05). The amount of flavonoids was increased significantly in accordance with the increase of MG concentration (P <0.05). Flavonoids were increased by two or three times as much as the concentration of dye matter compared to the control group. Total phenols play a significant role in the regulation of plant and algae metabolic processes and overall plant growth. It has been shown in some studies that synthesis of polyphenols depends on abiotic factors.

Enzymatic analysis
During phytoremediation processes, different plant enzymes (especially oxidoreductases) act on specific
recalcitrant pollutants to remove them by precipitation or transformation to other products. SOD neutralizes reactive superoxide radicals to hydrogen peroxide, which is detoxified by other antioxidative enzymes such as CAT and POD. After 2 h exposure to 7.5 and 15 ppm of MG, a signif-icant induction in the activity of SOD was observed in Chara sp., also the total SOD activity in the Chara significantly increased with increasing the MG concentration compared to the control. SOD is one of the ubiquitous enzymes in aerobic organisms and plays a key role in cellular defense mechanisms against ROS. Its activity modulates the relative amounts of O2.- and H2O2 and decreases the risk of OH-˙ radical formation. POD activity showed a similar pattern to the SOD activity, at high concentrations of MG, POD activity was increased up to 44% (compared to the control) (P < 0.05). Increased POD activity at high concentrations of MG after the increment of SOD activity probably reflects the high demand for detoxification of produced H2O2. The activity of CAT was also decreased in the presence of 7.5 and 15 ppm of MG. After 2 h exposure the activity of CAT was decreased 32% compared with control (P>0.05). In the present work, CAT activity was signif‌icantly decreased by MG. Therefore, this enzyme did not appear to be an eff‌icient scavenger of H2O2 produced during treatment of MG. The decline in CAT activity might be due to inhibition of enzyme synthesis or a change in the assembly of enzyme subunits in the presence of MG.

Conclusion
In the present study, the activity of antioxidant enzymes and other markers of oxidative stress and metabolites in Chara sp. were measured during the bioremediation of malachite green by macroalgae. The activity of peroxidase and superoxide dismutase was significantly increased, while the activity of other main antioxidant enzyme, catalase was declined. Concentration of malondialdehyde, as a final product of lipid peroxidation, doesn't changed significantly during the remediation process. On the other hand, the amount of photosynthetic pigments (chlorophyl a and b) was decreased, and phenolic compounds was significantly increased with increasing MG concentration. These data indicated that induction of oxidative stress during remediation of MG by Chara sp. affects the activity of antioxidant enzymes and some of the secondary metabolites in macroalgae. Finally, macroalgae Chara sp. increases antioxidant enzymes and non-enzyme metabolites to achieve hemostasis during bioremediation process.

Key Words: Macroalgae Chara sp., Synthetic dyes, Reactive Oxygen Species, Antioxidant Enzymes