Abstract: A study was carried out to explore the relationship between growth conditions and antioxidant properties of microalgae. Further, correlation between phenolics and antioxidant activities were studied to determine whether antioxidant activity depends on microalgal phenolics content under varying culture conditions. Total phenolics and antioxidant properties of Chlorella vulgaris and Scenedesmus obliquus grown under autotrophic, heterotrophic and mixotrophic conditions were evaluated. Domestic water, Bold’s medium and sewage water were used to cultivate the microalgae and the extracts were prepared in methanol and analyzed for biochemical (total phenolics) and antioxidant properties (DPPH assay, super oxide scavenging assay and antioxidant potential). The experiments were done in triplicates and significant correlation coefficients between antioxidant properties against phenolic content and growth conditions were interrelated. The amount of total phenolics content varied in growth conditions and ranged from 0.11-0.55 mg GAE g-1. Significant correlation co-efficient between phenolics and antioxidant properties of microalgae determined by DPPH, superoxide anion scavenging and total antioxidant activities were found in the study. The strongest positive correlation was found to be between total phenolics and DPPH activity in C. vulgaris (r = 0.997). In S. obliquus, the strongest positive correlation was between total phenolics and antioxidant potential (r = 0.091) at p<0.01 followed by superoxide scavenging (p<0.05). The findings indicated that phenolic compounds were the major contributors to the antioxidant properties of microalgae. The results demonstrated that strongest positive correlation was observed in mixotrophic conditions followed by autotrophic conditions in Chlorella whereas the correlation was significant under heterotrophic conditions in Scenedesmus followed by mixotrophic conditions.
INTRODUCTION
Oxidative stress due to the production of Reactive Oxygen Species (ROS) contribute to the pathogenesis of cardiovascular diseases, atherosclerosis and cancer (Dhalla et al., 2000; Finkel and Holbrook, 2000; Madhavi et al., 1996). Synthetic antioxidants such as Butylated Hydroxyl Toluene (BHT), Butylated Hydroxyl Anisole (BHA), α-tocopherol and propyl gallate have been used to reduce oxidative damages in the human body (Gulcin et al., 2002) but need to be replaced with natural antioxidants, as they were found to be toxic and carcinogenic in animal models (Ito et al., 1986; Safer and Nughamish, 1999).
Microalgae are being investigated for properties beneficial to the nutraceuticals and health foods industries and are promising alternative source of antioxidants (Li et al., 2007; Natrah et al., 2007; Rodriguez-Garcia and Guil-Guerrero, 2008; Chacon-Lee and Gonzalez-Marino, 2010; Lee et al., 2010). Microalgae exhibit adaptative responses to oxidative and radical stresses by producing potential chemicals via stimulation of their antioxidant defence system (Srivastava et al., 2005; Tsao and Deng, 2004). Microalgal antioxidant products are recognized as safe (Abe et al., 1999; El-Baz et al., 2002). Although many microalgal species have been reported widely for their antioxidant activity (Li et al., 2007; Rao et al., 2006; Duan et al., 2006; Wu et al., 2005; Herrero et al., 2006, 2005; Murthy et al., 2005; Tannin-Spitz et al., 2005; Kuda et al., 2005; Guzman et al., 2001; Mirada et al., 1998) there has been limited information on antioxidant levels of microalgae under varying culture conditions. Few studies reported that phenolic compounds had a high antioxidant capacity (Jaime et al., 2005; Geetha et al., 2010; Custodio et al., 2012), while another study found the opposite (Goh et al., 2010) hence it is not clear whether phenolic substances are important antioxidants in microalgae.
This research was attempted to explore the relationship between growth conditions and antioxidant properties of microalgae and also the correlation between phenolics and antioxidant activities. The experimental work encompassed screening of freshwater microalgae for antioxidant behaviour grown under autotrophic, heterotrophic and mixotrophic conditions. Phenolic content was determined and correlation coefficient was used to estimate and compare the contribution of phenolic compounds to the measured antioxidant activities.
MATERIALS AND METHODS
Sample collection and identification: Waste water was collected from Bangalore Water Sewerage and Supply Board (BWSSB), Bengaluru (13°04’N, 77°58’E), India and poured into a closed 250 mL bottle and exposed in sunlight for 3 weeks. The upper layer of the water was inoculated in BG11 medium enriched agar plates containing 200 μg mL–1 ampicillin. The plates were incubated at 25±2°C under cool white fluorescent light (40 μ mol photons m–2 sec–1; 15 h light/9 h dark) until algal growth was detected. Single green colour colonies were inoculated into BG11 medium and identified as Chlorella vulgaris and Scenedesmus obliquus according to Anderson (2005) and Round (1973).
Culture conditions: Microalgal cultivations were performed in 500 mL conical flasks in different growth medium namely normal water (autotrophic), Bold’s media (heterotrophic) and sewage water (mixotrophic).
Extract preparation: Microalgal extracts were prepared by centrifuging 10 mL of a 30 day-old cultures of Chlorella and Scenedesmus at 2500 rpm for 10 min. The pellet was then resuspended and homogenized in methanol (1:1 w/v) and then subjected to centrifugation at 4000 rpm for 5 min. The supernatant was collected and used for biochemical and antioxidant assays.
Determination of polyphenols: Total phenolics in the extracts were determined with Folin-Ciocalteau method (Javanmardi et al., 2003) using gallic acid as a standard phenolic compound (2-20 mg mL–1). Aliquots (200 μL) of each extract were added with 1.0 mL of Folin-Ciocalteau reagent and 800 μL of 7.5% sodium carbonate. The mixture was allowed to stand for 30 min in dark and the absorbance was measured at 765 nm. The total phenolic content was expressed as gallic acid equivalents (GAE g–1) dry weight of microalgae and calculated as mean value±SD.
Antioxidant evaluation assays
DPPH assay: Free radical scavenging activity of the samples was determined according to the modified methodology of Brand-Williams et al. (1995). Algal extracts (200 μL) were mixed with 1.8 mL of the methanolic DPPH solution (0.5 mM). The absorbance was measured at 517 nm immediately after mixing and after standing at room temperature for 30 min. The percent of scavenging has been calculated as the ratio of the absorption of the sample relative to the control DPPH solution without extract. The radical scavenging activity was calculated as the percentage of DPPH discoloration using the equation:
where, Asample is the absorbance of the solution when the sample solution has been added at a particular level and Acontrol is the absorbance of the DPPH solution.
Super oxide free radical scavenging activity: Measurement of superoxide anion scavenging activity of the samples was done based on the methodology of Nishikimi et al. (1972). Two hundred microliter aliquots of the extracts and ascorbic acid (2-20 mg mL–1) were added with 100 μL of Riboflavin solution (20 μg), 200 μL EDTA solution (12 mM), 200 μL methanol and 100 μL NBT (Nitro-blue tetrazolium) solution (0.1 mg). The absorbance of solution was measured at 590 nm using phosphate buffer as blank after illumination for 5 min.
Antioxidant potential assay: Antioxidant potential of the extracts has been assessed with the phosphomolybdenum reduction assay according to Prieto et al. (1999). The reagent solution contained ammonium molybdate (4 mM), sodium phosphate (28 mM) and sulphuric acid (600 mM) mixed with the extracts. The samples were incubated for 90 min at 90°C and the absorbance of the green phosphomolybdenum complex was measured at 695 nm. Ascorbic acid standard solutions (2-20 mg L–1) were used to plot the calibration curve and the reducing capacity of the extracts has been expressed as the ascorbic Acid Equivalent Antioxidant Content (AEAC).
Statistical analysis: The assays were carried out in triplicate and the results were expressed as mean values and the Standard Deviation (SD). The statistical differences represented by letters were obtained through one-way analysis of variance (ANOVA) (p<0.05). Correlations were established using Pearson’s correlation coefficient (r) in bivariate linear correlations (p<0.05 and p<0.01). These were carried out using Microsoft office Excel 2007 and SPSS version 16.0 program.
RESULTS AND DISCUSSION
Total phenolics content: The phenolic content of algal extracts were ranged from 0.11-0.55 mg GAE g–1 and extracts from autotrophic conditions showed low contents of phenolic compounds (<0.2 mg GAE g–1). Scenedesmus from mixotrophic had the highest phenolic content (0.55 mg GAE g–1) followed by Chlorella (0.39 mg GAE g–1). Phenolic contents under heterotrophic growth varied from 0.20 to 0.37 mg GAE g–1. In general, Scenedesmus has exhibited higher phenolics content than Chlorella.
Antioxidant activities of microalgae: As shown in Table 1, Chlorella exhibited highest DPPH free radical scavenging activity (52.3%) when grown under mixotrophic conditions whereas under autotrophic conditions, Scenedesmus had highest free radical scavenging activity (43.5%).
| Table 1: | Total levels of phenolics, DPPH, superoxide anion scavenging and antioxidant potential of microalgae under varying growth conditions |
| Data are expressed as mean±SD. In each column different letters indicate significant differences (p<0.05), DPPH: 2, 2-diphenyl-1-picrylhrazyl | |
| Table 2: | Correlation showing the interrelation among antioxidant activity in Chlorella vulgaris |
| **Correlation is significant at the 0.01 level (2-tailed), *Correlation is significant at the 0.05 level (2-tailed), DPPH: 2, 2-diphenyl-1-picrylhrazyl | |
| Table 3: | Correlation showing the interrelation among antioxidant activity in Scenedesmus obliquus |
| **Correlation is significant at the 0.01 level (2-tailed), *Correlation is significant at the 0.05 level (2-tailed), DPPH: 2, 2-diphenyl-1-picrylhrazyl | |
Lowest activity was observed under heterotrophic conditions in both algal extracts.
Among the growth conditions, mixotrophic and autotrophic growth exhibited considerable superoxide anion scavenging activity. Heterotrophic growth showed lower activity. Both Chlorella and Scenedesmus indicated variations in activities with 27.31 and 29.89% in autotrophic and 29 and 25% under mixotrophic conditions.
The total antioxidant activity of algal extracts was evaluated using phosphomolybdate method which is based on the reduction of Mo (IV) to Mo (V) by the sample analyte and the subsequent formation of green phosphate/Mo (V) compounds with a maximum absorption at 695 nm. Mixotrophic growth has significantly influenced the total antioxidant activity of both Chlorella and Scenedesmus (4322.5 and 5096.7 mg AEAE g–1).
Correlation between phenolic content and antioxidant properties: The correlation coefficient between the phenolic content and antioxidant activities of Chlorella and Scenedesmus grown under varying cultivating conditions were determined (Table 2 and 3). From the results, phenolic compounds have contributed to the antioxidant properties of microalgae irrespective of cultivating conditions. The strongest positive correlation was found to be between total phenolics and DPPH activity in C. vulgaris (r = 0.997). A strong positive correlation also exists between phenolics and antioxidant potential (r = 0.967). A significant correlation was obtained between phenolics and super oxide scavenging activity (p<0.05). In S. obliquus, the strongest positive correlation was between total phenolics and antioxidant potential (r = 0.091) at p<0.01 followed by superoxide (p<0.05). However, the interrelation among DPHH and superoxide was higher in Scenedesmus (r = 0.997).
| Table 4: | Correlation showing the interrelation among antioxidant activity in Chlorella vulgaris |
| ** correlation is significant at the 0.01 level (2-tailed), * Correlation is significant at the 0.05 level (2-tailed) | |
Correlation between growth conditions and antioxidant properties: The correlation between growth conditions and antioxidant properties were determined to find the influence of autotrophic, heterotrophic and mixotrophic conditions. Strongest positive correlation observed in mixotrophic conditions followed by autotrophic conditions in Chlorella (Table 4). However, the correlation was significant under heterotrophic conditions in Scenedesmus followed by mixotrophic conditions.
Reactive oxygen and free radicals are produced during oxygenic photosynthesis by microalgae. Antioxidant compounds are produced by microalgae as their defence to avoid oxidative damage (Lu and Foo, 1995) and are potent chemical blockers of UV radiation (Mata et al., 2010). Biochemical composition of microalgae can be fine tuned by cultural operations (Fabregas et al., 2001; Otero and Fabregas, 1997; Gigova and Ivanova, 2015) and determination of optimum culture conditions towards biomass growth and antioxidant synthesis in microalgae have been reported (Celekli and Yavuzatmaca, 2009). Influence of growth medium on antioxidant properties of cyanobacteria is reported by Tarko et al. (2012). Studies also show that antioxidant activities are influenced by illuminance while culturing (Madhyastha et al., 2009). The effect of pH and temperature on antioxidant productivity in Scenedesmus were assessed by Guedes et al. (2011) and found that pH plays an important role in antioxidant content. The effect of antioxidants on DPPH free radical scavenging was considered to be due to their hydrogen donating ability. In this study, extracts of both algae showed notable activities indicating the higher efficacy for scavenging of free radicals. Chlorella and Scenedesmus have exhibited higher free radical scavenging activity when cultivated under autotrophic and mixotrophic conditions respectively. A superoxide anion radical generally forms first and its effects can be exaggerated as it produces other kinds of cell damage inducing free radicals and oxidizing agents (Liu and Ng, 2000). In this study, significant super oxide scavenging activities of microalgae grown under autotrophic mixotrophic conditions were observed. Phenolic compounds donate a hydrogen atom or an electron in order to form stable radical intermediates thereby serves as important antioxidants. A number of phenolic compounds are present in microalgae (Klejdus et al., 2010; Kovacik et al., 2010) and were reported to responsible for microalgal antioxidant properties. But the contribution of phenolics to the antioxidant activity of microalgae needs to be understood in a better way. The amount of total phenolics by Folin-Ciocalteu method was varied in growth conditions. Correlation analyses indicated significant contribution of phenolics to antioxidant activity as measured by the DPPH, superoxide and total antioxidant assays. It was found that phenolic compounds are important contributors to antioxidant activities in Chlorella and Scenedesmus and the results are in accordance with previous studies (Hajimahmoodi et al., 2010; Goiris et al., 2012). In general, heterotrophic cultivation is having many advantages than autotrophic cultivation (Perez-Garcia et al., 2011) but in this study mixotrophic cultivation using municipal sewage water has increased antioxidant properties of microalgae. The use of wastewater for microalgal cultivation for higher biomass and lipid production has been reported widely (Clarens et al., 2010; Pittman et al., 2011) but to our knowledge, influence of municipal sewage water on antioxidant properties of microalgae is reported for the first time through this study.
CONCLUSION
Relationship between phenolics and antioxidant properties of microalgae grown under autotrophic, heterotrophic and mixotrophic conditions were studied. The results demonstrated that growth conditions play an important role in contributing antioxidant properties of microalgae. In general, mixotrophic culture using sewage water exhibited higher antioxidant activities followed by autotrophic culture. Correlation co-efficient studies revealed that antioxidant activity is depending on the phenolic content of microalgae which is varying under growth conditions.