Abstract: Background and Objective: The growth rate, primary and secondary metabolites are influenced by nitrogen concentration of media. This study aimed to determine the effect of different concentrations of KNO3 as a source of nitrogen on the growth, phytochemical components, cholesterol reduction, antioxidant and antibacterial activities of the green alga, Scenedesmus obliquus (S. obliquus). Materials and Methods: Scenedesmus obliquus was cultured in Kuhl’s medium and KNO3 was added with different concentrations in the medium at different concentration (0.12, 0.75, 1.5, 2.25 and 3 g L–1). The effect of different five concentrations of nitrogen on protein and carbohydrates was determined. Antioxidant activity, total phenolic content (TPC) of alga extracts, cholesterol reduction and antibacterial activity were evaluated. The data analyses were carried out using SPSS software version 16. Results: The results revealed that the best KNO3 concentration for algal growth and carbohydrate content is1.5 g L–1. Meanwhile, the high lipid content was obtained with KNO3 starvation. Medium containing 0.75 g L–1 of KNO3 has the highest effect on the protein production of S. obliquus. The contents of dry alga extracted with chloroform: methanol and the extracted contents were determined by GC/MS chromatogram; the major contents were 5-Hydroxymethylfurfural followed by hexadecanoic acid (palmitic acid), cis-9-octadecenoic acid (oleic acid) and hexadecanoic acid and methyl ester, respectively. Conclusion: Potassium nitrate limitation caused the highest effect on the total phenol content (TPC), increased antioxidant capacity, cholesterol reduction activity and also antibacterial activity against Staphylococcus aureus and Escherichia coli.
INTRODUCTION
Microalgae are good producers of a broad variety of valuable compounds like lipids, carbohydrates, proteins, vitamins, amino acids and Beta-carotene that are used as feedstock for energy production, feed additives, food, cosmetics and medicine1,2. Numerous applications of microalgae such as aquaculture feeding, manufacture of active ingredients for cosmetics and food formulations due to promising sources of fine chemicals3. Scenedesmus sp. contains high nutritional and bioactive metabolite contents. So, it is used in numerous biotechnological applications4. Hamouda et al.5 reported that S. obliquus produce high amount of carbohydrates over short periods. Also contains protein, all the essential amino acids, a good amount of minerals and lipid6. Antiproliferation and antioxidants agents could be used as ingredients that helps in health promotion and disease prevention had been extracted from green microalgae S. obliquus7. The favorable antioxidant compounds have been extracted from S. obliquus and used in food additives and active ingredients for therapeutics3. Green alga Scenedesmus showed antibacterial effect against Staphylococcus aureus and Bacillus subtilis8. Carotenoids and phenolic compounds are good potential source of natural antioxidants that had been extracted from microalgae Chlorella sp. and S. obliquus9. A major variety of microalgae commercialized for human nutrition such as Spirulina, Chlorella, Dunaliella salina and Aphanizomenon flos-aquae10.
The present research aimed to study the influence of KNO3 concentrations on S. obliquus growth, tannins, flavonoids, phenolic contents, cholesterol reduction effect, antibacterial and antioxidant activities those can be used in many biotechnological applications.
MATERIALS AND METHODS
The studying was carried out in the Microbial Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), University of Sadat City; Department of Bioprocess Development, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications, Alexandria, Egypt and Department of Botany, Faculty of Science, Menoufia University, Egypt (2016) and all chemicals were used from Sigma Aldrich.
Alga: Scenedesmus obliquus was collected from river Nile and then purified and identified according to the method of Prescott11. The axenic culture was maintained on Kuhl agar slants12 at 4°C
S. obliquus cultivation using different KNO3 (nitrogen) concentrations: Standard Kuhl's medium was prepared and used for growth of the microalgae12. KNO3 was added with different concentrations (0.12, 0.75, 1.5, 2.25 and 3 g L–1) to the medium.
Determination of algal growth parameters: Algal growth was followed by measuring optical density of growth using Unico UV-2000 spectrophotometer13. Cell numbers were estimated using Neubour Haemocytometer. Growth rate (μ) and doubling time (T2) were calculated by the following Eq:
where, N1, N2 cell number at Time T1 and T2 while T2 = 0.6391/μ. Total carbohydrate content, total soluble proteins and lipids were estimated after 18 days of incubation period14-16.
Analysis of extracted alga by GC/MS analysis: Oil content of alga was extracted by chloroform: methanol (1:1) was analyzed by GC/MS analysis17.
Influence of different KNO3 concentrations on antioxidant activity and total phenolic contents (TPC) of alga extracts: Half gram of each treatment of fine grind dried alga (S. obliquus) was soaked in 10 mL methanol for 48 h. The extracts were filtered and used for determination of total phenol content.
Cholesterol reduction effect: The cholesterol reduction by algal extracts was determined by using enzymatic colorimetric kit18.
Antibacterial activity of algal methanol extracts that grown under different nitrogen concentrations: The antibacterial activity of methanol extracts of alga in comparison with Vancomycin as positive control was assessed against both Escherichia coli and Staphylococcus aureus using agar well diffusion method according to Perez et al.19. Algal methanol extract was dissolved in dimethyl sulfoxide (DMSO) which also used as negative control. Exactly 200 μL from algal methanol extract (1 mg mL–1) was used for each well. The inhibition zones diameters were measured in mm after 24 h of incubation.
Statistical analysis: Results of the study were expressed as ±standard error of the mean. Significant differences between the means of parameters (LSD) were estimated using Duncan’s multiple range tests (p<0.05). All the above mentioned data analyses were carried out with SPSS software version 1620.
RESULTS AND DISCUSSION
Influence of KNO3 concentrations on S. obliquus growth: The effect of different concentrations of KNO3 on S. obliquus growth is shown in Fig. 1. The best growth of alga was at 1.5 g L–1 KNO3 after 13 days of cultivation. The lowest biomass was obtained in medium containing 0.75 g L–1 KNO3. The best biomass was obtained when alga cultivated in concentration of 1.5 g L–1 KNO3 followed by 2.25, 3, 0.75 and 0.12 g L–1 KNO3, respectively. The minimum amount of S. obliquus biomass was obtained with KNO3 limitation in Kuhl’s medium. Table 1 shows the specific growth rate and doubling time of alga that cultivated under various KNO3 concentrations. The exponential growth phase of alga grown in Kuhl’s medium was achieved at concentrations of 1.5 g L–1 of KNO3 at 10 days with specific growth rate of 0.427 and doubling time of 1.620. The green microalga Parietochloris incisa that grown on (+N) was possessed higher final biomass than the nitrogen-forbid (-N) cultures21.
Effect of different KNO3 concentrations on primary metabolites of S. obliquus: Table 1 shows the impact of KNO3 concentrations on total carbohydrates, protein and lipids contents of S. obliquus. The total carbohydrates and protein of alga are significantly reduced at low KNO3 concentrations, lead to an increase in lipids. The carbohydrate contents were 18.3, 17.49, 17.2, 15.31 and 9.65% of dry weight at 1.5, 2.25, 3.0, 0.75 and 0.12 g L–1 KNO3 of medium, respectively. The protein contents of S. obliquus under different concentrations of KNO3 were increased to 30.56 and 30.06 with 0.75 and 2.25 g L–1 of medium, respectively compared to control (1.5 g L–1 KNO3). The lowest concentration of KNO3 (0.12 g L–1) significantly increased the lipid content to double compared with control, the lipid content was 26% with 0.12 g L–1 KNO3 medium while it was 12% with control (1.5 g L–1 KNO3 of medium). Lipids content of S. obliquus was increased when KNO3 contents in media decrease, meanwhile total carbohydrate and protein contents were decreased. The lowest amount of protein was achieved with nitrogen limitation22 reported that nitrogen starvation is essentially characterized by a large reduce in the protein pool. Thompson23 reported that lipid accumulation of green algae increase of up to 2-3 folds might be expected under conditions of nitrogen deprivation. Nigam et al.24 demonstrated that lipid content rises as nitrogen concentration decrease in the medium.
| Table 1: | Effect of KNO3 concentrations on specific growth rate, doubling time carbohydrate, protein and lipids contents of S. obliquus |
| Fig. 1(a-b): | Influence of KNO3 concentrations on S. obliquus growth measured by (a) Optical density and (b) Cell numbers |
Values were taken as SE |
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| Table 2: | GC/MS chromatogram of chloroform: methanol extraction from S. obliquus |
| Fig. 2: | Type of tannins (tannic acid, polyphenol compounds) |
Nitrogen is the highest critical nutrient affecting in algae lipid metabolism. The accumulation of lipids, particularly TAG, related to nitrogen limitation has been noticed in a large number of species or strains of various microalgae25. In various microalgae, nitrogen starvation or limitation conditions are shown to enhance the biosynthesis and accumulation of lipids or carbohydrates or both26.
GC/MS chromatogram of chloroform: methanol extraction of S. obliquus: The results obtained by GC/MS chromatogram of methanol: chloroform extraction of S. obliquus indicate the presence of twelve components of extract. These components are 5-Hydroxymethylfurfural, 1-Octadecene,hexadecanoic acid methyl ester, hexadecanoic acid (palmitic acid), octadeconic acid methyl ester, cis-9-octadecenoic acid (oleic acid), octadecanoic acid (stearic acid), octadecadienoic acid (linoleic acid), acetohydrazide 2-(3-Hydroxy-2-pentyl cyclopentyl), hexadecanoic acid 2-Hydroxy-1-(hydroxymethyl), 1,2-Benzenedicarboxylic acid, diisooctyl ester and dioctyl phthalate (Table 2). Chloroform: methanol extract of dry S. obliquus biomass showed many compounds that had many biotechnological applications. 5-Hydroxymethylfurfural (HMF) used for the production of biofuels and plastics27. Also it has multi-functional compounds such as intermediate for polymers, pharmaceuticals, fine chemicals and for the synthesis of other organic derivatives28. Li et al.29 reported that 5-HMF has new marine natural antioxidant and prospective precursor for practical applications in the food, cosmetic and pharmaceutical fields. Hexadecanoic acid methyl ester has also been observed to cause autolysis of membranous structures, inhibit phagocytic activity, stimulate significant aortic dilation and nitric oxide production of various cells, diminish levels of tumor necrosis factor-alpha (TNF) and prostaglandin E2 (PGE2)30. Palmitic acid, oleic acid and linoleic acid were the three main compounds in the high-acid oil-biodiesel31. 1, 2-Benzenedicarboxylic acid, di-isooctyl ester has antimicrobial and antifouling32. Linton et al.33 stated that the octadecanoic acid (OA) methyl ester had antiviral activity against measles disease virus. Stearic acid is used in the manufacture of pharmaceutical products34. Also used for a cyclosporine-A drug carrier system35 and used for vanishing the bitter taste of pharmaceutical compounds36.
Influence of different KNO3 concentrations on tannins, flavonoids and phenolic contents of S. obliquus: Free radicals are controlled by natural products before attack cells and causes many diseases, these natural products are antioxidant. Tannins, phenolic compounds (Fig. 2) and flavonoids are accumulated by plants as secondary metabolites and considered antioxidant substances that advantage in the pharmaceutical industry.
| Fig. 3(a-c): | Influence of different KNO3 concentrations on bioactive components (a) Tannins, (b) Phenolic and (c) Flavonoids contents produced by of S. obliquus |
Different small letter(s) on the bars indicate significant differences (p>0.05) between KNO3 concentrations according to Duncan's multiple range test. Values were taken as SE |
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The effect of KNO3 concentrations on tannins contents of S .obliquus was clear in Fig. 3. Potassium nitrate limitation enhances tannic acid content of S. obliquus. The highest phenolic contents of S. obliquus were recorded when grown at 0.75 g L–1 KNO3 of Kuhl’s medium followed by 1.5, 3.0, 2.25 and 0.12 g L–1 KNO3, respectively. Figure 3 shows that the flavonoids content of S. obliquus was highest when alga cultivated with 1.5 g L–1 KNO3 medium and the stress conditions had no effect on flavonoids content. Flavonoids are secondary metabolites and have the ability to act as antioxidant, antibacterial, anti-inflammatory and anti-cancer agent37.
Antioxidant activity (DPPH), cholesterol reduction and antibacterial activity of methanol extract of S. obliquus grown under different concentrations of KNO3: Figure 4 represents influence of KNO3 concentrations on the antioxidant activity, cholesterol reduction and antibacterial activity of S. obliquus. The results revealed that the maximum amounts of antioxidant activity were present in alga grown under low amount of KNO3 (0.12 and 0.75 g L–1) followed by alga grown under a high amount of KNO3 (3.0 g L–1). Results clear that the high antioxidant activity present in S. obliquus cultivated under stress conditions with low and high concentrations of KNO3. The antioxidant activities of S. obliquus extracts around (59.8-64.3%) was recorded by Ali et al.9. Biochemical content of Scenedesmus sp. possessed antioxidant properties and used in the neutraceutical industry38.
The results clear that under KNO3 deprivation, Scenedesmus secondary metabolites have been affected and hence affect the cholesterol reduction. The high levels of cholesterol reduction (80.12 and 77.9%) were observed with culture grown under KNO3 concentrations at 0.12 and 0.75 g L–1 KNO3, respectively. The low concentrations of KNO3 in Kuhl’s medium were caused high lowering of cholesterol. The Scenedesmus acutus-enriched diet prevented an excessive deposition of cholesterol in the liver39.
The methanol extract of S. obliquus showed significant inhibition activities against Staphylococcus aureus and Escherichia coli. The highest zone of inhibition was observed at 0.75 g L–1 KNO3. Both concentrations of nitrogen (0.75 and 3.0 g L–1 KNO3) showed highest algal growth and also highest antibacterial activity against Staphylococcus aureus and Escherichia coli. Salem et al.8 reported that Scenedesmus sp. can serve as a potential antibacterial agent against food-borne pathogen of S. aureus.
| Fig. 4(a-c): | (a) DPPH radical scavenging activity (%), (b) Cholesterol reduction (%) and (c) Antibacterial activity (mm) of S. obliquus grown with different KNO3 concentrations |
Different small letter(s) on the bars indicate significant differences (p>0.05) between KNO3 concentrations according to Duncan's multiple range test. Values were taken as SE |
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CONCLUSION AND FUTURE RECOMMENDATIONS
KNO3 concentrations effect on the growth, primary metabolites (lipids, proteins and carbohydrates)and secondary metabolites (phenolic, tannins and flavonoids) of algae. Antibacterial activity, antioxidant activity and lowering cholesterol activity of S. obliquus varied with KNO3 concentration in the medium. The tannins content, antioxidant activity and also cholesterol reduction of alga are significantly increased at low KNO3 concentrations. The best concentration of KNO3 (0.75 g L–1 nitrogen medium) significantly increases production of phenol and antibacterial activity. 1.5 g L–1 of KNO3 medium) was best for flavonoids content. The extraction contents of S. obliquus were determined by GC/MS chromatogram and the major content present is 5-Hydroxymethylfurfural (HMF) that used for the production of various high-volume plastics, foods and treatments followed by palmitic acid (C16:0) that is display antioxidant.
That different concentration of nitrogen can enhance primary and secondary metabolites of Scenedesmus obliquus and significantly increases the production of bioactive compounds. Much study still needs to be done in such area.
SIGNIFICANCE STATEMENT
This study discovers the effect of various concentrations of KNO3 on the growth, production of primary and secondary metabolites of the micro green alga, Scenedesmus obliquus and also the effect of total phenolic content (TPC), antioxidant activities, cholesterol reduction and antibacterial activity against Staphylococcus aureus and Escherichia coli were studied that can be beneficial for studying the contents of S. obliquus extract that was determined by GC/MS analysis. This study will help the researchers to uncover the critical areas of that the potassium nitrate limitation effect on the contents of S. obliquus extract, that many researchers were not able to explore. Thus a new theory on the effect of potassium nitrate on the production of primary and secondary metabolites of the green alga, Scenedesmus obliquus may be arrived.