Selection of Nitrogen Source Affects the Growth and Metabolic Enzyme Activities of Chlorella Vulgaris (Beijerinck) Strain R-06/2 (Chlorophyta)
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Arch Biol Sci. 2020;72(2):291-300 https://doi.org/10.2298/ABS200219023V Selection of nitrogen source affects the growth and metabolic enzyme activities of Chlorella vulgaris (Beijerinck) strain R-06/2 (Chlorophyta) Ivanina A. Vasileva*, Juliana G. Ivanova and Liliana G. Gigova Laboratory of Experimental Algology, Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., bldg. 21, 1113, Sofia, Bulgaria *Corresponding author: [email protected] Received: February 19, 2020; Revised: April 27, 2020; Accepted: May 6, 2020; Published online: May 13, 2020 Abstract: The choice of nitrogen source in a cultivation medium can specifically affect the physiology and biochemistry of microalgae. To increase the production of low-cost valuable biomass, the preferred nitrogen form for each alga should be determined. The aim of our study was to analyze the effects of different nitrogen sources and cultivation times on the growth, biochemical composition and the activities of glutamine synthetase, glutamate synthase, glutamate dehydrogenase, malate dehydrogenase, aspartate aminotransferase and proteases of Chlorella vulgaris R-06/2. Media supplemented with urea or ammonium nitrate provided similarly (p>0.05) high growth rates for a short cultivation time (4 days). The two nitrogen compounds applied simultaneously ensured better biomass yield but for prolonged cultivation. In the exponential growth phase, ammonium nitrate stimulated (p<0.05) protein production, whereas urea enhanced (p<0.05) the carbohydrate content in older cultures as compared to the other nitrogen sources. The activity of each of the studied metabolic enzymes of C. vulgaris R-06/2 varied specifically depending on the nitrogen source and the growth phase, ensuring the maintenance of efficient, balanced metabolism under all cultivation conditions. When using large-scale cultivation to produce biomass for various useful applications, the selection of nitrogen source should be based on algal metabolism. Keywords: Chlorella sp.; growth rate; biochemical composition; nitrogen metabolism; in-gel enzyme activity Abbreviations: nitrogen (N), carbon (C), dry weight (DW), polyacrylamide gel electrophoresis (PAGE), glutamine syn- thetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (NAD-GDH), aspartate aminotransferase (AAT), malate dehydrogenase (NAD- MDH). INTRODUCTION growth, since it is required for the synthesis of amino acids, proteins, nucleic acids, pigments, coenzymes. The use of microalgae dates back thousands of years. Most algae can synthesize all cellular organic nitrog- In recent decades they have been rediscovered as an enous compounds from inorganic N sources such as excellent source of a vast range of valuable compounds nitrate, nitrite and ammonium. Some species may also due to the strong demand for natural, safe, eco-friendly use certain organic N compounds, including urea [5], and renewable products. The cultivation of microalgae is a promising process for obtaining their proteins, which as well as combinations of different N sources. To be contain all essential amino acids, carbohydrates, lipids able to utilize different N sources, microalgae have (especially essential fatty acids), pigments, vitamins, developed a variety of N assimilation pathways which etc., that have different potential applications in animal involve the action of a number of complex enzymes [6]. or human nutrition, nutraceuticals, pharmaceuticals All N sources taken up from the medium by the use of and cosmetics [1,2]. To produce large quantities of active membrane transporters have to be converted to low-cost biomass through autotrophic cultivation of ammonium inside the algal cells, as ammonium is the microalgae, it is important to achieve efficient usage of basic unit of N for amino acid synthesis. In a medium light [3] and nutrients [4]. Nitrogen (N) is considered with mixed N sources, ammonium is preferentially one of the most critical macronutrients for cellular utilized over nitrate or urea [7], mainly because of the © 2020 by the Serbian Biological Society How to cite this article: Vasileva IA, Ivanova JG, Gigova LG. Selection of nitrogen 291 source affects the growth and metabolic enzyme activities of Chlorella vulgaris (Beijerinck) strain R-06/2 (Chlorophyta). Arch Biol Sci. 2020;72(2):291-300. 292 Arch Biol Sci. 2020;72(2):291-300 lower energetic costs for its uptake and assimilation. MATERIALS AND METHODS In addition, ammonium both inhibits and represses the transport and assimilation of nitrate [8] and, in Algal strain and cultivation conditions some green algae, of urea [7,9]. Thus, ammonium ap- pears to be the most favorable N form for the growth C. vulgaris strain R-06/2, deposited in the collection of algae. The variety of regulatory mechanisms for N of the Laboratory of Experimental Algology, Institute assimilation depends on the species/strain; however, of Plant Physiology and Genetics, BAS, Sofia, Bul- the N status of the cells and some environmental garia, was used in this study. Non-axenic, monoalgal conditions allow specific preference of each alga to a cultures were grown in a modified mineral nutrient -1 particular N source. In addition to growth, the source medium composed of NH4NO3 (0.4 g L ), CO(NH2)2 -1 -1 of N in the nutrient medium can also specifically in- (0.3 g L ), MgSO4.7H2O (0.494 g L ), KH2PO4 (0.17 -1 -1 fluence the biochemical composition of algae [10,11]. g L ), NaHCO3 (2.0 g L ) and trace elements [21,22], Therefore, to increase the production of biomass and conditionally referred to as “standard medium”. To its valuable components for practical applications, it study the responses of the strain to different N sources, is important to select the most suitable N source for ammonium nitrate plus urea in the standard medium each biotechnologically promising strain. was substituted by only ammonium nitrate or only urea separately with equal final concentrations of N Chlorella is a widely commercialized eukaryotic (20 mM). An initial culture density of 0.8 g L-1 dry green alga used as a health food and animal feed in weight (DW) was used for all treatments. Experi- aquaculture, as well as in nutraceutical and pharma- ments were carried out at 30±1.0°C and continuous ceutical industries. In addition, Chlorella is one of lateral illumination with cool-white fluorescent lamps the microalgal genera widely used for greenhouse gas at a photon flux density of 132 μmol photons m-2 s-1 biomitigation and wastewater bioremediation [12]. measured at the surface of 200 mL flasks. A source of Although Chlorella species are among the best studied C was provided by bubbling sterile 2% CO2 (v/v) in air algae, the response of their N-metabolizing enzymes through the cultures. All experimental cultures were to changes in the N source in the nutrient medium is harvested after 96 h (during the exponential growth well characterized only when ammonium is replaced phase, as determined in preliminary experiments). by nitrate [13-16 and references therein]. Ammonium nitrate and urea cultures were harvested after 192 h and the ammonium nitrate plus urea culture Chlorella R-06/2, isolated from a geothermal spring after 480 h (at the early stationary phase). Cells were (42°C) in the region of Rupite village (SW Bulgaria) collected by centrifugation (5000 × g, 20 min), rinsed and classified as Chlorella vulgaris Beijerinck 1890 three times with distilled water, frozen, and stored at (Chlorophyta) [17], was recently characterized in -70°C until further analysis. terms of its responses to environmental stressors [18], as well as the biological activities of its cellular com- ponents [19,20]. In this study we examined the effects Determination of biomass dry weight and specific of different sources of N, urea, ammonium nitrate, growth rate and a combination of both, on the physiology and metabolism of C. vulgaris R-06/2 during its growth. The growth of C. vulgaris R-06/2 was evaluated by the -1 For this purpose, the growth rate, biomass production increment in algal biomass DW. The DW (g L ) was and biochemical composition as well as the isoenzyme determined gravimetrically. Algal suspensions (3 × 5 patterns and activities of some enzymes that link N and mL each) were filtered through Whatman GF/C glass carbon (C) metabolism were analyzed and compared. filters (Whatman International Ltd, Maidstone, UK), rinsed with tap water to eliminate salts and oven-dried at 105°C to a constant weight. The specific growth rate was calculated using the following formula: μ=ln(mt2/mt1)/t2–t1 [23], where mt1 and mt2 represent the DW at the start of the experiment nd th (t1) (t1=0) and on the 2 or 4 days of cultivation (t2). Arch Biol Sci. 2020;72(2):291-300 293 Biochemical composition analysis electrophoresis, the proteins were renatured in 2.5% (v⁄v) Triton X-100 for 15 min, two times prior to in- Frozen fresh biomass was used for all biochemical cubation, overnight in 50 mM Tris-HCl, pH 8, 10 mM analyses. Total protein content was measured as de- CaCl2 at 37°C. The gels were stained with 0.1% (w⁄v) scribed [24], with bovine serum albumin (BSA) as Coomassie Brilliant Blue G250 in 30% ethanol and standard. Total carbohydrates were quantified as glucose 10% (v⁄v) acetic acid. The reagents used for enzyme equivalents by the phenol-sulfuric acid method [25]. activity staining were purchased from Sigma (Sigma For determining the lipid content, the collected algal Inc., St. Louis, MO, USA). samples were repeatedly extracted with hot ethanol Gel patterns were photographed immediately under reflux until the extract became colorless, and after staining using the UVItec gel documentation then re-extracted with chloroform. The extract was system (Cambridge, UK). Image analysis of the gels released from the chloroform at 40-45°C on a rotary was performed on a PC using Gel-Pro32 Analyzer vacuum evaporator and the amount of lipid was de- software (Media Cybernetics, Bethesda, MD USA).