ФИЗИОЛОГИЯ РАСТЕНИЙ, 2015, том 62, № 5, с. 690-695
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
TEMPERATURE AND IRRADIANCE EFFECTS ON Rhodella reticulata GROWTH
AND BIOCHEMICAL CHARACTERISTICS1
© 2015 J. G. Ivanova*, L. V. Kabaivanova**, G. D. Petkov*
*Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Sofia, Bulgaria **Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria Received November 24, 2014
The red microalga Rhodella reticulata, a potential source of bioactive substances, was the subject of study of the irradiance and temperature effects on growth rate and biochemical composition of algal biomass. The optimum temperature for growth decreased from 28 to 26°C with increasing light intensity from 260 to 520 p.E/(m2 s). The maximal growth rate was 0.21/day at 28°C and lower light intensity (260 p.E/(m2 s)). Variations in these parameters also affected the fatty acid productivity, and proteins and carbohydrates content. At 34°C and high light intensity the quantity of carbohydrates was 1.16-fold higher than the quantity at the optimal temperature and low light intensity. Protein content was the highest at lower temperatures for both light intensities. Fatty acid profile showed the highest percent for the polyunsaturated eicosapentaenoic acid (EPA) at 28°C and both light intensities (46% from the whole fatty acid content), an important feature for this strain. This is a prerequisite for use of EPA as a supplement in food industry.
Keywords: Rhodella reticulata — irradiance — temperature — biochemical composition — eicosapentaenoic acid
DOI: 10.7868/S0015330315040107
INTRODUCTION
Over the past decade, algal biotechnology has grown steadily into a global industry with increasing numbers of entrepreneurs attempting to utilize its biochemical diversity for a wide array of applications [1]. The red microalgae of the genus Rhodophyta are potential sources of unique bioactive substances that can find different applications in medicine, pharmacy, cosmetics, agriculture, and food industry as food supplements, etc. [2]. Microalgae are rich in carbohydrates, pigments, and essential fatty acids that could be used as functional ingredients. Valuable products in the red microalgae are the polyunsaturated fatty acids (PUFAs) which are essential for humans [3]. Higher plants and most animals lack the required enzymes to synthesize PUFAs from more than 18 carbons, although they are absolutely necessary for good functioning, conferring flexibility, fluidity and selective permeability properties of membranes. Consequently, lipid profiles in microalgae play a vital role in maintaining the integrity of the world's aquatic food webs [4]. Nutritionally, EPA (20:5) is one of the most important fatty acids belonging to this group. These long-chain omega-3 fatty acids pro-
1 This text was submitted by the authors in English.
Abbreviations: ADW — absolutely dry weight; EPA — eicosapentaenoic acid; PUFAs — polyunsaturated fatty acids. Corresponding author: L. V. Kabaivanova. Institute of Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria, 26 Acad. G. Bonchev str.; e-mail: lkabaivanova@yahoo.com
vide significant health benefits to the human population, particularly in reducing cardiac diseases, stroke and high blood pressure, depression, rheumatoid arthritis, and asthma [5]. They have also been reported to inhibit tumor growth [6].
The physiology and biochemistry of Rhodella reticulata, producer of the above mentioned biologically active substances, are poorly studied. To enhance mi-croalgae production efficiency, it is necessary to optimize the growth conditions. Light and temperature are the major factors that affect the overall biomass productivity in nutrient unlimited culture conditions [7]. Temperature stress, in particular, influences the growth rate and chemical composition of microalgae and may limit nutrient interactions. Temperature has a major effect on the phase transition of membrane lipids, the kinetics of cellular enzymes, and active transport systems across membranes [8]. Meanwhile, light is also essential for the growth of phototrophic microalgae, as both light intensity and temperature affect their biomass productivity, metabolites formation, and production of some key enzymes associated with photosynthesis [9]. Understanding the combined effects of light and temperature on algal cultures will enable the optimization of growth in controlled production system by the implementation of temperature regulation as a function of irradiance [10].
The aim of this study was to explore growth and some biochemical characteristics of R reticulata, pro-
ducer ofbiologically active substances valuable for practice, under different temperatures and light intensities.
MATERIALS AND METHODS
Strain and cultivation conditions. The red mi-
croalga Rhodella reticulata (Rhodophyta), strain UTEX LB2320, was acquired in the Austin University, Texas, USA. It was isolated from brackish water in this region. It is maintained as a test culture from the laboratory collection of the Department of Experimental Algology, Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences.
Temperature range for growth was estimated using a gradient temperature block [11]. It permits to set the desired temperature, specifically, five different temperature conditions between 18 to 34°C simultaneously with two different light intensities 260 and 520 ^E/(m2 s). The other growth conditions were kept constant. Intensive cultivation was performed to test the effect of temperature and light on growth and biochemical productivity of R. reticulata culture. The experiments were conducted at two different permanent illuminations in special vessels (200 mL) and 2% CO2 supply. The optimal temperature was determined at pH 7.3. The algae were grown in a modified Brody Emerson nutrient medium for 96 h [12].
Dry weight measurement. The growth of the algal culture was estimated following the increase of its weight. 10 mL of the algal suspension were centrifuged at 6000 g for 20 min (Rrotofix 32A, "Hettich"). The supernatant was removed and the cells were dried at 105°C for 16 h (absolutely dry weight, ADW). Salts were eliminated by rinsing with tap water. Dry biomass content (g/L) was calculated according to MakareviC ien e
[13].
Specific growth rate. Specific growth rate (^/day) was calculated on dry weight:
^ = ln(N2/N)/(t2 - t1), where N1 and N2 represent the dry weight of algal cells at time t1 and t2 [14].
Protein determination. Protein quantity was assayed according to the Lowry method [15]. It was determined using the standard curve, prepared with albumin.
Carbohydrates analysis. Intracellular carbohydrates were determined from the pellet after sonica-tion. They were quantified as glucose equivalents by the phenol/sulfuric acid method of Dubois [16], using glucose as a standard.
The algal suspension was centrifuged. Then the residue was extracted with chloroform : methanol (2 : 1) solution by boiling and use of reverse condenser. Cell fragments were removed by filtration. It was followed by separation of chloroform and methanol with 11.5% NaCl solution (to 1/5 of the volume of the extract). The lipid extracts were released from the chloroform at 40-45°C on rotary vacuum evaporator, and re-extraction
followed. After solvent removal, drying was performed using Na2SO4. The quantity ofpure lipid extract was determined by weighing. Lipid content is presented as a percent from the ADW of the initial sample.
Fatty acids were analyzed by gas chromatography on a Perkin-Elmer instrument as previously described by Iliev and Petkov [17]. Fatty acids esterification was accomplished under boiling with reverse condenser for 1 h. The next step was the extraction with diethyl ether. The obtained methyl esters were purified by thin layer chromatography and analyzed afterwards using a gas chromatograph (Perkin-Elmer 900). The following fatty acids were used as standards: C14:0 — myristic acid, C16:0 — palmitic acid, C16:1 — palmitoleic acid, C16:2 — hexadecanoic acid, C18:0 — stearic acid, C18:1 — oleic acid, C18:2 — linoleic acid, C18:3 — lino-lenic acid, C20:3 — eicosatrienoic acid, C20:4 — arachi-donic acid, C20:5 — eicosapentaenoic acid.
All data are presented as means ± standard deviation. The significance of differences between the treatments was evaluated by one-way analysis of variance (ANOVA) and a Bonferroni post-hoc test using InStat ("GraphPad Software Inc.", La Jolla, CA, USA). Values of P < 0.05 were considered significant.
RESULTS AND DISCUSSION
Influence of temperature and light intensity on Rhodella reticulata growth
It is known that the growth rate will increase with the increase in temperature up to its optimum, and once it reaches its optimum, growth rate will decrease drastically with temperature increase. Under laboratory conditions, the investigated strain grew well at temperatures between 18 and 34°C only. The light and temperature ranges for R. reticulata growth are shown in fig. 1. Below 18°C growth was delayed, while above 34°C it was greatly suppressed. At these minimal and maximal temperatures for the strain growth, the values for cell number at the low light intensity were very similar (fig. 1a). Wherein, these values increased linearly with temperature increase up to 28°C. The high light intensity (fig. 1b) led to a shift in the temperature optimum for growth towards the lower temperature (26°C).
96 h after the start of the experiment, all algal cells remained vital for all variants, but cell growth at 34°C was lower. This fact could be explained by the sensitivity of the cells to high light intensity together with extreme temperatures. Vonshak [18] described how a definite regime of combined irradiance and temperature supplied to the culture resulted in stress conditions. The accumulation of algal biomass at 22°C and low light intensity was about 59% in compa
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