واکنش میزان رشد، محتوای پروتئین و اسیدچرب جلبک دسمودسموس کوناتئوس درشرایط کمبود و انباشتگی نیتروژن

Introduction

Microalgae are classified under a varied group of photoautotrophic organisms which play important role as primary producers for feeding of zooplanktons and other animals in the food chain of aquatic systems. The ability of microalgae in production of bioactive compounds such as pigments, vitamins, proteins and fatty acids make them a good source to use them as bio energy and pharmaceutical resources in aquaculture systems (1, 2, 3 and 4). The growth and biochemical composition of microalgae is affected by culture conditions (5, 6 and 7). Microalgal cells have shown different mechanisms (such as enzyme reaction, cell permeability, and cell composition) to survive and optimize their growth in response to strong variations in physicochemical conditions (7 and 8). As a nutrient, nitrogen is required in high concentration and is counted as one of the most important chemical nutrients that require microalgae growth. After carbon, nitrogen plays the second important role in production of biochemical compounds such as amino acids, nucleic acids and other nitrogen- enriched molecules in the grown microalga biomass. In recent years, the importance of nitrogen in microalgae biomass has been studied by many researchers (9, 10 and 11). In normal condition, the chlorophyll accumulation and cell division is. In contrast, depletion of nitrogen resources result in the changes in some biosynthesis pathway of microalgae such as a decline in nitrogenous photosynthetic pigments, reduction in photosynthetic efficiency, cell division prevention, lower growth rate, and a reduction in cell size (12 and 13). Further, nitrogen limitation leads to accumulation of carbon compounds such as polysaccharides, fats and reduction of carbohydrate production. Moreover, differences in fatty acid profile occurring response to various nitrogen concentrations (14 and 15). The nitrogen limitation also resulted in the higher saturation in lipid profiles (increase in C18:1, and decrease in C18:2 and C18:3) and fatty acid reduction (13 and 16). Numerous studies have shown different concentrations of nutrients that have a wide effect on the biochemical composition, growth and morphology of fresh water algae (12 and 17). Nitrogen starvation and limitation, on the other hand, cause very different nitrogen stresses which may lead to various physiological responses.

The green algae D. cuneatus is a chlorococcal coenobial (Scenedesmaceae, chlorococcales, chlorophyta) which is widely distributed in freshwaters with moderate temperature. The green algae are highly polymorphic (18). Accordingly in this study, the influence of different nitrogen concentrations on fatty acid profile, total protein, growth rate and chlorophyll content of Desmodesmus cuneatus was investigated.

Materials and methods

Desmodesmus cuneatus was obtained from algae bank Artemia and aquatic research institute, Urmia, Iran. Experiments were performed on culture of 450 ml, grown in 500 ml glass flasks. All the glassware and media were sterilized prior to inoculation. Culture was grown in BM medium (Table 1) (19). Stock culture of D. cuneatus was performed at 25°C under continuous fluorescent illumination (100 μmol photons m^-2 s^-1) and pH 7.5.

Table 1- The content of BM medium

Stress conditions

Initial density for D.cuneatus was set on 3.2 × 10⁵ algal cells. Cultures of cells were washed in double- distilled water (DDW) and resuspended in the medium with different concentrations of nitrogen (KNO₃) (5 mM, 2.5 mM, 1.25 mM, 0.62 mM and nitrogen free medium).

In order to assess the cell densities during culture period, cells were daily counted using a haemocytometer. Growth rates and doubling time (DT) were calculated on cell basis which were calculated based on following equation Guillard and Omori.

μg (day^-1) = (Ln (n₂/n₁) /t₂-t₁); (20)DT = log_e²/µ (21)

Where μ is the specific growth rate, N₂ and N₁ are biomass concentrations at the start (t₁) of exponential phase (cell.ml⁻¹) and at the end of experiment (t₂) respectively.

In order to determine chlorophyll a and b, 5ml acetone was added to each tube containing 5ml of culture and then filtered (using GF/C filters). Each sample was maintained in the dark at 4°C for 24 h, then cells were broken using an ultrasound (on ice) up to 90s and subsequently centrifuged at 12, 000×g for 5 min. Chlorophyll a and b was determined using the equation based on the method of Ritich (22).

Total protein was extracted according to Meijer and Wijffels' method (23). First 500

ml of the algal sample was centrifuged at 4°C at 500 g for 10 min, washed and centrifuged again and then freeze–dried. For protein analysis, 20 mg aliquots of the freeze–dried biomass were suspended for 20 min in 10 ml of lysis buffer in a Falcon tube and then resuspended in 10 ml phosphate buffer with 1% (w/v) SDS (sodium dodecyl sulfate) to facilitate the extraction of proteins. Total protein was determined by means of the method of Bradford, using bovine serum albumin standards (24).Quantitive measurement of fatty acids was performed using GAS chromatography (Agilent 6890 Gas Chromatography device) according to the method of Cohenet al. (25). Accordingly, Biomass was transmethylated with 2% H₂SO₄ in the dry methanol/toluene mixture (90:10, v/v) at 80°C for 1.5 h and heptadecanoic acid was added as an internal standard. FA methyl esters were determined by co- chromatography with authentic standards (Sigma) and by comparison of their equivalent chain length (26).

The SDS- PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) method was used for total protein analyses of Algae samples (27).

Two hundred milligrams of each sample were prepared for SDS- PAGE in liquid nitrogen and pulverized in 1.5 ml microtube by appropriate tips. Fifty milligrams of the resulting powder were transferred into 500 μl of protein extraction buffer (buffer K). The extraction buffer for SDS- PAGE contained: 5 mM MgCl₂, 5 mM nitrogen H₂PO4, 40mM Hepes, 70mM Potassium gluconate, and 150mM Sorbitol, and pH was set at 6.5 containing a protease inhibitor cocktail (Invitogene™ Mini from Roche Diagnostics GmbH) (28). Protein concentration was determined by the Bio photometer apparatus (Eppendorf). Samples were heated for 5 min at 95° C and subsequently cooled to room temperature in tap water. After low speed centrifugation (1600 g, 5 min) to remove insoluble fragments, supernatants were electrophoresed. Proteins were separated by 12.5% SDS- PAGE (27). Each lane was loaded with 15 μg total proteins run at 200 V for 45 min. Protein bands were stained with 0.125% Coomassie Brilliant Blue R- 250 in 40% methanol, 10% acetic acid for 1 h at a 50°C water bath with gentle agitation. Destaining was performed in 5% methanol, 7.5% acetic acid for 2 h at 50°C with gentle agitation.

The obtained growth rate data were analyzed by one- way ANOVA. Tukey’s test was performed to identify differences among the means, and differences were considered statistically significant at Pvalue

Results

The effect of five level of nitrogen concentrations on the growth of D. cuneatus was investigated for 12 days. The effect of nitrogen concentration was monitored by counting the cells number of D. cuneatus (Fig. 1). Based on Fig. 1, during stress, lowest growth rate (0.23 ± 0.06 μgday⁻¹) was observed in the nitrogen free treatment group. On the other hand, maximum specific growth rates (0.35 ± 0.09 μgday^-1) and (0.33 ± 0.09 day⁻¹), were observed in groups treated with 2.5 mM nitrogen and 5 mM nitrogen, respectively (Table 2). In 1.25 mM nitroge, the maximum growth rate (0.29 ± 0.05 μgday⁻¹) was observed with density of 22.72× 10⁵ cells ml⁻¹ (Fig. 1). In 0.62 mM nitrogen, the maximum growth rate was 0.27 ± 0.07 μgday⁻¹ (Table 2).

Fig. 1- Cell number of D. cuneatuscultured under different nitrogen concentrations

Table 2- Cell number of D. cuneatuscultured under different nitrogen concentrations

Values sharing the same letter in each row are not significantly different, ANOVA; P value>0.05

Table 3- Chlorophyll α, b and protein contents of D. cuneatus cultured under different nitrogen concentrations

Values sharing the same letter in each row are not significantly different, ANOVA; P value <0.05

High pigment values probably attributed to the high cell density. Content of chlorophyll α and b significantly varied (Pvalue<0.05) under various nitrogen concentrations. The maximum (8.01 mg.ml⁻¹) and the minimum (2.1 mg.ml⁻¹) chlorophyll a content were measured at 5 mM nitrogen and nitrogen free respectively (Table 3). The chlorophyll b content of the cells also was affected by different nitrogen concentrations. The maximum chlorophyll b (6.23 mg.l^-1) was measured at 5 mM nitrogen, while the minimum (1.3 mg.l^-1) was observed at nitrogen free treatment (Table 3). Different nitrogen concentrations positively affected protein content (Table 3). The maximum protein concentration was 36.81% at 5 mM nitrogen, and the minimum was 11.14% at nitrogen free.

D. cuneatus showed significant differences in total protein content at various level of nitrogen concentration (Fig. 2). A major protein of approximately 68 kDa was observed in stress conditions. In addition, several proteins with molecular weight of approximately 36, 48 and 60 kDa were observed in 5 and 2.5 mM of nitrogen concentration treatments (Fig. 2).

Fig. 2- SDS- PAGE gel electrophoresis of D.cuneatus cultured under different nitrogen concentrations

The fatty acid composition of D.cuneatus was measured in stationary phase of the cultured cells at different nitrogen concentration levels (Table 4). The most abundant saturated fatty acid was 16:0 (palmitic acid), which constituted 26.01% of the total fatty acids at the nitrogen free treatment. Among the PUFAs, 18:3 (n–3) was the major fatty acid (by 13.51%) of the total fatty acids. The total percentage of PUFA (Polyunsuturated fatty acids) decreased at low nitrogen concentration, so the minimum percentage of PUFA (17.01%) was observed at the nitrogen free treatment group. In contrast, the highest percentage of the PUFA (25.31%) was recorded at the maximum nitrogen concentration (5 mM nitrogen). As for SFA (Saturated fatty acids) and MUFA (Monounsaturated fatty acids), highernitrogen concentration was associated with low percentage of these groups of fatty acids. The maximum percentages of MUFA (22.10 and 23.00%) and SFA (29.89 and 31.54%) were observed at 0.62 mM nitrogen and nitrogen free respectively.

Table 4- Fatty acids (mean ± SD %) (Expressed on the percentage of total fatty acids) of D. cuneatuscultured under different nitrogen concentrations

Values sharing the same letter in each row are not significantly different, ANOVA; P value>0.05