Introduction

The physiology of microalgae is affected by physicochemical factors such as water temperature, salinity, light intensity, nutrient concentrations, and pH. It, however, is hard to identify the influence of environmental factors on the microalgae directly in nature. Thus, the physiological experiments are generally applied to understand the mechanism of environmental impacts on the different metabolic activities of microalgae through the laboratory culture studies (Cho et al., 1998).

 

Concentrations of cells in phytoplankton cultures are generally higher than those found in nature. Microalgae cultures must therefore be enriched with nutrients to make up for the deficiencies in the seawater. Macronutrients include nitrate, phosphate (in an approximate ratio of 6:1), and silicate. Silicate is specifically used for the growth of diatoms that utilize this compound for production of an external shell. Micronutrients consist of various trace metals and the vitamins like, thiamine (B1), cyanocobalamin (B12) and sometimes biotin.

 

Two enrichment media that have been used extensively and are suitable for the growth of most marine microalgae are Walne medium and Guillard’s F/2 medium. Generally, the most common nutrient medium used is F/2 standard described by Guillard and Ryther (1993). The complexity and cost of the above culture media often excludes their use for large scale culture operations. Alternative enrichment media that are suitable for mass production of microalgae in large-scale extensive systems contain only the most essential nutrients and are composed of agriculture grade rather than laboratory grade fertilizers (Lavens and Sorgeloos, 1996). Krichnavaruka et al. (2005) studied the optimal growth conditions and the cultivation of Chaetoceros calcitrans in airlift photobioreactor, and they found that a modified of standard F/2 medium with a two folds of silica and phosphorus concentrations was illustrated to result in a better growth of this diatom. Vitamin B12 in the range from 1 to 3 μg·l⁻¹ did not significantly affect the growth.

 

Growth rate of microalgae is influenced by environmental conditions (Renaud et al., 2002). Microalgae growth and composition may be influenced by nutrients like nitrate, phosphate and silicate (Paerl, 2009). The influence of nitrate and phosphate concentrations on growth, extracellular polysaccharide production, and fatty acid profile of marine diatoms has been reported (Liang et al., 2006). Modifications in culture medium such as nitrogen, phosphorus and silicate concentrations affect the growth rate of microalgae, cellular composition and fatty acid profile of the lipid fraction (Sanchez et al., 2000). Fried et al. (2003) evaluated the effects of nine different combinations of nitrate and phosphate concentrations on the algal growth by measuring relative chlorophyll levels. They concluded that both nitrates and phosphates have positive effects on the algal growth. However, these variables affect the algal growth independently of each other and there is no interaction between the two. This implies that both nitrates and phosphates are effective limiting nutrients that can be reduced to control algal profile. Hemalatha et al. (2012) studied the combined effects of temperature, nitrate and silicate on growth of the marine diatom, Chaetoceros simplex. The growth rate was directly proportional to nutrient concentration and temperature, whereas chlorophyll-a and biochemical composition were directly proportional to the nutrient concentrations. Karthikeyan et al. (2013) investigated the suitability of the marine diatoms, Chaetoceros curvisetus and Chaetoceros simplex for the removal of macronutrients from different wastewater. The growth and nitrate-phosphate removal properties were studied with nitrate, ammonium and urea nitrogen sources. The results indicated the Chaetoceros simplex was more efficient than Chaetoceros curvisetus and suitable for the removal of macronutrients when cultured with urea and nitrate nitrogen sources. Recently, Chaetoceros simplex was examined for its growth and biochemical compositions in different nitrate, phosphate and silicate concentrations by Hemalatha et al. (2014).

 

Aim of the work

The aim of the present work is to study the response of growth, photosynthetic pigments, carbohydrates, total soluble proteins and total lipids of Chaetoceros simplex Ostenfeld to different treatments of nitrates and phosphates.

 

Materials and Methods      

Isolation and purification of the organism

One liter of sub-surface seawaters was collected from Hurgada in carefully cleaned polyethylene bottle and passed immediately through plankton net of 100 μm mesh size to eliminate the macrozooplankton. This water was supplemented in the laboratory with F/2 medium (Guillard and Ryther, 1993). This is a common and widely used general enriched seawater medium designed for growing coastal marine algae, especially diatoms. This culture was poured in several conical flasks of 500 ml capacity (250 ml were added in each one) and incubated for 12 days at 25±1 °C under day light fluorescent lamps of about 6400 LUX. The algal cultures were supplied with dry air to provide CO₂ necessary for photosynthesis, to prevent the settling of the cells at the bottom of the containers and to maintain the algae in suspension without mechanical stress. The grown cells were examined under a binocular research microscope and identified as Chaetoceros simplex Ostenfeld. For accurate identification of the isolated cells; scanning electron microscope (SEM–JEOL-JSM5300) was used according to Ashour (2011) (Fig.1). Generally, the isolation and purification of C. simplex was carried out by the dilution method (Droop, 1954) and Picking up Capillary method (Stein, 1973).

 

Determination of growth

Algal cells were counted every 48 hours intervals with haemocytometer (0.1 mm depth) under a binuclear research microscope with a magnification power of 480x and the counts were expressed as cells·ml^-1.

 

Determination of different metabolic variables

The chlorophylls (a and c) were estimated according to the method recommended by Parsons and Strickland (1965). The total carbohydrate content was measured by the phenol-sulphuric acid method (Herbert et al., 1971). Total soluble protein was determined quantitatively using the method described by Lowry et al. (1951). The total lipids of algal cellswere extracted according to Bligh and Dyer (1959).

 

Statistical Analysis

The ANOVA analysis was carried out for the obtained data on the computer using the program of STATISTICA at p < 0.01.

 

Results and Discussion

The chemical well as to the experimental conditions applied like temperature, light content of microalgae can vary with culture age and with changes in culture conditions (Araújo and Garcia, 2005). The effect of variation of these parameters on many algal species has been studied by several workers (Uriarte et al., 1993; El-Sherif, 1993; Hemalatha et al., 2012, 2014; Abdel-Hamid et al., 2015). Data on the chemical composition of microalgae may also vary widely due to differences of the methods of measurement used (Barbarino and Lourenço, 2005); the physiological state of the microalgae, as intensity, medium cultivation or in outdoors conditions (Banerjee et al., 2011). In addition, due to the interaction of the organisms with the culture medium, a batch culture is under a continuous chemical change. These variations reflect on the cell metabolism and consequently on their chemical composition (Lourenço et al., 2002).

 

Growth of Chaetoceros simplex

Nutrients are among the most important factors controlling phytoplankton growth. However, nutrient contents in marine environments greatly change over space and time (Eker-Develi et al., 2006).In the present study, the concentrations of 1, 5, 50 and 100 μM of nitrate stimulated the algal growth of Chaetoceros simplex by 7, 14, 26 and 60%, respectively over the control culture in the 8^th day of culture. While, the values of 200 and 300 μM nitrate caused about 13 and 44 % growth reduction, respectively below the control culture. On the other hand, phosphate concentrations of 0.3, 1 and 5μM stimulated the growth of Chaetoceros simplex by 11, 23 and 32 %, respectively, while, the values of 25 and 50 μM exhibited a decrease in the algal growth by 19 and 49 %, respectively (Tables 1, 2 and Figs.2, 3). In general, the maximum growth of Chaetoceros simplex 330×10⁴ cell·ml^-1 and 310×10⁴ cell·ml^-1 were achieved at the value of 100 μM nitrate and 5 μM phosphates. While, the concentrations of 200 and 300 μM nitrate and 25 and 50 μM phosphate showed lower growth than the other treatments. These nutrients are usually involved in many biochemical reactions including proteins, nucleic acids, chlorophylls and amino acids biosynthesis in the photosynthetic organisms (Geider and La Roche, 2002; Van Mooy et al., 2009 and Van Mooy et al., 2009). However, the growth of Chaetoceros simplex decreased gradually after eight days of incubation may due to the expected gradual decrease of nitrate and phosphate concentrations with time as well as excretions of unfavorable metabolites in the culture media. Padhi et al. (2009)reported that the maximum growth of Chaetoceros curvisetus was foundat 3 mM NaNO₃ and slightly declined at 6 mM NaNO₃. Hemalatha et al. (2012) stated that the combination effect of temperature and nutrients on Chaetoceros simplex, recording the maximum cell density of 18.1×10⁵ cells·ml^-1 at 20 ºC under 1764 μM nitrate in the 10^th day of culture, whereas at 25ºC and 29 ºC, the maximum cell density reached 18.89×10⁵ cells·ml^-1 and 23.5 ×10⁵ cells·ml^-1, respectively. Also, Hemalatha et al.(2014) found that the maximum cell density of Chaetoceros simplex was reached in the culture treated with 1764 μM nitrate (18.23×10⁵ cells·ml^-1) and 72.4 μM phosphate (18.21×10⁵ cells·ml^-1). The growth rate was enhanced with increasing of nutrients up to the concentration 2646 μM nitrate and 90.5 μM phosphates. However, the concentrations of 1764 μM nitrate and 72.4 μM phosphate that persistent the highest cell densities of Chaetoceros simplex by Hemalatha et al. (2012, 2014) are extremely higher than recorded in the present study. This may be due to the combination effects of temperature and the nutrients; nitrate, phosphate and silicate in the culture F/2 media. This result may be also due to the differences in the environmental conditions, including salinity of seawater, pH value, dissolved oxygen concentrations and the nutrient salts of the filtered seawater that used in the culture F/2 media in the present study.

Chlorophylls are key compounds in plants for trapping light energy for photosynthesis, thus their quantitative determination is of great importance in the studies of photosynthesis, primary production and related subjects. Chlorophyll-a, chlorophyll-c and the accessory photosynthetic pigment Carotenoids were measured in this study.

 

The results in Fig. 4, 5, 6, 7, 8 and 9 indicated that, chl-a, chl-c and carotenoids content of Chaetoceros simplex followed a similar pattern of change to that of growth in response to different treatments of nitrate and phosphate. The addition of 100 μM nitrate and 5 μM phosphate to the culture medium recorded a maximum Chl-a of 0.72 ±0.02 μg·ml^-1 and 0.69±0.05 μg·ml^-1, respectively as compared with the control culture. While, the concentrations of 300 μM nitrate and 50 μM phosphate sustained the lowest chlorophyll-a of 0.35±0.02 μg·ml^-1 and 0.36±0.02 μg·ml^-1, respectively. This result is in accordance with that obtained by De la Curz et al. (2006) who reported that chlorophyll–a content was directly related to cellular density and indirectly to concentration of nutrients. In the present study, the chlorophyll-a content sustained its highest value at the early exponential growth phase, which coincided with the data reported by Hemalatha et al. (2014). The chl-c and carotenoid contents showed an increasing trend up to 100 μM nitrate and 5 μM phosphates. The addition of 100 μM nitrate to the culture medium recorded a maximum chlorophyll-c of 0.52±0.02 μg·ml^-1 and carotenoids value of 4.1±0.03 μg·ml^-1, while additions of 5 μM phosphate sustained chl-c of 0.49±0.03 μg·ml^-1 and carotenoids of 3.4±0.02 μg·ml^-1).

 

Carbohydrates

The main storage compounds of diatoms are lipids (TAGs) and a β-1, 3-linked carbohydrate known as chrysolaminarin, which are sun light driven cell factories that convert carbon dioxide to potential biofuels, foods and feeds (Walter et al., 2005). In the present study, the carbohydrates showed increasing trends with increase of nitrate and phosphate in the culture media up to 100 μM nitrate and 5 μM phosphates and then it gradually decreased. The addition of 5 μM phosphate to the culture medium of Chaetoceros simplex recorded a maximum carbohydrate of 54.6±3.5 μg·ml^-1 and the addition of 100 μM nitrate sustained the highest value of 50±5.3μg·ml^-1. Whereas, the minimum carbohydrate content was observed with the concentrations of 300 μM nitrate and 50 μM phosphate (Tables 3 and 4).