INTRODUCTION. food supplement, cosmetics, aquaculture feed, and most recently, for biofuel production.

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1 Acta Manilana 59 (2011), pp Printed in the Philippines ISSN: in two microalgal species, Botryococcus braunii and Fragilaria brevistriata by nitrogen limitation Susana F. Baldia 1,2,3, Marianne Kristine Arguel 3, Diane Sia 1, Celina Rebecca Tuazon 1, Alyssa Aya Yap 1, Rochelle Jane Ong 1, and Katrina Mariz Rodis 1 1 Department of Biological Sciences, College of Science, 2 Research Center for the Natural and Applied Sciences, 3 Graduate School, University of Santo Tomas, España Boulevard, 1015 Manila, Philippines Microalgae are one of the biological sources of energy which are renewable and biodegradable. They produce energy by the production of biogas (methane), generation of hydrogen, production of ethanol by fermentation, and the use of plant-derived oils. To date, algae have emerged as one of the promising sources for biodiesel production. In line with this, an was conducted to determine the growth and lipid production in two microalgal species Botryococcus braunii and Fragilaria brevistriata by nutrient limitation using different concentrations of nitrate-nitrogen (20, 10, 1.0, 0.5, and 0.1 mg N /L). Growth of F. brevistriata and B. braunii in terms of cell density increases with increasing nitrogen concentration, obtaining a maximum cell density of cells/ml and cells/ml, respectively at 20 mg N/L. With increasing nitrogen concentration, the dry weight and oil content decrease in both Botryococcus and Fragilaria. However, the oil content of the two algal species vary at different nitrogen concentrations, that is, 10 mg N/L for B. braunii and at 1 mg N/L for F. brevistriata. An oil content of mg/ml and mg/ml were obtained from F. brevistriata and B. braunii grown at 1 and 10 mg N/L, respectively. Keywords: Botryococcus braunii, Fragilaria brevistriata, nutrient limitation, nitrogen concentrations, oil content INTRODUCTION Algal biotechnology focuses on the production of high-value fine chemicals for *To whom correspondence should be addressed sfbaldia@yahoo.com food supplement, cosmetics, aquaculture feed, and most recently, for biofuel production. Biofuel is a type of fuel derived from the biomass of living organisms. It can be produced through any carbon source, thus Acta Manilana Volume 59 (2011)

2 Baldia SF et al. / Acta Manilana 59 (2011) photosynthetic plants are the commonly used material for production. To date, algae have emerged as one of the promising sources for biodiesel production. There are numerous reasons for considering the algal production system: (1) the process does not use fossil fuels, (2) uses marginal land for culturing, (3) does not compete with food crops in the use of land, (4) requires limited supplies of water (closed loop system), (5) high yield, (6) much lower cost than traditional biomass crops since large-scale cultures can be adjusted to various levels of operational skills, and (7) no pollution from fertilizers or pesticides. Moreover, biofuels specifically derived from microalgae are considered to be a technically viable alternative energy resource that is devoid of the major drawbacks associated with first and second generation biofuels. Though there are outstanding issues related to photosynthetic efficiencies and biomass output, micro-algae derived biofuels could progressively substitute a significant proportion of the fossil fuels required to meet the growing energy demand [1] and likewise, it could help in decreasing CO 2 production since combustion of fossil fuels could be prevented. With this important development in the field of microalgae, it becomes necessary to explore and evaluate algal strains that are renewable sources of energy and improve their performance in terms of their capacity to produce the desired algal product. Botryococcus braunii is a colonial green microalga which is capable of accumulating large amounts of hydrocarbons [2, 3] which range from 13% to 18% of its dry weight [4]. On the other hand, F. brevistriata is a diatom commonly found in the freshwater and brackish water habits. Diatoms have been identified as strong candidates for significant biodiesel production because they produce up to 60% of their cellular mass as triacylglycerols (TAG) which can easily be converted into biodiesel through transesterification reaction [5]. Culture conditions may affect the lipid content of algae and these may vary in different algal species. Lipid production in microalgae typically occurs during periods of environmental stress such as under nutrient-deficient conditions. Its growth and contents vary in accordance with culture conditions which can sometimes be enhanced by nitrogen starvation [6]. Nutrient limitation may influence hydrocarbon accumulation in Botryococcus. According to Ben-Amotz et al. [7], nitrate deficiency increases the amount of neutral hydrocarbons to about 8% of organic weight. Moreover, in nitrogen-limited situations, lipid content of algae usually increases because lipid-synthesizing enzymes are less susceptible to disorganization than carbohydrate synthesizing enzymes due to nitrogen deprivation; thus, the major proportion of carbon can be bound in lipids. However, biomass growth is often inhibited in nitrogen-lacking situations, so there is usually a lipid yield peak for each algal strain at certain nitrogen types and concentrations [8]. In this study, the growth and lipid content of B. braunii and F. brevistriata were compared under the situation of nitrogen limitation with the objective of determining the nutrient status for optimization of culture conditions geared towards the utilization of renewable energy. EXPERIMENTAL Acquisition of stock cultures. Botryococcus braunii and F. brevistriata were both acquired from the Culture Collection of the Research Center for the Natural and Applied Sciences, of the Thomas Aquinas Research Complex. Botryococcus braunii was originally 58

3 isolated from Paoay Lake, Ilocos Norte [9] while F. brevistriata was part of the Culture Collection of the Research Center for the Natural and Applied Sciences. Both al organisms were continuously renewed every days throughout the. These were incubated in culture shelves under cool white fluorescent lamps with a light intensity of 9 μmol photons m 2 s 1 at a temperature of 23±2 C. Preparation of culture media. Inorganic media (BRSP media) were used for both B. braunii and F. brevistriata, except the addition of sodium silicate for F. brevistriata [10]. The media were adjusted to ph 7.4 and sterilized at 15 lbs psi at 121 C for 15 min. Volumes of the said media depended on the amount of cultures necessary for the upscaling procedure. Up-scaling of cultures. About ten pieces of mm test tube cultures were used for storage and maintenance of stock cultures. These were transferred regularly every 12 days and up-scaled to three 125 ml flasks which were later on transferred to 250 ml flasks after 1 2 weeks. Then the same procedure was done to the stock cultures in the 250 ml flasks to 500 ml flasks. Cultures from 500 ml flasks were used as inocula for gallon bottles. Determination of exponential growth phase. During the initial phase of the study, the determination of the exponential growth phase of B. braunii and F. brevistriata was performed. Three replicates per algal species were used and the was conducted in 500 ml Erlenmeyer flasks. Growth was monitored every two days taking 1 ml sample from each of the replicates of both microalgal species. Samples were fixed with Lugol s solution and were counted using a haemacytometer [11] and a compound microscope. Nitrogen limitation. Five nitrogen concentrations (20, 10, 1, 0.5, and 0.1) with three replicates each were used for both B. braunii and F. brevistriata. Samples for growth and lipid content were harvested on the microalgae s exponential growth phase based on the data obtained from the first. Similarly during the exponential growth phase, the total algal biomass was harvested using a fine mesh cloth to separate the algal biomass from the media. This was kept in an aluminum foil and placed inside the freezer for lipid extraction. Dry weight determination and lipid extraction. For dry weight determination, each of the 10 ml samples were filtered in glass fiber filters. Samples were then placed inside the oven for an hour and dried into constant weight. During lipid extraction, each of the frozen biomass of both B. braunii and F. brevistriata were homogenized by using mortar and pestle. The homogenized biomass was then placed on centrifuge bottles. Then, 5 ml hexane was placed on each of the centrifuged bottles with homogenized algae and after which were centrifuged at 5000 rpm for 10 min. This was done twice for every centrifuge bottle. After centrifugation, the supernatant from the precipitate was separated. The supernatant was disposed off and the remaining biomass and oil were kept. The oil obtained from each of the replicate of each concentration was placed in scintillation bottles. Using the rotary evaporator, the remaining hexane in each of the oil obtained from the different concentrations was eliminated. After this process, the scintillation bottles were weighed with the extracted oil and were recorded as the final weight. 59

4 Baldia SF et al. / Acta Manilana 59 (2011) Statistical analysis. The relationship of dry weight and oil content was done using oneway analysis of variance to determine if there was significant difference between the dry weight and oil content obtained from the different nitrogen concentrations in both B. braunii and F. brevistriata. On the other hand, the Welch s F-test was used to identify if the oil obtained from algae of different concentrations are correlated. RESULTS AND DISCUSSIONS Exponential growth phase. Figure 1 shows the growth of F. brevistriata under optimum culture conditions, reaching its exponential growth phase on the eighth day of culture. Though a long lag phase was noted, it only needed one week for F. brevistriata to double its population. In the case of B. braunii, the exponential growth phase was attained in two weeks (Fig. 2). Botryococcus braunii is known to be a slow growing alga. This was likewise observed in the work of Dayananda et al. [4] in which it took B. braunii 12 days to reach its exponential growth under optimum culture conditions. Growth under conditions of nitrogen limitation. In F. brevistriata, growth in terms of cell density increases with increase in nitrogen concentration (Fig. 3). The highest cell density which is cells/ml was obtained in 20 mg N/L while the lowest ( cells/ml) was found in 0.1 mg N/L concentration. This suggested that growth was directly proportional to the concentration of nitrogen in the medium [12] Figure 1. Growth of Fragilaria brevistriata under optimum culture conditions day 2 day 4 day 6 day 8 day Figure 3. Growth of Fragilaria brevistriata in terms of cell density during nitrogen limitation mg N/L mg N/L Figure 2. Growth of Botryococcus braunii under optimum culture conditions Figure 4. Growth of Botryococcus braunii in terms of cell density during nitrogen limitation 60

5 Oil Content & Dry Weight (mg/ml) Dry Weight (mg/ml) Amount of Figure 5. Oil content and dry weight of Fragilaria brevistriata under different nitrogen concentrations Figure 7. Maximum oil content of Fragilaria brevistriata produced under nitrogen limitation Oil Content & Dry Weight (mg/ml) Dry Weight (mg/ml) Amount of Figure 6. Oil content and dry weight of Botryococcus braunii under different nitrogen concentrations Figure 8. Maximum oil content of Botryococcus braunii produced under nitrogen limitation The same trend was observed in B. braunii (Fig. 4). The highest cell density which is cells/ml was obtained in 20 mg N/L concentration while the lowest ( cells/ml) was found in 0.1 mg N/L In terms of dry weight, the concentration of 1 mg N/L gave the highest growth (0.34 mg/ml) in F. brevistriata (Fig. 5). However in B. braunii (Fig. 6), the higher concentration of 10 mg N/L gave the greatest amount of dry weight (0.23 mg/ ml). Relationship of dry weight and oil content. Figure 7 shows the maximum oil content of F. brevistriata grown in different nitrogen concentrations. The concentration of 1 mg N/L produced the highest oil content and the highest dry weight (Fig. 5). According to Borowitzka [13], dry weight and oil content are directly proportional. Similarly in B. braunii, the highest dry weight which was found in the concentration of 10 mg N/L gave the highest amount of oil, mg/ml (Fig. 8). Biomass growth is often inhibited in nitrogen-lacking situations, so there is usually a lipid peak for each algal strain at certain nitrogen types and concentrations [8]. Algal reproduction decreases significantly when exposed to environmental stress conditions. Therefore, even with higher lipid content, the net oil 61

6 Baldia SF et al. / Acta Manilana 59 (2011) Amount of Oil (mg/ml) Botryococcus braunii Fragilari brevistriata Figure 9. Comparison between the oil content of Fragilaria brevistriata and Botryococcus braunii produced under nitrogen limitation content gained may actually decrease in a nutrient deficient environment due to significantly reduced cell numbers [14]. Algal lipid content usually increases because lipid-synthesizing enzymes are less susceptible to disorganization than carbohydrate-synthesizing enzymes due to nitrogen deprivation. Thus, the major proportion of carbon can be bound in lipids [8] which contribute to the dry weight of the algae. It was demonstrated by Ramachandra et al. [12] that the lipid content yield of the diatom Didymosphenia germinate reached up to 70% of the dry weight which was 0.40 mg/ml. Furthermore, in the of Verma et al. [15], algal species such as B. braunii, Chlorella emersonii, and Chlorella minutissima yielded the most oil and that light, ph, and temperature are better limiting factors because they were able to yield 86% of algal oil. Based on statistical analysis by one-way analysis of variance, the relationship between dry weight and oil content in F. brevistriata and B. braunii is highly significant (F=15.79, r< ; F=21.17, r< ), thus, as the dry weight increases, the oil content also increases. Comparison between the oil content of Fragilaria and Botryococcus. A comparison between the oil content of B. braunii and F. brevistriata is shown in Figure 9. More oil was obtained from B. braunii than from F. brevistriata. Although B. braunii produced more oil, it needed more time and more nitrogen than F. brevistriata which produced a considerable amount of oil in a shorter period of time and with a lower nitrogen concentration. This is because diatoms produce oil which they need for buoyancy so that they can produce more oil in a shorter period of time [12]. CONCLUSION With increasing nitrogen concentration, the dry weight and oil content decrease in both B. braunii and F. brevistriata. However, the nitrogen and oil content of the two algal species vary at different concentrations, in which case at 10 mg N/L for B. braunii and at 1 mg N/L for F. brevistriata. Given different conditions of nitrogen limitation, F. brevistriata is a better source of oil because it takes only 1 mg N/L to produce the maximum oil content. While in B. braunii, more nitrogen, that is, 10 mg N/L is needed to obtain the maximum oil content. ACKNOWLEDGEMENT This study was part of the project of the senior author which was funded by the Research Center for the Natural and Applied Sciences, University of Santo Tomas. REFERENCES [1] Melis A & Mappe T. Hydrogen production. Green algae as a source of energy. Plant Physiology 2001; 127: [2] Brown AC, Knights B, & Conway E. Hydrocarbon content and its relationship to physiological state in the green algae Botryococcus braunii. Journal of Phytochemistry 1969; 8:

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