Bioresource Technology 156 (2014) 146–154

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Nitrogen stress triggered biochemical and morphological changes in the microalgae Scenedesmus sp. CCNM 1077 Imran Pancha a,b, Kaumeel Chokshi a,b, Basil George a, Tonmoy Ghosh a,b, Chetan Paliwal a,b, Rahulkumar Maurya a,b, Sandhya Mishra a,b,⇑ a b

Discipline of Salt & Marine Chemicals, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India Academy of Scientific & Innovative Research (AcSIR), CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 The nitrogen stress affects

morphology and biochemical composition of microalgae.  Scenedesmus sp. is able to grow at 80% reduction in nitrate compared to BG11.  Complete nitrate starvation gives 45.74% carbohydrate & 27.93% lipid accumulation.  Scenedesmus sp. is a potential feed stock for biodiesel and bio-ethanol production.  De-oiled biomass of Scenedesmus sp. can be used for bio-ethanol production.

a r t i c l e

i n f o

Article history: Received 23 November 2013 Received in revised form 4 January 2014 Accepted 6 January 2014 Available online 17 January 2014 Keywords: Microalgae Nitrogen stress Morphological changes Neutral lipid Carbohydrate

a b s t r a c t The aim of present study was to investigate the effects of nitrogen limitation as well as sequential nitrogen starvation on morphological and biochemical changes in Scenedesmus sp. CCNM 1077. The results revealed that the nitrogen limitation and sequential nitrogen starvation conditions significantly decreases the photosynthetic activity as well as crude protein content in the organism, while dry cell weight and biomass productivity are largely unaffected up to nitrate concentration of about 30.87 mg/L and 3 days nitrate limitation condition. Nitrate stress was found to have a significant effect on cell morphology of Scenedesmus sp. CCNM 1077. Total removal of nitrate from the growth medium resulted in highest lipid (27.93%) and carbohydrate content (45.74%), making it a potential feed stock for biodiesel and bio-ethanol production. This is a unique approach to understand morphological and biochemical changes in freshwater microalgae under nitrate limitation as well as sequential nitrate removal conditions. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Microalgae are oxygenic photosynthetic organisms, which utilize atmospheric CO2 and sunlight for their growth and produce various nutritional and bioactive compounds like carotenoids, ⇑ Corresponding author at: Academy of Scientific & Innovative Research (AcSIR), CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India. Tel.: +91 278 256 5801/3805x6160; fax: +91 278 256 6970/7562. E-mail address: [email protected] (S. Mishra). 0960-8524/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2014.01.025

phycobiliproteins, fatty acids, natural antioxidants and are also used as animal or aquaculture feed (Borowitzka, 1992). In recent years, due to various environmental issues, research has been focused on development of sustainable biofuel alternatives. Biodiesel and bio-ethanol are two of the most important renewable fuels that can replace fossil based fuels. Microalgal biomass have emerged as a good alternative to replace plant based biofuel feedstock due to its higher lipid and carbohydrate content (John et al., 2011; Chisti, 2007). Simple growth requirements, the ability to be grown on different kinds of water like seawater, brackish water or

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wastewater, high photosynthetic ability as compared to other terrestrial crops and very short regeneration time makes microalgae a good alternative for biofuel feed stock. To enhance the economic feasibility of microalgal based biofuel, biomass, lipid and carbohydrate productivity are the key parameters to be optimized. The biomass yield and biochemical composition of microalgae can be easily modified by changing medium ingredients or culture conditions to obtain a higher yield of desirable biomolecules (Procházková et al., 2013). Microalgal lipid and carbohydrate content can be enhanced by chemical stimuli like nitrogen and phosphate starvation or salinity stress, physical stimuli like changes in culture pH, temperature and light intensity or photoperiods. Among all above, nitrogen starvation is the most prominent technique to enhance microalgal biomass and biochemical constituents like lipid and carbohydrate. Studies have shown that instead of total nitrogen limitation strategy, intermediate nitrogen concentrations have also resulted in higher biomass and lipid productivity (Ördög et al., 2012). Apart from using intermediate nitrogen concentrations, another two stage nitrogen cultivation method has recently become popular, in which the microalgae is first grown in nitrogen rich conditions and then subsequently transferred into a nitrogen free medium which gives high lipid content with negligible biomass loss in many microalgal species (Chen et al., 2011). Scenedesmus sp. is commonly found in fresh and various types of wastewater streams. It mainly exists as unicells or multiples of two, with four or eight-celled coenobia and varies in morphological phenotype. The species differ mostly in the number and type of spines on the cells and the texture of cell wall. Morphological changes in Scenedesmus sp. are mainly induced by changes in nutrient concentration, pH or due to allochemicals released from grazers (Lürling, 1998). Scenedesmus sp. is regarded as one of the most important microalgae for biofuel feedstock due to its ability to grow in various types of wastewater along with its high biomass, lipid and carbohydrate content (Mata et al., 2012). In the present study, a fresh water Scenedesmus sp. CCNM 1077 was used as a model to study the effects of nitrate limitation and sequential nitrate starvation. Morphological and biochemical parameters including cell shape, size, growth rate, lipid, carbohydrate, protein, proline and photosynthetic pigments were used to understand the physiological mechanism of lipid and carbohydrate accumulation in microalgae. 2. Methods 2.1. Microalgae and growth conditions The microalgae used in this study was isolated from a rainwater pot hole near Bhavnagar (21.76°N, 72.15°E), Gujarat, India and identified as Scenedesmus sp. based on the following morphological characteristics (Philipose, 1967). Coenobia of 2–4 linear to slightly alternately arranged cells; cells 2.4–5 lm wide and 6–7 lm long; ellipsoid to broadly ovoid, with apices broadly rounded, marginal cells bearing a single large spine on each apex of coenobium, with 1–3 additional spines on outermost side of marginal cells which are shorter than main spines; Chloroplast parietal, almost covering entire wall, with a single prominent pyrenoid. It was cultivated in BG-11 containing (g/L) NaNO3, 1.5; K2HPO4, 0.03; MgSO47H2O, 0.075; CaCl22H2O, 0.036; citric acid, 0.006; ferric ammonium citrate, 0.006; EDTA, 0.001; Na2CO3, 0.020 and 1 ml of micronutrient solution containing (g/L) H3BO3, 2.86; MnCl24H2O, 1.81; ZnSO47H2O, 0.222; NaMoO45H2O, 0.390; CuSO45H2O, 0.0790; Co(NO3)26H2O, 0.0494 (Rippka et al., 1979). All the experiments were performed at 25 ± 2 °C with 12:12 h light dark period. The flasks were hand shaken twice daily (without sparging of air or CO2) to avoid adherence of the cells to the walls.

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For the nitrate limitation experiment, cells were grown with different initial nitrate concentrations of 247, 123.5, 61.75, 30.87, 15.43, and 0 mg/L. For the second sequential nitrate starvation experiment, cells were first grown in full nitrate concentration i.e., 247 mg/L. The cells were harvested after 6, 9 and 12 days of inoculation by centrifugation at 10,000 rpm for 10 min, washed twice with distilled water and re-inoculated in nitrate free medium. All the experiments were carried out in triplicates in 1 L conical flask containing 500 ml of culture medium inoculated with 10% of 10 day old culture. 2.2. Analytical procedures 2.2.1. Microscopy Light and fluorescence microscopy was carried out using a Carl Zeiss Axio A1 Imager equipped with Epi-fluorescence TRITC filter. For observing lipid body in algal cells, 1 ml of the culture was harvested by centrifugation at 10,000 rpm for 10 min and washed twice with normal saline. The cells were mixed with saline solution (0.5 ml) and 20 ll of Nile Red stain (0.1 mg/ml in acetone) was added to the cell suspension (1:100 v/v) and incubated for 10 min at room temperature. The cells were repeatedly washed to remove the excess stain and observed under fluorescent microscope using blue light as the excitation wavelength. 2.2.2. Determination of growth and biomass productivity Microalgal growth was monitored every 3rd day by measuring optical density at 750 nm. A calibration curve of OD750 vs. cell density was constructed from samples that ranged from OD750 0.2 to 2.0. Cell density was calculated using the equation: Cell density (mg/L) = 631.21  OD750 (R2 = 96.3). The biomass productivity (mg/L/day) was calculated according to equation P = (X2  X1) / (t2  t1), where X2 and X1 are the dry cell weight concentration (mg/L) at time t2 and t1, respectively. 2.2.3. Determination of pigments, lipids, carbohydrate and protein For determination of total pigment content, 2 ml of culture was taken in an eppendorf tube and centrifuged at 10,000 rpm for 5 min; the supernatant was discarded and 2 ml of 99.9% methanol was added to the pellet, mixed well and incubated at 45 °C for 24 h in dark. The total pigment content was calculated according to following equations: Chlorophyll a; Chl-a (lg/ml) = 16.72 A665.2 – 9.16 A652.4 Chlorophyll b; Chl-b (lg/ml) = 34.09 A652.4 – 15.28 A665.2 Carotenoids = (1000 A470  1.63 Chl-a  104.9 Chl-b)/221 (Lichtenthaler, 1987). Absorbencies at 470, 652.4 and 665.2 nm were corrected for turbidity by subtracting absorbance at 750 nm. For lipid extraction, cells were harvested by centrifugation at 10,000 rpm for 10 min, washed twice with distilled water and then dried in oven at 60 °C. Lipid was extracted from the dry biomass (at least 100 mg) by using Bligh and Dyer’s method (Bligh and Dyer, 1959). In detail, approximately 200 mg of biomass was vortexed thoroughly for 30 s, ultrasonicated for 30 min at ambient temperature in 30 ml chloroform:methanol (1:2, v/v), centrifuged (5 min at 3000 g) thrice and measured gravimetrically. Total lipid was further fractionated into neutral, glyco and phospholipids by method of Damiani et al. (2010) using 15 ml chloroform/acetic acid (9:1, v/ v) to collect neutral lipid, 20 ml acetone/methanol (9:1, v/v) to collect glyco lipids and 20 ml methanol to collect phospholipids. The content of neutral lipid, glycolipids and phospholipids were determined gravimetrically after drying at 60 °C. For carbohydrate determination, 100 mg lipid extracted biomass was mixed with 10 ml of 2% H2SO4 solution and hydrolyzed

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at 121 °C for 20 min. After hydrolysis, the mixture was neutralized with CaCO3 and diluted up to 100 ml with distilled water. This solution was centrifuged at 10,000 rpm for 5 min and the supernatant was used to determine total sugar content by Anthrone method (Yemm and Willis, 1954). For the estimation of crude protein, the total nitrogen content of microalgae was measured using a CHNS elemental analyzer (Perkin-Elmer Model 2 400, USA) calibrated using acetanilide as a reference standard. The crude protein concentration of the microalgae was estimated according to the following equation: ‘‘protein concentration = nitrogen content  6.25’’ (Becker, 1994). 2.2.4. Determination of proline content Proline was extracted with 3% sulphosalicylic acid and estimated by the method of Bates et al. (1973) using L-proline as a standard. 2.2.5. Residual nitrate estimation Nitrate concentration was determined using phenol disulphonic acid (PDA) method (Taras, 1950). The algal culture was filtered through a glass fiber filter paper and the supernatant was evaporated to dryness. After addition of 2 ml of PDA reagent, the system mixture was diluted with water and 10 ml of concentrated ammonium hydroxide was added. The system was diluted into a volumetric flask and color development was read at 410 nm. Nitrate concentration was determined from the standard curve prepared from standard nitrate solution ð5—500 mg=L N-NO 3 Þ. 2.2.6. Statistical analysis Each culture condition was analyzed on three biological replicates. The reported values are the means ± standard deviation of three values. Data were analyzed using one-way analysis of variance (ANOVA) using Microsoft Office Excel 2007 (Microsoft, USA). A significant difference was considered at the level of p < 0.05. 3. Results and discussion 3.1. Effect of nitrogen limitation and starvation on dry cell weight (DCW) and biomass productivity Microalgal biomass have been considered as a potential feedstock for liquid biofuels like biodiesel and bio-ethanol. A major technical bottleneck in commercial production is its low productivity. The biomass, lipid and carbohydrate productivity of microalgae is increased by changing the growth medium and cultivation conditions. Nitrogen is one of the most important elements for microalgae since it is a major component in many biological macromolecules like protein, chlorophyll, DNA etc. Nitrogen limitation or starvation leads to decrease in growth rate, protein synthesis, photosynthesis, cell size and increase in lipid and carbohydrate content (Simionato et al., 2013; Li et al., 2012). However, for economic production of lipid and carbohydrate rich microalgal biomass, the optimal nitrogen concentration has to be determined. In the present study, in 1st approach, Scenedesmus sp. CCNM 1077 was cultivated in a range of nitrate concentrations starting from 247 to 0 mg/L in order to find out the optimal nitrate concentration for the production of higher biomass, lipid and carbohydrate. In 2nd approach, the culture was sequentially transferred into nitrate free medium after 6, 9 and 12 days of culturing to find out the effective nitrogen limitation period for maximum biomass and lipid productivity in freshwater microalgae Scenedesmus sp. CCNM 1077. Fig. 1a shows the effect of nitrate limitation on dry cell weight and biomass productivity of microalgae Scenedesmus sp. CCNM 1077. The biomass content significantly decreased with a decrease

in nitrate concentration from 247 to 0 mg/L. The highest amount of DCW was found in cells grown in 123.5 mg/L nitrate (602.59 ± 2.98 g/L), then after a decrease in nitrate concentration in medium resulted in a decrease in DCW, which indicates that nitrogen starvation slows down the metabolic activity and cell division in Scenedesmus sp. CCNM 1077. Similar types of results have also been observed in many microalgal species like Chlorella sp. (Illman et al., 2000), Botryococcus braunii (Dayananda et al., 2007) etc. The total removal of nitrate from the growth medium slowed down the growth rate and resulted in only 332.85 ± 2.62 g/L DCW, which is about 44.77% lower compared to medium containing 123.5 mg/L nitrate. It was also observed that Scenedesmus sp. CCNM 1077 was able to grow in medium containing 80% less nitrate (30.87 mg/L) as compared to BG-11, which ultimately reduces the cultivation cost. In order to evaluate the effect of sequential nitrogen starvation on biomass yield and biochemical composition of Scenedesmus sp. CCNM 1077, culture was sequentially transferred into nitrate free medium after 6, 9 and 12 days of inoculation and grown for 15 days. Fig. 1b shows that biomass concentration was significantly affected by sequential nitrogen starvation. The results also indicate that 3 day nitrogen starvation does not significantly affect DCW (426.61 ± 7.09 mg/L) as compared to the control (481.42 ± 5.7 mg/L). However, 6 and 9 days nitrate starved cells showed significant reduction in dry biomass yield. The present study revealed that the sequential removal of nitrate from growth medium should be an effective technique to maintain sufficient biomass for subsequent biofuels production. Biomass productivity was largely unaffected in nitrate concentrations ranging from 247 to 30.87 mg/L nitrate; thereafter it gradually decreased and only 8.48 mg/L/day biomass productivity was observed in nitrate free media from the first day of cultivation. In comparison to nitrate starvation experiment, sequential nitrate removal had a significant effect on biomass productivity i.e. as compared to control (32.01 mg/L/day), 20% and 30% reduction in biomass productivity was observed in cultures after 6 (25.39 mg/ L/day) and 9 days (22.80 mg/L/day) nitrate starvation. The nitrate consumption trend for Scenedesmus sp. CCNM 1077 is presented in Fig. 2. As per our earlier observations, Scenedesmus sp. CCNM 1077, grown in BG-11 medium having 247 mg/L initial nitrate concentration, consumes only 60–70 mg/L (28% of total nitrate concentration) of nitrate throughout its 15 days of batch culture and the rest of it is still left in the medium. Therefore, in order to determine the effective nitrate concentration for algal growth, nitrogen limitation experiment was designed. Cells were grown with initial nitrate concentrations of 247, 123.5, 61.75, 30.87, 15.43 and 0 mg/L, respectively. Samples were harvested on 3, 6, 9, 12 and 15 days of culturing to check the nitrate consumption for each experimental nitrate concentration set. At the end of 15-days of cultivation, a certain amount of nitrate was still left in the medium, irrespective of the initial nitrate concentration (Fig. 2). This suggests that apart from the initial nitrate concentration certain other factors may limit microalgal growth (e.g., phosphate, temp, light intensity etc.). 3.2. Morphological changes during nitrogen limitation and starvation Scenedesmus is a pleomorphic strain which changes its morphology to produce unicells and coenobia under various environmental conditions (e.g., length of photoperiod, pH, nutrients etc.) and in response to predators. Several factors may influence the ecomorph expression in Scenedesmus species. Šetlík et al. (1972) also observed that the amount of energy stored in Scenedesmus is directly proportional to the number of cells in a colony. In the present study, it was observed that nitrate limitation as well as sequential nitrate removal triggers morphological changes in Scenedesmus sp. CCNM 1077. It was also observed that nitrate limited cell

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Fig. 1. (a) Effect of different nitrate concentration on DCW and biomass productivity of Scenedesmus sp. CCNM 1077. (b) Effect of sequential nitrate removal on DCW and biomass productivity of Scenedesmus sp. CCNM 1077.

Fig. 2. Nitrate consumption trend of Scenedesmus sp. CCNM 1077 in different initial nitrate concentration.

changes its morphology from unicell to 2 and 4 cell coeniobia with spines at terminal cells. The length and number of spines per cell was also found to increase with increase in nitrate limitation or

sequential nitrate removal from the medium. The present results are also in concordance with Gavis et al. (1979) in which he has reported similar types of 4 and 8 celled coenobia formation under

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nitrate limited conditions. Apart from this, a change in cell size was also observed. When cultured in nitrate starved condition, the normal cell size (4.5 lm) was found to increase to around 5.3 lm. The cell width decreased from 3.36 to 2.44 lm. Thus, one of the possible explanations of why most of the lab grown Scenedesmus are unicellular is due to high concentration of nitrate and phosphate in growth medium, which may be limited in natural conditions like oligotrophic lakes or other environmental conditions. 3.3. Effect of nitrogen limitation and starvation on pigment composition Nitrogen starvation triggers many metabolic responses in microalgae like degradation of nitrogenous compounds like protein, chlorophyll, DNA etc. and accumulation of energy rich compounds like lipids and carbohydrates. Changes in photosynthetic pigment content were measured to study the effects of nitrogen starvation on photosynthesis. Nitrogen limitation and sequential nitrogen starvation leads to decrease in all photosynthetic pigments viz. Chl-a, Chl-b and total carotenoid content, which was also observed by change in the color of culture from green to yellow and light yellow in 9 days of nitrate starved condition as well as in completely nitrate free culture medium (Supporting information Fig. S1). As shown in Table 1, a decrease in nitrogen concentration from 123 to 0 mg/L strongly decreased the content of Chl-a and b as well as carotenoids (p < 0.05). Reduction in nitrate in cultivation medium from 247 to 0 mg/L has reduced the total chlorophyll by about 75.43% (p < 0.05). A similar type of pattern was also found with carotenoid content. However, the ratio of Chl a/b and Caro/total Chl was found to increase in nitrogen limited or starved cultures, thus indicating the decrease in light harvesting complex and PS II activity; nevertheless, the microalgae maintains a balance between all pigments for efficient utilization of carbon and energy. It has been suggested that higher accumulation of carotenoids and increase in Chl a/b, Caro/total Chl ratio under nitrogen starvation serves as a protective function against oxidative stress induced lipid production in microalgae (Zhang et al., 2013). The result indicated that as chlorophyll is a nitrogenous compound, its content and composition are greatly influenced by nitrate concentration in the growth medium. Sequential nitrogen starvation also has similar pigment profile and therefore has similar photosynthetic ability to nitrogen limitation strategy (Table 2). 3.4. Effect of nitrogen limitation and starvation on biochemical composition Effect of nitrogen limitation on lipid, crude protein and carbohydrate content is represented in Fig. 3a. As nitrogen is one of the most important components for protein synthesis, decreasing the nitrogen concentration or sequential removal of nitrogen from growth medium has significantly decreased the crude protein content in Scenedesmus sp. CCNM 1077 (p < 0.05). Re-addition of nitrate in growth medium has been reported to be a slow enhancer

of protein content in microalgae (Giordano et al., 2001). But, in certain microalgae like Chlamydomonas reinhardtii and Scenedesmsus subspicatus, nitrogen limitation results in only 15–18% reduction in total protein content (Dean et al., 2010). Present results show that decreasing the nitrate concentration in the growth medium from 247 to 0 mg/L resulted in a decrease in crude protein content from 47.75% to 16.87%, which was about 60% less than the nitrogen rich culture. It was also found that sequential removal of nitrate from the growth medium after 3, 6 and 9 days also resulted in decrease in crude protein content of about 27%, 38% and 46%, respectively. A possible explanation towards decrease in protein content in both the cultivation strategy is that the cells might have degraded the nitrogenous compounds to maintain intracellular nitrogen quota for their normal metabolic function. In microalgae during nutrient limitation conditions, photosynthetic carbon flow changes into different ways to channel metabolic energy into various energy rich compounds like carbohydrates and lipids and there is a competition between synthesis of such compounds (Siaut et al., 2011). Lipids and carbohydrates are the preferred storage products in various stress conditions because they are hydrophobic in nature, have highly reduced states, are efficiently packed in small compartment of cells and can also be used during adverse conditions for cell survival and proliferation (Courchesne et al., 2009). Fig. 3a shows that decrease in nitrate concentration in growth medium significantly enhanced the lipid and carbohydrate content in Scenedesmus sp. CCNM 1077. The total lipid content increased from 18.87% to 27.93% after 15 days of cultivation in nitrogen rich and nitrogen deficient conditions, respectively. The results also indicate that a decrease in nitrate concentration in growth medium has a positive co-relation with the cellular lipid and carbohydrate content. A similar type of trend in lipid accumulation was also observed in Chlorella minutissima (Ördög et al., 2012). Microalgal total lipid is composed of neutral, glyco and phospholipids. Neutral lipids (NL) are mainly composed of TAGs, phospholipids (PL) are mainly composed of phosphatidyl glycerols (PG), phosphatidyl ethanolamines (PE), phosphatidylcholines (PC) whereas glycolipids (GL) contains mono-galatosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DG DG). PL and GL are important components of external membrane and membrane of chloroplast and the endoplasmic reticulum. Increase in neutral lipid and decrease in cellular protein content in microalgae are one of the most obvious effects of nitrogen starvation in various microalgae. These strategies are mainly used to generate biodiesel feed stock from various oleaginous microalgae and diatoms like Chlorella vulgaris, Scenedesmus sp., Nannochloropsis sp., Cylindrotheca fusiformis, Phaeodactylum tricornutum etc. (Griffiths et al., 2012). Therefore, detailed information about microalgal lipid class in nitrogen starvation or other stress conditions is an important criterion for selection of microalgae for their further use in production of biodiesel or for their nutraceutical applications (Olmstead et al., 2013). To find out the effects of nitrogen starvation on lipid class, total lipid was further fractionated into neutral, glycol- and

Table 1 Effect of various initial nitrate concentration on pigments composition of Scenedesmus sp. CCNM 1077.

a b c

Treatments (mg/L)

Chl-a (lg/ml)a

Chl-b (lg/ml)b

Chl a+b (lg/ml)

Caro (lg/ml)c

Chl a/b

Caro/Chl a+b

247 123.5 61.75 30.87 15.43 0

7.03 ± 0.40 7.13 ± 0.21 6.69 ± 0.52 5.67 ± 0.19 5.87 ± 0.11 1.89 ± 0.08

4.20 ± 0.25 4.48 ± 0.05 4.36 ± 0.11 3.41 ± 0.15 3.52 ± 0.26 0.87 ± 0.08

11.23 ± 0.64 11.61 ± 0.26 11.05 ± 0.07 9.08 ± 0.34 9.39 ± 0.37 2.76 ± 0.07

1.54 ± 0.07 1.65 ± 0.06 1.44 ± 0.29 1.40 ± 0.05 1.42 ± 0.06 0.64 ± 0.01

1.67 ± 0.03 1.59 ± 0.03 1.54 ± 0.14 1.66 ± 0.01 1.67 ± 0.09 2.19 ± 0.28

0.14 ± 0.0 0.14 ± 0.0 0.13 ± 0.02 0.15 ± 0.0 0.15 ± 0.01 0.23 ± 0.05

Chl-a: Chlorophyll-a. Chl-b: Chlorophyll-b. Caro: Carotenoids.

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I. Pancha et al. / Bioresource Technology 156 (2014) 146–154 Table 2 Effect of sequential nitrate starvation on pigments composition of Scenedesmus sp. CCNM 1077.

a b c

Treatments

Chl-a (lg/ml)a

Chl-b (lg/ml)b

Chl a+b (lg/ml)

Caro (lg/ml)c

Chl a/b

Caro/Chl a+b

Control 3 Day N6 Day N9 Day N-

5.43 ± 0.39 4.23 ± 0.25 3.42 ± 0.12 1.95 ± 0.04

2.28 ± 0.37 1.74 ± 0.20 1.35 ± 0.11 0.74 ± 0.01

7.71 ± 0.73 5.97 ± 0.40 4.77 ± 0.21 2.69 ± 0.04

1.53 ± 0.02 1.18 ± 0.09 0.97 ± 0.02 0.61 ± 0.01

2.35 ± 0.30 2.45 ± 0.22 2.55 ± 0.15 2.66 ± 0.08

0.19 ± 0.02 0.20 ± 0.01 0.20 ± 0.01 0.23 ± 0.01

Chl-a: Chlorophyll-a. Chl-b: Chlorophyll-b. Caro: Carotenoids.

Fig. 3. (a) Effect of nitrate concentration on biochemical composition of Scenedesmus sp. CCNM 1077. (b) Effect of sequential nitrate removal on biochemical composition of Scenedesmus sp. CCNM 1077.

phospholipids by column chromatography. Scenedesmus sp. CCNM 1077 cells accumulate 86.36% NL (Supporting information Table S1) in total nitrate starved condition, which was 1.27 fold higher than the NL content of cells grown in medium containing 247 mg/L nitrate. Neutral lipid accumulation was also confirmed through fluorescence microscopy; increased neutral lipid accumulation (yellow fluorescence) was observed with decreasing the nitrate concentration (Supporting information Fig. S1). Decreasing the nitrate concentration in the medium significantly decreased the polar GL and PL contents which are major components of cell and intracellular organ membranes; GL content decreases from 16.21% to 4.54% and PL decreases from 16.21% to 9.09% in nitrate

rich and nitrogen starved condition, respectively. In sequential nitrate starvation approach, there was no significant difference in total lipid of control and 3 day starved cells was observed; however, decrease in total lipid from 20.27% to 17.17% in 9 days starved cells was observed. A drastic change in the lipid class composition was observed in sequential starvation of Scenedesmus sp. CCNM 1077. For 3, 6 and 9 days nitrate starved cultures, an increase in NL content (81.81%, 76.47% and 84.84%, respectively) was observed, which indicates that 3 day nitrogen starved cultures have a similar amount of NL with 9 day starved culture with significantly higher DCW. Thus, 3 day nitrogen starvation is a better approach to enhance biomass and neutral lipid content in

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Fig. 4. (a) Effect of nitrate concentration on proline content of Scenedesmus sp. CCNM 1077. (b) Effect of sequential nitrate removal on proline content of Scenedesmus sp. CCNM 1077.

Scenedesmus sp. CCNM 1077, which can be effectively utilized for biodiesel production. The present study also indicates that changes in initial nitrate concentration had negligible effect on the neutral lipid composition in microalgae as compared to sequential nitrate removal approach. Thus, it is proposed that sequential nitrogen limitation is a suitable approach to enhance the neutral lipid content in microalgae. In sequential nitrogen starvation, the GL content was decreased from 16.21% in control to 11.76% and 9% in 6 and 9 days of nitrogen starvation condition, respectively. Similarly, PL content was also decreased from 16.21% in control to 6% in 9 days nitrate starved culture. It is well documented that accumulation of lipid in microalgae in nitrogen starved condition is due to increase in NL content of total lipid. Increase in NL is mainly due to the accumulation of TAGs, which are synthesized by two pathways; one from the glycerol pathway with the help of acyl-CoA-dependent diacylglycerol acyltransferase enzyme, which catalyzes acetyl Co-A to manolyl CoA, which is subsequently used for fatty acid synthesis. Another pathway is acyl CoA-independent pathway mainly found in higher plants and yeast (Xiao et al., 2013). The decrease in PL content in both the approaches indicates that Scenedesmus sp. CCNM 1077 contain phospholipid: diacylglycerol acyltransferase (PDAT) which converts PL into the TAGs.

Recently, it has been known that microalgal carbohydrates are a potential resource for bio-ethanol production because they mainly contain cellulose in their cell wall and its cytoplasm mainly contains starch. They can be easily converted into fermentable sugars for subsequent bio-ethanol production. In addition, microalgal carbohydrates do not contain lignin, which is mainly found in plants and seaweeds, so their hydrolysis is easy and does not require harsh pretreatment. Furthermore, microalgae mainly contain hexose sugars, thus dispensing with the need of pentose fermentation pathway which economizes the process (Yeh and Chang, 2011; Markou et al., 2012). From Fig. 3a and b, it can be observed that nitrate in cultivation medium significantly affects the carbohydrate production and its biosynthesis is dominant over lipid production in nitrogen starvation or limitation conditions. Hence, we can infer that excess metabolic carbon is first synthesized into starch followed by TAG accumulation. Similar results are also found in C. reinhardtii when cultivated in various nutrient limitation conditions showing the dominance of starch biosynthesis over oil accumulation. Consequently, higher rates of TAG synthesis occur only when carbon supply exceeds the capacity of starch synthesis (Fan et al., 2012). Present results show that decrease in nitrate concentration from 247 to 30.87 mg/L resulted in 1.39 fold

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increase in total carbohydrate content while it did not affect the biomass and lipid content of microalgae. Thus, 30.87 mg/L of nitrate in growth medium is best for generation of both lipid and carbohydrate rich microalgal biomass for subsequent biodiesel and bio-ethanol production. However, total removal of nitrate from the growth medium enhances carbohydrate content from 18% to 45.74%. In sequential nitrate removal approach, no significant difference in carbohydrate content was observed in control and 3 days starved cultures; however, 6 and 9 day starved cultures subsequently enhanced carbohydrate content from 16.96% in control to 24.38% and 28.19% in 6 and 9 day starved culture, respectively. 3.5. Effect of nitrogen limitation and starvation on proline content Proline is a known osmolyte for maintaining osmo-regulation in plant and algae under high salinity and other abiotic stresses. To determine the effects of nitrogen limitation on osmolyte production, the concentration of proline in microalgae was measured. It was hypothesized that as lipid plays a role as an osmolyte, its content directly regulates the production of other osmolytes in the cell. Fig. 4a shows effect of nitrate limitation on proline content of Scenedesmus sp. CCNM 1077. It was observed that a decrease in nitrate concentration in growth medium decreases the proline content of the cell. About 10 fold decrease in proline content was observed in nitrate starved medium compared to medium having 123 mg/L nitrate. In sequential nitrate removal approach a similar type of trend was observed (Fig. 4b). About 2.22 and 2.61 fold decrease in proline content was observed in 3 and 9 days nitrate starved cultures, respectively, which indicates that lipid can also act as an osmolyte during nitrate limitation or starvation condition. A similar trend of decreased proline content under the nitrogen deprivated condition was also observed in marine microalgae Nannochloropsis oceanic IMET1 (Xiao et al., 2013) and diatom Thalassiosira pseudonana (Bromke et al., 2013).

4. Conclusion In summary, the results of the present study show that freshwater microalgae Scenedesmus sp. CCNM 1077 is able to grow in the face of an 80% reduction in initial nitrate concentration as well as withstand nitrogen limitation up to 9 days. Sequential nitrate limitation for 3 days was the best approach to enhance biomass, neutral lipid and carbohydrate accumulation in Scenedesmus sp. CCNM 1077. Our findings therefore have importance not only for understanding of the regulation of carbon portioning in carbohydrates and lipid biosynthesis, but also for biochemical strategies to develop microalgae-based integrated biodiesel and bio-ethanol production systems. Acknowledgements IM, T.G., C.P. and R.M. would like to acknowledge CSIR for awarding Senior Research Fellowship. The authors gratefully acknowledge CSIR-MoES-NMITLI for providing the financial support. The authors would also like to thank Dr. P.K. Ghosh, Director, CSIR-CSMCRI, for his constant encouragement. The continuous support from Dr. Arvind Kumar, DC, SMC and the entire staff of the division is gratefully acknowledged. The authors would like to thank Dr. Parimal Paul, DC, ADCIF, CSIR-CSMCRI, Bhavnagar for their help during the analysis and Dr. Arup Ghosh for his help in scrutinizing the statistical analysis. I.P., K.C., T.G., C.P. and R.M. wish to acknowledge AcSIR for Ph.D. enrollment.

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014.01. 025. References Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water–stress studies. Plant Soil 39, 205–207. Becker, E.W., 1994. Microalgae: Biotechnology and Microbiology. Cambridge University Press, Cambridge. Bligh, E., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Borowitzka, M.A., 1992. Algal biotechnology products and processes matching science and economics. J. Appl. Phycol. 4, 267–279. Bromke, M.A., Giavalisco, P., Willmitzer, L., Hesse, H., 2013. Metabolic analysis of adaptation to short-term changes in culture conditions of the marine diatom Thalassiosira pseudonana. PLoS One 8, e67340. Chen, M., Tang, H., Ma, H., Holland, T.C., Ng, K.Y., Salley, S.O., 2011. Effect of nutrients on growth and lipid accumulation in the green algae Dunaliella tertiolecta. Bioresour. Technol. 102, 1649–1655. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306. Courchesne, N.M.D., Parisien, A., Wang, B., Lan, C.Q., 2009. Enhancement of lipid production using biochemical, genetic and transcription factor engineering approaches. J. Biotechnol. 141, 31–41. Damiani, M.C., Popovich, C.A., Constenla, D., Leonardi, P.I., 2010. Lipid analysis in Haematococcus pluvialis to assess its potential use as a biodiesel feedstock. Bioresour. Technol. 101, 3801–3807. Dayananda, C., Sarada, R., Usha Rani, M., Shamala, T.R., Ravishankar, G.A., 2007. Autotrophic cultivation of Botryococcus braunii for the production of hydrocarbons and exopolysaccharides in various media. Biomass Bioenergy 31, 87–93. Dean, A.P., Sigee, D.C., Estrada, B., Pittman, J.K., 2010. Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae. Bioresour. Technol. 101, 4499–4507. Fan, J., Yan, C., Andre, C., Shanklin, J., Schwender, J., Xu, C., 2012. Oil accumulation is controlled by carbon precursor supply for fatty acid synthesis in Chlamydomonas reinhardtii. Plant Cell Physiol. 53, 1380–1390. Gavis, J., Chamberlin, C., Lystad, L.D., 1979. Coenobial cell number in Scenedesmus quadricauda (chlorophyceae) as a function of growth rate in nitrate-limited chemostats. J. Phycol. 15, 273–275. Giordano, M., Kansiz, M., Heraud, P., Beardall, J., Wood, B., McNaughton, D., 2001. Fourier transform infrared spectroscopy as a novel tool to investigate changes in intracellular macromolecular pools in the marine microalga Chaetoceros muellerii (Bacillariophyceae). J. Phycol. 37, 271–279. Griffiths, M.J., van Hille, R.P., Harrison, S.T., 2012. Lipid productivity, settling potential and fatty acid profile of 11 microalgal species grown under nitrogen replete and limited conditions. J. Appl. Phycol. 24, 989–1001. Illman, A.M., Scragg, A.H., Shales, S.W., 2000. Increase in Chlorella strains calorific values when grown in low nitrogen medium. Enzyme Microb. Technol. 27, 631–635. John, R.P., Anisha, G.S., Nampoothiri, K.M., Pandey, A., 2011. Micro and macroalgal biomass: a renewable source for bioethanol. Bioresour. Technol. 102, 186–193. Li, Y., Fei, X., Deng, X., 2012. Novel molecular insights into nitrogen starvationinduced triacylglycerols accumulation revealed by differential gene expression analysis in green algae Micractinium pusillum. Biomass Bioenergy 42, 199–211. Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol. 148, 350–382. Lürling, M., 1998. Effect of grazing-associated infochemicals on growth and morphological development in Scenedesmus acutus (chlorophyceae). J. Phycol. 34, 578–586. Markou, G., Angelidaki, I., Georgakakis, D., 2012. Microalgal carbohydrates: an overview of the factors influencing carbohydrates production, and of main bioconversion technologies for production of biofuels. Appl. Microbiol. Biotechnol. 96, 631–645. Mata, T.M., Melo, A.C., Simões, M., Caetano, N.S., 2012. Parametric study of a brewery effluent treatment by microalgae Scenedesmus obliquus. Bioresour. Technol. 107, 151–158. Olmstead, I.L., Hill, D.R., Dias, D.A., Jayasinghe, N.S., Callahan, D.L., Kentish, S.E., Martin, G.J., 2013. A quantitative analysis of microalgal lipids for optimization of biodiesel and omega-3 production. Biotechnol. Bioeng. 110, 2096–2104. Ördög, V., Stirk, W.A., Bálint, P., van Staden, J., Lovász, C., 2012. Changes in lipid, protein and pigment concentrations in nitrogen-stressed Chlorella minutissima cultures. J. Appl. Phycol. 24, 907–914. Philipose, M.T., 1967. Chlorococcales, eighth ed. Indian Council of Agricultural Research, New Delhi. Procházková, G., Brányiková, I., Zachleder, V., Brányik, T., 2013. Effect of nutrient supply status on biomass composition of eukaryotic green microalgae. J. Appl. Phycol.. http://dx.doi.org/10.1007/s10811-013-0154-9. Rippka, R., Derulles, J., Waterburt, J.B., Herdman, M., Stanier, R.Y., 1979. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111, 1–61.

154

I. Pancha et al. / Bioresource Technology 156 (2014) 146–154

Šetlík, I., Berková, E., Doucha, J., Kubín, Š., Vendlová, J., Zachleder, V., 1972. The coupling of synthetic and reproduction processes in Scenedesmus quadricauda. Arch. Hydrobiol. Algolog. Stud. 7, 172–217. Siaut, M., Cuine, S., Cagnon, C., Fessler, B., Nguyen, M., Carrier, P., Beyly, A., Beisson, F., Triantaphylides, C., Li-Beisson, Y.H., Peltier, G., 2011. Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves. BMC Biotechnol. 11, 7. Simionato, D., Block, M.A., La Rocca, N., Jouhet, J., Maréchal, E., Finazzi, G., Morosinotto, T., 2013. The response of Nannochloropsis gaditana to nitrogen starvation includes de novo biosynthesis of triacylglycerols, a decrease of chloroplast galactolipids, and reorganization of the photosynthetic apparatus. Eukaryot. Cell 12, 665–676. Taras, M.J., 1950. Phenoldisulfonic acid method of determining nitrate in water. Photometric study. Anal. Chem. 22, 1020–1022.

Xiao, Y., Zhang, J., Cui, J., Feng, Y., Cui, Q., 2013. Metabolic profiles of Nannochloropsis oceanic IMET1 under nitrogen-deficiency stress. Bioresour. Technol. 130, 731– 738. Yeh, K.L., Chang, J.S., 2011. Nitrogen starvation strategies and photobioreactor design for enhancing lipid content and lipid production of a newly isolated microalga Chlorella vulgaris ESP-31: implications for biofuels. Biotechnol. J. 6, 1358–1366. Yemm, E.W., Willis, A.J., 1954. The estimation of carbohydrates in plant extracts by anthrone. Biochem. J. 57, 508–514. Zhang, Y.M., Chen, H., He, C.L., Wang, Q., 2013. Nitrogen starvation induced oxidative stress in an oil-producing green alga Chlorella sorokiniana C3. PLoS One 8, e69225.