Bioresource Technology 193 (2015) 315–323
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Bicarbonate supplementation enhanced biofuel production potential as well as nutritional stress mitigation in the microalgae Scenedesmus sp. CCNM 1077 Imran Pancha, Kaumeel Chokshi, Tonmoy Ghosh, Chetan Paliwal, Rahulkumar Maurya, Sandhya Mishra ⇑ 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 Bicarbonate additions enhance biomass and biochemical content of microalgae. 0.6 g/L was optimum bicarbonate concentration. Bicarbonate supplementation helps in nutritional stress ameroliation in microalgae. Nitrate starvation with bicarbonate addition produced highest carbohydrate and lipid. Scenedesmus sp. is a potential feedstock for biodiesel and bioethanol production.
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Article history: Received 25 May 2015 Received in revised form 18 June 2015 Accepted 19 June 2015 Available online 25 June 2015 Keywords: Microalgae Sodium bicarbonate Nutritional starvation Neutral lipid Carbohydrate
a b s t r a c t The aim of the present study was to find out the optimum sodium bicarbonate concentration to produce higher biomass with higher lipid and carbohydrate contents in microalgae Scenedesmus sp. CCNM 1077. The role of bicarbonate supplementation under different nutritional starvation conditions was also evaluated. The results clearly indicate that 0.6 g/L sodium bicarbonate was optimum concentration resulting in 20.91% total lipid and 25.56% carbohydrate along with 23% increase in biomass production compared to normal growth condition. Addition of sodium bicarbonate increased the activity of nutrient assimilatory enzymes, biomass, lipid and carbohydrate contents under different nutritional starvation conditions. Nitrogen starvation with bicarbonate supplementation resulted in 54.03% carbohydrate and 34.44% total lipid content in microalgae Scenedesmus sp. CCNM 1077. These findings show application of bicarbonate grown microalgae Scenedesmus sp. CCNM 1077 as a promising feedstock for biodiesel and bioethanol production. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Microalgae are considered as green bio-factories for the production of various bioactive compounds like pigments, fatty acids, vitamins etc. Apart from its nutraceutical importance, microalgae are recently considered as a promising renewable energy feed stock due to their high growth rate, photosynthetic ability, high lipid and carbohydrate contents as well as their ability to obtain nutrients from wastewaters (Chisti, 2007). To make microalgal based biofuel economically viable, downstream processing like ⇑ Corresponding author at: Discipline of Salt & Marine Chemicals, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India. Tel.: +91 278 256 5801/256 3805x6160; fax: +91 278 256 6970/256 7562. E-mail address: [email protected] (S. Mishra). http://dx.doi.org/10.1016/j.biortech.2015.06.107 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
harvesting, dewatering and biodiesel production must be optimized; at the same time, it is also important to improve biomass, lipid and carbohydrate productivity of microalgae to generate higher biomass with high lipid and carbohydrate contents (Wijffels and Barbosa, 2010). Cultural conditions like nutrients concentration and composition, light intensity and photoperiod, pH, temperature, etc. significantly affect the growth and biochemical composition of microalgae (George et al., 2014). Many cultivation strategies like nitrogen starvation, salinity stress and temperature stress significantly enhanced the lipid and carbohydrate contents in microalgae (Pancha et al., 2014; Pancha et al., 2015a; Chokshi et al., 2015). However, these stress conditions resulted in lower growth rate and biomass productivity, which ultimately increase the biofuel production cost. On the other hand, heterotrophic and mixotrophic
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growth utilizing various types of organic sources have been resulted in higher biomass along with higher lipid and carbohydrate contents in microalgae (Pancha et al., 2015b). However, all microalgae are unable to grow under such growth conditions; moreover, these conditions result in high initial operating cost due to utilization of costly organic substrates. Supply of inorganic carbon in the form of CO2 is important for active photosynthesis, carbon dioxide fixation and generation of biomass with desired products in microalgae. Many microalgae have the ability to uptake dissolved inorganic carbon (DIC) from the surrounding for active photosynthesis by CO2 concentrating mechanism (El-Ansari and Colman, 2015). In various microalgae addition of sodium bicarbonate to the culture medium resulted in high pH as well as high DIC in the growth medium which increased their growth and higher TAG accumulation (Gardner et al., 2012; White et al., 2013). However, the role of bicarbonate supplementation under various nutritional starvation conditions like nitrate and phosphate starvations as well as its involvement in active uptake of nutrients, lipid and carbohydrate accumulation in microalgae is still unclear. In the present study, freshwater microalgae Scenedesmus sp. CCNM 107 was first grown in the range (0–1.5 g/L) of sodium bicarbonate concentrations to find out the optimum concentration to obtain higher biomass, lipid and carbohydrate contents. Next, the combined effect of bicarbonate addition and nutritional starvation on morphological and biochemical parameters including cell morphology, dry cell weight, lipid, carbohydrate, protein, nutrients assimilatory enzymes and photosynthetic pigments were used to understand the physiological mechanism of bicarbonate supplementation on lipid and carbohydrate accumulation in the microalgae Scenedesmus sp. CCNM 1077. 2. Methods 2.1. Microalgae and experimental conditions The microalgae used in this study, Scenedesmus sp. CCNM 1077, was cultured in BG-11 medium as described previously (Pancha et al., 2014). For the determination of ideal bicarbonate concentration, microalgae Scenedesmus sp. CCNM 1077 was first grown in BG-11 medium with different concentrations of sodium bicarbonate i.e. 0, 0.3, 0.6, 0.9, 1.2 and 1.5 g/L. After obtaining best bicarbonate concentration, its effect on nitrate starvation (NP+), phosphate starvation (N+P) and combined nitrate phosphate starvation (NP) was evaluated under bicarbonate supplemented and non-supplemented conditions. All the experiments were conducted in triplicate in 1 L flasks containing 500 ml of respective culture medium and inoculated with 10% actively growing culture of Scenedesmus sp. CCNM 1077. All the experiments were performed in batch culture at 25 ± 2 °C with 12:12 h light dark period under 60 lmol m2 s1 light intensity. The flasks were manually shaken thrice a day. 2.2. Biomass and biochemical composition analysis 2.2.1. Determination of growth and biomass productivity Microalgal growth was monitored every 5th day by measuring optical density at 750 nm. 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 (DCW) (mg/L) at time t2 and t1, respectively. 2.2.2. Determination of pigments For the determination of pigments content, 2 ml microalgal culture was centrifuged at 10,000 rpm for 5 min, supernatant was
discarded and pellet was mixed with 99.9% methanol and incubated in dark for 24 h at 45 °C. After incubation, pigments content was determined using the following formulas:
Chlorophyll a; Chl-a ðl g=mlÞ ¼ 16:72 A665:2 9:16 A652:4 Chlorophyll b; Chl-b ðl g=mlÞ ¼ 34:09 A652:4 15:28 A665:2 Carotenoids ¼ ð1000 A470 1:63 Chl-a 104:9 Chl-bÞ=221 Absorbances at 470, 652.4 and 665.2 nm were corrected for turbidity by subtracting absorbance at 750 nm (Lichtenthaler, 1987). 2.2.3. Determination of lipid, protein and carbohydrate contents Lipid was extracted using chloroform:methanol (1:2 v/v) and determined gravimetrically (Bligh and Dyer, 1959). Total lipid was further fractionated into neutral lipid using chloroform/acetic acid (9:1, v/v), glycolipids using acetone/methanol (9:1, v/v) and phospholipids using methanol as described in Pancha et al. (2014). CHNS content in microalgal biomass was determined through elemental analysis using a CHNS analyzer (Perkin-Elmer Model 2 400, USA). Crude protein content of microalgae was calculated according to the following equation (Becker, 1994):
Crude protein ð%Þ ¼ N ð%Þ 6:25 For the determination of carbohydrate content in microalgae, 50 mg of lipid extracted biomass was first hydrolyzed with 500 ll of 72% (w/v) H2SO4 for 1 h. Concentration of H2SO4 was reduced to 4% by the addition of distilled water and autoclaved at 121 °C for 1 h. After cooling down to room temperature, total volume was made up to 50 ml with distilled water (Van Wychen and Laurens, 2013). The resulting solution was centrifuged at 10,000 rpm for 10 min and the supernatant was used for sugar estimation by phenol sulfuric acid method (Dubois et al., 1956) using D-glucose as a standard. 2.2.4. Determination of nutrients concentrations Residual nitrate concentration in the microalgal culture medium was estimated using salicylic acid and sodium hydroxide (Cataldo et al., 1975). Residual phosphate concentration in the microalgal culture medium was estimated using ascorbic method (Grasshoff et al., 1999). 2.2.5. Determination of nutrients assimilatory enzymes Nitrate reductase (NR) activity of microalgal cells was determined according to the method described by Li et al. (2011). Alkaline (ALP) and acid phosphates (AP) activities were determined according to Lee (2000). Total soluble protein content was measured using bovine serum albumin as a standard (Bradford, 1976). 2.3. Statistical analysis All results are expressed as mean values ± standard deviation. The statistical differences between experimental groups were assessed by analysis of variance (ANOVA) using the Info stat software package (2012). The mean values were compared with LSD test (p < 0.05). 3. Results and discussion 3.1. Effect of sodium bicarbonate supplementation on dry cell weight (DCW) and biomass productivity Generally, microalgae grow photoautotrophically utilizing atmospheric CO2 as an inorganic carbon source. Since CO2 has very
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low solubility in water, bicarbonate addition is carried out in open pond large scale microalgal cultivation to provide desired inorganic carbon to generate more biomass. Addition of sodium bicarbonate to the growth medium has been reported to significantly increase the biomass and biochemical composition of various microalgae (White et al., 2013). In the present study, addition of sodium bicarbonate to the growth medium significantly enhanced the DCW and biomass productivity of microalgae Scenedesmus sp. CCNM 1077 (Fig. 1a) which indicates that sodium bicarbonate supplementation enhances the cell division and metabolic process in microalgae. Addition of sodium bicarbonate (0–1.2 g/L) increased DCW and biomass productivity in a dose dependent manner and no significant difference (p < 0.05) in DCW production was observed beyond 0.6 g/L sodium bicarbonate concentration. Biomass productivity of Scenedesmus sp. CCNM 1077 was largely unaffected in the range of 0.6–1.5 g/L sodium bicarbonate supplemented growth medium. The highest biomass productivity was found in cultures grown with 1.2 g/L sodium bicarbonate (28.32 ± 0.77 mg/L/day) which was 1.4 folds higher than the only BG-11 grown culture (19.92 ± 0.26 mg/L/day). In a similar study, Peng et al. (2014) also found higher biomass production in diatom Phaeodactylum tricornutum cultivated in the presence of sodium bicarbonate. In the present study, nitrate and phosphate consumption trend was recorded to correlate the effect of sodium bicarbonate
supplementation on active uptake of nutrients by microalgae Scenedesmus sp. CCNM 1077 (Fig. 2). The results indicate that uptake of nitrate and phosphate from the growth medium increased with the increase in bicarbonate addition to the growth medium. Addition of only 0.3 g/L sodium bicarbonate to the growth medium resulted in 66 ± 1.87% phosphate uptake and 33 ± 1.71% nitrate uptake from the growth medium, which was higher than those of the control culture. Lee and Lee (2002) reported that high CO2 grown Chlorella kessleri culture had higher nitrate consumption possibly due to enhanced nitrate absorption efficacy of the cells. In the present study, highest consumption of nitrate (37.58 ± 2.39%) and phosphate (78.58 ± 0.56%) was found in cells cultivated under 0.9 g/L sodium bicarbonate. Highest DCW (549.15 ± 13.13 mg/L) production was found in cells cultivated with 1.2 g/L sodium bicarbonate. However, further increase in bicarbonate concentration to 1.5 g/L showed no significant difference in DCW production (547 ± 16.42 mg/L) but showed reduced nutrient consumptions (63.55 ± 0.13% phosphate and 35.12 ± 1.33% nitrate) by Scenedesmus sp. CCNM 1077. Lower nutrient consumption under high bicarbonate supplementation might be due to increase in pH of the growth medium since it has been known that many salts of the marine culture medium gets precipitated and become unavailable for consumption by microalgae (Guihéneuf and Stengel, 2013).
Fig. 1. (a) Effect of different NaHCO3 concentration on DCW and biomass productivity of Scenedesmus sp. CCNM 1077. (b) Effect of different NaHCO3 concentration on biochemical composition of Scenedesmus sp. CCNM 1077 (Values with different letters represent significant difference at p < 0.05 between treatments).
Fig. 2. (a) Nitrate consumption trend of Scenedesmus sp. CCNM 1077 in different initial NaHCO3 concentration. (b) Phosphate consumption trend of Scenedesmus sp. CCNM 1077 in different initial NaHCO3 concentration.
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3.2. Effect of sodium bicarbonate supplementation on biochemical composition Effect of initial sodium bicarbonate concentrations on lipid, crude protein and carbohydrate content of microalgae Scenedesmus sp. CCNM 1077 is shown in Fig. 1b. Crude protein content of microalgae Scenedesmus sp. CCNM 1077 increased in a dose dependent manner. Highest crude protein content (50.12 ± 0.23%) was observed in cells grown with 1.5 g/L sodium bicarbonate followed by 1.2 (49.46 ± 0.41%) and 0.9 g/L (49.56 ± 0.37%) sodium bicarbonate supplemented cultures. Carbon availability and supply is a key metabolic regulator which governs the carbohydrate and lipid synthesis in microalgae (Fan et al., 2012). Fig. 1b also shows that addition of 0.9 g/L sodium bicarbonate to the growth medium increased the carbohydrate content from 18.55 ± 0.79 to 30.88 ± 1.47%. The results also indicate a positive correlation between sodium bicarbonate supplementation and cellular carbohydrate content in microalgae Scenedesmus sp. CCNM 1077. It has been reported that high availability of inorganic carbon results in high activity of Ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) which is a key enzyme for the conversion of 3-phosphoglycerate, a substrate for the biosynthesis of carbohydrate and fatty acid in plants and microalgae (Peng et al., 2014). There was no significant difference (p < 0.05) in carbohydrate accumulation pattern in cells cultivated with 0.3 g/L (24.57 ± 0.72%) and 0.6 g/L (25.56 ± 0.84%) sodium bicarbonate. Many recent studies show that microalgal carbohydrates are better than plant and seaweed derived carbohydrates due to high starch content, which can be easily converted into hexose sugar. Moreover, microalgal carbohydrates do not contain lignin which significantly reduces the pretreatment cost for bio-ethanol production (Yeh and Chang, 2011; Markou et al., 2012; Harun and Danquah, 2011). In the present study, carbohydrate content was estimated from the left over de-oiled microalgal biomass making its application towards biodiesel and bioethanol coproduction which will ultimately reduce their production cost. Similar to carbohydrate, addition of sodium bicarbonate also enhanced the total lipid content in microalgae Scenedesmus sp. CCNM 1077 (Fig. 1b). There was no significant difference (p < 0.05) in total lipid content of cells cultivated with 0.6 g/L (20.91 ± 0.31%) and 0.9 g/L (20.89 ± 0.35%) sodium bicarbonate. Guihéneuf and Stengel (2013) showed that under supply of inorganic carbon in the form of sodium bicarbonate microalgae Pavlova lutheri produce high amount of LC-PUFA rich TAGs which are mainly generated from de novo synthesis rather than membrane remodeling. In the present study, no significant difference in lipid content was observed in 1.2 g/L (19.36 ± 0.23%) and 1.5 g/L (19.07 ± 0.29%) sodium bicarbonate supplemented cultures. In order to understand the role of sodium bicarbonate addition on lipid class composition, total lipid content of microalgae Scenedesmus sp. CCNM 1077 was fractionated into neutral lipid, glycolipids and phospholipids utilizing silica gel G column chromatography. Addition of sodium bicarbonate to the growth medium increased the NL content and reduced the GL and PL contents of microalgae Scenedesmus sp. CCNM 1077 (Fig. 3). Addition of 0.6 g/L sodium bicarbonate to the growth medium resulted in 75.8 ± 0.85% NL of total lipid, which was about 1.12 folds higher than that of the BG-11 grown culture (67.24 ± 0.55% of TL). Reduction in GL and PL contents indicate changes in cell membrane composition of microalgae Scenedesmus sp. CCNM 1077. Addition of bicarbonate to the culture medium enhances its pH and microalgae change their membrane composition to withstand this alkaline stress. GL content decreased from 16.4 ± 0.14% to 14.9 ± 0.1% in 1.2 g/L bicarbonate grown cultures; similarly, PL content also decreased from 15.6 ± 0.18% to 8.13 ± 0.28% in 0.9 g/L bicarbonate grown cultures. Results of this section show that sodium
bicarbonate at concentration of 0.6 g/L is optimum for higher biomass generation along with higher lipid and carbohydrate contents in microalgae Scenedesmus sp. CCNM 1077. 3.3. Effect of sodium bicarbonate supplementation on pigments composition Addition of sodium bicarbonate to the growth medium increased all the photosynthetic pigments viz. Chl-a, Chl-b and total carotenoids content of microalgae Scenedesmus sp. CCNM 1077 (Table 1). Increase in sodium bicarbonate concentration from 0 to 1.2 g/L significantly (p < 0.05) enhanced the Chl-a and b as well as total carotenoids contents. Highest amount of Chl-a (6.11 ± 1.14 lg/ml) was found in 0.9 g/L sodium bicarbonate grown culture, which was about 1.17 folds higher than the BG-11 grown culture (5.18 ± 0.32 lg/ml). Similar to our results, White et al. (2013) also observed 2.24 and 1.91 folds increase in Chl-a content in Tetraselmis suecica and Nannochloropsis salina, respectively, when cultivated under 1 g/L sodium bicarbonate supplementation compared to non bicarbonate supplemented cultures. Addition of sodium bicarbonate (up to 1.2 g/L) resulted in the increase in total carotenoids of the cells in dose dependent manner. Highest amount of total carotenoids (1.53 ± 0.10 lg/ml) was found in 1.2 g/L sodium bicarbonate supplemented culture followed by 0.9 g/L (1.45 ± 0.15 lg/ml) and 0.6 g/L (1.38 ± 0.09) sodium bicarbonate supplemented cultures. Addition of sodium bicarbonate (up to 1.2 g/L) to the growth medium increased the ratios of Chl a/b as well as Caro/total Chl. In this study, we supplemented bicarbonate to the growth medium that increase the culture alkalinity and pH, which might be the reason for higher ratio of Chl a/b, Caro/total Chl in microalgae Scenedesmus sp. CCNM 1077. In a similar study, no significant difference in pigments ratio was found when T. suecica was grown with supplementation of sodium bicarbonate, while in case of N. salina, carotenoids: Chl-a ratio increased with the increase in sodium bicarbonate in the growth medium (White et al., 2013). 3.4. Effect of sodium bicarbonate supplementation on DCW and biomass productivity under nutritional starvation conditions Microalgal lipid and carbohydrate contents generally increase in various nutritional stress conditions like nitrate and phosphate starvation. However, these nutritional starvation conditions result in lower DCW and biomass productivity in microalgae. In this study, addition of inorganic carbon in the form of sodium bicarbonate not only increased the biomass content of Scenedesmus sp. CCNM 1077 but also resulted in higher accumulation of neutral lipid and carbohydrate. To check the role of inorganic carbon supplementation under nutrition starvation condition, Scenedesmus sp. CCNM 1077 was grown under nitrate starvation (NP+), phosphate starvation (N+P) and combined nitrate and phosphate starvation (NP) with and without 0.6 g/L sodium bicarbonate supplementation. Fig. 4a shows effect of sodium bicarbonate addition on DCW and biomass productivity under nutrient starvation conditions. From the data it is clear that removal of either nitrate or phosphate or both from the growth medium significantly affects the metabolic process of microalgae Scenedesmus sp. CCNM 1077 and results in lower DCW and biomass productivity. However, addition of 0.6 g/L sodium bicarbonate reduced the stress effects with the increase in DCW and biomass production under all the tested nutritional stress conditions. Our earlier study with microalgae Scenedesmus sp. CCNM 1077 (Pancha et al., 2014) showed that removal of nitrate from the growth medium resulted in 46% reduction in DCW compared to control culture. However, in the present study, addition of 0.6 g/L bicarbonate helped in reducing the stress
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Fig. 3. Changes in neutral lipids (NL), glycolipids (GL) and phospholipids (PL) concentration of Scenedesmus sp. CCNM 1077 under different NaHCO3 concentration ((a) 0 g/L; (b) 0.3 g/L; (c) 0.6 g/L; (d) 0.9 g/L; (e) 1.2 g/L; (f) 1.5 g/L).
Table 1 Effect of different sodium bicarbonate concentration on pigments composition of Scenedesmus sp. CCNM 1077. NaHCO3 (g/L) 0 0.3 0.6 0.9 1.2 1.5
Chl-a* (lg/ml) a
5.18 ± 0.32 5.81 ± 0.23a 6.02 ± 0.48a 6.11 ± 1.14a 6.10 ± 0.16a 5.19 ± 1.05a
Chl-b** (lg/ml) b
3.29 ± 0.25 3.56 ± 0.22ab 3.51 ± 0.28ab 3.53 ± 0.79ab 3.67 ± 0.45ab 4.13 ± 0.59a
Chl a + b (lg/ml) a
8.47 ± 0.56 9.37 ± 0.44a 9.53 ± 0.76a 9.64 ± 1.94a 9.78 ± 0.61a 9.32 ± 1.05a
Caro*** (lg/ml)
Chl a/b
Caro/Chl a + b
1.02 ± 0.06d 1.25 ± 0.01bc 1.38 ± 0.09abc 1.45 ± 0.15ab 1.53 ± 0.10a 1.15 ± 0.27cd
1.58 ± 0.03a 1.63 ± 0.05a 1.72 ± 0.01a 1.74 ± 0.06a 1.67 ± 0.15a 1.28 ± 0.33b
0.12 ± 0.00c 0.13 ± 0.007abc 0.14 ± 0.00ab 0.15 ± 0.01a 0.16 ± 0.02a 0.12 ± 0.03bc
Different superscript letters within column indicates significant differences at p < 0.05. * Chl-a: chlorophyll-a. ** Chl-b: chlorophyll-b. *** Caro: carotenoids.
effects and 38% reduction in DCW was observed. Removal of both, nitrate and phosphate, from the growth medium resulted in 178 ± 1.67 mg/L DCW, while addition of 0.6 g/L bicarbonate augmented DCW production i.e. 221.13 ± 6.07 mg/L under combined nutrient starvation. Similar to our study, Chu et al. (2013) also found reduction in total DCW production by microalgae Chlorella vulgaris in nitrate (95.4 mg/L/day) and phosphate (38.3 mg/L/day) starved cultures compared to control culture (100.4 mg/L/day). In the present study, DCW production was not much affected under phosphate starvation and only 19% reduction in DCW (320.87 ± 11.68 mg/L) was observed. Biomass productivity was largely unaffected in phosphate starvation condition (15.74 ± 0.84 mg/L/day) as well as bicarbonate supplemented phosphate starvation conditions (18.08 ± 0.31 mg/L/day) compared to control culture (20.77 ± 0.28 mg/L/day). Removal of nitrate or nitrate and phosphate, both, from the growth medium resulted in very low biomass productivity i.e. 8.25 ± 0.21
and 6.48 ± 0.07 mg/L/day, respectively. The result of the present section shows that nitrate is a vital nutrient for normal growth and metabolism of microalgae Scenedesmus sp. CCNM 1077 and addition of bicarbonate has nutrient stress ameliorating effects which helps in improving its DCW and biomass productivity. Our earlier observations show that microalgae Scenedesmus sp. CCNM 1077 is a pleomorphic strain, which changes its morphology from single cell to coenobium of 2 and 4 under nitrate starvation condition (Pancha et al., 2014). In the present study too, nitrate or combined nitrate and phosphate starvations resulted in 4 cells coenobium with spines at terminal cells. However, size of 4 cells coenobium was smaller in combined stress compared to only nitrogen stressed culture. Addition of bicarbonate did not show any significant effect on cell morphology under starvation conditions. Compared to control culture, cells grown under phosphate starvation did not show any significant change in their morphology; however, addition of bicarbonate resulted in slightly larger
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Fig. 4. (a) Effect of sodium bicarbonate supplementation on DCW and biomass productivity under nutritional starvation conditions. (b) Effect of sodium bicarbonate supplementation on biochemical composition under nutritional starvation conditions (Values with different letters represent significant difference at p < 0.05 between treatments).
cell size. It has been reported that larger cell has slow metabolic rate which helps in saving energy from damage and repair as well as the amount of energy stored is directly propositional to cell size of microalgae (Šetlík et al., 1972).
3.5. Effect of sodium bicarbonate supplementation on nutrient consumption and nutrient assimilatory enzymes Fig. 5 shows the effect of sodium bicarbonate addition on nutrient consumption trend of microalgae Scenedesmus sp. CCNM 1077 under different nutritional starvation conditions. Addition of 0.6 g/L sodium bicarbonate resulted in 46.97 ± 0.67% phosphate consumption in nitrogen-starved culture, which was 38% higher than those of the cultures without sodium bicarbonate supplementations. More utilization of phosphate is also evident from higher activities of AP and ALP in bicarbonate grown cultures (Fig. 5c). In the present study, activity of AP was higher than ALP. Similarly, Kruskopf and Du Plessis (2004) reported higher activity of AP than ALP in Chlorella and Chlamydomonas under nitrate and phosphate starvation conditions. AP helps in liberation of intracellular P reserve whereas ALP helps to dissolve organic P from the growth medium. In the present study, addition of bicarbonate did not show any effect on AP and ALP activities in phosphate starved cultures. This suggests that presence of phosphate is important for the induction of both, AP and ALP, in microalgae Scenedesmus sp. CCNM 1077.
Fig. 5. (a) Nitrate consumption trend of Scenedesmus sp. CCNM 1077 with sodium bicarbonate supplementation under nutritional starvation conditions. (b) Phosphate consumption trend of Scenedesmus sp. CCNM 1077 with sodium bicarbonate supplementation under nutritional starvation conditions. (c) Changes in nutrient assimilatory enzymes of Scenedesmus sp. CCNM 1077 with sodium bicarbonate supplementation under nutritional starvation conditions (Values with different letters represent significant difference at p < 0.05 between treatments.) (Abbreviations: NP WTout; NP+ without NaHCO3, NP WT; NP+ with 0.6 g/L NaHCO3, NP WTout; N+P without NaHCO3, NP WT; N+P with 0.6 g/L NaHCO3, NP WTout; NP without NaHCO3, NPWT; NP 0.6 g/L NaHCO3).
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Rate of nitrate assimilation was also increased by addition of sodium bicarbonate to the growth medium. BG-11 grown culture consumed 30 ± 0.44% of nitrate while cultures grown under phosphate starvation with and without bicarbonate addition consumed 28 ± 1.4% and 29.39 ± 1.1% nitrate, respectively. Chu et al. (2014) showed significant reduction in nitrate assimilation under phosphate starvation by microalgae S. obliquus FACHB-417. Nitrate reductase activity was significantly (p < 0.05) affected in various stress conditions. Removal of both nitrate and phosphate from the growth medium did not show any significant difference in nitrate reductase activity. However, addition of sodium bicarbonate under phosphate starvation resulted in NR activity of 0.83 ± 0.0 lmol min1 mg1 protein which was almost double than that of the culture without bicarbonate supplementation (0.42 ± 0.01 lmol min1 mg1 protein). Culture under nitrate starvation with sodium bicarbonate supplementation resulted in NR activity of 0.21 ± 0.01 lmol min1 mg1 protein which was significantly lower than that of the culture without bicarbonate supplementation, but similar to that of the nitrate and phosphate starved cultures (0.20 ± 0.02 lmol min1 mg1 protein). 3.6. Effect of sodium bicarbonate supplementation on biochemical composition under nutritional starvation conditions Effect of bicarbonate addition on lipid, carbohydrate and protein contents of microalgae Scenedesmus sp. CCNM 1077 is shown in Fig. 4b. As protein is nitrogenous compound, removal of nitrogen from the growth medium significantly (p < 0.05) reduced the crude protein content from 47.19 ± 0.26% to 11.95 ± 0.14% in combined stress and to 10.63 ± 0.44% in nitrogen starvation condition. Addition of bicarbonate resulted in 12.93 ± 0.11% and 11.76 ± 0.25% crude protein content in combined and nitrogen starvation conditions, respectively. Similarly, Chu et al. (2014) also found sharp decrease in protein content in microalgae S. obliquus FACHB-417 during combine nitrate and phosphate starvation. The degradation of protein helps in maintaining the intracellular nitrogen pool in microalgae, thereby helping in normal functioning of cellular metabolism. Phosphate starvation did not show any significant effect on crude protein content of microalgae Scenedesmus sp. CCNM 1077 and resulted in 39.89 ± 0.22% crude protein. Lipid and carbohydrate are the two most reduced storage products in microalgae, generally accumulated under various nutritional stress conditions due to their hydrophobic nature, more reduced state and efficient storage in smaller compartments of the cells (Courchesne et al., 2009). Fig. 4b also shows effect of bicarbonate addition on carbohydrate content of microalgae Scenedesmus sp. CCNM 1077. As per our earlier observation, nutrient limitation results in significantly higher accumulation of carbohydrate and its synthesis is dominated over lipid production in microalgae Scenedesmus sp. CCNM 1077 (Pancha et al., 2014). Highest accumulation of carbohydrate (54.03 ± 1.08%) was found under nitrogen limitation with bicarbonate supplementation followed by combined nutrient starvation with bicarbonate supplementation (51.25 ± 0.67%). Total removal of phosphate from the growth medium resulted in 27.47 ± 0.49% carbohydrate which further increased up to 29.86 ± 0.26% during 0.6 g/L bicarbonate supplementation. These results indicate that addition of bicarbonate under various nutritional stress conditions significantly enhanced the carbohydrate content in microalgae Scenedesmus sp. CCNM 1077. Among all, phosphate starvation was found to be a good alternative as it resulted in comparatively lesser biomass loss with relatively higher lipid and carbohydrate contents in microalgae Scenedesmus sp. CCNM 1077. Nutrients limitations like nitrate, phosphate or combined nitrate and phosphate limitations have resulted in higher
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accumulation of lipid in various microalgae (Chu et al., 2013, 2014; Li et al., 2014). However, only few researchers have studied the effects of bicarbonate addition under nitrate limited growth conditions (White et al., 2013; Guihéneuf and Stengel, 2013) and there are no reports on effects of bicarbonate addition under phosphate or combined nitrate phosphate stress conditions in microalgae. Total lipid content of microalgae Scenedesmus sp. CCNM 1077 was increased from 17.85 ± 0.14% to 34.42 ± 0.27% under nitrogen starvation with bicarbonate addition, which was significantly higher than that of without bicarbonate addition (28.05 ± 0.08%). This indicates that inorganic carbon supply under nitrogen starvation helps in de novo synthesis of microalgal lipids. Similar to our study, Guihéneuf and Stengel (2013) also found high TAGs in bicarbonate supplemented nitrogen starved culture of marine haptophyte Pavlova lutheri. In the present study, Scenedesmus sp. CCNM 1077 grown under combined nutrition starvation with bicarbonate supplementation did not show any significant increase in total lipid content (33.29 ± 0.42%) compared to that of nitrate starved bicarbonate supplemented culture. Li et al. (2014) have reported higher lipid accumulation in Chlorella during nitrate phosphate co-deficiency compared to only nitrate starvation. In the present study, phosphate starvation with bicarbonate addition resulted in 27.97 ± 0.24% total lipid, which was similar to that of nitrogen starved culture. This treatment can be used as an efficient approach to produce microalgal based biofuel as it resulted in limited loss of biomass with higher lipid and carbohydrate contents, which can be converted into biodiesel and bioethanol, respectively. Sodium bicarbonate supplementation had significant effect on lipid class composition of microalgae Scenedesmus sp. CCNM 1077 (Fig. S1). Neutral lipid content of 88.69 ± 2.1% of total lipid was observed in nitrate starved bicarbonate supplemented cells which was 1.3 folds higher than that of the BG-11 grown culture (67.2 ± 0.55%). Nitrate and phosphate starvation significantly reduced the GL and PL content of microalgae Scenedesmus sp. CCNM 1077. NL is mainly composed of TAGs which are important for the production of microalgal based biodiesel, while GL and PL are the components of cell membranes (Olmstead et al., 2013). Addition of sodium bicarbonate under all tested nutritional starvation conditions resulted in higher NL content compared to that of the cultures grown without bicarbonate addition, indicating positive role of sodium bicarbonate in NL accumulation in microalgae Scenedesmus sp. CCNM 1077.
3.7. Effect of sodium bicarbonate supplementation on pigments under nutritional starvation conditions Pigments content and composition are most affected during various nutritional stress conditions like nitrogen and salinity stress since pigments are mainly composed of nitrogen rich compounds (Pancha et al., 2014). To gain insight into the photosynthetic process under various stress conditions, Chl-a, Chl-b and total carotenoids content were measured (Table 2). All the stress conditions resulted in lower Chl-a content, which was also evident from the color of cultures from green to yellow and light yellow in phosphate and nitrate combined stress conditions. As shown in Table 2, removal of nitrate from the culture medium resulted in 0.15 ± 0.02 lg/ml Chl-a. However, addition of sodium bicarbonate resulted in 0.58 ± 0.11 lg/ml Chl-a, which was about 3.8 folds higher than that of the cells without bicarbonate supplementation. Addition of sodium bicarbonate did not show much effect on pigment content under combined stress conditions. Pigments content of phosphate starved cultures were least affected i.e. 2.59 ± 0.19 and 3.13 ± 0.12 lg/ml Chl-a in cultures without and with bicarbonate supplementation, respectively.
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Table 2 Effect of sodium bicarbonate supplementation on pigments composition under nutritional starvation conditions. Treatments Control NP WTout NP WT NP WTout NP WT NP WTout NP WT
Chl-a* (lg/ml) a
5.26 ± 0.24 0.15 ± 0.02e 0.58 ± 0.11d 2.59 ± 0.19c 3.13 ± 0.12b 0.25 ± 0.06e 0.21 ± 0.02e
Chl-b** (lg/ml) a
3.31 ± 0.27 0.20 ± 0.11c 0.41 ± 0.12c 2.0 ± 0.15b 1.91 ± 0.04b 0.18 ± 0.02c 0.24 ± 0.04c
Chl a + b (lg/ml) a
8.57 ± 0.50 0.35 ± 0.12e 0.98 ± 0.23d 4.60 ± 0.29c 4.04 ± 0.16b 0.43 ± 0.07e 0.45 ± 0.04e
Caro*** (lg/ml) a
1.18 ± 0.03 0.02 ± 0.09e 0.23 ± 0.03d 0.48 ± 0.16c 0.78 ± 0.01b 0.11 ± 0.03de 0.12 ± 0.03de
Chl a/b
Caro/Chl a + b a
1.59 ± 0.06 0.94 ± 0.6b 1.44 ± 0.17a 1.30 ± 0.12ab 1.64 ± 0.03a 1.43 ± 0.3b 0.90 ± 0.15b
0.14 ± 0.01a 0.11 ± 0.36a 0.23 ± 0.03a 0.10 ± 0.03a 0.15 ± 0.01a 0.26 ± 0.04a 0.26 ± 0.06a
Different superscript letters within column indicates significant differences at p < 0.05. Abbreviations: NP WTout; NP+ without NaHCO3, NP WT; NP+ with 0.6 g/L NaHCO3, NP WTout; N+P without NaHCO3, NP WT; N+P with 0.6 g/L NaHCO3, NP WTout; NP without NaHCO3, NP WT; NP 0.6 g/L NaHCO3. * Chl-a: chlorophyll-a. ** Chl-b: chlorophyll-b. *** Caro: carotenoids.
4. Conclusion In summary, results of the present study show that addition of 0.6 g/L sodium bicarbonate to the growth medium (BG-11) significantly enhance the biomass, lipid and carbohydrate content of microalgae Scenedesmus sp. CCNM 1077. Addition of sodium bicarbonate under nitrate and phosphate starvation conditions also show stress ameliorating effects by improving biomass productivity. Among all the tested conditions phosphate starvation under bicarbonate supplementation was best approach due to higher carbohydrate and lipid production with marginal loss in biomass production compared to BG-11 grown cultures. This suggests an efficient approach for the sequential production of microalgal based biodiesel and bioethanol. Acknowledgements CSIR-CSMCRI Registration Number: 036/2015. IP, TG, CP and RM would like to acknowledge CSIR for awarding Senior Research Fellowship. The authors gratefully acknowledge CSC 0203 for providing the financial support. 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 Mr. Satyavver Gothwal, ADCIF, CSIR-CSMCRI, Bhavnagar for his help during the CHNS analysis. IP, KC, TG, CP and RM wish to acknowledge AcSIR for Ph.D. enrollment. 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.2015.06. 107. References 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. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Cataldo, D.A., Maroon, M., Schrader, L.E., Youngs, V.L., 1975. Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid 1. Commun. Soil Sci. Plant 6, 71–80. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306. Chokshi, K., Pancha, I., Trivedi, K., George, B., Maurya, R., Ghosh, A., Mishra, S., 2015. Biofuel potential of the newly isolated microalgae Acutodesmus dimorphus under temperature induced oxidative stress conditions. Bioresour. Technol. 180, 161–171. Chu, F.F., Chu, P.N., Cai, P.J., Li, W.W., Lam, P.K., Zeng, R.J., 2013. Phosphorus plays an important role in enhancing biodiesel productivity of Chlorella vulgaris under nitrogen deficiency. Bioresour. Technol. 134, 341–346.
Chu, F.F., Chu, P.N., Shen, X.F., Lam, P.K., Zeng, R.J., 2014. Effect of phosphorus on biodiesel production from Scenedesmus obliquus under nitrogen-deficiency stress. Bioresour. Technol. 152, 241–246. 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. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. El-Ansari, O., Colman, B., 2015. Inorganic carbon acquisition in the acid-tolerant alga Chlorella kessleri. Physiol. Plant. 153, 175–182. 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. Gardner, R.D., Cooksey, K.E., Mus, F., Macur, R., Moll, K., Eustance, E., Peyton, B.M., 2012. Use of sodium bicarbonate to stimulate triacylglycerol accumulation in the chlorophyte Scenedesmus sp. and the diatom Phaeodactylum tricornutum. J. Appl. Phycol. 24, 1311–1320. George, B., Pancha, I., Desai, C., Chokshi, K., Paliwal, C., Ghosh, T., Mishra, S., 2014. Effects of different media composition, light intensity and photoperiod on morphology and physiology of freshwater microalgae Ankistrodesmus falcatus – a potential strain for bio-fuel production. Bioresour. Technol. 171, 367–374. Grasshoff, K., Ehrhardt, M., Kremling, K., 1999. Methods of Seawater Analysis, third ed. Verlag Chemie, Weinheim, Germany. Guihéneuf, F., Stengel, D.B., 2013. LC-PUFA-enriched oil production by microalgae: accumulation of lipid and triacylglycerols containing n-3 LC-PUFA is triggered by nitrogen limitation and inorganic carbon availability in the marine haptophyte Pavlova lutheri. Mar. Drugs 11, 4246–4266. Harun, R., Danquah, M.K., 2011. Enzymatic hydrolysis of microalgal biomass for bioethanol production. Chem. Eng. J. 168, 1079–1084. Kruskopf, M.M., Du Plessis, S., 2004. Induction of both acid and alkaline phosphatase activity in two green-algae (chlorophyceae) in low N and P concentrations. Hydrobiologia 513, 59–70. Lee, C.G., Lee, C.G., 2002. Nitrogen removal from wastewaters by microalgae without consuming organic carbon sources. J. Microbiol. Biotechnol. 12, 979– 985. Lee, T.M., 2000. Phosphate starvation induction of acid phosphatase in Ulva lactuca L. (Ulvales, Chlorophyta). Bot. Bull. Acad. Sin., 41 Li, Y., Han, D., Sommerfeld, M., Hu, Q., 2011. Photosynthetic carbon partitioning and lipid production in the oleaginous microalga Pseudochlorococcum sp. (Chlorophyceae) under nitrogen-limited conditions. Bioresour. Technol. 102, 123–129. Li, Y., Han, F., Xu, H., Mu, J., Chen, D., Feng, B., Zeng, H., 2014. Potential lipid accumulation and growth characteristic of the green alga Chlorella with combination cultivation mode of nitrogen (N) and phosphorus (P). Bioresour. Technol. 174, 24–32. Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol. 148, 350–382. 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. 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. Pancha, I., Chokshi, K., George, B., Ghosh, T., Paliwal, C., Maurya, R., Mishra, S., 2014. Nitrogen stress triggered biochemical and morphological changes in the microalgae Scenedesmus sp. CCNM 1077. Bioresour. Technol. 156, 146–154. Pancha, I., Chokshi, K., Maurya, R., Trivedi, K., Patidar, S.K., Ghosh, A., Mishra, S., 2015a. Salinity induced oxidative stress enhanced biofuel production potential of microalgae Scenedesmus sp. CCNM 1077. Bioresour. Technol. 189, 341–348. Pancha, I., Chokshi, K., Mishra, S., 2015b. Enhanced biofuel production potential with nutritional stress amelioration through optimization of carbon source and light intensity in Scenedesmus sp. CCNM 1077. Bioresour. Technol. 179, 565–572.
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Bicarbonate supplementation enhanced biofuel production potential as well as nutritional stress mitigation in the microalgae Scenedesmus sp. CCNM 1077 Imran Pancha, Kaumeel Chokshi, Tonmoy Ghosh, Chetan Paliwal, Rahulkumar Maurya, Sandhya Mishra ⇑ 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 Bicarbonate additions enhance biomass and biochemical content of microalgae. 0.6 g/L was optimum bicarbonate concentration. Bicarbonate supplementation helps in nutritional stress ameroliation in microalgae. Nitrate starvation with bicarbonate addition produced highest carbohydrate and lipid. Scenedesmus sp. is a potential feedstock for biodiesel and bioethanol production.
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Article history: Received 25 May 2015 Received in revised form 18 June 2015 Accepted 19 June 2015 Available online 25 June 2015 Keywords: Microalgae Sodium bicarbonate Nutritional starvation Neutral lipid Carbohydrate
a b s t r a c t The aim of the present study was to find out the optimum sodium bicarbonate concentration to produce higher biomass with higher lipid and carbohydrate contents in microalgae Scenedesmus sp. CCNM 1077. The role of bicarbonate supplementation under different nutritional starvation conditions was also evaluated. The results clearly indicate that 0.6 g/L sodium bicarbonate was optimum concentration resulting in 20.91% total lipid and 25.56% carbohydrate along with 23% increase in biomass production compared to normal growth condition. Addition of sodium bicarbonate increased the activity of nutrient assimilatory enzymes, biomass, lipid and carbohydrate contents under different nutritional starvation conditions. Nitrogen starvation with bicarbonate supplementation resulted in 54.03% carbohydrate and 34.44% total lipid content in microalgae Scenedesmus sp. CCNM 1077. These findings show application of bicarbonate grown microalgae Scenedesmus sp. CCNM 1077 as a promising feedstock for biodiesel and bioethanol production. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Microalgae are considered as green bio-factories for the production of various bioactive compounds like pigments, fatty acids, vitamins etc. Apart from its nutraceutical importance, microalgae are recently considered as a promising renewable energy feed stock due to their high growth rate, photosynthetic ability, high lipid and carbohydrate contents as well as their ability to obtain nutrients from wastewaters (Chisti, 2007). To make microalgal based biofuel economically viable, downstream processing like ⇑ Corresponding author at: Discipline of Salt & Marine Chemicals, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India. Tel.: +91 278 256 5801/256 3805x6160; fax: +91 278 256 6970/256 7562. E-mail address: [email protected] (S. Mishra). http://dx.doi.org/10.1016/j.biortech.2015.06.107 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
harvesting, dewatering and biodiesel production must be optimized; at the same time, it is also important to improve biomass, lipid and carbohydrate productivity of microalgae to generate higher biomass with high lipid and carbohydrate contents (Wijffels and Barbosa, 2010). Cultural conditions like nutrients concentration and composition, light intensity and photoperiod, pH, temperature, etc. significantly affect the growth and biochemical composition of microalgae (George et al., 2014). Many cultivation strategies like nitrogen starvation, salinity stress and temperature stress significantly enhanced the lipid and carbohydrate contents in microalgae (Pancha et al., 2014; Pancha et al., 2015a; Chokshi et al., 2015). However, these stress conditions resulted in lower growth rate and biomass productivity, which ultimately increase the biofuel production cost. On the other hand, heterotrophic and mixotrophic
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growth utilizing various types of organic sources have been resulted in higher biomass along with higher lipid and carbohydrate contents in microalgae (Pancha et al., 2015b). However, all microalgae are unable to grow under such growth conditions; moreover, these conditions result in high initial operating cost due to utilization of costly organic substrates. Supply of inorganic carbon in the form of CO2 is important for active photosynthesis, carbon dioxide fixation and generation of biomass with desired products in microalgae. Many microalgae have the ability to uptake dissolved inorganic carbon (DIC) from the surrounding for active photosynthesis by CO2 concentrating mechanism (El-Ansari and Colman, 2015). In various microalgae addition of sodium bicarbonate to the culture medium resulted in high pH as well as high DIC in the growth medium which increased their growth and higher TAG accumulation (Gardner et al., 2012; White et al., 2013). However, the role of bicarbonate supplementation under various nutritional starvation conditions like nitrate and phosphate starvations as well as its involvement in active uptake of nutrients, lipid and carbohydrate accumulation in microalgae is still unclear. In the present study, freshwater microalgae Scenedesmus sp. CCNM 107 was first grown in the range (0–1.5 g/L) of sodium bicarbonate concentrations to find out the optimum concentration to obtain higher biomass, lipid and carbohydrate contents. Next, the combined effect of bicarbonate addition and nutritional starvation on morphological and biochemical parameters including cell morphology, dry cell weight, lipid, carbohydrate, protein, nutrients assimilatory enzymes and photosynthetic pigments were used to understand the physiological mechanism of bicarbonate supplementation on lipid and carbohydrate accumulation in the microalgae Scenedesmus sp. CCNM 1077. 2. Methods 2.1. Microalgae and experimental conditions The microalgae used in this study, Scenedesmus sp. CCNM 1077, was cultured in BG-11 medium as described previously (Pancha et al., 2014). For the determination of ideal bicarbonate concentration, microalgae Scenedesmus sp. CCNM 1077 was first grown in BG-11 medium with different concentrations of sodium bicarbonate i.e. 0, 0.3, 0.6, 0.9, 1.2 and 1.5 g/L. After obtaining best bicarbonate concentration, its effect on nitrate starvation (NP+), phosphate starvation (N+P) and combined nitrate phosphate starvation (NP) was evaluated under bicarbonate supplemented and non-supplemented conditions. All the experiments were conducted in triplicate in 1 L flasks containing 500 ml of respective culture medium and inoculated with 10% actively growing culture of Scenedesmus sp. CCNM 1077. All the experiments were performed in batch culture at 25 ± 2 °C with 12:12 h light dark period under 60 lmol m2 s1 light intensity. The flasks were manually shaken thrice a day. 2.2. Biomass and biochemical composition analysis 2.2.1. Determination of growth and biomass productivity Microalgal growth was monitored every 5th day by measuring optical density at 750 nm. 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 (DCW) (mg/L) at time t2 and t1, respectively. 2.2.2. Determination of pigments For the determination of pigments content, 2 ml microalgal culture was centrifuged at 10,000 rpm for 5 min, supernatant was
discarded and pellet was mixed with 99.9% methanol and incubated in dark for 24 h at 45 °C. After incubation, pigments content was determined using the following formulas:
Chlorophyll a; Chl-a ðl g=mlÞ ¼ 16:72 A665:2 9:16 A652:4 Chlorophyll b; Chl-b ðl g=mlÞ ¼ 34:09 A652:4 15:28 A665:2 Carotenoids ¼ ð1000 A470 1:63 Chl-a 104:9 Chl-bÞ=221 Absorbances at 470, 652.4 and 665.2 nm were corrected for turbidity by subtracting absorbance at 750 nm (Lichtenthaler, 1987). 2.2.3. Determination of lipid, protein and carbohydrate contents Lipid was extracted using chloroform:methanol (1:2 v/v) and determined gravimetrically (Bligh and Dyer, 1959). Total lipid was further fractionated into neutral lipid using chloroform/acetic acid (9:1, v/v), glycolipids using acetone/methanol (9:1, v/v) and phospholipids using methanol as described in Pancha et al. (2014). CHNS content in microalgal biomass was determined through elemental analysis using a CHNS analyzer (Perkin-Elmer Model 2 400, USA). Crude protein content of microalgae was calculated according to the following equation (Becker, 1994):
Crude protein ð%Þ ¼ N ð%Þ 6:25 For the determination of carbohydrate content in microalgae, 50 mg of lipid extracted biomass was first hydrolyzed with 500 ll of 72% (w/v) H2SO4 for 1 h. Concentration of H2SO4 was reduced to 4% by the addition of distilled water and autoclaved at 121 °C for 1 h. After cooling down to room temperature, total volume was made up to 50 ml with distilled water (Van Wychen and Laurens, 2013). The resulting solution was centrifuged at 10,000 rpm for 10 min and the supernatant was used for sugar estimation by phenol sulfuric acid method (Dubois et al., 1956) using D-glucose as a standard. 2.2.4. Determination of nutrients concentrations Residual nitrate concentration in the microalgal culture medium was estimated using salicylic acid and sodium hydroxide (Cataldo et al., 1975). Residual phosphate concentration in the microalgal culture medium was estimated using ascorbic method (Grasshoff et al., 1999). 2.2.5. Determination of nutrients assimilatory enzymes Nitrate reductase (NR) activity of microalgal cells was determined according to the method described by Li et al. (2011). Alkaline (ALP) and acid phosphates (AP) activities were determined according to Lee (2000). Total soluble protein content was measured using bovine serum albumin as a standard (Bradford, 1976). 2.3. Statistical analysis All results are expressed as mean values ± standard deviation. The statistical differences between experimental groups were assessed by analysis of variance (ANOVA) using the Info stat software package (2012). The mean values were compared with LSD test (p < 0.05). 3. Results and discussion 3.1. Effect of sodium bicarbonate supplementation on dry cell weight (DCW) and biomass productivity Generally, microalgae grow photoautotrophically utilizing atmospheric CO2 as an inorganic carbon source. Since CO2 has very
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low solubility in water, bicarbonate addition is carried out in open pond large scale microalgal cultivation to provide desired inorganic carbon to generate more biomass. Addition of sodium bicarbonate to the growth medium has been reported to significantly increase the biomass and biochemical composition of various microalgae (White et al., 2013). In the present study, addition of sodium bicarbonate to the growth medium significantly enhanced the DCW and biomass productivity of microalgae Scenedesmus sp. CCNM 1077 (Fig. 1a) which indicates that sodium bicarbonate supplementation enhances the cell division and metabolic process in microalgae. Addition of sodium bicarbonate (0–1.2 g/L) increased DCW and biomass productivity in a dose dependent manner and no significant difference (p < 0.05) in DCW production was observed beyond 0.6 g/L sodium bicarbonate concentration. Biomass productivity of Scenedesmus sp. CCNM 1077 was largely unaffected in the range of 0.6–1.5 g/L sodium bicarbonate supplemented growth medium. The highest biomass productivity was found in cultures grown with 1.2 g/L sodium bicarbonate (28.32 ± 0.77 mg/L/day) which was 1.4 folds higher than the only BG-11 grown culture (19.92 ± 0.26 mg/L/day). In a similar study, Peng et al. (2014) also found higher biomass production in diatom Phaeodactylum tricornutum cultivated in the presence of sodium bicarbonate. In the present study, nitrate and phosphate consumption trend was recorded to correlate the effect of sodium bicarbonate
supplementation on active uptake of nutrients by microalgae Scenedesmus sp. CCNM 1077 (Fig. 2). The results indicate that uptake of nitrate and phosphate from the growth medium increased with the increase in bicarbonate addition to the growth medium. Addition of only 0.3 g/L sodium bicarbonate to the growth medium resulted in 66 ± 1.87% phosphate uptake and 33 ± 1.71% nitrate uptake from the growth medium, which was higher than those of the control culture. Lee and Lee (2002) reported that high CO2 grown Chlorella kessleri culture had higher nitrate consumption possibly due to enhanced nitrate absorption efficacy of the cells. In the present study, highest consumption of nitrate (37.58 ± 2.39%) and phosphate (78.58 ± 0.56%) was found in cells cultivated under 0.9 g/L sodium bicarbonate. Highest DCW (549.15 ± 13.13 mg/L) production was found in cells cultivated with 1.2 g/L sodium bicarbonate. However, further increase in bicarbonate concentration to 1.5 g/L showed no significant difference in DCW production (547 ± 16.42 mg/L) but showed reduced nutrient consumptions (63.55 ± 0.13% phosphate and 35.12 ± 1.33% nitrate) by Scenedesmus sp. CCNM 1077. Lower nutrient consumption under high bicarbonate supplementation might be due to increase in pH of the growth medium since it has been known that many salts of the marine culture medium gets precipitated and become unavailable for consumption by microalgae (Guihéneuf and Stengel, 2013).
Fig. 1. (a) Effect of different NaHCO3 concentration on DCW and biomass productivity of Scenedesmus sp. CCNM 1077. (b) Effect of different NaHCO3 concentration on biochemical composition of Scenedesmus sp. CCNM 1077 (Values with different letters represent significant difference at p < 0.05 between treatments).
Fig. 2. (a) Nitrate consumption trend of Scenedesmus sp. CCNM 1077 in different initial NaHCO3 concentration. (b) Phosphate consumption trend of Scenedesmus sp. CCNM 1077 in different initial NaHCO3 concentration.
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3.2. Effect of sodium bicarbonate supplementation on biochemical composition Effect of initial sodium bicarbonate concentrations on lipid, crude protein and carbohydrate content of microalgae Scenedesmus sp. CCNM 1077 is shown in Fig. 1b. Crude protein content of microalgae Scenedesmus sp. CCNM 1077 increased in a dose dependent manner. Highest crude protein content (50.12 ± 0.23%) was observed in cells grown with 1.5 g/L sodium bicarbonate followed by 1.2 (49.46 ± 0.41%) and 0.9 g/L (49.56 ± 0.37%) sodium bicarbonate supplemented cultures. Carbon availability and supply is a key metabolic regulator which governs the carbohydrate and lipid synthesis in microalgae (Fan et al., 2012). Fig. 1b also shows that addition of 0.9 g/L sodium bicarbonate to the growth medium increased the carbohydrate content from 18.55 ± 0.79 to 30.88 ± 1.47%. The results also indicate a positive correlation between sodium bicarbonate supplementation and cellular carbohydrate content in microalgae Scenedesmus sp. CCNM 1077. It has been reported that high availability of inorganic carbon results in high activity of Ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) which is a key enzyme for the conversion of 3-phosphoglycerate, a substrate for the biosynthesis of carbohydrate and fatty acid in plants and microalgae (Peng et al., 2014). There was no significant difference (p < 0.05) in carbohydrate accumulation pattern in cells cultivated with 0.3 g/L (24.57 ± 0.72%) and 0.6 g/L (25.56 ± 0.84%) sodium bicarbonate. Many recent studies show that microalgal carbohydrates are better than plant and seaweed derived carbohydrates due to high starch content, which can be easily converted into hexose sugar. Moreover, microalgal carbohydrates do not contain lignin which significantly reduces the pretreatment cost for bio-ethanol production (Yeh and Chang, 2011; Markou et al., 2012; Harun and Danquah, 2011). In the present study, carbohydrate content was estimated from the left over de-oiled microalgal biomass making its application towards biodiesel and bioethanol coproduction which will ultimately reduce their production cost. Similar to carbohydrate, addition of sodium bicarbonate also enhanced the total lipid content in microalgae Scenedesmus sp. CCNM 1077 (Fig. 1b). There was no significant difference (p < 0.05) in total lipid content of cells cultivated with 0.6 g/L (20.91 ± 0.31%) and 0.9 g/L (20.89 ± 0.35%) sodium bicarbonate. Guihéneuf and Stengel (2013) showed that under supply of inorganic carbon in the form of sodium bicarbonate microalgae Pavlova lutheri produce high amount of LC-PUFA rich TAGs which are mainly generated from de novo synthesis rather than membrane remodeling. In the present study, no significant difference in lipid content was observed in 1.2 g/L (19.36 ± 0.23%) and 1.5 g/L (19.07 ± 0.29%) sodium bicarbonate supplemented cultures. In order to understand the role of sodium bicarbonate addition on lipid class composition, total lipid content of microalgae Scenedesmus sp. CCNM 1077 was fractionated into neutral lipid, glycolipids and phospholipids utilizing silica gel G column chromatography. Addition of sodium bicarbonate to the growth medium increased the NL content and reduced the GL and PL contents of microalgae Scenedesmus sp. CCNM 1077 (Fig. 3). Addition of 0.6 g/L sodium bicarbonate to the growth medium resulted in 75.8 ± 0.85% NL of total lipid, which was about 1.12 folds higher than that of the BG-11 grown culture (67.24 ± 0.55% of TL). Reduction in GL and PL contents indicate changes in cell membrane composition of microalgae Scenedesmus sp. CCNM 1077. Addition of bicarbonate to the culture medium enhances its pH and microalgae change their membrane composition to withstand this alkaline stress. GL content decreased from 16.4 ± 0.14% to 14.9 ± 0.1% in 1.2 g/L bicarbonate grown cultures; similarly, PL content also decreased from 15.6 ± 0.18% to 8.13 ± 0.28% in 0.9 g/L bicarbonate grown cultures. Results of this section show that sodium
bicarbonate at concentration of 0.6 g/L is optimum for higher biomass generation along with higher lipid and carbohydrate contents in microalgae Scenedesmus sp. CCNM 1077. 3.3. Effect of sodium bicarbonate supplementation on pigments composition Addition of sodium bicarbonate to the growth medium increased all the photosynthetic pigments viz. Chl-a, Chl-b and total carotenoids content of microalgae Scenedesmus sp. CCNM 1077 (Table 1). Increase in sodium bicarbonate concentration from 0 to 1.2 g/L significantly (p < 0.05) enhanced the Chl-a and b as well as total carotenoids contents. Highest amount of Chl-a (6.11 ± 1.14 lg/ml) was found in 0.9 g/L sodium bicarbonate grown culture, which was about 1.17 folds higher than the BG-11 grown culture (5.18 ± 0.32 lg/ml). Similar to our results, White et al. (2013) also observed 2.24 and 1.91 folds increase in Chl-a content in Tetraselmis suecica and Nannochloropsis salina, respectively, when cultivated under 1 g/L sodium bicarbonate supplementation compared to non bicarbonate supplemented cultures. Addition of sodium bicarbonate (up to 1.2 g/L) resulted in the increase in total carotenoids of the cells in dose dependent manner. Highest amount of total carotenoids (1.53 ± 0.10 lg/ml) was found in 1.2 g/L sodium bicarbonate supplemented culture followed by 0.9 g/L (1.45 ± 0.15 lg/ml) and 0.6 g/L (1.38 ± 0.09) sodium bicarbonate supplemented cultures. Addition of sodium bicarbonate (up to 1.2 g/L) to the growth medium increased the ratios of Chl a/b as well as Caro/total Chl. In this study, we supplemented bicarbonate to the growth medium that increase the culture alkalinity and pH, which might be the reason for higher ratio of Chl a/b, Caro/total Chl in microalgae Scenedesmus sp. CCNM 1077. In a similar study, no significant difference in pigments ratio was found when T. suecica was grown with supplementation of sodium bicarbonate, while in case of N. salina, carotenoids: Chl-a ratio increased with the increase in sodium bicarbonate in the growth medium (White et al., 2013). 3.4. Effect of sodium bicarbonate supplementation on DCW and biomass productivity under nutritional starvation conditions Microalgal lipid and carbohydrate contents generally increase in various nutritional stress conditions like nitrate and phosphate starvation. However, these nutritional starvation conditions result in lower DCW and biomass productivity in microalgae. In this study, addition of inorganic carbon in the form of sodium bicarbonate not only increased the biomass content of Scenedesmus sp. CCNM 1077 but also resulted in higher accumulation of neutral lipid and carbohydrate. To check the role of inorganic carbon supplementation under nutrition starvation condition, Scenedesmus sp. CCNM 1077 was grown under nitrate starvation (NP+), phosphate starvation (N+P) and combined nitrate and phosphate starvation (NP) with and without 0.6 g/L sodium bicarbonate supplementation. Fig. 4a shows effect of sodium bicarbonate addition on DCW and biomass productivity under nutrient starvation conditions. From the data it is clear that removal of either nitrate or phosphate or both from the growth medium significantly affects the metabolic process of microalgae Scenedesmus sp. CCNM 1077 and results in lower DCW and biomass productivity. However, addition of 0.6 g/L sodium bicarbonate reduced the stress effects with the increase in DCW and biomass production under all the tested nutritional stress conditions. Our earlier study with microalgae Scenedesmus sp. CCNM 1077 (Pancha et al., 2014) showed that removal of nitrate from the growth medium resulted in 46% reduction in DCW compared to control culture. However, in the present study, addition of 0.6 g/L bicarbonate helped in reducing the stress
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Fig. 3. Changes in neutral lipids (NL), glycolipids (GL) and phospholipids (PL) concentration of Scenedesmus sp. CCNM 1077 under different NaHCO3 concentration ((a) 0 g/L; (b) 0.3 g/L; (c) 0.6 g/L; (d) 0.9 g/L; (e) 1.2 g/L; (f) 1.5 g/L).
Table 1 Effect of different sodium bicarbonate concentration on pigments composition of Scenedesmus sp. CCNM 1077. NaHCO3 (g/L) 0 0.3 0.6 0.9 1.2 1.5
Chl-a* (lg/ml) a
5.18 ± 0.32 5.81 ± 0.23a 6.02 ± 0.48a 6.11 ± 1.14a 6.10 ± 0.16a 5.19 ± 1.05a
Chl-b** (lg/ml) b
3.29 ± 0.25 3.56 ± 0.22ab 3.51 ± 0.28ab 3.53 ± 0.79ab 3.67 ± 0.45ab 4.13 ± 0.59a
Chl a + b (lg/ml) a
8.47 ± 0.56 9.37 ± 0.44a 9.53 ± 0.76a 9.64 ± 1.94a 9.78 ± 0.61a 9.32 ± 1.05a
Caro*** (lg/ml)
Chl a/b
Caro/Chl a + b
1.02 ± 0.06d 1.25 ± 0.01bc 1.38 ± 0.09abc 1.45 ± 0.15ab 1.53 ± 0.10a 1.15 ± 0.27cd
1.58 ± 0.03a 1.63 ± 0.05a 1.72 ± 0.01a 1.74 ± 0.06a 1.67 ± 0.15a 1.28 ± 0.33b
0.12 ± 0.00c 0.13 ± 0.007abc 0.14 ± 0.00ab 0.15 ± 0.01a 0.16 ± 0.02a 0.12 ± 0.03bc
Different superscript letters within column indicates significant differences at p < 0.05. * Chl-a: chlorophyll-a. ** Chl-b: chlorophyll-b. *** Caro: carotenoids.
effects and 38% reduction in DCW was observed. Removal of both, nitrate and phosphate, from the growth medium resulted in 178 ± 1.67 mg/L DCW, while addition of 0.6 g/L bicarbonate augmented DCW production i.e. 221.13 ± 6.07 mg/L under combined nutrient starvation. Similar to our study, Chu et al. (2013) also found reduction in total DCW production by microalgae Chlorella vulgaris in nitrate (95.4 mg/L/day) and phosphate (38.3 mg/L/day) starved cultures compared to control culture (100.4 mg/L/day). In the present study, DCW production was not much affected under phosphate starvation and only 19% reduction in DCW (320.87 ± 11.68 mg/L) was observed. Biomass productivity was largely unaffected in phosphate starvation condition (15.74 ± 0.84 mg/L/day) as well as bicarbonate supplemented phosphate starvation conditions (18.08 ± 0.31 mg/L/day) compared to control culture (20.77 ± 0.28 mg/L/day). Removal of nitrate or nitrate and phosphate, both, from the growth medium resulted in very low biomass productivity i.e. 8.25 ± 0.21
and 6.48 ± 0.07 mg/L/day, respectively. The result of the present section shows that nitrate is a vital nutrient for normal growth and metabolism of microalgae Scenedesmus sp. CCNM 1077 and addition of bicarbonate has nutrient stress ameliorating effects which helps in improving its DCW and biomass productivity. Our earlier observations show that microalgae Scenedesmus sp. CCNM 1077 is a pleomorphic strain, which changes its morphology from single cell to coenobium of 2 and 4 under nitrate starvation condition (Pancha et al., 2014). In the present study too, nitrate or combined nitrate and phosphate starvations resulted in 4 cells coenobium with spines at terminal cells. However, size of 4 cells coenobium was smaller in combined stress compared to only nitrogen stressed culture. Addition of bicarbonate did not show any significant effect on cell morphology under starvation conditions. Compared to control culture, cells grown under phosphate starvation did not show any significant change in their morphology; however, addition of bicarbonate resulted in slightly larger
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Fig. 4. (a) Effect of sodium bicarbonate supplementation on DCW and biomass productivity under nutritional starvation conditions. (b) Effect of sodium bicarbonate supplementation on biochemical composition under nutritional starvation conditions (Values with different letters represent significant difference at p < 0.05 between treatments).
cell size. It has been reported that larger cell has slow metabolic rate which helps in saving energy from damage and repair as well as the amount of energy stored is directly propositional to cell size of microalgae (Šetlík et al., 1972).
3.5. Effect of sodium bicarbonate supplementation on nutrient consumption and nutrient assimilatory enzymes Fig. 5 shows the effect of sodium bicarbonate addition on nutrient consumption trend of microalgae Scenedesmus sp. CCNM 1077 under different nutritional starvation conditions. Addition of 0.6 g/L sodium bicarbonate resulted in 46.97 ± 0.67% phosphate consumption in nitrogen-starved culture, which was 38% higher than those of the cultures without sodium bicarbonate supplementations. More utilization of phosphate is also evident from higher activities of AP and ALP in bicarbonate grown cultures (Fig. 5c). In the present study, activity of AP was higher than ALP. Similarly, Kruskopf and Du Plessis (2004) reported higher activity of AP than ALP in Chlorella and Chlamydomonas under nitrate and phosphate starvation conditions. AP helps in liberation of intracellular P reserve whereas ALP helps to dissolve organic P from the growth medium. In the present study, addition of bicarbonate did not show any effect on AP and ALP activities in phosphate starved cultures. This suggests that presence of phosphate is important for the induction of both, AP and ALP, in microalgae Scenedesmus sp. CCNM 1077.
Fig. 5. (a) Nitrate consumption trend of Scenedesmus sp. CCNM 1077 with sodium bicarbonate supplementation under nutritional starvation conditions. (b) Phosphate consumption trend of Scenedesmus sp. CCNM 1077 with sodium bicarbonate supplementation under nutritional starvation conditions. (c) Changes in nutrient assimilatory enzymes of Scenedesmus sp. CCNM 1077 with sodium bicarbonate supplementation under nutritional starvation conditions (Values with different letters represent significant difference at p < 0.05 between treatments.) (Abbreviations: NP WTout; NP+ without NaHCO3, NP WT; NP+ with 0.6 g/L NaHCO3, NP WTout; N+P without NaHCO3, NP WT; N+P with 0.6 g/L NaHCO3, NP WTout; NP without NaHCO3, NPWT; NP 0.6 g/L NaHCO3).
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Rate of nitrate assimilation was also increased by addition of sodium bicarbonate to the growth medium. BG-11 grown culture consumed 30 ± 0.44% of nitrate while cultures grown under phosphate starvation with and without bicarbonate addition consumed 28 ± 1.4% and 29.39 ± 1.1% nitrate, respectively. Chu et al. (2014) showed significant reduction in nitrate assimilation under phosphate starvation by microalgae S. obliquus FACHB-417. Nitrate reductase activity was significantly (p < 0.05) affected in various stress conditions. Removal of both nitrate and phosphate from the growth medium did not show any significant difference in nitrate reductase activity. However, addition of sodium bicarbonate under phosphate starvation resulted in NR activity of 0.83 ± 0.0 lmol min1 mg1 protein which was almost double than that of the culture without bicarbonate supplementation (0.42 ± 0.01 lmol min1 mg1 protein). Culture under nitrate starvation with sodium bicarbonate supplementation resulted in NR activity of 0.21 ± 0.01 lmol min1 mg1 protein which was significantly lower than that of the culture without bicarbonate supplementation, but similar to that of the nitrate and phosphate starved cultures (0.20 ± 0.02 lmol min1 mg1 protein). 3.6. Effect of sodium bicarbonate supplementation on biochemical composition under nutritional starvation conditions Effect of bicarbonate addition on lipid, carbohydrate and protein contents of microalgae Scenedesmus sp. CCNM 1077 is shown in Fig. 4b. As protein is nitrogenous compound, removal of nitrogen from the growth medium significantly (p < 0.05) reduced the crude protein content from 47.19 ± 0.26% to 11.95 ± 0.14% in combined stress and to 10.63 ± 0.44% in nitrogen starvation condition. Addition of bicarbonate resulted in 12.93 ± 0.11% and 11.76 ± 0.25% crude protein content in combined and nitrogen starvation conditions, respectively. Similarly, Chu et al. (2014) also found sharp decrease in protein content in microalgae S. obliquus FACHB-417 during combine nitrate and phosphate starvation. The degradation of protein helps in maintaining the intracellular nitrogen pool in microalgae, thereby helping in normal functioning of cellular metabolism. Phosphate starvation did not show any significant effect on crude protein content of microalgae Scenedesmus sp. CCNM 1077 and resulted in 39.89 ± 0.22% crude protein. Lipid and carbohydrate are the two most reduced storage products in microalgae, generally accumulated under various nutritional stress conditions due to their hydrophobic nature, more reduced state and efficient storage in smaller compartments of the cells (Courchesne et al., 2009). Fig. 4b also shows effect of bicarbonate addition on carbohydrate content of microalgae Scenedesmus sp. CCNM 1077. As per our earlier observation, nutrient limitation results in significantly higher accumulation of carbohydrate and its synthesis is dominated over lipid production in microalgae Scenedesmus sp. CCNM 1077 (Pancha et al., 2014). Highest accumulation of carbohydrate (54.03 ± 1.08%) was found under nitrogen limitation with bicarbonate supplementation followed by combined nutrient starvation with bicarbonate supplementation (51.25 ± 0.67%). Total removal of phosphate from the growth medium resulted in 27.47 ± 0.49% carbohydrate which further increased up to 29.86 ± 0.26% during 0.6 g/L bicarbonate supplementation. These results indicate that addition of bicarbonate under various nutritional stress conditions significantly enhanced the carbohydrate content in microalgae Scenedesmus sp. CCNM 1077. Among all, phosphate starvation was found to be a good alternative as it resulted in comparatively lesser biomass loss with relatively higher lipid and carbohydrate contents in microalgae Scenedesmus sp. CCNM 1077. Nutrients limitations like nitrate, phosphate or combined nitrate and phosphate limitations have resulted in higher
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accumulation of lipid in various microalgae (Chu et al., 2013, 2014; Li et al., 2014). However, only few researchers have studied the effects of bicarbonate addition under nitrate limited growth conditions (White et al., 2013; Guihéneuf and Stengel, 2013) and there are no reports on effects of bicarbonate addition under phosphate or combined nitrate phosphate stress conditions in microalgae. Total lipid content of microalgae Scenedesmus sp. CCNM 1077 was increased from 17.85 ± 0.14% to 34.42 ± 0.27% under nitrogen starvation with bicarbonate addition, which was significantly higher than that of without bicarbonate addition (28.05 ± 0.08%). This indicates that inorganic carbon supply under nitrogen starvation helps in de novo synthesis of microalgal lipids. Similar to our study, Guihéneuf and Stengel (2013) also found high TAGs in bicarbonate supplemented nitrogen starved culture of marine haptophyte Pavlova lutheri. In the present study, Scenedesmus sp. CCNM 1077 grown under combined nutrition starvation with bicarbonate supplementation did not show any significant increase in total lipid content (33.29 ± 0.42%) compared to that of nitrate starved bicarbonate supplemented culture. Li et al. (2014) have reported higher lipid accumulation in Chlorella during nitrate phosphate co-deficiency compared to only nitrate starvation. In the present study, phosphate starvation with bicarbonate addition resulted in 27.97 ± 0.24% total lipid, which was similar to that of nitrogen starved culture. This treatment can be used as an efficient approach to produce microalgal based biofuel as it resulted in limited loss of biomass with higher lipid and carbohydrate contents, which can be converted into biodiesel and bioethanol, respectively. Sodium bicarbonate supplementation had significant effect on lipid class composition of microalgae Scenedesmus sp. CCNM 1077 (Fig. S1). Neutral lipid content of 88.69 ± 2.1% of total lipid was observed in nitrate starved bicarbonate supplemented cells which was 1.3 folds higher than that of the BG-11 grown culture (67.2 ± 0.55%). Nitrate and phosphate starvation significantly reduced the GL and PL content of microalgae Scenedesmus sp. CCNM 1077. NL is mainly composed of TAGs which are important for the production of microalgal based biodiesel, while GL and PL are the components of cell membranes (Olmstead et al., 2013). Addition of sodium bicarbonate under all tested nutritional starvation conditions resulted in higher NL content compared to that of the cultures grown without bicarbonate addition, indicating positive role of sodium bicarbonate in NL accumulation in microalgae Scenedesmus sp. CCNM 1077.
3.7. Effect of sodium bicarbonate supplementation on pigments under nutritional starvation conditions Pigments content and composition are most affected during various nutritional stress conditions like nitrogen and salinity stress since pigments are mainly composed of nitrogen rich compounds (Pancha et al., 2014). To gain insight into the photosynthetic process under various stress conditions, Chl-a, Chl-b and total carotenoids content were measured (Table 2). All the stress conditions resulted in lower Chl-a content, which was also evident from the color of cultures from green to yellow and light yellow in phosphate and nitrate combined stress conditions. As shown in Table 2, removal of nitrate from the culture medium resulted in 0.15 ± 0.02 lg/ml Chl-a. However, addition of sodium bicarbonate resulted in 0.58 ± 0.11 lg/ml Chl-a, which was about 3.8 folds higher than that of the cells without bicarbonate supplementation. Addition of sodium bicarbonate did not show much effect on pigment content under combined stress conditions. Pigments content of phosphate starved cultures were least affected i.e. 2.59 ± 0.19 and 3.13 ± 0.12 lg/ml Chl-a in cultures without and with bicarbonate supplementation, respectively.
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Table 2 Effect of sodium bicarbonate supplementation on pigments composition under nutritional starvation conditions. Treatments Control NP WTout NP WT NP WTout NP WT NP WTout NP WT
Chl-a* (lg/ml) a
5.26 ± 0.24 0.15 ± 0.02e 0.58 ± 0.11d 2.59 ± 0.19c 3.13 ± 0.12b 0.25 ± 0.06e 0.21 ± 0.02e
Chl-b** (lg/ml) a
3.31 ± 0.27 0.20 ± 0.11c 0.41 ± 0.12c 2.0 ± 0.15b 1.91 ± 0.04b 0.18 ± 0.02c 0.24 ± 0.04c
Chl a + b (lg/ml) a
8.57 ± 0.50 0.35 ± 0.12e 0.98 ± 0.23d 4.60 ± 0.29c 4.04 ± 0.16b 0.43 ± 0.07e 0.45 ± 0.04e
Caro*** (lg/ml) a
1.18 ± 0.03 0.02 ± 0.09e 0.23 ± 0.03d 0.48 ± 0.16c 0.78 ± 0.01b 0.11 ± 0.03de 0.12 ± 0.03de
Chl a/b
Caro/Chl a + b a
1.59 ± 0.06 0.94 ± 0.6b 1.44 ± 0.17a 1.30 ± 0.12ab 1.64 ± 0.03a 1.43 ± 0.3b 0.90 ± 0.15b
0.14 ± 0.01a 0.11 ± 0.36a 0.23 ± 0.03a 0.10 ± 0.03a 0.15 ± 0.01a 0.26 ± 0.04a 0.26 ± 0.06a
Different superscript letters within column indicates significant differences at p < 0.05. Abbreviations: NP WTout; NP+ without NaHCO3, NP WT; NP+ with 0.6 g/L NaHCO3, NP WTout; N+P without NaHCO3, NP WT; N+P with 0.6 g/L NaHCO3, NP WTout; NP without NaHCO3, NP WT; NP 0.6 g/L NaHCO3. * Chl-a: chlorophyll-a. ** Chl-b: chlorophyll-b. *** Caro: carotenoids.
4. Conclusion In summary, results of the present study show that addition of 0.6 g/L sodium bicarbonate to the growth medium (BG-11) significantly enhance the biomass, lipid and carbohydrate content of microalgae Scenedesmus sp. CCNM 1077. Addition of sodium bicarbonate under nitrate and phosphate starvation conditions also show stress ameliorating effects by improving biomass productivity. Among all the tested conditions phosphate starvation under bicarbonate supplementation was best approach due to higher carbohydrate and lipid production with marginal loss in biomass production compared to BG-11 grown cultures. This suggests an efficient approach for the sequential production of microalgal based biodiesel and bioethanol. Acknowledgements CSIR-CSMCRI Registration Number: 036/2015. IP, TG, CP and RM would like to acknowledge CSIR for awarding Senior Research Fellowship. The authors gratefully acknowledge CSC 0203 for providing the financial support. 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 Mr. Satyavver Gothwal, ADCIF, CSIR-CSMCRI, Bhavnagar for his help during the CHNS analysis. IP, KC, TG, CP and RM wish to acknowledge AcSIR for Ph.D. enrollment. 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.2015.06. 107. References 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. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Cataldo, D.A., Maroon, M., Schrader, L.E., Youngs, V.L., 1975. Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid 1. Commun. Soil Sci. Plant 6, 71–80. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306. Chokshi, K., Pancha, I., Trivedi, K., George, B., Maurya, R., Ghosh, A., Mishra, S., 2015. Biofuel potential of the newly isolated microalgae Acutodesmus dimorphus under temperature induced oxidative stress conditions. Bioresour. Technol. 180, 161–171. Chu, F.F., Chu, P.N., Cai, P.J., Li, W.W., Lam, P.K., Zeng, R.J., 2013. Phosphorus plays an important role in enhancing biodiesel productivity of Chlorella vulgaris under nitrogen deficiency. Bioresour. Technol. 134, 341–346.
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