Bioresource Technology 193 (2015) 315–323

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Bioresource Technology

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Bicarbonate supplementation enhanced biofuel production potentialas well as nutritional stress mitigation in the microalgae Scenedesmussp. CCNM 1077

http://dx.doi.org/10.1016/j.biortech.2015.06.1070960-8524/� 2015 Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Discipline of Salt & Marine Chemicals, CSIR-CentralSalt 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: smishra@csmcri.org (S. Mishra).

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, IndiaAcademy 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.

a r t i c l e i n f o

Article history:Received 25 May 2015Received in revised form 18 June 2015Accepted 19 June 2015Available online 25 June 2015

Keywords:MicroalgaeSodium bicarbonateNutritional starvationNeutral lipidCarbohydrate

a b s t r a c t

The aim of the present study was to find out the optimum sodium bicarbonate concentration to producehigher 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 eval-uated. The results clearly indicate that 0.6 g/L sodium bicarbonate was optimum concentration resultingin 20.91% total lipid and 25.56% carbohydrate along with 23% increase in biomass production comparedto normal growth condition. Addition of sodium bicarbonate increased the activity of nutrient assimila-tory enzymes, biomass, lipid and carbohydrate contents under different nutritional starvation conditions.Nitrogen starvation with bicarbonate supplementation resulted in 54.03% carbohydrate and 34.44% totallipid content in microalgae Scenedesmus sp. CCNM 1077. These findings show application of bicarbonategrown microalgae Scenedesmus sp. CCNM 1077 as a promising feedstock for biodiesel and bioethanolproduction.

� 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Microalgae are considered as green bio-factories for the produc-tion of various bioactive compounds like pigments, fatty acids,vitamins etc. Apart from its nutraceutical importance, microalgaeare recently considered as a promising renewable energy feedstock due to their high growth rate, photosynthetic ability, highlipid and carbohydrate contents as well as their ability to obtainnutrients from wastewaters (Chisti, 2007). To make microalgalbased biofuel economically viable, downstream processing like

harvesting, dewatering and biodiesel production must be opti-mized; at the same time, it is also important to improve biomass,lipid and carbohydrate productivity of microalgae to generatehigher biomass with high lipid and carbohydrate contents(Wijffels and Barbosa, 2010).

Cultural conditions like nutrients concentration and composi-tion, light intensity and photoperiod, pH, temperature, etc. signifi-cantly affect the growth and biochemical composition ofmicroalgae (George et al., 2014). Many cultivation strategies likenitrogen starvation, salinity stress and temperature stress signifi-cantly 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 rateand biomass productivity, which ultimately increase the biofuelproduction cost. On the other hand, heterotrophic and mixotrophic

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growth utilizing various types of organic sources have beenresulted in higher biomass along with higher lipid and carbohy-drate contents in microalgae (Pancha et al., 2015b). However, allmicroalgae are unable to grow under such growth conditions;moreover, these conditions result in high initial operating costdue to utilization of costly organic substrates.

Supply of inorganic carbon in the form of CO2 is important foractive photosynthesis, carbon dioxide fixation and generation ofbiomass with desired products in microalgae. Many microalgaehave the ability to uptake dissolved inorganic carbon (DIC) fromthe surrounding for active photosynthesis by CO2 concentratingmechanism (El-Ansari and Colman, 2015). In various microalgaeaddition of sodium bicarbonate to the culture medium resultedin high pH as well as high DIC in the growth medium whichincreased their growth and higher TAG accumulation (Gardneret al., 2012; White et al., 2013). However, the role of bicarbonatesupplementation under various nutritional starvation conditionslike nitrate and phosphate starvations as well as its involvementin active uptake of nutrients, lipid and carbohydrate accumulationin 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 sodiumbicarbonate concentrations to find out the optimum concentrationto obtain higher biomass, lipid and carbohydrate contents. Next,the combined effect of bicarbonate addition and nutritional starva-tion on morphological and biochemical parameters including cellmorphology, dry cell weight, lipid, carbohydrate, protein, nutrientsassimilatory enzymes and photosynthetic pigments were used tounderstand the physiological mechanism of bicarbonate supple-mentation on lipid and carbohydrate accumulation in the microal-gae 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 (Panchaet al., 2014). For the determination of ideal bicarbonate concentra-tion, microalgae Scenedesmus sp. CCNM 1077 was first grown inBG-11 medium with different concentrations of sodium bicarbon-ate i.e. 0, 0.3, 0.6, 0.9, 1.2 and 1.5 g/L. After obtaining best bicarbon-ate concentration, its effect on nitrate starvation (N�P+),phosphate starvation (N+P�) and combined nitrate phosphatestarvation (N�P�) was evaluated under bicarbonate supplementedand non-supplemented conditions.

All the experiments were conducted in triplicate in 1 L flaskscontaining 500 ml of respective culture medium and inoculatedwith 10% actively growing culture of Scenedesmus sp. CCNM1077. All the experiments were performed in batch culture at25 ± 2 �C with 12:12 h light dark period under 60 lmol m�2 s�1

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 productivityMicroalgal 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 pigmentsFor the determination of pigments content, 2 ml microalgal cul-

ture was centrifuged at 10,000 rpm for 5 min, supernatant was

discarded and pellet was mixed with 99.9% methanol and incu-bated in dark for 24 h at 45 �C. After incubation, pigments contentwas 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 tur-bidity by subtracting absorbance at 750 nm (Lichtenthaler, 1987).

2.2.3. Determination of lipid, protein and carbohydrate contentsLipid was extracted using chloroform:methanol (1:2 v/v) and

determined gravimetrically (Bligh and Dyer, 1959). Total lipidwas further fractionated into neutral lipid using chloroform/aceticacid (9:1, v/v), glycolipids using acetone/methanol (9:1, v/v) andphospholipids using methanol as described in Pancha et al. (2014).

CHNS content in microalgal biomass was determined throughelemental analysis using a CHNS analyzer (Perkin-Elmer Model 2400, USA). Crude protein content of microalgae was calculatedaccording 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 with500 ll of 72% (w/v) H2SO4 for 1 h. Concentration of H2SO4 wasreduced to 4% by the addition of distilled water and autoclavedat 121 �C for 1 h. After cooling down to room temperature, totalvolume was made up to 50 ml with distilled water (Van Wychenand Laurens, 2013). The resulting solution was centrifuged at10,000 rpm for 10 min and the supernatant was used for sugarestimation by phenol sulfuric acid method (Dubois et al., 1956)using D-glucose as a standard.

2.2.4. Determination of nutrients concentrationsResidual nitrate concentration in the microalgal culture med-

ium was estimated using salicylic acid and sodium hydroxide(Cataldo et al., 1975). Residual phosphate concentration in themicroalgal culture medium was estimated using ascorbic method(Grasshoff et al., 1999).

2.2.5. Determination of nutrients assimilatory enzymesNitrate reductase (NR) activity of microalgal cells was deter-

mined according to the method described by Li et al. (2011).Alkaline (ALP) and acid phosphates (AP) activities were determinedaccording to Lee (2000). Total soluble protein content was mea-sured 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 wereassessed by analysis of variance (ANOVA) using the Info stat soft-ware package (2012). The mean values were compared with LSDtest (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 utilizingatmospheric CO2 as an inorganic carbon source. Since CO2 has very

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low solubility in water, bicarbonate addition is carried out in openpond large scale microalgal cultivation to provide desired inorganiccarbon to generate more biomass. Addition of sodium bicarbonateto the growth medium has been reported to significantly increasethe biomass and biochemical composition of various microalgae(White et al., 2013). In the present study, addition of sodium bicar-bonate to the growth medium significantly enhanced the DCW andbiomass productivity of microalgae Scenedesmus sp. CCNM 1077(Fig. 1a) which indicates that sodium bicarbonate supplementationenhances the cell division and metabolic process in microalgae.Addition of sodium bicarbonate (0–1.2 g/L) increased DCW andbiomass productivity in a dose dependent manner and no signifi-cant difference (p < 0.05) in DCW production was observed beyond0.6 g/L sodium bicarbonate concentration. Biomass productivity ofScenedesmus sp. CCNM 1077 was largely unaffected in the range of0.6–1.5 g/L sodium bicarbonate supplemented growth medium.The highest biomass productivity was found in cultures grownwith 1.2 g/L sodium bicarbonate (28.32 ± 0.77 mg/L/day) whichwas 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) alsofound higher biomass production in diatom Phaeodactylum tricor-nutum cultivated in the presence of sodium bicarbonate.

In the present study, nitrate and phosphate consumption trendwas recorded to correlate the effect of sodium bicarbonate

Fig. 1. (a) Effect of different NaHCO3 concentration on DCW and biomassproductivity of Scenedesmus sp. CCNM 1077. (b) Effect of different NaHCO3

concentration on biochemical composition of Scenedesmus sp. CCNM 1077 (Valueswith different letters represent significant difference at p < 0.05 betweentreatments).

supplementation on active uptake of nutrients by microalgaeScenedesmus sp. CCNM 1077 (Fig. 2). The results indicate thatuptake of nitrate and phosphate from the growth mediumincreased with the increase in bicarbonate addition to the growthmedium. Addition of only 0.3 g/L sodium bicarbonate to thegrowth medium resulted in 66 ± 1.87% phosphate uptake and33 ± 1.71% nitrate uptake from the growth medium, which washigher than those of the control culture. Lee and Lee (2002)reported that high CO2 grown Chlorella kessleri culture had highernitrate consumption possibly due to enhanced nitrate absorptionefficacy of the cells. In the present study, highest consumption ofnitrate (37.58 ± 2.39%) and phosphate (78.58 ± 0.56%) was foundin cells cultivated under 0.9 g/L sodium bicarbonate. HighestDCW (549.15 ± 13.13 mg/L) production was found in cells culti-vated with 1.2 g/L sodium bicarbonate. However, further increasein bicarbonate concentration to 1.5 g/L showed no significant dif-ference in DCW production (547 ± 16.42 mg/L) but showedreduced nutrient consumptions (63.55 ± 0.13% phosphate and35.12 ± 1.33% nitrate) by Scenedesmus sp. CCNM 1077. Lower nutri-ent consumption under high bicarbonate supplementation mightbe due to increase in pH of the growth medium since it has beenknown that many salts of the marine culture medium gets precip-itated and become unavailable for consumption by microalgae(Guihéneuf and Stengel, 2013).

Fig. 2. (a) Nitrate consumption trend of Scenedesmus sp. CCNM 1077 in differentinitial 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 biochemicalcomposition

Effect of initial sodium bicarbonate concentrations on lipid,crude protein and carbohydrate content of microalgaeScenedesmus sp. CCNM 1077 is shown in Fig. 1b. Crude protein con-tent of microalgae Scenedesmus sp. CCNM 1077 increased in a dosedependent manner. Highest crude protein content (50.12 ± 0.23%)was observed in cells grown with 1.5 g/L sodium bicarbonate fol-lowed by 1.2 (49.46 ± 0.41%) and 0.9 g/L (49.56 ± 0.37%) sodiumbicarbonate supplemented cultures.

Carbon availability and supply is a key metabolic regulatorwhich governs the carbohydrate and lipid synthesis in microalgae(Fan et al., 2012). Fig. 1b also shows that addition of 0.9 g/L sodiumbicarbonate to the growth medium increased the carbohydratecontent from 18.55 ± 0.79 to 30.88 ± 1.47%. The results also indi-cate a positive correlation between sodium bicarbonate supple-mentation and cellular carbohydrate content in microalgaeScenedesmus sp. CCNM 1077. It has been reported that high avail-ability of inorganic carbon results in high activity of Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) which is a keyenzyme for the conversion of 3-phosphoglycerate, a substrate forthe biosynthesis of carbohydrate and fatty acid in plants andmicroalgae (Peng et al., 2014). There was no significant difference(p < 0.05) in carbohydrate accumulation pattern in cells cultivatedwith 0.3 g/L (24.57 ± 0.72%) and 0.6 g/L (25.56 ± 0.84%) sodiumbicarbonate. Many recent studies show that microalgal carbohy-drates are better than plant and seaweed derived carbohydratesdue to high starch content, which can be easily converted into hex-ose sugar. Moreover, microalgal carbohydrates do not contain lig-nin which significantly reduces the pretreatment cost forbio-ethanol production (Yeh and Chang, 2011; Markou et al.,2012; Harun and Danquah, 2011). In the present study, carbohy-drate content was estimated from the left over de-oiled microalgalbiomass making its application towards biodiesel and bioethanolcoproduction which will ultimately reduce their production cost.

Similar to carbohydrate, addition of sodium bicarbonate alsoenhanced 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 inor-ganic carbon in the form of sodium bicarbonate microalgaePavlova lutheri produce high amount of LC-PUFA rich TAGs whichare mainly generated from de novo synthesis rather than mem-brane remodeling. In the present study, no significant differencein lipid content was observed in 1.2 g/L (19.36 ± 0.23%) and1.5 g/L (19.07 ± 0.29%) sodium bicarbonate supplemented cultures.In order to understand the role of sodium bicarbonate addition onlipid class composition, total lipid content of microalgaeScenedesmus sp. CCNM 1077 was fractionated into neutral lipid, gly-colipids and phospholipids utilizing silica gel G column chromatog-raphy. Addition of sodium bicarbonate to the growth mediumincreased the NL content and reduced the GL and PL contents ofmicroalgae Scenedesmus sp. CCNM 1077 (Fig. 3). Addition of0.6 g/L sodium bicarbonate to the growth medium resulted in75.8 ± 0.85% NL of total lipid, which was about 1.12 folds higherthan that of the BG-11 grown culture (67.24 ± 0.55% of TL).Reduction in GL and PL contents indicate changes in cell membranecomposition of microalgae Scenedesmus sp. CCNM 1077. Addition ofbicarbonate to the culture medium enhances its pH and microalgaechange their membrane composition to withstand this alkalinestress. GL content decreased from 16.4 ± 0.14% to 14.9 ± 0.1% in1.2 g/L bicarbonate grown cultures; similarly, PL content alsodecreased from 15.6 ± 0.18% to 8.13 ± 0.28% in 0.9 g/L bicarbonategrown cultures. Results of this section show that sodium

bicarbonate at concentration of 0.6 g/L is optimum for higher bio-mass generation along with higher lipid and carbohydrate contentsin microalgae Scenedesmus sp. CCNM 1077.

3.3. Effect of sodium bicarbonate supplementation on pigmentscomposition

Addition of sodium bicarbonate to the growth mediumincreased all the photosynthetic pigments viz. Chl-a, Chl-b andtotal carotenoids content of microalgae Scenedesmus sp. CCNM1077 (Table 1). Increase in sodium bicarbonate concentration from0 to 1.2 g/L significantly (p < 0.05) enhanced the Chl-a and b as wellas total carotenoids contents. Highest amount of Chl-a(6.11 ± 1.14 lg/ml) was found in 0.9 g/L sodium bicarbonate grownculture, which was about 1.17 folds higher than the BG-11 grownculture (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 contentin Tetraselmis suecica and Nannochloropsis salina, respectively,when cultivated under 1 g/L sodium bicarbonate supplementationcompared to non bicarbonate supplemented cultures. Addition ofsodium bicarbonate (up to 1.2 g/L) resulted in the increase in totalcarotenoids of the cells in dose dependent manner. Highestamount of total carotenoids (1.53 ± 0.10 lg/ml) was found in1.2 g/L sodium bicarbonate supplemented culture followed by0.9 g/L (1.45 ± 0.15 lg/ml) and 0.6 g/L (1.38 ± 0.09) sodium bicar-bonate supplemented cultures. Addition of sodium bicarbonate(up to 1.2 g/L) to the growth medium increased the ratios of Chla/b as well as Caro/total Chl. In this study, we supplemented bicar-bonate to the growth medium that increase the culture alkalinityand pH, which might be the reason for higher ratio of Chl a/b,Caro/total Chl in microalgae Scenedesmus sp. CCNM 1077. In a sim-ilar study, no significant difference in pigments ratio was foundwhen T. suecica was grown with supplementation of sodium bicar-bonate, while in case of N. salina, carotenoids: Chl-a ratio increasedwith the increase in sodium bicarbonate in the growth medium(White et al., 2013).

3.4. Effect of sodium bicarbonate supplementation on DCW andbiomass productivity under nutritional starvation conditions

Microalgal lipid and carbohydrate contents generally increasein various nutritional stress conditions like nitrate and phosphatestarvation. However, these nutritional starvation conditions resultin lower DCW and biomass productivity in microalgae. In thisstudy, addition of inorganic carbon in the form of sodium bicarbon-ate not only increased the biomass content of Scenedesmus sp.CCNM 1077 but also resulted in higher accumulation of neutrallipid and carbohydrate. To check the role of inorganic carbon sup-plementation under nutrition starvation condition, Scenedesmussp. CCNM 1077 was grown under nitrate starvation (N�P+), phos-phate starvation (N+P�) and combined nitrate and phosphate star-vation (N�P�) with and without 0.6 g/L sodium bicarbonatesupplementation.

Fig. 4a shows effect of sodium bicarbonate addition on DCWand biomass productivity under nutrient starvation conditions.From the data it is clear that removal of either nitrate or phosphateor both from the growth medium significantly affects the meta-bolic process of microalgae Scenedesmus sp. CCNM 1077 andresults in lower DCW and biomass productivity. However, additionof 0.6 g/L sodium bicarbonate reduced the stress effects with theincrease in DCW and biomass production under all the testednutritional stress conditions. Our earlier study with microalgaeScenedesmus sp. CCNM 1077 (Pancha et al., 2014) showed thatremoval of nitrate from the growth medium resulted in 46% reduc-tion in DCW compared to control culture. However, in the presentstudy, addition of 0.6 g/L bicarbonate helped in reducing the stress

Table 1Effect of different sodium bicarbonate concentration on pigments composition of Scenedesmus sp. CCNM 1077.

NaHCO3 (g/L) Chl-a* (lg/ml) Chl-b** (lg/ml) Chl a + b (lg/ml) Caro*** (lg/ml) Chl a/b Caro/Chl a + b

0 5.18 ± 0.32a 3.29 ± 0.25b 8.47 ± 0.56a 1.02 ± 0.06d 1.58 ± 0.03a 0.12 ± 0.00c

0.3 5.81 ± 0.23a 3.56 ± 0.22ab 9.37 ± 0.44a 1.25 ± 0.01bc 1.63 ± 0.05a 0.13 ± 0.007abc

0.6 6.02 ± 0.48a 3.51 ± 0.28ab 9.53 ± 0.76a 1.38 ± 0.09abc 1.72 ± 0.01a 0.14 ± 0.00ab

0.9 6.11 ± 1.14a 3.53 ± 0.79ab 9.64 ± 1.94a 1.45 ± 0.15ab 1.74 ± 0.06a 0.15 ± 0.01a

1.2 6.10 ± 0.16a 3.67 ± 0.45ab 9.78 ± 0.61a 1.53 ± 0.10a 1.67 ± 0.15a 0.16 ± 0.02a

1.5 5.19 ± 1.05a 4.13 ± 0.59a 9.32 ± 1.05a 1.15 ± 0.27cd 1.28 ± 0.33b 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.

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).

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effects and 38% reduction in DCW was observed. Removal of both,nitrate and phosphate, from the growth medium resulted in178 ± 1.67 mg/L DCW, while addition of 0.6 g/L bicarbonate aug-mented DCW production i.e. 221.13 ± 6.07 mg/L under combinednutrient starvation. Similar to our study, Chu et al. (2013) alsofound reduction in total DCW production by microalgae Chlorellavulgaris 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). Inthe present study, DCW production was not much affectedunder phosphate starvation and only 19% reduction in DCW(320.87 ± 11.68 mg/L) was observed.

Biomass productivity was largely unaffected in phosphatestarvation condition (15.74 ± 0.84 mg/L/day) as well as bicarbonatesupplemented phosphate starvation conditions (18.08 ± 0.31mg/L/day) compared to control culture (20.77 ± 0.28 mg/L/day).Removal of nitrate or nitrate and phosphate, both, from the growthmedium 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 presentsection shows that nitrate is a vital nutrient for normal growthand metabolism of microalgae Scenedesmus sp. CCNM 1077 andaddition of bicarbonate has nutrient stress ameliorating effectswhich 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 morphologyfrom single cell to coenobium of 2 and 4 under nitrate starvationcondition (Pancha et al., 2014). In the present study too, nitrateor combined nitrate and phosphate starvations resulted in 4 cellscoenobium with spines at terminal cells. However, size of 4 cellscoenobium was smaller in combined stress compared to onlynitrogen stressed culture. Addition of bicarbonate did not showany significant effect on cell morphology under starvation condi-tions. Compared to control culture, cells grown under phosphatestarvation did not show any significant change in their morphol-ogy; however, addition of bicarbonate resulted in slightly larger

Fig. 4. (a) Effect of sodium bicarbonate supplementation on DCW and biomassproductivity under nutritional starvation conditions. (b) Effect of sodium bicar-bonate supplementation on biochemical composition under nutritional starvationconditions (Values with different letters represent significant difference at p < 0.05between treatments).

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cell size. It has been reported that larger cell has slow metabolicrate which helps in saving energy from damage and repair as wellas the amount of energy stored is directly propositional to cell sizeof microalgae (Šetlík et al., 1972).

Fig. 5. (a) Nitrate consumption trend of Scenedesmus sp. CCNM 1077 with sodiumbicarbonate supplementation under nutritional starvation conditions. (b)Phosphate consumption trend of Scenedesmus sp. CCNM 1077 with sodiumbicarbonate supplementation under nutritional starvation conditions. (c) Changesin nutrient assimilatory enzymes of Scenedesmus sp. CCNM 1077 with sodiumbicarbonate supplementation under nutritional starvation conditions (Values withdifferent letters represent significant difference at p < 0.05 between treatments.)(Abbreviations: N�P WTout; N�P+ without NaHCO3, N�P WT; N�P+ with 0.6 g/LNaHCO3, NP�WTout; N+P�without NaHCO3, NP�WT; N+P�with 0.6 g/L NaHCO3,N�P� WTout; N�P� without NaHCO3, N�P�WT; N�P� 0.6 g/L NaHCO3).

3.5. Effect of sodium bicarbonate supplementation on nutrientconsumption and nutrient assimilatory enzymes

Fig. 5 shows the effect of sodium bicarbonate addition on nutri-ent consumption trend of microalgae Scenedesmus sp. CCNM 1077under different nutritional starvation conditions. Addition of0.6 g/L sodium bicarbonate resulted in 46.97 ± 0.67% phosphateconsumption in nitrogen-starved culture, which was 38% higherthan those of the cultures without sodium bicarbonate supplemen-tations. More utilization of phosphate is also evident from higheractivities 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 activityof AP than ALP in Chlorella and Chlamydomonas under nitrate andphosphate starvation conditions. AP helps in liberation of intracel-lular P reserve whereas ALP helps to dissolve organic P from thegrowth medium. In the present study, addition of bicarbonatedid not show any effect on AP and ALP activities in phosphatestarved cultures. This suggests that presence of phosphate isimportant for the induction of both, AP and ALP, in microalgaeScenedesmus sp. CCNM 1077.

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Rate of nitrate assimilation was also increased by addition ofsodium bicarbonate to the growth medium. BG-11 grown cultureconsumed 30 ± 0.44% of nitrate while cultures grown under phos-phate starvation with and without bicarbonate addition consumed28 ± 1.4% and 29.39 ± 1.1% nitrate, respectively. Chu et al. (2014)showed significant reduction in nitrate assimilation under phos-phate starvation by microalgae S. obliquus FACHB-417. Nitratereductase activity was significantly (p < 0.05) affected in variousstress conditions. Removal of both nitrate and phosphate fromthe growth medium did not show any significant difference innitrate reductase activity. However, addition of sodium bicarbon-ate under phosphate starvation resulted in NR activity of0.83 ± 0.0 lmol min�1 mg�1 protein which was almost double thanthat of the culture without bicarbonate supplementation(0.42 ± 0.01 lmol min�1 mg�1 protein). Culture under nitrate star-vation with sodium bicarbonate supplementation resulted in NRactivity of 0.21 ± 0.01 lmol min�1 mg�1 protein which was signif-icantly lower than that of the culture without bicarbonate supple-mentation, but similar to that of the nitrate and phosphate starvedcultures (0.20 ± 0.02 lmol min�1 mg�1 protein).

3.6. Effect of sodium bicarbonate supplementation on biochemicalcomposition under nutritional starvation conditions

Effect of bicarbonate addition on lipid, carbohydrate and pro-tein contents of microalgae Scenedesmus sp. CCNM 1077 is shownin Fig. 4b. As protein is nitrogenous compound, removal of nitrogenfrom the growth medium significantly (p < 0.05) reduced the crudeprotein content from 47.19 ± 0.26% to 11.95 ± 0.14% in combinedstress and to 10.63 ± 0.44% in nitrogen starvation condition.Addition of bicarbonate resulted in 12.93 ± 0.11% and11.76 ± 0.25% crude protein content in combined and nitrogenstarvation conditions, respectively. Similarly, Chu et al. (2014) alsofound sharp decrease in protein content in microalgae S. obliquusFACHB-417 during combine nitrate and phosphate starvation.The degradation of protein helps in maintaining the intracellularnitrogen pool in microalgae, thereby helping in normal functioningof cellular metabolism. Phosphate starvation did not show any sig-nificant effect on crude protein content of microalgae Scenedesmussp. CCNM 1077 and resulted in 39.89 ± 0.22% crude protein.

Lipid and carbohydrate are the two most reduced storage prod-ucts in microalgae, generally accumulated under various nutri-tional stress conditions due to their hydrophobic nature, morereduced state and efficient storage in smaller compartments ofthe cells (Courchesne et al., 2009). Fig. 4b also shows effect ofbicarbonate addition on carbohydrate content of microalgaeScenedesmus sp. CCNM 1077. As per our earlier observation, nutri-ent limitation results in significantly higher accumulation of carbo-hydrate and its synthesis is dominated over lipid production inmicroalgae Scenedesmus sp. CCNM 1077 (Pancha et al., 2014).Highest accumulation of carbohydrate (54.03 ± 1.08%) was foundunder nitrogen limitation with bicarbonate supplementation fol-lowed by combined nutrient starvation with bicarbonate supple-mentation (51.25 ± 0.67%). Total removal of phosphate from thegrowth medium resulted in 27.47 ± 0.49% carbohydrate which fur-ther increased up to 29.86 ± 0.26% during 0.6 g/L bicarbonate sup-plementation. These results indicate that addition of bicarbonateunder various nutritional stress conditions significantly enhancedthe carbohydrate content in microalgae Scenedesmus sp. CCNM1077. Among all, phosphate starvation was found to be a goodalternative as it resulted in comparatively lesser biomass loss withrelatively higher lipid and carbohydrate contents in microalgaeScenedesmus sp. CCNM 1077.

Nutrients limitations like nitrate, phosphate or combinednitrate and phosphate limitations have resulted in higher

accumulation of lipid in various microalgae (Chu et al., 2013,2014; Li et al., 2014). However, only few researchers have studiedthe effects of bicarbonate addition under nitrate limited growthconditions (White et al., 2013; Guihéneuf and Stengel, 2013) andthere are no reports on effects of bicarbonate addition under phos-phate or combined nitrate phosphate stress conditions in microal-gae. Total lipid content of microalgae Scenedesmus sp. CCNM 1077was increased from 17.85 ± 0.14% to 34.42 ± 0.27% under nitrogenstarvation with bicarbonate addition, which was significantlyhigher than that of without bicarbonate addition (28.05 ± 0.08%).This indicates that inorganic carbon supply under nitrogen starva-tion helps in de novo synthesis of microalgal lipids. Similar to ourstudy, Guihéneuf and Stengel (2013) also found high TAGs in bicar-bonate supplemented nitrogen starved culture of marine hapto-phyte Pavlova lutheri. In the present study, Scenedesmus sp.CCNM 1077 grown under combined nutrition starvation withbicarbonate supplementation did not show any significant increasein total lipid content (33.29 ± 0.42%) compared to that of nitratestarved bicarbonate supplemented culture. Li et al. (2014) havereported higher lipid accumulation in Chlorella during nitrate phos-phate co-deficiency compared to only nitrate starvation. In the pre-sent study, phosphate starvation with bicarbonate additionresulted in 27.97 ± 0.24% total lipid, which was similar to that ofnitrogen starved culture. This treatment can be used as an efficientapproach to produce microalgal based biofuel as it resulted in lim-ited loss of biomass with higher lipid and carbohydrate contents,which can be converted into biodiesel and bioethanol, respectively.Sodium bicarbonate supplementation had significant effect onlipid class composition of microalgae Scenedesmus sp. CCNM1077 (Fig. S1). Neutral lipid content of 88.69 ± 2.1% of total lipidwas observed in nitrate starved bicarbonate supplemented cellswhich was 1.3 folds higher than that of the BG-11 grown culture(67.2 ± 0.55%). Nitrate and phosphate starvation significantlyreduced the GL and PL content of microalgae Scenedesmus sp.CCNM 1077. NL is mainly composed of TAGs which are importantfor the production of microalgal based biodiesel, while GL and PLare the components of cell membranes (Olmstead et al., 2013).Addition of sodium bicarbonate under all tested nutritional starva-tion conditions resulted in higher NL content compared to that ofthe cultures grown without bicarbonate addition, indicating posi-tive role of sodium bicarbonate in NL accumulation in microalgaeScenedesmus sp. CCNM 1077.

3.7. Effect of sodium bicarbonate supplementation on pigments undernutritional starvation conditions

Pigments content and composition are most affected duringvarious nutritional stress conditions like nitrogen and salinitystress since pigments are mainly composed of nitrogen rich com-pounds (Pancha et al., 2014). To gain insight into the photosyn-thetic process under various stress conditions, Chl-a, Chl-b andtotal carotenoids content were measured (Table 2). All the stressconditions resulted in lower Chl-a content, which was also evidentfrom the color of cultures from green to yellow and light yellow inphosphate and nitrate combined stress conditions. As shown inTable 2, removal of nitrate from the culture medium resulted in0.15 ± 0.02 lg/ml Chl-a. However, addition of sodium bicarbonateresulted in 0.58 ± 0.11 lg/ml Chl-a, which was about 3.8 foldshigher than that of the cells without bicarbonate supplementation.Addition of sodium bicarbonate did not show much effect on pig-ment content under combined stress conditions. Pigments contentof phosphate starved cultures were least affected i.e. 2.59 ± 0.19and 3.13 ± 0.12 lg/ml Chl-a in cultures without and with bicarbon-ate supplementation, respectively.

Table 2Effect of sodium bicarbonate supplementation on pigments composition under nutritional starvation conditions.

Treatments Chl-a* (lg/ml) Chl-b** (lg/ml) Chl a + b (lg/ml) Caro*** (lg/ml) Chl a/b Caro/Chl a + b

Control 5.26 ± 0.24a 3.31 ± 0.27a 8.57 ± 0.50a 1.18 ± 0.03a 1.59 ± 0.06a 0.14 ± 0.01a

N�P WTout 0.15 ± 0.02e 0.20 ± 0.11c 0.35 ± 0.12e 0.02 ± 0.09e 0.94 ± 0.6b 0.11 ± 0.36a

N�P WT 0.58 ± 0.11d 0.41 ± 0.12c 0.98 ± 0.23d 0.23 ± 0.03d 1.44 ± 0.17a 0.23 ± 0.03a

NP� WTout 2.59 ± 0.19c 2.0 ± 0.15b 4.60 ± 0.29c 0.48 ± 0.16c 1.30 ± 0.12ab 0.10 ± 0.03a

NP� WT 3.13 ± 0.12b 1.91 ± 0.04b 4.04 ± 0.16b 0.78 ± 0.01b 1.64 ± 0.03a 0.15 ± 0.01a

N�P� WTout 0.25 ± 0.06e 0.18 ± 0.02c 0.43 ± 0.07e 0.11 ± 0.03de 1.43 ± 0.3b 0.26 ± 0.04a

N�P� WT 0.21 ± 0.02e 0.24 ± 0.04c 0.45 ± 0.04e 0.12 ± 0.03de 0.90 ± 0.15b 0.26 ± 0.06a

Different superscript letters within column indicates significant differences at p < 0.05.Abbreviations: N�P WTout; N�P+ without NaHCO3, N�P WT; N�P+ with 0.6 g/L NaHCO3, NP�WTout; N+P� without NaHCO3, NP�WT; N+P� with 0.6 g/L NaHCO3, N�P�WTout; N�P� without NaHCO3, N�P� WT; N�P� 0.6 g/L NaHCO3.

* Chl-a: chlorophyll-a.** Chl-b: chlorophyll-b.

*** Caro: carotenoids.

322 I. Pancha et al. / Bioresource Technology 193 (2015) 315–323

4. Conclusion

In summary, results of the present study show that addition of0.6 g/L sodium bicarbonate to the growth medium (BG-11) signif-icantly enhance the biomass, lipid and carbohydrate content ofmicroalgae Scenedesmus sp. CCNM 1077. Addition of sodium bicar-bonate under nitrate and phosphate starvation conditions alsoshow stress ameliorating effects by improving biomass productiv-ity. Among all the tested conditions phosphate starvation underbicarbonate supplementation was best approach due to higher car-bohydrate and lipid production with marginal loss in biomass pro-duction compared to BG-11 grown cultures. This suggests anefficient approach for the sequential production of microalgalbased biodiesel and bioethanol.

Acknowledgements

CSIR-CSMCRI Registration Number: 036/2015. IP, TG, CP andRM would like to acknowledge CSIR for awarding SeniorResearch Fellowship. The authors gratefully acknowledge CSC0203 for providing the financial support. The continuous supportfrom Dr. Arvind Kumar, DC, SMC and the entire staff of the divisionis gratefully acknowledged. The authors would like to thankMr. Satyavver Gothwal, ADCIF, CSIR-CSMCRI, Bhavnagar for hishelp during the CHNS analysis. IP, KC, TG, CP and RM wish toacknowledge AcSIR for Ph.D. enrollment.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2015.06.107.

References

Becker, E.W., 1994. Microalgae: Biotechnology and Microbiology. CambridgeUniversity 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 ofmicrogram 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 colorimetricdetermination 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 dimorphusunder 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 animportant role in enhancing biodiesel productivity of Chlorella vulgaris undernitrogen 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 onbiodiesel production from Scenedesmus obliquus under nitrogen-deficiencystress. Bioresour. Technol. 152, 241–246.

Courchesne, N.M.D., Parisien, A., Wang, B., Lan, C.Q., 2009. Enhancement of lipidproduction using biochemical, genetic and transcription factor engineeringapproaches. J. Biotechnol. 141, 31–41.

Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P., Smith, F., 1956. Colorimetricmethod 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 algaChlorella kessleri. Physiol. Plant. 153, 175–182.

Fan, J., Yan, C., Andre, C., Shanklin, J., Schwender, J., Xu, C., 2012. Oil accumulation iscontrolled by carbon precursor supply for fatty acid synthesis inChlamydomonas 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 inthe 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 onmorphology 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, thirded. 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 triggeredby nitrogen limitation and inorganic carbon availability in the marinehaptophyte Pavlova lutheri. Mar. Drugs 11, 4246–4266.

Harun, R., Danquah, M.K., 2011. Enzymatic hydrolysis of microalgal biomass forbioethanol production. Chem. Eng. J. 168, 1079–1084.

Kruskopf, M.M., Du Plessis, S., 2004. Induction of both acid and alkaline phosphataseactivity 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 microalgaewithout consuming organic carbon sources. J. Microbiol. Biotechnol. 12, 979–985.

Lee, T.M., 2000. Phosphate starvation induction of acid phosphatase in Ulva lactucaL. (Ulvales, Chlorophyta). Bot. Bull. Acad. Sin., 41

Li, Y., Han, D., Sommerfeld, M., Hu, Q., 2011. Photosynthetic carbon partitioning andlipid 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 lipidaccumulation and growth characteristic of the green alga Chlorella withcombination cultivation mode of nitrogen (N) and phosphorus (P). Bioresour.Technol. 174, 24–32.

Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: pigments of photosyntheticbiomembranes. Methods Enzymol. 148, 350–382.

Markou, G., Angelidaki, I., Georgakakis, D., 2012. Microalgal carbohydrates: anoverview of the factors influencing carbohydrates production, and of mainbioconversion 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 optimizationof 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 themicroalgae 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 potentialof microalgae Scenedesmus sp. CCNM 1077. Bioresour. Technol. 189, 341–348.

Pancha, I., Chokshi, K., Mishra, S., 2015b. Enhanced biofuel production potential withnutritional stress amelioration through optimization of carbon source and lightintensity in Scenedesmus sp. CCNM 1077. Bioresour. Technol. 179, 565–572.

I. Pancha et al. / Bioresource Technology 193 (2015) 315–323 323

Peng, X., Liu, S., Zhang, W., Zhao, Y., Chen, L., Wang, H., Liu, T., 2014. Triacylglycerolaccumulation of Phaeodactylum tricornutum with different supply of inorganiccarbon. J. Appl. Phycol. 26, 131–139.

Šetlík, I., Berková, E., Doucha, J., Kubín, Š., Vendlová, J., Zachleder, V., 1972. Thecoupling of synthetic and reproduction processes in Scenedesmus quadricauda.Arch. Hydrobiol. Algolog. Stud. 7, 172–217.

Van Wychen, S., Laurens, L.M.L., 2013. Determination of Total Carbohydrates inAlgal Biomass: Laboratory Analytical Procedure (LAP) (No. NREL/TP-5100-60957). National Renewable Energy Laboratory (NREL), Golden CO.

White, D.A., Pagarette, A., Rooks, P., Ali, S.T., 2013. The effect of sodium bicarbonatesupplementation on growth and biochemical composition of marine microalgaecultures. J. Appl. Phycol. 25, 153–165.

Wijffels, R.H., Barbosa, M.J., 2010. An outlook on microalgal biofuels. Science 5993,796–799.

Yeh, K.L., Chang, J.S., 2011. Nitrogen starvation strategies and photobioreactordesign for enhancing lipid content and lipid production of a newly isolatedmicroalga Chlorella vulgaris ESP-31: implications for biofuels. Biotechnol. J. 6,1358–1366.