Development of sustainable, clean, and renewable energy sources is currently one of most daunting challenge faced by the world. At present, approximately 90% of global energy demand is fulfilled by fossil fuels, which are on the verge of depletion along with an escalation in greenhouse gas emissions affecting the well-being of all the life forms (Maity et al. 2014). Biofuels are viable alternatives which have the capacity to replace conventional petro-based fuels. Currently, biofuels including bioethanol and biodiesel are being produced from terrestrial-based feed stocks (food and non-food) such as corn, sugarcane molasses/bagasse, soya bean, rapeseed, canola, jatropha, and palm oil (Doshi et al. 2016). However, these feed stocks require large cultivation area, nutrients, and water thereby directly competing with the agriculture resources (Ward et al. 2014). Photosynthetic microalgae capable of growing in waste water, sea water, and river water represent a viable alternative to plant-based fuels due to their high biomass and oil productivity, year round production, marginal use of agricultural land, no requirement of any herbicide/pesticide treatment, and biofixation of atmospheric as well as excess CO2 emissions (Pittman et al. 2011). Microalgae can accumulate ~ 60–70% of lipids (mostly as triacylglycerols) under adverse conditions such as nutritional limitations, temperature/pH/salinity/light fluctuations, and heavy metal stress (Sharma et al. 2015). These triacylglycerols (TAGs) can be converted to biodiesel via a simple transesterification process. However, the economic evaluation of algae biodiesel indicated a selling cost of $5–10.31/gal which is higher than the cost of conventional biodiesel ($ 4.21/gal) (Zhang et al. 2017). Therefore, to reduce the cost of algal biodiesel production, various improvements in algal cultivation encompassing the feedstock cost, bioprospecting of potential high lipid-accumulating strains, and downstream production are quintessential.

Currently, the large-scale cultivation of microalgae is based on large, outdoor open ponds, and closed tubular photobioreactors covering vast land (Perez-Garcia et al. 2011). The main disadvantage associated with such systems is the shelf-shading of microalgal cultures that limits the light availability thereby decreasing the biomass production (Arora et al. 2016b). Strategies to improve the growth of microalgae are to deploy heterotrophic, photoheterotrophic, and mixotrophic cultivation modes. Among these, in mixotrophic culture regime, the microalga assimilates both the inorganic carbon (CO2) and organic carbon sources, i.e., both respiratory and photosynthetic metabolism occur concurrently (Cheirsilp and Torpee 2012). Cultivation of microalgae under mixotrophic cultivation mode has shown to increase the biomass and lipid yields (Mandal and Mallick 2009; Wan et al. 2011; Ramanna et al. 2014). Considering the chemical composition of a microalga (CH1.7O0.4N0.15P0.00094), nitrogen contributes to almost 10% (w/w), while the content of carbon is ~ 20% (w/w) of the total biomass, thus making it vital for the selection of both nitrogen and carbon sources for enhancing the biomass under mixotrophy (Cho et al. 2011). In the present investigation, six different organic and inorganic nitrogen sources such as ammonium chloride (NH4Cl), ammonium bicarbonate (NH4HCO3), sodium nitrite (NaNO2), sodium nitrate (NaNO3), ammonium nitrate (NH4NO3), yeast extract, and urea at a concentration of 0.25 g/L, and eight organic carbon sources (glucose, mannose, maltose, arabinose, lactose, sucrose, galactose, xylose) and two inorganic carbons (sodium acetate, sodium bicarbonate) at a concentration of 2 g/L both individually and in synergy for maximum lipid accumulation in Scenedesmus sp. IITRIND2 were studied. The effects of these nitrogen and carbon sources on microalga’s fatty acid profile were compared to estimate its potential for biodiesel.

Scenedesmus sp. IITRIND2 (Gene bank Accession no. KT932960) isolated previously by our group from a fresh water lake was used as model organism to evaluate the efficiency of different carbon and nitrogen sources cultivated under mixotrophy (Arora et al. 2016a). Bold’s Basal Medium (BBM) was taken as base medium, since it is a simple media containing no inorganic/carbon source and only one nitrogen source as sodium nitrate (Guarnieri et al. 2011). To study the feasibility of six different organic and inorganic nitrogen sources for cultivating Scenedesmus sp. IITRIND2, 0.25 g/L of the listed nitrogen sources was added to the BBM and cultivated at 25 °C and 140 rpm with photoperiod of 18:6-h light/dark cycle under white light illumination (200 μmol/m2/s) for 10 days. Similarly, the microalga was also grown in different organic carbon sources and inorganic carbons. The organic nutrients (2 g/L) were separately sterilized by filtration through 0.22 μm membranes and added in BBM. The microalga was grown in 250 mL of Erlenmeyer flasks containing 75 mL culture in triplicate with initial inoculum of 0.02 g/L (fresh weight) for 10 days. Dry cell weight (DCW; g/L) was evaluated by harvesting the microalgal biomass on the 10th day by centrifugation at 10,000 rpm for 5 min and the dried in hot air oven at 105 °C for 12 h (Arora et al. 2015). The effect of different nitrogen and carbon sources on the biochemical composition of Scenedesmus sp. IITRIND2 was analyzed by estimating changes in total lipid content and yield, total protein, carbohydrate content, and photosynthetic pigments respectively. The total lipid content (g/L) was extracted and estimated using our earlier established protocol (Arora et al. 2015). The total lipid yield was then calculated using the following equation:

$$ \mathrm{Total}\ \mathrm{lipid}\ \mathrm{yield}\ \left(\%\right)=\mathrm{total}\ \mathrm{lipid}\ \mathrm{content}\ \left(\mathrm{g}/\mathrm{L}\right)\times 100/\mathrm{DCW}\ \left(\mathrm{g}/\mathrm{L}\right). $$

The total protein content was measured using elemental CHNS analyzer (Thermo Fischer, USA) while the total carbohydrate content was estimated by phenol sulfuric method using d-glucose as standard (Arora et al. 2016a). The photosynthetic pigments (chlorophyll a, chlorophyll b, and carotenoids) were estimated by preparing methanolic extracts and measuring absorbance at 470, 652.4, 665.2, and 750 nm (to exclude impurities) (Lichtenthaler 1987). Acid-catalyzed transesterification was carried out using H2SO4/methanol (6%) method (Arora et al. 2015). The fatty acid profile was analyzed by GC-MS (Agilent technologies, USA) equipped with DB-5 capillary column (30 mm 0.25 mm 1 μm) and helium (1 ml/min) was used as carrier gas (Arora et al. 2015).

Nitrogen is an essential component for synthesis of biological molecules and can act as a key player in understanding dynamics of microalgal growth and relative change in biochemical composition (Chandra et al. 2019). Scenedesmus sp. IITRIND2 was able to adapt and grow in all the nitrogen sources including NH4Cl, NH4HCO3, NaNO2, NaNO3, NH4NO3, yeast extract, and urea (Fig. 1a). However, depending on the nitrogen source, the DCW, biochemical, and fatty acid methyl ester (FAME) composition varied. The maximum DCW (1.96 ± 0.03 g/L) and total lipid content (0.77 ± 0.02 g/L) were observed in NH4HCO3 followed by NH4NO3> yeast extract = urea > NaNO2> NaNO3> NH4Cl. The reason for higher biomass and lipid accumulation in ammonium sources (NH4HCO3, NH4NO3) could be due to its direct incorporation in the microalga without any prior redox reaction. On the other hand, in case of nitrate, it first reduces to nitrite catalyzed by nitrate reductase, followed by its subsequent conversion to ammonia by nitrite reductase and ferrodoxin thereby decreasing the cellular NADPH levels and imposing burden on the microalgal cells (Ramanna et al. 2014). Among the ammonium sources, the microalga showed enhanced biomass and lipid production in NH4HCO3 due the presence of an additional carbon source apart from the atmospheric CO2. It has been reported that presence of bicarbonate in the growth medium increases the dissolved inorganic carbon (DIC) which boosts the microalgal growth and lipid content (Pancha et al. 2015). Interestingly, despite NH4Cl being an ammonium source, minimum biomass and lipid accumulation were observed (Fig.1a). On analyzing the pH of the medium, a decline in pH (4.2) was recorded on day 3 compared with other cultures whose pH ranged between 7.5 and 9 (data not shown). Indeed, the breakdown of NH4Cl generates positive NH4+ ion which are taken up in exchange of H+, resulting in the formation of HCl. Similar results showing the toxic effects of NH4Cl have been reported for Chlorella saccharophila and Chlorella sorokiniana (Isleten-Hosoglu et al. 2012; Ramanna et al. 2014). Such toxicity imposes a barrier for wastewater utilization by the microalgal strains as most of the industrial wastewaters contain NH4Cl as nitrogen source. However, the microalgae are able to uptake the NH4+ from these wastewaters without significantly affecting the pH due to the presence of high amounts of beneficial salts such as Na+ and K+ which stimulates the assimilation of ammonium ions (Wu et al. 2013). The two organic nitrogen sources (yeast extract and urea) showed moderate effects on both DCW and lipid accumulation (Fig. 1a). Urea dissociates to form CO2 along with ammonia, catalyzed by urease and ATP: urea amidolyase enzymes, while in order to metabolize yeast extract, it requires ATP and NADH for its metabolism (Ramanna et al. 2014; Leftley and Syrett 1973). Indeed, requirement of energy for the metabolism of organic nitrogen sources impedes the growth rate of the microalgal cells and in turn lipid accumulation. Similar results have been reported in various microalgae such as Scenedesmus bijugatus, Scenedesmus sp., Ellipsoidan sp., Isochrysis galbana, and Chlorella sp. (Fidalgo et al. 1998; Xu et al. 2001; Lin and Lin 2011; Ren et al. 2013). However, it is crucial to note here that the utilization of respective nitrogen source for growth and lipid accumulation is species specific and can vary in different microalgal strains.

Fig. 1
figure 1

Effect of different nitrogen sources at concentration of 0.25 g/L (1) NH4Cl, (2) NH4HCO3, (3) NaNO2, (4) NaNO3, (5) NH4NO3, (6) yeast extract, and (7) urea on the a DCW (g/L) and total lipid content (g/L), b biochemical composition (%), c photosynthesis pigments (μg/ml) and PSII efficiency, and d FAME profile of Scenedesmus sp. IITRIND2 on the 10th day

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In order to decipher the effects of different nitrogen sources on biochemical composition of Scenedesmus sp. IITRIND2, changes in the total protein and total carbohydrate content were recorded (Fig. 1b). The maximum carbohydrate content (~ 46%) was recorded in cells supplemented with organic nitrogen (yeast extract and urea) followed by NH4NO3 (~ 33%), NH4Cl (~ 32%), NaNO3 (~ 31%), NH4HCO3 (~ 22%) and NaNO2 (~ 21%) respectively. Such an increase in carbohydrate in organic nitrogen cultures as these sources not only provide nitrogen but also organic carbon, vitamins, and trace elements which might have triggered carbohydrate metabolism in the microalga. Further, high carbohydrate content in NH4Cl cultures could be a stress response caused by the acidic medium. On the other hand, the protein content showed an inverse correlation with carbohydrate content, i.e., maximum protein content (~ 47%) was attained in NaNO2> NaNO3> NH4HCO3> NH4Cl> yeast extract = urea > NH4NO3 (Fig. 1b).

Photosynthetic pigments (chlorophyll a, chlorophyll b, and carotenoids) are indicators of microalgae health. Apart from NH4Cl cultures, all the cultures showed healthy state of microalgal cells (Fig. 1c). The maximum chlorophyll content (a + b) was observed in NH4NO3 cultures (~ 11 mg/ml) which were closely followed by NaNO3 and NH4HCO3 cultures (Fig. 1c). The possible reason for a decrease in pigment content in NH4HCO3 grown could be due to presence of inorganic carbon (bicarbonate) which reduces the photosynthetic efficiency while still enhancing the biomass production in microalgae (Wan et al. 2011). On a similar note, the chlorophyll (a + b) content decreased by ~ 20% compared with inorganic sources, apart from NH4Cl due to the presence of complex organic carbon sources. The maximum carotenoid content (~ 2 mg/ml) was obtained in yeast extract and urea cultures closely followed by NH4Cl (Fig. 1c). Further, the ratio of carotenoids/chlorophyll (a + b) was comparatively low (~ 0.11) in all the inorganic nitrogen cultures, apart from NH4Cl as opposed to organic nitrogen sources (~ 0.24). A high carotenoids/chlorophyll (a + b) indicates a decrease in PSII activity (Arora et al. 2015).

The ideal FAME component for good quality biodiesel should be rich in C16 and C18 fatty acids with other proportions of low fatty acids (Maheshwari et al. 2019). The FAME analysis showed the dominance of C14:0 (myristric acid), C16:0 (palmitic acid), C16:1 (7, 10 hexadecadienoic acid), C18:0 (stearic acid), C18:1 (oleic acid), and C18:2 (linoleic acid) as depicted in Fig. 1d. The ratio of oleic acid to total FAMEs in the microalga was the maximum irrespective of the nitrogen source utilized. High amounts of oleic acid in the biodiesel can improve the oxidative stability (shelf life) and cold filter plugging properties (low temperature operability) thereby leading to a good-quality biodiesel. Further, the proportions of unsaturated fatty acids (UFAs) and saturated fatty acids (SFAs) are crucial for determining the key biodiesel physical properties including cetane number, kinematic viscosity, cold filter plugging property (CFPP), and shelf life (Qu et al. 2019). For instance, an increase in the UFAs favors better viscosity (flow properties) and applicability at low temperatures (Maheshwari et al. 2019). The FAME profile of lipid extracted from Scenedesmus sp. IITRIND2 cultivated in the best nitrogen sources (NH4HCO3 and NH4NO3) showed 25–35% UFAs implicating a vehicular quality biodiesel. Higher percentages of SFAs increase the resistant to degradation and oxidation thereby improving the shelf life (Vishwakarma et al. 2019). However, presence of SFAs can lead to high viscosity and poor atomization of the biodiesel (Deshmukh et al. 2019). The biodiesel obtained from the microalgae cultivated in NH4HCO3 and NH4NO3 contained high amounts of 30–40% SFAs with low polyunsaturated fatty acids (5–6%) implicating a high cetane number, which not only improves the ignition property of the biodiesel but also lowers down the NOx emissions (Saraf and Thomas 2007).

The suitability of 10 different carbon sources (organic and inorganic) was evaluated in comparison with atmospheric CO2 for the biomass and lipid generation in Scenedesmus sp. IITRIND2 (Fig. 2a). The obtained results suggested the unique capability of the microalga to adapt to all the tested carbon sources apart from xylose in which the microalga did not survive. All the carbon sources (organic and inorganic) showed an increase in DCW as well as lipid accumulation. This could be due to the synchronized respiration and photosynthesis occurring during the mixotrophy, which promotes both biomass and lipid accumulation in the microalga. Similar results have been reported for various microalgae belonging to diverse genera including Chlorella, Scenedesmus, Dunaliella, and Nannochloropsis (Bhatnagar et al. 2011; Cho et al. 2011; Wan et al. 2011; Ren et al. 2013). The maximum DCW (2.8 ± 0.04 g/L) and total lipid content (1.18 ± 0.03 g/L) were recorded in cultures supplemented with mannose which did not significantly differ from glucose cultures (Fig. 2a). These results were closely followed by microalga grown in BBM supplemented with sodium acetate > sucrose = sodium bicarbonate > maltose > arabinose = lactate >galactose> CO2 respectively (Fig. 2 a and b). Mannose is a C-2 epimer of glucose and its metabolism has been demonstrated in a thermo-acidophilic rhodophyte, Galdieria sulphuraria via an ATP-dependent fructokinase which phosphorylates mannose but not glucose (Heilmann et al. 1997). On the other hand, glucose is the final product of photosynthesis and its uptake by microalgae takes place using hexose transporters namely HUP 1, 2, and 3 (hexose uptake) (Morales-Sánchez et al. 2015). Furthermore, an increase in DCW and lipid accumulation in glucose cultures compared with sodium acetate-supplemented cells was due to the higher energy production (~ 2.8 kJ/mol) during glucose metabolism compared with acetate (0.8 kJ/mol) (Wang et al. 2012). The results indicated that Scenedesmus sp. IITRIND2 preferred C6 sugars (mannose and glucose) compared with C5 (arabinose) or C4 (galactose) and disaccharides (sucrose, maltose, and lactose) respectively. Similar results have been reported with other Scenedesmus sp. which showed high growth and lipid production on addition of glucose compared with other carbon sources (He et al. 2019; Ma et al. 2019). Further, growth of the microalga in arabinose compared with xylose indicated the selective metabolism of the former.

Fig. 2
figure 2

Effect of different carbon sources at concentration of 2 g/L (1) glucose, (2) mannose, (3) maltose, (4) arabinose, (5) lactose, (6) sucrose, (7) galactose, (8) CO2, (9) sodium acetate, (10) sodium bicarbonate on a DCW (g/L) and total lipid content (g/L), b biochemical composition (%), c photosynthesis pigments (μg/ml) and PSII efficiency, and d FAMEs profile of Scenedesmus sp. IITRIND2 on the 10th day

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Evaluation of the changes in the total carbohydrate content of the microalgae with respect to different carbon sources indicated a similar accumulation pattern with maximum in C5 sugar (44%) followed by disaccharides (~ 34%) and ~ 30% in C6 and inorganic sugars (acetate and bicarbonate) respectively (Fig. 2b). On the other hand, a varied response was observed for the total protein content with maximum synthesis in lactose (~ 44%) followed by sucrose = galactose> bicarbonate > glucose = mannose = acetate = arabinose > maltose (Fig. 2b). Addition of carbon source particularly simple sugars to the growth medium provides additional pool of carbon which triggers the synthesis of acetyl-CoA and NADPH thereby enhancing the lipid and carbohydrate accumulation in microalgae (Wan et al. 2011).

An apparent reduction (~ 30–45%) in the photosynthesis pigments (chlorophyll a + b) in the microalgal cells supplemented with organic carbon sources compared with CO2 indicated decline in photosynthetic efficiency which was also evident by the increase in carotenoids/chlorophyll (a + b) ratio (Fig. 2c). This could be due to less dependence of the microalga on light for its growth. It has been reported in G. sulphuraria that glucose inhibited the biosynthesis of chlorophyll a by partially blocking the transformation of chlorophyll a precursor molecule, coproporphyrin III (Smith et al. 2015). The results were in line with earlier reported studies on Phaeodactylum tricornutum and C. vulgaris (Cerón García et al. 2005; Cheirsilp and Torpee 2012).

The FAME composition of the microalga cultivated in different carbon sources showed more diversification in the profile compared with the nitrogen sources (Fig. 2d). The data indicated that the FAME profile depended on the number of carbon atoms present in the sugar moiety as the C6, C5, and inorganic carbon sources showed presence of C14:0, C16:0, C16:2, C18:0, C18:1, and C18:2 while in case of disaccharides and galactaose (C4 sugar), small amounts of C15:0 (2–4%) were also detected (Fig. 2d). Further, in sucrose cultures, the lipid showed presence of C16:1 (1.47%) and C17:0 (1.29%) while galactose showed absence of C16:2. Such kind of fluctuations in the FAME profile could be attributed to the metabolic adaptation occurring in the microalgae in response to carbon source and its catabolism. Alteration in the fatty acid profile is responsible for changing the permeability of the cell membrane of the microalga which may contribute to the uptake of the carbon source from the growth medium. Interestingly, the addition of external carbon particularly the best carbon sources (glucose, mannose, and acetate) showed increase in the UFAs (40–45%) and PUFAs (7–11%) as depicted in Fig. 2d. The SFAs were ~ 46% in lipid derived from microalgae cultivated in mannose and glucose while ~ 35% when acetate was used as a carbon source.

Based on the above results obtained, two nitrogen sources (NH4NO3 and NH4HCO3) and three carbon sources (glucose, mannose, and sodium acetate) were selected to study their synergistic effects on Scenedesmus sp. IITRIND2 growth and lipid accumulation (Fig. 3a). All the six combinations (NH4NO3 + glucose; NH4NO3 + mannose, NH4NO3 + acetate, NH4HCO3 + glucose, NH4HCO3 + mannose, and NH4HCO3 + acetate) showed ~ 2–3-folds higher DCW compared with their individual counterparts (Figs. 1a, 2a, and 3a). Among the tested nitrogen and carbon combinations, the maximum DCW (4.04 ± 0.03 g/L) was attained in NH4HCO3 + mannose which was closely followed by NH4HCO3 + glucose and NH4HCO3 + acetate respectively (Fig. 3a). However, the lipid accumulation in the synergistic studies was attained in the following order: NH4HCO3 + acetate > NH4HCO3 + glucose = NH4HCO3 + mannose > NH4NO3 + acetate = NH4NO3 + mannose > NH4NO3 + glucose (Fig. 3 a and b). The results indicate that though acetate alone was not sufficient to generate maximum lipid accumulation, its combination with NH4HCO3 elevated the DCW and lipid content of the microalga. Acetate is believed to be metabolically oxidized in the glyoxylate cycle, generating malate in the microalga (Morales-Sánchez et al. 2015). Further, addition of bicarbonate in the growth media causes a metabolic shift from growth state to product formation state thereby increasing the overall lipid accumulation (Gardner et al. 2013).

Fig. 3
figure 3

Synergistic effect of different nitrogen (0.25 g/L) and carbon sources (2 g/L) (1) NH4NO3 + glucose, (2) NH4NO3 + mannose, (3) NH4NO3 + sodium acetate, (4) NH4HCO3 + glucose, (5) NH4HCO3 + mannose, (6) NH4HCO3 + sodium acetate on the a DCW (g/L) and total lipid content (g/L), b biochemical composition (%) c photosynthesis pigments (μg/ml) and PSII efficiency, and d FAME profile of Scenedesmus sp. IITRIND2 on the 10th day

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Interestingly, the total protein and carbohydrate content in the synergistic cultures did not show much variation as the protein content which ranged from ~ 35–30% while the total carbohydrate content ranged from ~ 38–31% respectively (Fig. 3b). Furthermore, the photosynthetic pigments (chlorophyll a + b) were higher in inorganic carbon sources irrespective of the nitrogen source compared with the organic carbon sources. The ratio of carotenoids/chlorophyll (a + b) was also equivalent to the autotrophic cultures (CO2) indicating the healthy state of PS II (Fig. 3c). The fatty acid composition of the synergistic cultures showed maximum amounts of C16:0 and C18:1 while lower proportions of C14:0, C16:2, C18:0, and C18:2 signify the potential utility of the derived biodiesel in combustion engines (Fig. 3d). A comparison of the FAMES profile of Scenedesmus sp. IITRID2 with previously published reports of various Scenedesmus sp. cultivated in different nitrogen and carbon sources indicated that C16:0 and C18:1 were the predominant fatty acids in all the microalgae (Table 1). Further, the C18:3 content was > 12% in most all microalgae with the exception of FAMEs obtained from Scenedesmus sp. and Scenedesmus dimorphous. Overall, the FAME profile of Scenedesmus sp. IITRIND2 cultivated in the best carbon and nitrogen sources, i.e. NH4HCO3 + glucose/mannose/acetate indicated right proportions of SFAs and UFAs with appropriate amount of PUFs indicating it to be good quality biodiesel.

Table 1 Comparison of FAME profile of different Scenedesmus sp. cultivated in various nitrogen and carbon sources with the present study
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In conclusion, the study demonstrated the unique capability of Scenedesmus sp. IITRIND2 to utilize various complex organic and inorganic nitrogen and carbon sources under mixotrophic mode. The microalga grown in the medium containing ammonium salts (nitrate and bicarbonate) along with C-6 sugars and acetate cells stimulated high biomass (3–4 g/L) and lipid accumulation (33–35%) compared with their individual counterparts.