Specific uptake kinetics of glucose and nitrate in carbon-limited and nitrogen-limited C:N ratio under photoheterotrophic cultural conditions for Botryococcus braunii growth and lipid production

Bioresource Technology Reports 8 (2019) 100337

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Specific uptake kinetics of glucose and nitrate in carbon-limited and nitrogen-limited C:N ratio under photoheterotrophic cultural conditions for Botryococcus braunii growth and lipid production Shailendra Singh Khichi, Deepak Dohare, S. Rohith, Sharika Sachin, Sanjoy Ghosh

T

⁎

Biochemical Engineering Lab, Department of Biotechnology, Indian Institute of Technology, Roorkee, Roorkee 247667, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Uptake kinetics C:N ratio Microalgae Botryococcus braunii Lipid productivity

Carbon and nitrogen are the most significant parameters for microalgal growth and an effective C:N ratio is critical to assess the biomass and lipid productivities of Botryococcus braunii. In this study, the effect of the C:N ratio on specific uptake kinetics of glucose and nitrate was evaluated using substrate kinetic equation, under glucose limited and nitrate limited photoheterotrophic conditions. In glucose limited conditions, the maximum specific growth rate, biomass and lipid productivity of 0.0873 h−1, 1.11 g L−1 d−1 and 0.390 g L−1 d−1 were observed respectively for C:N ratio of 29:1. Maximum specific nitrate uptake rate of 0.190 mM gb−1 h−1 was observed under glucose limited C:N ratio of 61:1 (i.e. carbon sufficient), while the maximum glucose uptake rate of 0.74 mM gb−1 h−1 was observed in nitrate limited C:N ratio of 13:1 (i.e. nitrogen sufficient). This suggests that carbon and nitrogen metabolism is linked together and significantly affects the specific uptake kinetics of the each other.

1. Introduction Microalgae are simple aquatic organisms that photosynthesize solar energy to produce carbohydrates and small amount of liquid biofuel under autotrophic conditions (Chen and Jiang, 2017). However, under autotrophic conditions the maximum cell densities reaches up to 30 g L−1 of dry cell biomass which is lower than the maximum cells densities of 50–100 g L−1 of dry cell biomass under heterotrophic conditions (Chen and Jiang, 2017). To conquer the limitations of autotrophic conditions, microalgae should be grown in heterotrophic, photoheterotrophic and/or mixotrophic cultivations. In particular, the biolipid content and growth of the microalgae is affected by the carbon and nitrogen source in the nutrient medium under heterotrophic, photoheterotrophic and mixotrophic cultivations (Isleten-Hosoglu et al., 2012). Heterotrophic, photoheterotrophic or mixotrophic cultivation of microalgae offers higher specific growth rates in comparison to photoautotrophic cultivation and resulted in substantiate biomass and lipid productivities which subsequently minimized the process harvesting cost (Brennan and Owende, 2010). However, photoheterotrophic and mixotrophic cultivation of microalgae is attractive,

because light limitation is no longer issue in these conditions (Guldhe et al., 2017). In this study we have focused on photoheterotrophic cultivation of microalgae to analyze the role of carbon and nitrogen, on the uptake of each other. Photoheterotrophic cultivation of microalgae requires light and an organic substrate which act as an energy and carbon source, respectively (Guldhe et al., 2017). Microalgal growth rate under photoheterotrophic mode of nutrition significantly depends on the strain type, culture conditions, light intensity, pH, carbon or/and nitrogen sources (Bekirogullari et al., 2017). Notably, the carbon source uptake in photoheterotrophic culture conditions is not only regulated by the diffusion or transport of carbon source across the cellular membrane (Morales-Sánchez et al., 2013), but also highly affected by the nitrogen source and its concentration in the nutrient medium (Gopalakrishnan et al., 2015). Carbon and nitrogen metabolism in microorganisms is generally linked by the α-KG intermediate of the TCA cycle (Bren et al., 2016). In nitrogen limiting conditions, the accumulation of α-KG intermediate inhibits the Enzyme I (i.e the first step of the phosphotransferase system) which subsequently blocks the glucose uptake ability of the

Abbreviations: ATP, adenosine triphosphate; BG-11, blue-green medium; DCW, dry cell weight; DNS, 3,5-dinitrosalicylic acid; GL, glucose limited; GUR, glucose uptake rate; NADPH, nicotinamide adenine dinucleotide phosphate; NL, nitrate limited; NUR, nitrate uptake rate; PEPCase, phosphoenolpyruvate carboxylase; PPP, pentose phosphate pathway; TCA, tricarboxylic acid cycle; α-KG, α-Ketoglutarate ⁎ Corresponding author at: Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, India. E-mail address: [email protected] (S. Ghosh). https://doi.org/10.1016/j.biteb.2019.100337 Received 23 September 2019; Received in revised form 19 October 2019; Accepted 20 October 2019 Available online 22 October 2019 2589-014X/ © 2019 Published by Elsevier Ltd.

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μmax qGmax

Nomenclature Mx0 Mx(t) Ms0 Ms(t)

Biomass concentration at t = 0 (g L−1) Biomass concentration at different time interval (g L−1) Initial concentration of substrate (glucose or nitrogen) (g L−1) Substrate concentration (glucose or nitrate) at different time (mM)

qNmax Plipid Clipid R2

microorganism (Doucette et al., 2011). Also, in these conditions, excess ATP is determined, which inhibits the glycolytic pathway and reduces glucose assimilation efficiency of the microalgae (Pagnanelli et al., 2014). Moreover, in these conditions, the relative activity of pentose phosphate pathway (PPP) is found to be higher than that of glycolysis, reflecting excess NADPH requirements for lipid biosynthesis (Xiong et al., 2010). Notably, it is evident that the PPP is preferred for glucose metabolism in nitrogen limiting conditions, while glycolytic pathway is favoured over PPP for glucose metabolism in nitrogen sufficient (carbon limited) conditions (Gopalakrishnan et al., 2015). Furthermore, in carbon limited (nitrogen sufficient) conditions, the relative activity of phosphoenolpyruvate carboxylase (PEPCase) increases which assists glucose consumption through TCA cycle and subsequently favors carbon skeleton generation for improved carbon and nitrogen assimilation into protein synthesis. It also provides reducing equivalents to meet growth demands (Gopalakrishnan et al. 2015). Nitrogen utilization pathway is affected by a regulatory molecule (α KG) which serves both as a TCA intermediate and as the carbon backbone of glutamate and glutamine. In addition, α KG together with the glutamine regulates the nitrogen assimilation system (Bren et al., 2016). Moreover, nitrogen assimilation (nitrate or/and nitrite) in green microalgae is regulated at three levels: (1) the activity and capacity of nitrate and nitrite transport systems, (2) the activity of nitrate reductase and nitrite reductase, and (3) the amount of nitrate and nitrite reductases in the cells (Fernandez and Cardenas, 1989). Furthermore, the nitrate uptake rate (NUR) is also stimulated in the presence of glucose, and a 5 fold increase in NUR of Chlorella vulgaris was observed when the cells were pre-incubated with glucose (Schlee et al., 1985). To date, our understanding of the substrate (i.e. glucose and nitrate) uptake kinetics in C– limited and N-limited photoheterotrophic cultivation condition is limited. While previous research manifested that B. braunii can be grown under different organic carbon source and inorganic nitrogen source, little is known about the role of carbon and nitrogen on the specific uptake rates of each other, in C– limited and Nlimited photoheterotrophic cultivation conditions. Therefore, we have compared the specific uptake kinetics of B. braunii in C– limited medium with sufficient NO3− as the sole source of N, with the Nlimited medium supplied with sufficient glucose as the sole C source. In addition, the effect of individual C:N ratio on biomass growth and lipid accumulation were also studied in these conditions.

Specific growth rate (h−1) Maximum specific substrate consumption rate of glucose (mM gb−1 h−1) Maximum specific substrate consumption rate of nitrate (mM gb−1 h−1) Lipid concentration (g L−1) Lipid content of cells (g g−1) Coefficient of determination (−)

Table 1A Glucose limited (GL) C:N ratio. GL C:N ratio

Glucose conc. (g L−1)

Nitrate conc. (g L−1)

13:1 21:1 29:1 37:1 61:1 Control 1

4.015 6.49 8.96 11.43 18.84 –

0.75 0.75 0.75 0.75 0.75 0.75

2.1.1. Set I (glucose limited C:N ratio) Five batch experiments were conducted with varying glucose concentration (i.e., 4.015 to 18.84 g L−1) and fixed nitrate concentration (i.e. NaNO3− = 0.75 g L−1) to attain the C:N ratios of 13:1, 21:1, 29:1, 37:1 and 61:1 respectively according to Table 1A. One control experiment (i.e. control 1) was also designed in which glucose was completely absent (Table 1A). 2.1.2. Set II (nitrate limited C:N ratio) In this group the required C:N ratios of 13:1, 21:1, 29:1, 37:1 and 61:1 were obtained by varying the nitrate concentration (i.e., 1.211 to 0.26 g L−1) at fixed glucose concentration of 6.49 g L−1 according to Table 1. In the case of control experiment (i.e. control 2), nitrate was completely absent (Table 1). All the experiments were conducted in triplicates. 2.2. Biomass and nitrate estimation Microalgal biomass was determined as an optical density function measured at 750 nm wavelength (Khichi et al., 2019) using a UV–Visible spectrophotometer (Carry 60, Agilent). Extracellular nitrate concentration was quantified in the supernatant collected by centrifugation of 1 ml sample at 10,000 rpm for 6 min (Khichi et al., 2018). 2.3. Sugar estimation Residual sugars were estimated by modified DNS (3,5Dinitrosalicylic Acid) method (Miller et al., 1960). To estimate the residual glucose concentration, 1 ml of sample was taken from each flask in every 12 h interval. Supernatant was separated by centrifugation at 10,000 rpm for 6 min. Then, 100 μl of diluted supernatant was added to 900 μl of deionized water, 1 ml of DNS and 333 μl of Rochelle's salt

2. Material and methods 2.1. Strain and culture medium

Table 1B Nitrate limited (NL) C:N ratio.

The strain of B. braunii was collected from the Institute of Bioresources and Sustainable Development (IBSD, Takyelpat, Imphal), and its growth was maintained in modified BG-11 medium (Khichi et al., 2018). The temperature, pH and rpm of the inoculum medium were set to 27 ± 1 °C, 8 and 130 rpm, respectively in the light based incubator shaker. For experimental studies 3–4 days old inoculum was used in which initial biomass concentration was in the range of 0.3–0.5 g L−1. Experimental studies were conducted in two different sets. 2

NL C:N ratio

Glucose conc. (g L−1)

Nitrate conc. (g L−1)

13:1 21:1 29:1 37:1 61:1 Control 2

6.49 6.49 6.49 6.49 6.49 6.49

1.211 0.75 0.54 0.43 0.26 –

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(40% w/v). The mixture was then heated in boiling water bath for 5 min and cooled to room temperature, and the absorbance of the sample was measured at 540 nm against the blank.

Ms (t ) − Ms0 =

−1

Maximum specific growth rate of B. braunii in batch cultivation was calculated by applying the biomass mass balance (2)

Biomass mass balance can be solved by separation of variables followed by integration ⎟

(3)

( ) M

A plot between ln M x vs t gives the slope as μmax which is the x0 maximum specific growth rate of microalgae. 2.6. Maximum specific substrate uptake rates Specific substrate uptake kinetics was measured by applying the substrate mass balance to a batch culture of B. braunii. (4)

dMs dt

is the total consumption rate of nitrogen, t is the fermenwhere, tation time, qsmax is maximum specific substrate consumption rate, Mx is biomass concentration at time t. Biomass formation kinetics can be expressed as Eq. (5).

dMx = μmax Mx dt

(5)

Table 2 Kinetic parameters of B. braunii in glucose limited C:N ratio (i.e. maximum biomass (Xmax), maximum specific growth rate (μmax), maximum specific glucose uptake rate (qGmax) and maximum specific nitrate uptake rate (qNmax)).

After integrating Eq. (5), it was written as Eq. (6) as follows.

Mx = Mx 0. exp(μmax t )

(6)

After substituting Mx in Eq. (4)

dMs = qsmax Mx 0. exp(μmax t ) dt

(7)

Integrating Eq. (7) from t = 0 to t and Ms0 to Ms(t)

μmax

was the initial concentration

B. braunii growth in two sets of GL (Set I, i.e., fixed NaNO3− = 0.75 g L−1, with varying glucose) and NL (Set II, i.e., fixed glucose = 6.49 g L−1, with varying nitrate) with five different C:N ratios (13:1, 21:1, 29:1, 37:1 and 61:1) under photoheterotrophic conditions were studied in 250 ml Erlenmeyer flasks. The effect of varying GL C:N ratio (i.e. Set I) on B. braunii biomass production was evaluated based on the growth pattern of the strain under photoheterotrophic cultural conditions. As shown in Tables 2 and 4, the maximum biomass concentration, maximum biomass productivity and maximum specific growth rate of 4.44 g L−1, 1.11 g L−1 d−1 and 0.087 h−1, were observed at 29:1C:N ratio on the 4th day of cultivation, respectively. At GL C:N ratio of 13:1, 21:1, 37:1, 61:1 maximum biomass concentration, maximum biomass productivity and maximum specific growth rate were 2.76 g L−1, 0.69 g L−1 d−1 and 0.068 h−1; 4.20 g L−1, 1.05 g L−1 d−1 and 0.081 h−1; 4.04 g L−1, 1.01 g L−1 d−1 and 0.084 h−1; 3.57 g L−1, 0.89 g L−1 d−1 and 0.082 h−1 respectively (Tables 2 and 4). Moreover, as depicted in Fig. 1A, the biomass yield obtained in glucose-supplemented modified BG-11 medium was higher than that observed in photoautotrophic cultivation condition. This agreed with the previous studies and suggests that photoheterotrophic cultural condition significantly enhance the growth potential of some algal strains compared to the photoautotrophic conditions (Selvakumar and Umadevi, 2014). Moreover, experimental studies also suggest that the growth rate of B. braunii increased with the increment in GL C:N ratio, in the range of 13:1 to 29:1, which implied that the organic carbon source provided with nutrient medium was a significant factor affecting the growth rate of microalgae in photoheterotrophic conditions. However, with a further increase in GL C:N ratio, a gradual decline in biomass was observed for 37:1 and 61:1 GL C:N ratios. This indicates that at higher than optimum glucose concentration microalgae growth reduced significantly. In addition, Gao et al. (2019) observed similar effect on growth pattern and demonstrated that when the C:N ratio increased from 24 to 30, the organic carbon source was no longer considered a limiting factor in the nutrient medium. Gim et al., 2014 also reported that optimum growth of B. braunii was attained at 10 g L−1 glucose concentration; however with higher than that at 20 g L−1 of glucose concentration, 30–40% reduction in biomass

2.5. Maximum specific growth rate

dMs = qsmax Mx dt

s0

3.1. Algal growth

d , Clipid is lipid content of where Plipid is lipid productivity in g L cells or lipid yield of the cells in g g−1, DCW is dry cell weight g L−1, and Time is the cultivation period in days.

M ln ⎛ x ⎞ = μmax t M ⎝ x0 ⎠

μmax

3. Results and discussion

−1

dMx = μmax Mx dt

(8)

of glucose or nitrogen (mM), Ms(t) was glucose or nitrate substrate concentration at different time (mM), Mx0 was initial biomass concentration and Mx(t) was biomass concentration at different time points.

(1)

Time

qsmax Mx 0

qsmax

( ), where M

Clipid × DCW

Ms (t ) − Ms0 =

. [Mx (t ) − Mx 0]

A plot between [Ms(t) − Ms0] vs [Mx(t) − Mx0] was drawn, and

Extraction of lipid was done by modified Bligh and Dyer method with chloroform and methanol as solvents, and water as co-solvent (Bligh and Dyer, 1959). The cells were harvested by centrifugation at 10,000 rpm for 10 min at 4 °C. After centrifugation, the pellet was dried in the oven for 2 h at 80 °C. The chloroform/methanol (2:1 v/v) solvent system was used to extract the lipid from dried algal cells. The layers were separated by centrifugation for 10 min at 2000 rpm. The lower layer was separated and the procedure was repeated with the pellet. The lipid content was measured gravimetrically and expressed as dry weight percentage. The lipid productivity was calculated by the following equation

⎜

μmax

slope was calculated i.e.

2.4. Lipid extraction and estimation

Plipid =

qsmax

. [exp (μmax . t ) − 1] 3

GL C:N ratio

Xmax (g L−1)

13:1 21:1 29:1 37:1 61:1 Control 1

2.76 4.20 4.44 4.04 3.57 0.66

± ± ± ± ± ±

μmax (h−1) 0.41 0.46 0.41 0.52 0.66 0.07

0.063 0.069 0.073 0.072 0.071 0.014

± ± ± ± ± ±

0.009 0.007 0.006 0.009 0.010 0.001

qGmax (GUR) (mM gb−1 h−1)

qNmax (NUR) (mM gb−1 h−1)

0.44 0.56 0.59 0.65 0.46 –

0.168 0.160 0.150 0.153 0.187 0.094

± ± ± ± ±

0.065 0.061 0.053 0.084 0.076

± ± ± ± ± ±

0.025 0.018 0.014 0.021 0.031 0.008

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Fig. 1. (A). Growth profile of B. braunii in glucose limited C:N ratio (Set I) with varying glucose concentration 13:1 (glucose = 4.015 g L−1), 21:1 (glucose = 6.49 g L−1), 29:1 (glucose = 8.96 g L−1), 37:1 (glucose = 11.43 g L−1), and 61:1 (glucose = 18.84 g L−1) and fixed nitrate concentration (NaNO3− = 0.75 g L−1), Control 1 (glucose = 0 g L−1, NaNO3− = 0.75 g L−1); (B). Growth profile of B. braunii in nitrate limited C:N ratio (Set II) with varying nitrate concentration 13:1 (NaNO3− = 1.21 g L−1), 21:1 (NaNO3− = 0.75 g L−1), 29:1 (NaNO3− = 0.54 g L−1), 37:1 (NaNO3− = 0.43 g L−1), and 61:1 (NaNO3− = 0.26 g L−1) and fixed glucose concentration (glucose = 6.49 g L−1), Control 2 (glucose = 6.49 g L−1, NaNO3− = 0 g L−1).

are in accordance with Yang et al. (2011), who reported similar decrease in sugar consumption pattern using glycerin as a carbon source for Chlorella minutissima. Li et al. (2016) showed that glucose was completely consumed when initial glucose concentration was 5 g L−1 while about 60% and 30% of glucose consumption was observed with initial glucose concentration of 10 and 20 g L−1 respectively. The results reported in present study has also shown similar trend of glucose consumption under different GL C:N ratios. Extracellular glucose profile in NL C:N ratio was also obtained under photoheterotrophic culture conditions (Fig. 2B). In NL culture conditions, the GUR rate was calculated by Eq. (8), and the effect of extracellular nitrate on GUR in NL C:N ratio was analyzed. Remarkably, the GUR is highly dependent on the external nitrate concentration available in the culture solution. In particular, the GUR was decreased with the increase in NL C:N ratio (i.e. decrease in nitrate concentration). Maximum specific glucose uptake rate (qGmax) of 0.74 mM gb−1 h−1 was observed under 13:1 NL C:N ratio. At NL C:N ratios of 21:1, 29:1, 37:1 and 61:1, the observed qGmax was 0.73 mM mM gb−1 h−1, 0.61 mM gb−1 h−1, 0.54 mM gb−1 h−1and 0.49 mM gb−1 h−1 respectively (Table 3). These results are consistent with Li et al. (2016) who observed that with initial NaNO3 at a low value of 0.1 g L−1 about 6 g L−1 glucose (glucose initial = 10 g L−1) remained in the culture medium while glucose (i.e. 10 g L−1) was completely consumed when initial NaNO3 concentration was 1.5 and 3.5 g L−1 in the culture medium, implying that glucose consumption ability of microalgae depends upon the initial nitrate concentration in the culture medium. This study suggests that the lower nitrogen content in nutrient medium reduces glucose consumption Similarly Gopalakrishnan et al. (2015) also observed that sub-optimal level of nitrogen in the cultivation medium redirects the metabolic flux of glucose from glycolysis to PPP, which subsequently reduces glucose consumption.

production from B. braunii was observed. Therefore, in GL photoheterotrophic cultivation conditions, excess glucose concentration had an inhibitory effect on microalgae growth when the GL C:N ratio of the growth medium increased from 29:1 to 37:1 or 61:1. The growth pattern of B. braunii with varying nitrate concentrations and constant organic carbon source (i.e. NL C:N ratios, or Set II) are depicted in Fig. 1B. After 5 days of cultivation, a moderate increase in algal growth was observed with initial increase in NL C:N ratio from 13:1 to 21:1 respectively (Fig. 1B). However, the further increment in NL C:N ratio consequently reduces the microalgae growth. In this case (i.e. Set II) the maximum biomass concentration, maximum biomass productivity and maximum specific growth rate of 4.20 g L−1, 1.05 g L−1 d−1 and 0.081 h−1 were obtained at 21:1 NL C:N ratio on 4th of day of cultivation (Tables 3 and 5). The biomass growth potential of the B. braunii was significantly affected by the extracellular nitrogen content present in the cultivation medium. However, biomass limitation under nitrogen deficient conditions is a major concern, as lower nitrogen content in the cultivation medium tends to reduce the biomass productivities. Furthermore, Choi et al. (2010) reported that, lower nitrate concentration in the cultivation medium (i.e. > 0.04 mM) significantly reduced the growth rate of the B. braunii to 0.090 d−1 compared to the 0.159 d−1 which was achieved at moderate nitrate concentration of 0.37 mM in the nutrient medium (Choi et al., 2010). This suggests that nitrogen limitation is negatively correlated with biomass productivities and, it has a negative impact on microalgae cell division, metabolic activity, biomass and lipid productivity (Ördög et al., 2012). Nitrogen limitation reduced biomass growth and lipid productivity because availability of nitrogen containing molecules reduced under the influence of sub optimal nitrogen level in the medium, which affected cell growth (Wu and Miao, 2014). 3.2. Specific glucose uptake kinetics

3.3. Specific nitrate uptake kinetics

Residual glucose pattern under GL C:N ratios was recorded in photoheterotrophic cultivation mode (Fig. 2A). To quantitatively analyze the effect of GL C:N ratio on glucose consumption, the maximum specific uptake rate of glucose (qGmax) was calculated by Eq. (8). As shown in Table 2, an elevation in the maximum specific glucose uptake rate (qGmax) was observed with the increment in GL C:N ratio in the range of 13:1 to 37:1. Maximum value of qGmax was 0.83 mM gb−1 h−1 obtained for GL C:N ratio of 37:1, while the lowest value of qGmax (i.e. 0.53 mM gb−1 h−1) was observed for the GL C:N ratio of 13:1. This suggests that the GUR is proportional to the glucose level present in the nutrient medium in the range of 13:1 to 37:1 GL C:N ratio, and the glucose consumption was decreased at higher than optimum glucose level in culture solutions. In this research, glucose consumption rates of 91.41%, 92.68%, 76.84%, 65.88% and 36.94% were observed for GL C:N ratios of 13:1, 21:1, 29:1, 37:1 and 61:1 respectively. These results

Nitrogen depletion pattern in GL C:N ratio was also observed. As Table 3 Kinetic parameters of B. braunii in nitrate limited C:N ratio (i.e. maximum biomass (Xmax), maximum specific growth rate (μmax), maximum specific glucose uptake rate (qGmax) and maximum specific nitrate uptake rate (qNmax)).

4

NL C:N ratio

Xmax (g L−1)

μmax (h−1)

13:1 21:1 29:1 37:1 61:1 Control 2

4.10 ± 0.33 4.20 ± 0.46 3.59 ± 0.41 3.014 ± 0.37 2.82 ± 0.21 0.35 ± 0.04

0.060 0.069 0.061 0.057 0.056 0.023

± ± ± ± ± ±

0.008 0.007 0.007 0.006 0.004 0.002

qGmax (GUR) (mM gb−1 h−1)

qNmax (NUR) (mM gb−1 h−1)

0.57 ± 0.073 0.56 ± 0.061 0.49 ± 0.054 0.48 ± 0.031 0.41 ± 0.060 0.021 ± 0.02

0.178 0.160 0.130 0.115 0.074 –

± ± ± ± ±

0.023 0.018 0.016 0.013 0.005

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Fig. 2. (A). Glucose consumption profile in glucose limited C:N ratio (Set I) with varying glucose concentration 13:1 (glucose = 4.015 g L−1), 21:1 (glucose = 6.49 g L−1), 29:1 (glucose = 8.96 g L−1), 37:1 (glucose = 11.43 g L−1), and 61:1 (glucose = 18.84 g L−1) and fixed nitrate concentration (NaNO3− = 0.75 g L−1); (B). Glucose consumption profile in nitrate limited C:N ratio (Set II) with varying nitrate concentration 13:1 (NaNO3− = 1.21 g L−1), 21:1 (NaNO3− = 0.75 g L−1), 29:1 (NaNO3− = 0.54 g L−1), 37:1 (NaNO3− = 0.43 g L−1), and 61:1 (NaNO3− = 0.26 g L−1) and fixed glucose concentration (glucose = 6.49 g L−1), Control 2 (glucose = 6.49 g L−1, NaNO3− = 0 g L−1).

reduction (i.e. 62.83% reduction) in NUR was observed (i.e 0.226 mM gb−1 h−1 to 0.084 mM gb−1 h−1). Since nitrate consumption to form amino acids requires energy provided by the carbon metabolism, and depletion of the nitrate concentration in culture medium reduces the ATP consumption in microalgae which inhibits the glucose assimilation capabilities of microalgae (Pagnanelli et al., 2014).

shown in Fig. 3A, the extracellular nitrate concentration exponentially decreased after 24 h of cultivation. The perceptible change in nitrate depletion was quantified using specific nitrate uptake rate (NUR) by Eq. (8). Notably, as depicted in Table 2, the regulation of nitrate uptake was governed by the extracellular glucose present in the culture solution. Particularly, the NUR was decreased with increase in GL C:N ratio from 13:1 to 29:1 (i.e. from 0.182 mM gb−1 h−1 to 0.180 mM gb−1 h−1) (Table 2). As shown in Fig. 3A, and Table 2, the NUR was decreased with increase in biomass concentration for 13:1 to 29:1 GL C:N ratio. With a further increase in GL C:N ratio, an elevation in NUR was observed for 37:1 C:N ratio (0.185 mM gb−1 h−1), while the highest value (i.e.,0.190 mM gb−1 h−1) of NUR was observed at 61:1 GL C:N ratio. Similar results were obtained by Yan et al. (2013) who observed the highest TN removal efficiency of 92.85% in GL C:N ratio of 5:1 for Chlorella vulgaris. In addition, it has been previously reported that addition of glucose in the culture solution induces nitrogen transport systems (i.e. short chain neutral amino acids, basic amino acids) in microalgal cells (Schlee et al., 1985). Nitrate consumption pattern (Fig. 3B) and nitrate uptake rate (Table 3) in NL C:N ratio was also recorded. Maximum NUR of 0.226 mM gb−1 h−1 was observed for 13:1 NL C:N ratio (i.e. NaNO3− = 1.21 g L−1). At NL C:N ratio 21:1 (i.e. NaNO3− = 0.75 g L−1), 29:1 (i.e. NaNO3− = 0.54 g L−1), 37:1 (i.e. NaNO3− = 0.43 g L−1), 61:1 (i.e. NaNO3− = 0.26 g L−1) the observed maximum NUR was 0.188 mM gb−1 h−1, 0.164 mM gb−1 h−1, 0.133 mM gb−1 h−1, 0.084 mM gb−1 h−1 respectively. The obtained result shows that the NUR in NL C:N ratio is regulated by the external nitrate concentration available in the culture solution. In addition, as the nitrate concentration was decreased from 1.21 to 0.26 g L−1 (i.e. 78.51% decrease), a significant

3.4. Lipid productivities Lipid content in GL C:N ratio was examined as represented in Fig. 4A. Noteworthily, an upsurge in the lipid content was noticed in the range of GL C:N ratio of 13:1 to 37:1, while a decrease in lipid content was observed, above the optimal glucose concentration (i.e. 61:1C:N ratio). Although the maximum lipid content of 0.38 g g−1 was attained at 37:1 GL C:N ratio, the maximum lipid productivity of 0.39 g L−1 d−1 (lipid content = 0.35 g g−1) was achieved at 29:1 GL C:N ratio (Table 4). However, this observation demonstrates that the lipid content was increased with increase in glucose concentration (i.e. up to 11.43 g L−1 or 37:1C:N ratio) in the culture solution. It must be noted that lipid productivity is considered as a function of biomass productivity, while the lipid content of the microalgal cell is related to the external carbon source present in the GL photoheterotrophic cultivation. So that, the excessive glucose concentration (37:1C:N ratio) increases the lipid content but remarkably decreases the biomass productivity (i.e. 1.01 g L−1 d−1, Table 4) which consequently reduces the overall lipid productivity at high GL C:N ratio of 37:1. The lipid content and lipid productivity of 0.23 g g−1 and 0.16 g L−1 d−1; 0.33 g g−1 and 0.35 g L−1 d−1; 0.31 g g−1 and 0.28 g L−1 d−1 were observed for 13:1, 21:1, 61:1 respectively. Moreover, the lipid productivities obtained

Fig. 3. (A). Nitrate consumption profile in glucose limited C:N ratio (Set I) with varying glucose concentration 13:1 (glucose = 4.015 g L−1), 21:1 (glucose = 6.49 g L−1), 29:1 (glucose = 8.96 g L−1), 37:1 (glucose = 11.43 g L−1), and 61:1 (glucose = 18.84 g L−1) and fixed nitrate concentration (NaNO3− = 0.75 g L−1), Control 1 (glucose = 0 g L−1, NaNO3− = 0.75 g L−1); (B). Nitrate consumption profile in nitrate limited C:N ratio (Set II) with varying nitrate concentration 13:1 (NaNO3− = 1.21 g L−1), 21:1 (NaNO3− = 0.75 g L−1), 29:1 (NaNO3− = 0.54 g L−1), 37:1 (NaNO3− = 0.43 g L−1), and 61:1 (NaNO3− = 0.26 g L−1) and fixed glucose concentration (glucose = 6.49 g L−1). 5

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Fig. 4. (A). Maximum biomass and lipid content in glucose limited C:N ratio (Set I) with varying glucose concentration 13:1 (glucose = 4.015 g L−1), 21:1 (glucose = 6.49 g L−1), 29:1 (glucose = 8.96 g L−1), 37:1 (glucose = 11.43 g L−1), and 61:1 (glucose = 18.84 g L−1) and fixed nitrate concentration (NaNO3− = 0.75 g L−1), Control 1 (glucose = 0 g L−1, NaNO3− = 0.75 g L−1); (B). Maximum biomass and lipid content in nitrate limited C:N ratio (Set II) with varying nitrate concentration 13:1 (NaNO3− = 1.21 g L−1), 21:1 (NaNO3− = 0.75 g L−1), 29:1 (NaNO3− = 0.54 g L−1), 37:1 (NaNO3− = 0.43 g L−1), and 61:1 (NaNO3− = 0.26 g L−1) and fixed glucose concentration (glucose = 6.49 g L−1), Control 2 (glucose = 6.49 g L−1, NaNO3− = 0 g L−1). Table 4 Effect of GL C:N ratio on biomass productivity, lipid productivity, lipid Content, and lipid yield on glucose (YP/Glu). GL C:N ratio

Biomass productivity (g L−1 d−1)

Lipid productivity (g L−1 d−1)

Lipid content (g g−1)

YP/Glu

13:1 21:1 29:1 37:1 61:1 Control 1

0.69 ± 0.103 1.059 ± 0.120 1.11 ± 0.101 1.01 ± 0.131 0.89 ± 0.135 0.17 ± 0.0145

0.16 ± 0.024 0.35 ± 0.038 0.39 ± 0.034 0.38 ± 0.049 0.28 ± 0.032 0. 03 ± 0.002

0.23 0.33 0.35 0.38 0.31 0.18

0.172 ± 0.025 0.23 ± 0.026 0.225 ± 0.02 0.219 ± 0.028 0.159 ± 0.027 –

± ± ± ± ± ±

0.039 0.036 0.032 0.048 0.051 0.016

Table 5 Effect of NL C:N ratio on biomass productivity, lipid productivity, lipid Content, and lipid yield on glucose (YP/Glu). NL C:N ratio

Biomass productivity (g L−1 d−1)

Lipid productivity (g L−1 d−1)

Lipid content (g g−1)

YP/Glu

13:1 21:1 29:1 37:1 61:1 Control 2

1.02 1.05 0.90 0.75 0.71 0.09

0.28 ± 0.047 0.35 ± 0.038 0.31 ± 0.030 0.26 ± 0.029 0.30 ± 0.022 0.023 ± 0.002

0.28 0.33 0.34 0.35 0.43 0.26

0.249 0.230 0.250 0.226 0.340 –

± ± ± ± ± ±

0.132 0.0118 0.110 0.090 0.053 0.010

± ± ± ± ± ±

0.036 0.037 0.042 0.039 0.032 0.029

± ± ± ± ±

0.031 0.026 0.030 0.026 0.025

Table 6 Comparable literature values of biomass productivity and lipid productivity under different C:N ratios. Microalgae

C: N ratio

Biomass productivity (g L−1 day−1)

Lipid productivity (g L−1 day−1)

Data

Reference

B. braunii B. braunii N. oleoabundans N.oleoabundans B.braunii (GL) B.braunii (NL)

47.35 72.85 17 278 29 21

0.13 0.163 0.344 1.022 1.11 1.05

– 0.0645 0.0826 0.528 0.39 0.35

Calculated Calculated Provided Provided Calculated Calculated

Zhang et al. (2011) Yeesang and Cheirsilp (2014) Morales-Sánchez et al. (2013) Morales-Sánchez et al. (2013) Present studies Present studies

under GL C:N ratio of 29:1 (i.e. 0.35 g L−1 d−1) is higher than the other B. braunii strains for different C:N ratios (Table 6). However, the lipid productivity of N. oleoabundans under C:N ratio of 278 was higher than the present studies (Morales-Sánchez et al., 2013), this might be due to the different types of strains and/or different cultivation systems used for the production of biomass and lipid adopted in their studies. In NL C:N ratio, the trend for lipid content in Fig. 4B, and lipid productivity of B. branui in Table 5 were exemplified. As depicted in Table 5, the elevation in lipid content was observed with increase in NL C:N ratio (i.e. decrease in nitrate concentration) in culture solution. Remarkably, the trend for maximum lipid productivity was found exactly reverse to the lipid content trend (i.e. lipid productivity decreased

with increase in NL C:N ratio). In addition, it must be noteworthy that the lower nitrate concentration present in the culture solution reduces the biomass productivity while it increases the lipid accumulation in microalgal cells in NL photoheterotrophic cultivation. In NL C:N ratio, the lipid content increased with decrease in nitrate concentration (i.e. increase in NL C:N ratio) and maximum lipid yield of 0.43 g g−1 was found for the NL C:N ratio of 61:1. The biolipid production trend for B. braunii under NL C:N ratios was as follows: 37:1 (i.e. 34.70%) > 29:1 (34.31%) > 21:1 (33%) > 13:1 (28%). The results reported in this study (i.e. biolipid production trend) are consistent with Wu and Miao (2014), who reported quite similar trend of biolipid production under varying nitrate concentration (i.e. 0.3 g L−1 (39.16%) > 0.6 g L−1 6

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(36.56%) > 0.9 g L−1 (34.92%) > 1.5 g L−1 (32.53%)) for S. obliquus. This suggests that maintaining an intermediate concentration of nitrate in the nutrient medium is more suitable to produce the high biomass and lipid content for B. braunii in photoheterotrophic cultivation conditions.

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4. Conclusion Carbon and nitrogen sources are the most significant nutrients for the photoheterotrophic growth of B. braunii and their interlinking metabolic activity is regulated by the availability of C or N source in the medium. In this study, we have measured the specific uptake rate of glucose and nitrate in GL and NL conditions. It suggests that GUR ability of B. braunii is regulated by the external nitrate level in the medium, while the NUR is associated with the external glucose concentration. Hence, the optimum range of glucose (4.015 to 8.96 g L−1) and nitrate (0.75 to 1.211 g L−1) are required for efficient growth and lipid production. A disclosure/conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organization that could inappropriately influence or the bias the content of the paper. It is to specifically state that “No Competing interests are at stake and there is No Conflict of Interest” with other people or organizations that could inappropriately influence or bias the content of the paper. 1. All the authors mutually agreed for submitting this manuscript to BITEB. 2. The manuscript is original work. 3. The manuscript has not been submitted earlier to Bioresource Technology Reports. Acknowledgements We gratefully acknowledge the financial support for Department of Biotechnology (DBT), Government of India for providing JRF fellowship to SSK (Fellowship Grant No.: 8798-35-044) for completion of the project (Contingency Grant No.: 8798-35-061). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biteb.2019.100337. References Bekirogullari, M., Fragkopoulos, I.S., Pittman, J.K., 2017. C. theodoropoulos production of lipid-based fuels and chemicals from microalgae: an integrated experimental and model-based optimization study. Algal Res. 23, 78–87. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Canadian J. Biochem. Physiol. 37, 911–917. Bren, A., Park, J.O., Towbin, B.D., Dekel, E., Rabinowitz, J.D., Alon, U., 2016. Glucose becomes one of the worst carbon sources for E. coli on poor nitrogen sources due to suboptimal levels of cAMP. Sci. Rep. 6, 24834. Brennan, L., Owende, P., 2010. Biofuels from microalgae—a review of technologies for production, processing, and extractions of biofuels and co-products. Renew. Sust. Energ. Rev. 14 (2), 557–577.

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