Appl Microbiol Biotechnol (2014) 98:2345–2356 DOI 10.1007/s00253-013-5442-4

BIOENERGY AND BIOFUELS

The effect of nitrogen limitation on lipid productivity and cell composition in Chlorella vulgaris Melinda J. Griffiths & Robert P. van Hille & Susan T. L. Harrison

Received: 12 August 2013 / Revised: 26 November 2013 / Accepted: 27 November 2013 / Published online: 12 January 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Chlorella vulgaris accumulates lipid under nitrogen limitation, but at the expense of biomass productivity. Due to this tradeoff, improved lipid productivity may be compromised, despite higher lipid content. To determine the optimal degree of nitrogen limitation for lipid productivity, batch cultures of C. vulgaris were grown at different nitrate concentrations. The growth rate, lipid content, lipid productivity and biochemical and elemental composition of the cultures were monitored for 20 days. A starting nitrate concentration of 170 mg L−1 provided the optimal tradeoff between biomass and lipid production under the experimental conditions. Volumetric lipid yield (in milligram lipid per liter algal culture) was more than double that under nitrogen-replete conditions. Interpolation of the data indicated that the highest volumetric lipid concentration and lipid productivity would occur at nitrate concentrations of 305 and 241 mg L−1, respectively. There was a strong correlation between the nitrogen content of the cells and the pigment, protein and lipid content, as well as biomass and lipid productivity. Knowledge of the relationships between cell nitrogen content, growth, and cell composition assists in the prediction of the nitrogen regime required for optimal productivity in batch or continuous culture. In addition to enhancing lipid productivity, nitrogen limitation improves the lipid profile for biodiesel production and reduces the requirement for nitrogen fertilizers, resulting in cost and energy savings and a reduction in the environmental burden of the process. Keywords Algal biodiesel . Nutrient stress . Oil yield . Lipid productivity . Nitrogen limitation . Algal cell composition

M. J. Griffiths : R. P. van Hille : S. T. L. Harrison (*) Centre for Bioprocess Engineering Research (CeBER), University of Cape Town, Rondebosch, 7701 Cape Town, South Africa e-mail: [email protected]

Introduction Microalgae are an attractive alternative source of oil. Their simple, unicellular structure allows a potentially higher productivity than traditional oilseed crops. They do not compete for agricultural resources and can be grown in brackish or salt water, utilizing only sunlight, CO2, and essential nutrients such as nitrogen and phosphorous (Rodolfi et al. 2009). There is the potential for coproduction of valuable products such as pigments, antioxidants, and pharmaceuticals, as well as biomass for animal feed or fertilizer (Mata et al. 2010). Products from algal oil include biodiesel, specialty oils, cosmetics, nutraceuticals, and food and feeds with a high calorific value (Apt and Behrens 1999). One of the major challenges that need to be overcome in order for algal biodiesel to become economically viable and environmentally desirable is improvement in algal biomass and lipid productivity, as well as the maximum volumetric lipid concentration attainable (Borowitzka 1992; Sheehan et al. 1998; Tsukahara and Sawayama 2005). Improvements in algal lipid productivity lead to lower culture volumes, and hence lower cost per unit product. Lipid productivity is influenced by both biomass and lipid production by the algae. Productive strains and culture conditions able to produce cells with a high growth rate and lipid content are required (Rodolfi et al. 2009). Several environmental factors, such as temperature (Converti et al. 2009), light (Rodolfi et al. 2009), and nutrient (e.g., phosphate, nitrate, and silicate) availability (Shifrin and Chisholm 1981) affect microalgal lipid composition and productivity. Nitrogen (N) limitation is the most frequently reported method of enhancing lipid content. It is inexpensive and easy to manipulate and has a reliable and strong influence on lipid content in many species (Chelf 1990; Rodolfi et al. 2009; Shifrin and Chisholm 1981). However, stress conditions that enhance lipid content, such as N limitation, also decrease the growth rate, and therefore may not improve overall lipid productivity

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(Lardon et al. 2009; Griffiths et al. 2012; Breuer et al. 2012; Lv et al. 2010). Under N-limiting conditions, the cells’ ability to synthesize N-containing compounds, such as proteins, nucleic acids, and chlorophyll, is compromised. As these compounds are necessary for cell growth and division, the growth rate becomes dependent on the intracellular N concentration. Growth eventually ceases, but, given conducive conditions, photosynthesis continues. Metabolic flux of carbon assimilated during photosynthesis is diverted from protein synthesis to lipid or carbohydrate production (Li et al. 2008). Storage lipids, primarily composed of triacylglycerol (TAG), are a compact and efficient cellular store of carbon and energy. They are relatively inert and can be packed into lipid vesicles easily. Lipids generate more energy than carbohydrates on oxidation, providing an excellent reserve for biomass production once N becomes available (Roessler 1990). Synthesis of TAGs also serves as an electron sink under photooxidative stress conditions (Hu et al. 2008). Production of lipids, a dense, highly reduced storage compound that does not contain N, reduces oxidative damage, while allowing the cell to continue photosynthesising (Klok et al. 2013; Lacour et al. 2012). Although N limitation is well known to enhance lipid content in many microalgae, its effect on growth rate, lipid content, and overall lipid productivity is species specific. The majority of studies have tested only the two extremes of N-replete and Ndepleted cultures, usually in a two-stage batch process (Breuer et al. 2012; Griffiths et al. 2012; Illman et al. 2000; Shifrin and Chisholm 1981; Su et al. 2011). For example, Breuer et al. (2012) examined the total fatty acid and TAG productivity of nine species in two-stage (N replete followed by N depleted) batch culture. All species except Dunaliella tertiolecta showed an increase in total fatty acid and TAG content under N limitation. It was also found that all species continued to increase in biomass concentration after transfer to N-free media. The main difference between species was the duration of retention of biomass productivity and the amount of biomass produced. Few studies have investigated the effect of intermediate degrees of N limitation (Hsieh and Wu 2009; Lv et al. 2010; Piorreck et al. 1984; Stephenson et al. 2010). Piorreck et al. (1984) investigated the growth of Chlorella and Scenedesmus and four species of cyanobacteria at six different concentrations between 0.0003 and 0.1 % NH4Cl or KNO3 (equivalent to between 2 and 1,159 mg L−1 nitrate in terms of moles N provided). They reported that higher N concentrations led to greater biomass, while low N levels led to an increase in total lipid content, with up to 70 % made up of neutral lipids such as TAG, as well as a lower protein and pigment content. The main focus of their work was to determine the effect of different N levels on cell, lipid and fatty acid composition, and the authors did not calculate lipid productivity or propose an optimal degree of N limitation. Stephenson et al. (2010) investigated the effect of nitrate (10, 100, 200, and 550 mg L−1) on Chlorella vulgaris

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cultures, while Hsieh and Wu (2009) examined the effect of urea (25, 50, 100, 150, and 200 mg L−1, equivalent to 52, 103, 207, 310, and 413 mg L−1 nitrate in terms of moles N provided) on cultures of a marine Chlorella species. Both studies showed an inverse relationship between lipid content and biomass concentration at different N levels. The optimal N concentration for lipid productivity was 200 mg L−1 nitrate and 100 mg L−1 urea (equivalent to 207 mg L−1 nitrate), respectively. In neither study were cultures shown to be N replete at the maximum N concentration tested, therefore lipid productivity under N limited conditions could not be compared to that under N-replete conditions. Lv et al. (2010) reported that maximum lipid productivity in C. vulgaris (40 mg L−1 day−1) occurred at a starting concentration of 1 mM KNO3 (equivalent to 62 mg L−1 nitrate). Some studies have investigated N limited continuous culture at an intermediate degree of N stress (Klok et al. 2013; Pruvost et al. 2009; San Pedro et al. 2013). Klok et al. (2013) hypothesized that creating an energy imbalance by reducing N supply while maintaining light supply to the cells would allow cell division to continue while stimulating TAG accumulation. They reported that Neochloris oleoabundans, cultivated in a turbidostat under continuous operation, reached maximum TAG productivity under high light conditions at a N feed rate of 35 mg L−1 day−1 (equivalent to 155 mg L−1 day−1 of nitrate). At lower N feed rates, TAG productivity decreased as the increase in TAG content no longer compensated for the loss in biomass productivity. However, San Pedro et al. (2013) found no increase in lipid content in Nannochloropsis gaditana in N-limited continuous cultures that were able to maintain biomass growth. The authors returned to two-stage batch culture to enhance lipid productivity. Pruvost et al. (2009) reported a similar lipid productivity in N. oleoabundans across all cultivation protocols tested. In studies of the effect of nutrient stress on microalgae, lipid content has often been measured at only a few points in the growth cycle, e.g., before and after N limitation. A temporal profile of lipid accumulation and growth rate with time allows a more accurate calculation of lipid productivity at specific time points in the growth cycle. This assists in determining the optimal cultivation strategy and harvesting times. The aim of this study was to develop an understanding of the interaction between biomass productivity, lipid accumulation, and cell biochemistry across various degrees of N limitation in C. vulgaris. The temporal profile of biomass and lipid accumulation over the culture period was recorded over a range of nitrate concentrations extending from N replete to the absence of N in the cultivation medium. The pigment, protein, and carbohydrate content and elemental composition of the cells was also measured to investigate the interaction between cell N content and key growth and lipid parameters. An improved understanding of the species-specific optimal degree of N limitation in C. vulgaris will assist in designing

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batch and continuous culture regimes that lead to optimal lipid productivities.

Materials and methods Cultures C. vulgaris UTEX 395 was grown in batch culture in airlift photobioreactors. The glass and steel reactors were 60 cm high with an external diameter of 10 cm, a draft tube of 5 cm external diameter, and a working volume of 3.2 L. Air enriched with 0.29 % CO2, reported to provide sufficient carbon for C. vulgaris growth in these reactors (Langley et al. 2012), was sparged at 2 L min−1, resulting in a circulation time of approximately 7 s and an overall mass transfer coefficient for CO2 of 0.0094±0.00026 s−1 (Langley et al. 2012). Light (250 μmol m−2 s−1 at the reactor surface) was provided by three cool white 18 W fluorescent light bulbs (Osram). Culture temperature was monitored daily and remained constant at 25±1 °C. All cultures were grown in 3 N BBM medium (Bold 1949) with adjusted levels of sodium nitrate. C. vulgaris was grown in 500 mL glass bottles for 7 to 10 days, with an initial nitrate concentration of 570 mg L−1, before being used to inoculate the airlift reactors at a biomass concentration of 0.05 g L−1, at starting nitrate concentrations of 0, 40, 70, 100, 170, 420, 570, 1,200, and 2,000 mg L−1. Sterile, distilled water was added daily to replace that lost to evaporation. Antifoam (20 μl) was added to each reactor to reduce foaming. The error in replicate growth experiments was investigated in five replicate cultures of C. vulgaris at 570 mg L−1 nitrate. The average relative error in biomass concentration across the growth curve was less than 5 %. Assays The nitrate concentration in the media was monitored daily by spectrophotometry. Filtered samples were diluted to less than 12 mg L−1 nitrate. Optical density (OD) at 220 nm was measured in a quartz cuvette and quantified using a NaNO3 standard curve (Clesceri et al. 1998). The spectrophotometric method was verified by comparing the results to nitrate concentration measured by high-performance liquid chromatography. Results from the two different methods were found to differ by an average of 12 % over the course of a growth curve. Biomass was measured by OD at 750 nm every day, while pigment content and dry weight (DW) were quantified every third day (Griffiths et al. 2011). Individual calibration curves of OD as a function of DW were constructed for each culture, and used to calculate DW from OD. Cell lipid content (taken to be equivalent to total fatty acid content) was measured daily by direct transesterification (Griffiths et al. 2010). Other cell components were quantified

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every third day. The protein content of the cells was determined by extraction into NaOH (Rausch 1981) and quantified by the bicinchoninic acid method of Walker (1994). Carbohydrate content was measured by the phenol-sulfuric acid method of Dubois et al. (1956). The carbon (C), hydrogen (H), and nitrogen (N) composition of the algal cells was determined by elemental analysis using a Thermo Flash EA 1112 series elemental analyzer. Pigments were extracted from algal cells using dimethyl sulfoxide (DMSO, 99 %, Saarchem). Culture samples (2 ml) were centrifuged in Eppendorf tubes at 10,000 rpm for 3 min and the supernatant discarded. Hot (60 °C) DMSO (2 ml) was added and cells resuspended by vortexing. Samples were incubated at 60 °C, with occasional shaking, for 10 min before centrifugation. The supernatant pigment extract was removed and diluted with DMSO to an OD of less than 1. The OD at 649, 665, and 480 nm was determined and the total pigment content was calculated as the sum of the chlorophyll a and b and total carotenoid content according to the equations of Wellburn (1994). Calculations Instantaneous biomass productivity (Q X , in gram per liter per day) was calculated as the change in biomass concentration (X , in gram per liter) per unit time between two consecutive sampling times. Specific growth rate (μ , in per day) was determined across each pair of sample points from the slope of the natural logarithm of biomass concentration as a function of time. Volumetric lipid concentration (P VOL, in milligram per liter) was calculated as the product of biomass concentration (X , in gram per liter) and lipid content (P, fraction of DW) (Eq. 1). Instantaneous lipid productivity (Q P INST, in milligram per liter per day) was calculated as the change in P VOL as a function of time (Eq. 2), and average lipid productivity (Q P AVE, in milligram per liter per day) as P VOL at the time point of interest divided by the time from inoculation (Eq. 3). Maximum parameters were defined as the highest value reached within the 14-day culture period. For the calculation of μ max, Q Xmax and Q P INST max, three consecutive instantaneous values of μ , Q X , and Q P INST were averaged across the time course to provide a rolling average, and the maximum defined as the highest average value reached. PVOL ¼ X  P

ð1Þ

QP

INST

¼

PVOLðnÞ −PVOLðn−1Þ t ðnÞ −t ðn−1Þ

ð2Þ

QP

AVE

¼

PVOLðnÞ t ðnÞ

ð3Þ

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The nitrate uptake rate was similar in cultures with different starting nitrate concentrations. The initial nitrate uptake rate (up to day 4) was between 96 and 100 mg L−1 day−1 in all cultures with more than 100 mg L−1 nitrate (Fig. 1a). Nitrate uptake rate decreased over time to an overall average of between 34 and 89 mg L−1 day−1 in all cultures. Therefore,

the nitrate uptake rate per unit biomass decreased with culture age and residual nitrate concentration. The greater the starting nitrate concentration, the greater the final biomass concentration (Fig. 1b) and the lower the final lipid content (measured as total fatty acid content; Fig. 1c). The time at which the nitrate became exhausted in the medium, along with the maximum specific growth rate, and maximum biomass and lipid productivities, are shown as a function of initial nitrate concentration in Table 1. Cultures with a nitrate concentration less than 170 mg L−1 exhausted the nitrate in the

Fig. 1 a Residual nitrate concentration in the media, b biomass concentration, c lipid content, d volumetric lipid concentration, and e average lipid productivity of C. vulgaris batch cultures over 20 days at nitrate concen-

trations of 0 (open circle), 40 (filled square), 70 (open diamond), 100 (filled triangle), 170 (multiplication symbol), 420 (open triangle), 570 (filled diamond), 1,200 (open square), and 2,000 (filled circle) mg L−1

Results Growth and lipid characteristics

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Table 1 The time of nitrogen exhaustion in the medium, maximum specific growth rate (μ max), maximum biomass productivity (Q X max), and maximum instantaneous lipid productivity (Q P INST max) of C. vulgaris batch cultures at different starting nitrate concentrations Initial nitrate N exhaustion μ max (day−1) concentration (days from −1 inoculation) (mg L )

Q X max Q P INST max (g L−1 day−1) (mgL−1 day−1)

0 40 70 100 170 420 570 1,200 2,000

0 1.2 1.7 2.0 2.2 4.9

0.4 1.5 1.5 1.5 1.4 1.4

0.016 0.149 0.200 0.265 0.326 0.325

3 30 48 53 57 55

8.6 19.7 –

1.4 1.3 1.1

0.352 0.366 0.373

48 29 31

medium during exponential growth (before day 2.2, i.e., nitrate was the primary limiting nutrient). Cultures with 420 and 570 mg L−1 nitrate depleted the nitrate in the media during linear growth (days 5 and 9, respectively, i.e., light limitation preceded nitrate limitation). In the culture with an initial 1,200 mg L−1 nitrate, nitrate was depleted after 20 days. With an initial 2,000 mg L−1 nitrate, the culture still had a concentration of 660 mg L−1 nitrate at the end of the experiment. The maximum specific growth rate (μ max) was highest (1.5 day−1) in cultures with between 40 and 100 mg L−1 nitrate, and decreased slightly with increasing nitrate concentrations (to 1.1 day−1 at 2,000 mg L−1, Table 1). This is suggestive of substrate inhibition at high nitrate concentrations. In the culture with 0 mg L−1 nitrate, the μ max was 0.4 day−1, indicating that the Monod saturation constant (K S ) for nitrate in C. vulgaris is between 20 and 30 mg L−1. The maximum biomass concentration measured was 2.4 g L−1 in N-replete cultures, between 1.7 and 1.9 g L−1 in cultures where N became exhausted during linear growth or early stationary phase, and less than 1.5 g L−1 in cultures depleted of N during exponential growth (Fig. 1b). The culture with no initial nitrate had a very low maximum biomass concentration (0.8 g L−1). N-replete cultures (starting nitrate concentrations of 1,200 and 2,000 mg L−1) maintained a stable lipid content of between 10 and 12 % throughout the growth period (Fig. 1c). Cultures with 420 and 570 mg L−1 nitrate at inoculation showed a gradual increase in lipid content, beginning 4 to 6 days after N exhaustion in the medium. The lipid content of these cultures reached 35 and 28 % DW, respectively, at 20 days and was still rising linearly with time. Cultures with an initial nitrate concentration of 100 and 170 mg L−1 increased in lipid content steadily over 20 days, approaching a maximum of between 50 and 55 % DW. Cultures which became N limited during the first two days of growth

(70 mg L−1 and below) showed an immediate and rapid increase in lipid content, reaching a maximum of between 55 and 65 % DW from day 16. P VOL was highest throughout the experiment in the culture grown in medium containing 170 mg L−1 nitrate (Fig. 1d). This culture maintained a constant rate of increase in P VOL over 20 days. The 70 and 100 mg L−1 nitrate cultures maintained an equivalent P VOL up to days 8 and 14, respectively. Lipid productivity declined thereafter due to the cessation of growth and the attainment of maximum lipid content. Cultures with nitrate concentrations of 40, 420, 570, 1,200, and 2,000 mg L−1 all had very similar P VOL up to day 8. From day 9, the P VOL of the 420 mg L−1 nitrate culture began to increase due to an increase in lipid content. By day 20, it had nearly matched the P VOL of the 170 mg L−1 nitrate culture. The culture with a starting nitrate concentration of 570 mg L−1 followed a similar pattern 6 days later. Q P INST is influenced by both biomass productivity and lipid content. The 170 and 100 mg L−1 nitrate cultures gave the most consistent Q P INST across 20 days due to a steady increase in both biomass and lipid (time course data not shown, maximum values shown in Table 1). The 420 and 570 mg L−1 nitrate cultures showed intermediate lipid productivity for the first few days due to the low lipid content and high growth rate, but reached a higher productivity after N limitation due to the rapid increase in lipid content with a constant, but high biomass. The 40 and 70 mg L−1 nitrate cultures both had high initial lipid productivities due to rapid lipid accumulation, but productivity decreased steadily to zero at day 18 as their lipid content reached a plateau at 55 to 60 % and biomass no longer increased. The 0 mg L−1 culture had a very low lipid productivity throughout, due to low biomass productivity. The 2,000 and 1,200 mg L−1 nitrate cultures maintained a constant lipid content and therefore the lipid productivity was dictated by the biomass productivity which decreased with time, following the onset of light limitation. Q P AVE indicates the overall productivity of a culture at a certain time point. At day 3, most cultures had a similar Q P −1 AVE (Fig. 1e), except for the 0 mg L nitrate culture which had a very low lipid productivity due to a low biomass concentration. By day 5, the cultures with starting concentrations of 70, 100, and 170 mg L−1 nitrate had been more productive. The culture with 170 mg L−1 nitrate maintained the highest Q P AVE of between 35 and 40 mg L−1 day−1 from days 5 to 20. The culture with 100 mg L−1 nitrate also maintained this Q P AVE from day 6, but began to decrease from day 14. Maximum biomass concentration and biomass productivity increased with increasing nitrate concentration. The maximum lipid content showed an opposite trend (Fig. 2a), illustrating the tradeoff between biomass concentration and lipid content. The optimal starting nitrate concentration tested, in terms of lipid productivity, was 170 mg L−1. Interpolating the data by fitting a second order polynomial through the points

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Fig. 2 a The tradeoff between maximum biomass concentration (X max) and lipid content (P max) achieved over 20 days in batch cultures of C. vulgaris at different starting nitrate concentrations and b interpolation

of maximum volumetric lipid concentration (P VOL max) and average lipid productivity (Q P AVE max) to find the optimal starting nitrate concentration

from 70 to 570 mg L−1 nitrate, the starting nitrate concentrations expected to achieve maximum volumetric lipid concentration and lipid productivity were estimated as 305 and 241 mg L−1, respectively (Fig. 2b). Below this nitrate concentration, cells achieved a high lipid content, but the biomass concentration was too low for optimal productivity. Above this nitrate concentration, the higher biomass concentration of cultures was offset by a lower lipid content, and there was a lag phase between N exhaustion and lipid accumulation (Fig. 1c).

that became N limited in stationary phase, or remained N replete (cultures 420–2,000 mg L−1), had an average C content of 48 %. The C content of cultures at 100 and 170 mg L−1 changed from 46 to 64 and 60 %, respectively, over the course of the growth cycle. The higher C content in N-limited cultures was probably due to a relative increase in C-containing compounds such as lipid in the cells. A very similar pattern was seen with the hydrogen (H) content (up to 9 % DW in N limited cultures, and between 5 and 7 % DW in N-replete cultures), as expected, because C-containing compounds such as lipids and carbohydrates are H-rich compounds (data not shown). The N content of cells decreased with time and N limitation (Fig. 4b). A slow decrease occurred in the N replete cultures (1,200 and 2,000 mg L−1). In the N-limited cultures, a sharp decrease in N content was observed immediately following the exhaustion of N in the medium. Cells reached a minimum N content of between 0.5 and 1 %. Pigment and protein content of the cultures declined gradually with culture age and rapidly with the onset of N

Cell biochemistry Nitrogen limited cultures continued to increase in DW for 2 to 5 days after the exhaustion of nitrate in the medium (Fig. 1a, b). In many cases, the increase in biomass after nitrate depletion was greater than that during N-replete growth. A large proportion (>50 %) of the additional biomass was made up of lipid. In the N-limited cultures (0 to 420 mg L−1 nitrate), lipid contributed over 63 % of the increase in DW after N depletion. The relative increase in non-lipid biomass after nitrate depletion in the medium was greatest in cultures that ran out of N earliest (Fig. 3). In the 0 and 570 mg L−1 nitrate cultures, some of the non-lipid biomass formed before N depletion was apparently converted to lipid postnitrate-depletion, causing the negative values for non-lipid biomass production (Fig. 3, solid gray bars). Lipid produced before N depletion was greatest in cultures with a high starting nitrate concentration, but that produced after depletion was greatest at an intermediate starting nitrate concentration (100 and 170 mg L−1). The C content of cells in the cultures with 0 or more than 420 mg L−1 nitrate remained fairly stable over the course of the growth cycle, while cultures with between 40 and 170 mg L−1 nitrate varied more (Fig. 4a). Those that became N limited early in exponential growth (cultures 0 to 70 mg L−1) had an average C content of 61 %, while those

Fig. 3 Non-lipid biomass (solid bars) and lipid (hatched bars) produced before (black ) and after (gray ) nitrate depletion in the medium in C. vulgaris cultures with different starting nitrate concentrations

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Fig. 5 Lipid (diamond), protein (square), carbohydrate (circle), and pigment (triangle) content on day 20 of C. vulgaris cultures containing 0, 40, 70, 100, 170, 420, 570, 1,200, and 2,000 mg L−1 nitrate

Fig. 4 a C content and b N content of cells of C. vulgaris cultures containing 0 (open circle), 40 (filled square), 70 (open diamond), 100 (filled triangle), 170 (multiplication symbol), 420 (open triangle), 570 (filled diamond), 1,200 (open square), and 2,000 (filled circle) mg L−1 nitrate

limitation, following a very similar trend to the N content of cells (data not shown). Carbohydrate content decreased with culture age in N-limited cultures (0 to 170 mg L−1) from an average of 26 % at day 3, to 13 % after 20 days. In the 420 and 570 mg L−1 nitrate cultures, the carbohydrate content increased from 19 to 38 and 35 %, respectively, over the first 12 days, before declining gradually to 33 and 34 %, respectively, at day 20. N-replete cultures (1,200 and 2,000 mg L−1 nitrate) showed a gradual increase in carbohydrate content from 16 and 18 to 27 and 28 %, respectively (data not shown). With increasing N limitation, lipid content increased, along with a decrease in all other major cell components (Fig. 5). Correlations of cell biochemistry with cell nitrogen content The pigment content of cells was proportional to their N content. The slope of the regression line was 0.69, with an R 2 of 0.95 (Fig. 6a). Biomass productivity during exponential growth decreased with a decrease in the N content of the cells (Fig. 6b). There was a critical minimum cellular N content required for growth (between 1 and 2 % DW). Above this point, the productivity increased with increasing N content, showing a typical Monod growth response, approaching a maximum Q X of between 0.35 and 0.37 g L−1 day−1. At

high N availability, there was a maximum N content of the cells (9 % DW). Lipid content was inversely correlated with N content (Fig. 6c). Above a cell N content of 5 %, lipid content was independent of N content and remained constant at approximately 10 % DW (zero-order relationship). At cell N content below 5 %, lipid content was a function of the N content (firstorder relationship). Between a cell N content of 2 and 5 %, there was a transition zone where cell lipid content increased with a decrease in N content, up to intermediate levels (13– 35 % DW). The slope of the linear regression line was −6.5 (R 2 =0.65). At a cell N content of below 2 %, cell lipid content was between 30 and 64 % DW. In this region, lipid content was a stronger function of cell N content, demonstrating a linear correlation with a slope of −17.8 (R 2 =0.70). Overall average lipid productivity was at a maximum (34– 40 mg L−1 day−1) at a cell N content of between 1 and 2 % (Fig. 6d), owing to the tradeoff between lipid content and biomass productivity. At cellular N contents greater than 3 %, the lipid productivities were in the range of 14– 26 mg L−1 day−1, owing to a higher biomass productivity, but lower lipid content. Below a cell N content of 1 %, there was a large variation in lipid productivity, due to the range of biomass productivities in cultures that experienced N limitation before or during exponential growth (0, 40, 70, and 100 mg L−1 nitrate), although similar lipid content resulted under these conditions.

Discussion The similar μ max (1.4–1.5 day−1) in cultures with a starting nitrate concentration between 40 and 570 mg L−1 (Table 1) was due to the fact that μ max occurred early in the growth curve (between days 0.8 and 1.2), when there was still sufficient nitrate in the media. The culture containing 0 mg L−1 nitrate experienced immediate N limitation, resulting in a

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Fig. 6 a Pigment content, b biomass productivity during the exponential growth phase, c lipid content, and d average lipid productivity, plotted against the nitrogen content of cells of C. vulgaris cultures with starting nitrate concentrations of 0 (open circle), 40 (filled square), 70 (open

triangle), 100 (filled triangle), 170 (multiplication symbol), 420 (open triangle), 570 (filled diamond), 1,200 (open square), and 2,000 (filled circle) mg L−1

lower μ max (0.4 day−1). The lower μ max of the 1,200 and 2,000 mg L−1 cultures (1.3 and 1.1 day−1) may have been due to substrate inhibition. These results are similar to those of Hsieh and Wu (2009) who reported a μ max of 1.4 day−1 in a marine Chlorella species at urea concentrations between 100 and 200 mg L−1 (equivalent to 207 and 413 mg L−1 nitrate in terms of moles of N supplied). Below 100 mg L−1 urea, μ max was reported to decrease. In this work, C. vulgaris was able to maintain μ max at lower starting levels of equivalent N availability. This may be due to the differences in species or substrate used. X max in the 2,000 mg L−1 nitrate culture (2.3 g L−1) was no higher than the 1,200 mg L−1 culture (Fig. 1b), indicating that these cultures were N replete. Hsieh and Wu (2009), using a marine species of Chlorella, reported a similar X max of 2 g L−1 under conditions of maximum urea supply (200 mg L−1, equivalent to 413 mg L−1 nitrate in terms of moles of N supplied, Table 2). The final lipid contents measured in this work (between 12 and 14 % for N replete and up to 65 % for N limited; Table 2) were similar to previous reports, where the lipid content of C. vulgaris varied between 12 and 30 % under high N conditions, and 40 and 66 % under low N conditions (Breuer et al. 2012; Hsieh and Wu 2009; Illman et al. 2000; Piorreck et al. 1984; Widjaja et al. 2009).

Despite a lower lipid content, Breuer et al. (2012) reported average volumetric lipid productivities (77 mg L−1 day−1 under N replete and 130 mg L−1 day−1 under N-deficient conditions, Table 2) more than three times higher than those found in this work. This is due to much higher average growth rates which may have been due to the different reactor types and cultivation conditions used. Stephenson et al. (2010) also report lower lipid contents, but significantly higher biomass concentrations, and found the highest average lipid productivity (71 mg L−1 day−1) at an intermediate starting nitrate concentration similar to that found in this work (200 mg L−1). Widjaja et al. (2009) and Converti et al. (2009) reported lower average lipid productivities (10–13 and 8 mg L−1 day−1 under N replete conditions and 6–10 and 20 mg L−1 day−1 under N depleted condition, respectively). This could be due to the lower light intensities used (30 and 70 μmol m−2 s−1, respectively, Table 2). In this work, additional total biomass accumulated in the absence of any external N was up to six times the DW at the time of nitrate exhaustion (cultures containing 40 and 70 mg L−1 nitrate; gray-filled and striped bars, Fig. 3). This is within the range of 1.4- (Isochrysis galbana) to 7.8-fold (Scenedesmus obliquus) reported by Breuer et al. (2012) who found an increase in biomass concentration after N removal in

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Table 2 Comparison of maximum biomass concentration (X max), lipid content (P max), volumetric lipid concentration (P VOL max), and average and instantaneous lipid productivity (Q P AVE max and Q P INST max) Reference

Species

Breuer et al. (2012)

C. vulgaris

Starting concentration

33.6 mM KNO3 Transfer to no nitrogen Converti et al. (2009) C. vulgaris 1.5 g L−1 NaNO3 0.75 g L−1 NaNO3 0.375 g L−1 NaNO3 Hsieh and Wu (2009) Chlorella sp. 0.025 g L−1 urea 0.05 g L−1 urea 0.1 g L−1 urea 0.15 g L−1 urea 0.2 g L−1 urea Lv et al. (2010) C. vulgaris 5 mM KNO3 3 mM KNO3 1 mM KNO3 0.2 mM KNO3 Piorreck et al. (1984) C. vulgaris 0.0003 % KNO3 0.001 % KNO3 0.003 % KNO3 0.01 % KNO3 0.03 % KNO3 0.1 % KNO3 Stephenson et al. (2010) C. vulgaris 0 mg L−1 nitrate 10 mg L−1 nitrate 100 mg L−1 nitrate 200 mg L−1 nitrate 550 mg L−1 nitrate Widjaja et al. (2009) C. vulgaris 70.02 mg L−1 nitrogen 7 days nitrogen free 17 days nitrogen free This study C. vulgaris 0 mg L−1 nitrate 40 mg L−1 nitrate 70 mg L−1 nitrate 100 mg L−1 nitrate 170 mg L−1 nitrate 420 mg L−1 nitrate 570 mg L−1 nitrate 1,200 mg L−1 nitrate 2,000 mg L−1 nitrate

reported in the literature for C. vulgaris grown in batch culture under nitrogen-limiting conditions

Nitrogen Light X max P max P VOL max Q P AVE max Q P INST max (mmol L−1) (μmol m−2 s−1) (g L−1) (% DW) (mg L−1) (mg L−1 day−1) (mg L−1 day−1)

33.6 0.0 17.6 8.8 4.4 0.8 1.7 3.3 5.0 6.7 5.0 3.0 1.0 0.2 0.03 0.1 0.3 1.0 3.0 9.9 0.0 0.2 1.6 3.2 8.9 5.0 0.0 0.0 0.0 0.6 1.1 1.6 2.7 6.8 9.2 19.4 32.3

all species tested. These authors found that the increase in biomass was partly (42–69 %) explained by an increase in fatty acid content. In this work, the additional biomass was found to be 52–70 % fatty acids. Li et al. (2008) also observed that biomass concentration continued to increase after exhaustion of N in the media of N. oleoabundans cultures. As the cell content of N-containing compounds, such as chlorophyll and protein, also decreased with N limitation, they concluded that the additional growth must be supported by the redistribution of these intracellular N pools.

150 150 70 70 70 600 600 600 600 600 60 60 60 60 ∼11 ∼11 ∼11 ∼11 ∼11 ∼11 165 165 165 165 165 30 30 30 250 250 250 250 250 250 250 250 250

11 3.8

0.464 0.849 1.422 1.785 2.027 0.4 0.77 1.05 1.2 0.017 0.057 0.077 0.212 0.293 0.287 0.72 0.84 2.17 3.11 5.21 0.86

15 46.1 5.9 14.37 15.31 66.1 60.2 52.2 36.5 32.6 15.9 18.5 20 22.5 57.9 62.9 42.7 22 21.8 22.6 20 22 39 46 18 30

0.08 0.48 0.83 1.19 1.48 1.87 1.68 2.35 2.3

65 60 56 52 54 35 28 12 14

91 173

77 130 8.16 20.44 20.3 51 85 124 109 110 35 40

128 864 1,320 334

43 274 458 624 790 659 451 281 302

20 21 65 111 24 12.77 8 10 3 30 48 53 57 55 48 29 31

0.1 7 45 71 18

9 27 35 39 41 33 24 25 26

It was found in this work that the N content of nitratelimited cells decreased dramatically, from a maximum of 9 % to a minimum of about 1 % DW. Lipid productivity was highest in cells with a N content of between 1 and 3 %. This is the region of N limitation where the tradeoff between lipid content and biomass productivity produced maximum lipid productivity. This provides supporting evidence for the work by Rodolfi et al. (2009) who reported that lipid content in Nannochloropsis increased substantially only when N was fed to the culture in amounts equal to between 2.5 and

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1.25 % of the biomass productivity. Breuer et al. (2012) reported an initial N content for S. obliquus and Chlorella zofingiensis of 8–10 %. This decreased steadily with time from N depletion to a minimum N content of 1.3–1.6 % (estimated from the initial N content and increase in biomass concentration, assuming a constant volumetric protein content). San Pedro et al. (2013) defined the specific nitrate input (SNI) as the amount of nitrate supplied per unit of biomass per unit time. They found that, in N. gaditana, the SNI required to trigger fatty acid accumulation was below 1.5 mmol nitrate g−1 biomass day−1. The starting nitrate concentration estimated to result in the highest lipid productivity through data interpolation (241 mg L−1) is similar to the optimal initial nitrate concentration of 200 mg L−1 reported by Stephenson et al. (2010) for C. vulgaris. It also supports the work by Li et al. (2008), who investigated the effect of N concentrations between 3 and 20 mM NaNO3 (equivalent to 186 to 1,240 mg L−1 nitrate) on lipid productivity in N. oleoabundans . They reported that maximum overall lipid productivity (133 mg L−1 day−1) occurred at an intermediate NaNO3 concentration (5 mM, equivalent to 310 mg L−1 nitrate). Hsieh and Wu (2009) reported lipid productivity in a marine strain of Chlorella to be maximum (124 mg L−1 day−1) at 100 mg L−1 urea. This is equivalent, in terms of moles of N, to 207 mg L−1 of nitrate. Lardon et al. (2009) have highlighted the importance of decreasing fertilizer and energy use in achieving a positive energy balance in microalgal biodiesel production. Nitrogen fertilizer has a large embodied energy due to the extreme process conditions required to generate combined N in the manufacturing process. A decrease in the process demand for N has the potential to decrease the cost and environmental burden of the process. Growth of algal cultures under Nlimited conditions increases the yield of lipid per unit N (data not shown). Cultures with starting nitrate concentrations of 170 mg L−1 or below yielded over 20 g lipid per gram N taken up at the end of 20 days, while cultures with 420 mg L−1 nitrate or above yielded below 6 g lipid per gram N taken up (calculated as volumetric lipid concentration divided by N removed from the medium). Therefore, the yield of lipid on N can be improved by at least 300 % under N-limited conditions. In addition, starting a batch culture with the optimal limiting N concentration, as opposed to transferring cells from N-replete to N-free media, removes the need to harvest the cells or change culture medium during cultivation, leading to savings in energy, water, and media components. In order to optimize the process, it is necessary to combine information on the financial and energy cost of N provision, with the cost associated with reduction in the duration of sustained productivity at reduced N addition. This analysis will allow the relative benefit of N addition in the range 100 to 400 mg L−1 to be determined.

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Cultures that became N exhausted later in the growth cycle reached higher biomass concentrations, with associated higher biomass productivities, but the onset of lipid accumulation was delayed, and initially slower than cultures that ran out of N earlier. There was a significant delay in lipid accumulation in those cultures (420 and 570 mg L−1 nitrate) which ran out of N during the linear or early stationary phase, compared to those that ran out during exponential growth (Fig. 1c). This could have been due to cultures becoming N limited at greater cell densities and cells experiencing greater mutual shading and hence less metabolic flux from photosynthesis for lipid accumulation. Lipid synthesis requires significant metabolic resources in terms of carbon compounds as well as energycarrying metabolites such as ATP and NADPH. The substrates for lipid synthesis are provided by both the light and carbon sources available to the culture, as well as the carbon and energy already contained within the cells as macromolecules at the end of the growth period. Further experiments (data not shown) have shown that dilution of cultures in the N limitation phase led to more rapid lipid accumulation, confirming that light availability limits lipid production. Klok et al. (2013) also reported that increased light availability under the same N limitation conditions led to an increase in TAG production. This is a challenge to achieving high lipid productivity, as the higher the biomass concentration, the greater the mutual shading, the lower the light availability and the slower the lipid accumulation. C. vulgaris cell physiology changed dramatically with N limitation. Increasing N limitation resulted in a higher lipid content and lower protein, pigment, and carbohydrate content. These changes were evident in the elemental composition of the cells, with the C and H content increasing and the N content decreasing with increased N limitation. An empirical correlation was found between N content and pigment content. In the absence of other causes of chlorosis, and given the relationships demonstrated between cellular N content, biomass productivity and lipid content, cell pigment content could be used as an indicator of intracellular N content, from which the metabolic state of the cell (i.e., growth mode (>5 % N), transition mode (2–5 % N), or lipid storage mode (1–2 % N)) could be determined. As pigment content is relatively rapid and easy to measure, this could be used as a qualitative indicator to facilitate monitoring of algal cultures. In addition to enhanced lipid productivity, the benefits of N-limited algal cultivation for biodiesel production include reduced use of N, and improved lipid profile. Under N limitation, the proportion of total lipid made up of TAG, the storage form of lipid most suited to biodiesel production, has been reported to increase. Stephenson et al. (2010) reported that, in C. vulgaris after 12 days of N limitation, over 50 % of the total lipid was TAG, compared to approximately 3 % under N-replete conditions. The fatty acid composition of algal

Appl Microbiol Biotechnol (2014) 98:2345–2356

lipids has also been reported to change with N limitation. In previous work (Griffiths et al. 2012), C. vulgaris metabolism shifted from the production of polyunsaturated fatty acids (C18:2 and C18:3) to the production of mainly saturated or monounsaturated fatty acids (C18:0 and C18:1) under N limitation. This shift in fatty acid composition resulted in the biodiesel from C. vulgaris lipid theoretically meeting the European biodiesel standards (EN 14214) under N-limited conditions. This was not the case under N-replete conditions. When applying N limitation in microalgal production, its effect not only on lipid productivity, but also on the overall energy balance and cost of the process (affected by factors such as nutrient supply, ease of downstream processing, and product range and quality) should be taken into account. In addition to determining product content, N limitation affects final biomass concentration and is expected to alter cell properties (e.g., density), which would influence the choice of harvesting method. One of the most attractive ways of improving the economics of an algal biodiesel process would be the coproduction of other valuable products in a biorefinery approach. In this case, the radical changes in cell composition under N limitation would have consequences for the suite of products that could be produced. Ideally, decisions made about the choice of an algal species or cultivation conditions should be based on a holistic view of the production process. This work investigated the interaction between biomass and lipid accumulation in C. vulgaris batch cultures grown under various degrees of N stress. An optimal degree of N limitation for maximum lipid productivity was found to occur at an estimated starting nitrate concentration of between 241 and 305 mg L−1, and a cell N content of between 1 and 3 %, corresponding to the optimal tradeoff between enhanced lipid content and decreased growth rate. This has practical application in the design of culture regimes and the prediction of optimal N feed rates and cell harvesting times in order to optimize microalgal lipid production. Microalgal lipids can be used for a variety of products such as biofuels, oils for food or feed, lubricants, cosmetics, or omega-3 fatty acids. Enhancing the lipid content of algae also increases their calorific content as a fuel in other energy applications (e.g., hydrogenation, gasification, or combustion), or as feed for livestock or aquaculture (Apt and Behrens 1999; Converti et al. 2009). Acknowledgments This work is based upon research supported by the South African National Energy Development Institute (SANEDI), the South African Research Chairs Initiative (SARChI) of the Department of Science and Technology, the National Research Foundation (NRF), and the Technology Innovation Agency (TIA). The financial assistance of these organizations is hereby acknowledged. Opinions expressed and conclusions arrived at are those of the authors and are not necessarily to be attributed to SANEDI, SARChI, TIA, or the NRF.

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