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Influence of nitrogen-limitation regime on the production by Chlorella vulgaris of lipids for biodiesel feedstocks
Biofuels (2010) 1(1), 4758

Anna L Stephenson1, John S Dennis1, Christopher J Howe2, Stuart A Scott3 & Alison G Smith4
Microalgae have the potential to provide large amounts of lipid-containing biomass that could be used as a renewable oil feedstock for biofuel use. Many fast-growing algae, such as Chlorella vulgaris, when grown under nutrient-sufficient conditions, have a lipid profile that is unsuitable for biodiesel use, since the triacylglyceride content is too low. However, growth under nitrogen limitation has been shown to increase triacylglycerides. Here we investigate the most effective nitrogen-limitation regime and find that rather than transferring cells to a medium without nitrogen, the maximal triacylglyceride productivity (46mg/l/day) is achieved by allowing the cells to deplete the nitrogen naturally. Detailed analysis of the fatty acid composition of the lipids indicated that there was a high proportion of a-linolenic acid (C18:3) under all conditions, so that it would have to be blended with other feedstocks in order to adhere to international standards for biodiesel.

The transport sector is a major contributor to the anthropogenic emission of greenhouse gases because of its reliance on liquid fuels derived from crude oil. One way of reducing this dependence is to substitute a fraction of the fuels made from crude oil with liquid fuels made from renewable biological resources, such as biodiesel. This research article is concerned with the expression in microalgae of lipids and lipid-like molecules that could be processed into diesel fuel (biodiesel). Microalgae have the potential to provide suitable biodiesel feedstock, since there are many species that contain high levels of lipid molecules. For example, the green alga Botryococcus braunii can have 60 wt% lipid [1] . However, this species is very slow growing. In contrast, many fast-growing algal species have relatively low lipid levels, and the major fraction of these are membrane lipids. For example, in the green alga Chlamydomonas reinhardtii, Vieler et al. reported that under nutrient-sufficient conditions, the glycolipids monogalactosyl-diacylglycerol (MGDG),
1

digalactosyldiacylglycerol (DGDG) and sulphoquinovosyl-diacylglycerol (SQDG), which are the major constituents of the photosynthetic membranes, the thylakoids, represented approximately 85 wt% of the total content of lipid [2] . Another 13 wt% of the total lipids were the phospholipids phosphatidyl glycerol (PG; the major phospholipid in thylakoids), and phosphatidyl inositol (PI), phosphatidyl ethanolamine (PE) and phosphatidyl choline (PC) present elsewhere in the cells, including mitochondria and the endomembrane system [2] . By contrast, only a small fraction of the total content of lipid comprises triacyl glycerides (TAGs), which are the most suitable lipids for biodiesel production because they contain three fatty acid (FA) chains, each of which can be transesterified into biodiesel, leaving only the glycerol backbone [3] . Phospholipids, on the other hand, must be removed from the oil if they are to be used for biodiesel production as they promote the accumulation of water in the biodiesel product, increase catalyst consumption

Author for correspondence Department of Chemical Engineering & Biotechnology, University of Cambridge, Pembroke Street, Cambridge, CB2 3RA, UK; Tel.: +44 122 333 4788; Fax:+44 122 333 4796; E-mail: als53@cam.ac.uk 2 Department of Biochemistry, University of Cambridge, Downing Site, Cambridge, CB2 1QW, UK 3 Department of Engineering, University of Cambridge, Trumpington Street, Cambridge, CB2 1PZ, UK 4 Department of Plant Sciences, University of Cambridge, Downing Site, Cambridge, CB2 3EA, UK

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10.4155/BFS.09.1 2010 Future Science Ltd

ISSN 1759-7269

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Stephenson, Dennis, Howe, Scott & Smith achieving the highest level of lipid per cell as possible, so cells containing only a modest amount of lipid could easily render the downstream processing unviable. Hence, two-stage strategies for growth have emerged. For example, Rodolfi et al. reported that when the microalga Nannochloropsis was first grown with sufficient nutrients to produce as high a biomass concentration as possible, and then the nutrients supplied to the biomass were limited, the cells contained approximately 60 wt% lipid and overall lipid productivity was higher [11] . Apart from the inter-relationship between productivity and lipid content per unit mass of cells, the composition of the FAs in algal oil is of key importance if it is to be used for diesel fuel. To adhere to EN 14214, the linolenic methyl ester (produced from C18:3 FA) content of biodiesel must be less than 12 wt%, the polyunsaturated methyl ester (at least four double bonds) less than 1 wt%, and the iodine value, (determined by the degree of saturation of the methyl esters) less than 120 g of iodine per 100 g [4] . Accordingly, whether the profile of FAs can be adjusted by varying the conditions of growth is an important issue. The aim of the research presented in this research article is to explore, using the two-stage approach, the tradeoff between the high biomass productivities of C. vulgaris when grown under nitrogen-sufficient conditions, and its ability to accumulate high proportions of TAG when grown under nitrogen-limited conditions. Very little information exists on the time-course of these reactions and yet temporal information is critical in the scaled-up design of appropriate bioreactors for the production of TAG for biofuels: this paper makes an initial attack on this problem. Particular areas of investigation are:
Determination of the impact of the initial concentra-

during the alkaline-catalyzed transesterification of the oil and act as emulsifiers, impeding phase separation during transesteriMicroalgae: Microscopic, single-celled fication [3] . Phospholipids also organisms that can photosynthesize increase the phosphorus content Triacylglyceride: Three fatty acid chains that are attached to a glycerol of the fuel, whilst the glycolipid backbone SQDG increases its sulfur conChlorella vulgaris: Single-celled green tent; both of these compounds alga capable of rapid growth must be below 10 ppm by mass to Biofuel: Fuel produced from plants meet the international standard EN or algae 14214 [4] . It is therefore important Nitrogen depletion: Cultivation of to maximize TAG production in microalgae in media containing low microalgae, if these organisms are levels of biologically available nitrogen to be used for the production of biodiesel feedstock. For a number of microalgal species, TAG production has been shown to be increased by growth in stress conditions, such as reduced levels of nitrogen or phosphate, high concentrations of Fe(III) or high temperature [5] . Species of Chlorella [6,7,8] , Dunaliella [9,10] , Nannochloropsis [11] and Neochloris [12] alter their metabolism and divert the flow of fixed carbon from protein to lipid such as TAGs, which act as a store of energy and carbon, and are deposited in the cytosol of the algal cell, in the form of densely packed lipid bodies [13] . Nitrogen-limitation regimes to increase TAG production have either involved reducing the nitrogen level in the medium in which the cells are grown [6] , or growing the cells heterotrophically with organic carbon sources such as glucose or acetate in the medium, thereby reducing the ratio of nitrogen to carbon [14] . The green microalga Chlorella vulgaris is a suitable species for biofuel feedstock because, when grown under nitrogensufficient conditions, it has relatively high rates of biomass production, of approximately 0.170.18 g/l/day, on a dry cell basis [15,11] . Under these conditions, total lipid production can be as high as 37 mg/l/day, albeit consisting predominantly of phospholipids and glycolipids [11] . Under nitrogen limitation, contents of total lipid of up to 63 wt% have been reported, consisting primarily of TAGs, and therefore appropriate for the production of biodiesel [16] . However, under nitrogen limitation, cell growth rate is also reduced, so that increased oil accumulation per cell does not necessarily lead to an increase in total oil productivity [6,17] . For example, Piorreck et al. achieved a lipid productivity of only 1.2 mg/l/day when growing C. vulgaris in limited nitrogen [16] . A more recent study reported 10 mg/l/day [18] , significantly less than the lipid productivity of approximately 37 mg/l/day achieved when C. vulgaris is grown in nitrogen sufficiency [11] . Productivity is not the only measure of interest: the economics of the subsequent extraction of the intracellular lipids from the cells are critically dependent on
Key terms Biodiesel: Fatty acid alkyl ester that can substitute for fossil diesel fuel

tion of cells in the culture on the subsequent accumulation of TAG during nitrogen depletion ;
The effect of the profile of nitrate in the medium

during nitrogen depletion on the production of TAG (i.e., whether it is more beneficial to remove nitrate from the medium abruptly or to let the algal cells deplete it themselves);
The FA composition of the lipids of C. vulgaris before

and after nitrogen depletion, to determine whether the resulting biodiesel could meet the specification on unsaturated FAs. Experimental
Definitions

In the context of this work, nitrogen-sufficient growth is defined as the autotrophic growth of microalgae in 3N-BBM+V medium, with nitrogen being supplied in the form of sodium nitrate of concentration 750 mg/l (equivalent to 550 mg/l nitrate). Nitrogen limitation is defined as

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Influence of nitrogen-limitation regime on lipid production by C. vulgaris the growth of cells in medium containing lower nitrogen levels than those employed for nitrogen-sufficient growth. Nitrogen deprivation is defined as the state achieved when the exogenous supply is completely exhausted.
Algal strain & cultivation

Research Article

C. vulgaris, strain 211/11B, was obtained from the Culture Collection of Algae and Protozoa at Dunstaffnage Marine Laboratory, Oban, Scotland. It was routinely cultured under continuous illumination in 3N-BBM+V medium that contained (per liter): 750 mg NaNO3 (equivalent to 550 mg nitrate), 25 mg CaCl 22H 2O, 75 mg MgSO47H 2O, 75 mg K 2HPO43H2O, 175 mg KH2PO4, 25 mg NaCl, 4.5 mg Na2EDTA, 582 g FeCl36H2O, 246 g MnCl24H2O, 30 g ZnCl 26H 2O, 12 g CoCl 26H 2O, 24 g Na 2MoO42H2O, 1.2 mg thiamine hydrochloride (vitamin B1) and 10 g cyanocobalamin (vitamin B12). The algae were cultivated in either continuous or batch systems, depending on the experiment, as described below.
Continuous culture in fermenters

of various nitrate concentrations and either grown in continuous culture in the fermenters or in batch culture in conical flasks, depending on the experiment. Once the culture had been grown to the required cell density of 5 107 cells/ml (equivalent to a dry cell mass of approximately 0.4 g/l), it was removed from the fermenters and centrifuged at 5000 g. The supernatant was discharged and the pellet of algal cells was washed several times in distilled water to remove all traces of the original medium. The pellets were then resuspended in new media, with various known nitrate concentrations. To achieve different initial cell concentrations, the pellets were diluted to different extents: for example, where an initial cell number of 2.5 107 cells/ml was required, the culture was diluted approximately twofold to achieve a dry cell mass of approximately 0.2 g/l. For the continuous system, the algae were re-inoculated into the 3 l fermenters and 75-ml samples were taken every 12 days and replaced with 75 ml fresh media. For the batch system, the algae were inoculated into conical flasks and the content of each flask was used for a single time point.
Dry cell mass & cell number

C. vulgaris was grown in 3 l fermenters, of (ungassed) liquid depth 0.23 m and diameter 0.13 m, illuminated from below using fluorescent lighting (Osram 860 bulbs), which produced light with spectra imitating daylight at 68 mol photons/m2/s, when measured at the bottom surface of the fermenter. The cultures were agitated at 300 rpm using an impeller with a diameter of 45 mm. The fermenters were supplied with air, enriched in CO2 to 5 vol%, at an average flow rate of 80 ml/min, as measured at 1 atm and 25C. The pH of the culture was monitored and kept stable (between pH 6.0 and 6.5) by altering the gas flow rate, therefore ensuring the provision of adequate CO2 for cell growth. Each fermenter had a water jacket linked to a temperature controller to maintain the temperature inside the fermenters at 22 1C.
Batch culture

Dry cell mass was determined by centrifuging a known volume of algal culture (1050 ml) at 5000 g, discarding the supernatant and washing the cells in distilled water. The algal cells were then filtered through a preweighed glass-fiber filter paper (Whatman GF/C 45 m), which had been previously stored under moisture-free conditions in a desiccator. The filter paper with the algal cells was dried at 80C for 24 h and left in a desiccator for 24 h before reweighing to determine the dry cell weight. Cell number was determined using a hemocytometer: the cells were diluted with deionized water to a concentration of 2501250 cells/mm 2 before counting, and at least 250 cells were counted per sample, to ensure suitable accuracy.
Lipid extraction

Alternatively, C. vulgaris was grown in 100 ml conical flasks, containing either 50 or 75 ml of algal culture with a depth of liquid of 30 and 43 mm, respectively, in an incubator provided with an atmosphere consisting of 10 vol% CO2, 71 vol% N2 and 19 vol% O2, and shaken at 120 rpm. The temperature was maintained between 20 and 23C. The flasks were under continuous illumination of 165 mol photons/m2/s from above, as measured from the platform holding the conical flasks, and provided using fluorescent tubes as above (Osram 860 bulbs).
Nitrogendepletion experiments

Initial inocula were grown in the fermenters, as described, under nitrogen sufficiency for approximately 20 days. The inocula were then introduced into media

Lipids were extracted from the algal biomass using a modified version of the Folch procedure [19] . Samples of algal culture (volume: 3050 ml) were centrifuged at 5000 g, and the resulting pellet was sonicated in 12 ml methanol for 10 min, in a bath sonicator at 20C. The samples were then heated under reflux for 10 min, 24 ml chloroform was added and the solution was refluxed for a further 60 min. The resulting extract was washed by shaking vigorously for 1 min with 20-ml saturated sodium chloride solution (6.2 M), before being centrifuged for 10 min at 5000 g. The lower, nonaqueous layer was removed and the washing repeated. The chloroform and methanol were finally removed from the lipid sample using a rotary evaporator.

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Stephenson, Dennis, Howe, Scott & Smith were dissolved in 50-l heptane with known concentrations of an internal standard of methyl heptadecanoate (Sigma Chemicals), to which peak areas of FA esters were compared for quantification. The retention times of FA esters were compared with standards (C16:0, C16:1, C18:0, C18:3, C20:0, C20:1 and C22:1, Sigma Chemicals) for identification.
Protein determination

Separation of lipids by thinlayer chromatography

Solvent systems for thin-layer chromatography (TLC) were made in approximately 1-l batches. For each solvent system, 100 ml was added to 20 20 cm TLC developing tanks lined with chromatography paper. The tanks were left for 2 h to equilibrate before the plates were run. Algal lipid extracts were dissolved in a known volume of chloroform (0.251 ml) and Hamilton syringes (25 l) were used to aspirate between 20- and 80-l portions of the lipid extract on to 20 20 cm silica gel 60 TLC plates (Fisher, UK). 1D TLC was used to separate the neutral lipids (including TAGs) and free FAs from the other lipids present, employing a solvent system of hexane:diethyl ether:acetic acid (70:30:2 v/v) [20] . 2D TLC was used to separate the polar lipids, including MGDG, DGDG, SQDG, PI, PG, PE and PC. Plates were run first in chloroform:methanol:water (65:25:4 v/v), then rotated 90 and run in chloroform: acetone:methanol:acetic acid:water (50:20:10:10:5 v/v) [21] . On completion, the TLC plates were sprayed with 0.2% 8-anilino-1-naphthalenesulphonic acid in methanol. The resulting complexes were examined under UV light of wavelength 366 nm, and the individual classes of lipids identified by comparison with standards (Lipid Products, UK).
Quantification of lipids & FAs

To determine the total content of protein in the algal cells, 1-ml samples were centrifuged (5000 g for 2 min): the cell pellet was resuspended in 1 ml 1 M NaOH and boiled for 5 min. The protein content was determined using the Bradford assay, with bovine serum albumin as the standard [22] .
Chlorophyll determination

The total content of chlorophyll in the cells, in wt% of the dry cell mass, was determined using the method of Arnon, using 1-ml samples of the algal culture under ana lysis [23] .
Nitrate concentration

The silica containing each of the lipids was scraped off the plates and each fraction was placed in a 12-ml screw neck test tube provided with a polytetrafluoroethylenelined cap (Fisher). FAs were methylated by adding 3 ml of 3 vol% H2SO4 (18.4 M) in methanol to each test tube, vortexing for 30 s and incubating at 100C. After 1 h, the reaction was quenched by the addition of 3 ml of deionized water and 3 ml hexane. The test tubes were vortexed for 30 s and centrifuged at 5000 g for 2 min in order to separate and remove the layer of hexane, which contained the FA methyl esters. A further 3 ml of hexane was added to the silica solution and the extraction process repeated to improve the recovery of the ester. The hexane was removed from the esters using a rotary evaporator. The recovery of the ester was monitored qualitatively; 1D TLC of the final product, employing a solvent system of hexane:diethyl ether:acetic acid (70:30:2 v/v), was carried out to ensure that all the lipids had been converted to esters. The FA esters were quantified and identified using GC (Agilent Technology, model 6850). The GC column was made from polyethylene glycol and was 30 m in length, with 320 m internal diameter and 0.25-m film thickness (Agilent 19091N-613E). The GC was operated at a temperature of 200C, a pressure of 12 psi and used helium as the carrier gas, with a constant flow of 1.4 ml/min as measured at 1 atm and 25C. Samples

The concentration of nitrate in the media was determined by colorimetry, using a nitrate assay supplied by Hach Lange Gmbh (Nitrate kit code: LCK 339), employing 1 ml of medium after removal of algal cells by centrifugation.
Error analysis

Six technical replicates were performed for each experimental procedure in order to determine the errors associated with each data point. Results & discussion
Composition of lipids in Chlorella vulgaris grown in continuous culture
Nitrogen-sufficient conditions

The total lipid content, lipid composition and FAs were measured in C. vulgaris cells grown in continuous culture under nitrogen-sufficient conditions over a period of 20 days using the fermenter system. The cell number increased during this period from 1 106 to 1.2 108 cells/ml whilst the lipid composition stayed fairly constant. Figure 1 illustrates the composition of the lipid fraction over this period, which averaged 13.7% of the total dry cell mass. It can be seen that a significant proportion (~27 wt%) of the lipids are phospholipids, which cannot be used for the production of biodiesel, whilst only approximately 3 wt% are TAGs. The principal FAs of the total extracted lipids were found to be palmitic acid (C16:0, 19 wt%), linoleic acid (C18:2, 23 wt%) and a-linolenic acid (C18:3, 21 wt%) (Figure2A) .

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Influence of nitrogen-limitation regime on lipid production by C. vulgaris

Nitrogendeprived conditions
20 wt% of total dry cell mass

Research Article

Total: 19.4 wt%

By contrast, when C. vulgaris was grown in the fermenters in medium devoid of nitrogen, after 12 days the lipid content had increased to almost 20 wt% of total dry cell mass and, moreover, the major fraction (over half) was now TAGs, whilst the proportion of phospholipids and glycolipids fell. In addition, the FA composition of the TAGs was also altered under nitrogen-limitation conditions (Figure 2B) , so that the major FA was now oleic acid (C18:1, 3237 wt%), while the proportions of the more highly unsaturated FAs, C18:2, C18:3 and C16:2, were all reduced compared with levels under nutrient-sufficient conditions. For example a-linolenic acid (C18:3) fell from 21 wt% to 15 wt%. Interestingly, levels of the saturated FA, stearic acid (C18:0) were also reduced in nitrogen limitation, indicating that the alterations were not due simply to lower rates of unsaturation, but rather a shift in overall FA production.
Strategies to enhance lipid productivity ofC.vulgaris

15

Total: 13.7 wt%

10

Other TAG FFA MGDG DGDG SQDG PE PG PI PC B

wt% of total FA content

Although nitrogen-deprivation increased TAG levels, these were still much below the proportion that would allow economic downstream processing. We therefore investigated an alternative experimental setup, in which cultures were grown under batch conditions in 100-ml conical flasks (total culture volume: 75 ml). This employed smaller depths of liquid (0.04 vs 0.23 m) and higher light levels (167 vs 68 mol photons/m2/s) compared with conditions in the fermenters; therefore, conditions were somewhat closer to those in industrial production in ponds or bioreactors, where a high productivity of biomass is sought.
Nitrogen-sufficient conditions

Figure1. Lipid composition of Chlorella vulgaris when grown in continuous culture. (A) Average composition over 20days of growth in nitrogensufficient medium with an increase in cell count from 1 106 to 1.2 108 cells/ml. (B) Lipid composition after growth in medium containing no nitrogen for 12days, with initial cell density of 5 107 cells/ml. DGDG: Digalactosyl-diacylglycerol; FFA: Free fatty acid; MGDG: Monogalactosyl diacylglycerol; PC: Phosphatidyl choline; PE: Phosphatidyl ethanolamine; PG:Phosphatidyl glycerol; PI: Phosphatidyl inositol; SQDG: Sulphoquinovosyl diacylglycerol; TAG:Triacylglyceride.
100

80 Other C18:3 C18:2 C18:1 C18:0 C16:3 C16:2 C16:1 C16:0 A B C

The change in cell number over time of C. vulgaris grown under nitrogen-sufficient conditions in batch culture is illustrated in Figure 3. The culture reached its maximum cell density after approximately 9 days (~4.2 g/l, 8.5 108 cells/ml), with the average biomass productivity being 480 mg/l/day. The total lipid productivity is therefore 66 mg/l/day, when assuming the same lipid composition as reported in the Nitrogen-sufficient conditions section above. However, when considering the useful lipids to be TAGs, it was found that only approximately 0.5 wt% of C. vulgaris, when grown under nitrogen-sufficient conditions, are the most appropriate lipids for biodiesel production. Useful lipid productivity is therefore calculated to be only 2 mg/l/day.
Nitrogen-limited conditions

60

40

20

In order to investigate lipid productivity under nitrogen-limited conditions, different methods of nitrogen deprivation were employed to determine the most effective.

Figure2. Average FA compositions of (A) the total lipid produced when grown in nitrogen-sufficient conditions over a timeframe of 20days in continuous culture, with an increase in cell count from 1 106 to 1.2 108 cells/ml; (B) the TAG produced when grown under nitrogen deprivation in continuous culture; and (C) the TAG produced when grown under nitrogen deprivation in batch culture. FA: Fatty acid; TAG: Triacylglyceride.

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12 Cell number (108 cells/ml) 10 8 6 4 2 0

Stephenson, Dennis, Howe, Scott & Smith the TAG content in the cells had ceased to increase, so the increase in dry cell mass implies the production of other cell components (e.g., carbohydrates). Table1 presents the values at each time interval of the experiment, allowing both the TAG and biomass productivity achieved after the cells were introduced to the new medium to be calculated. It is clear from these data, and from Figure 4C, that in the first 19 days the TAG content (in wt%) is higher in the cultures that were initially more dilute; a students t-test confirmed that the values for cultures initiated at 1.25 107 cells/ml were significantly different (95% confidence) from those in cultures initiated at 10 107 cells/ml at time points 5, 9 and 19 days. One possible reason for this is that more light is available per cell, therefore more metabolic flux generated from photosynthesis is available for lipid accumulation. Although a low initial cell density resulted in higher concentrations of TAG in the cells, TAG productivity (in mg/l/day) was lower than in cultures with higher initial cell concentrations, and the maximal productivity of 49 mg/l/day was achieved in the most dense culture. However, in this experiment, the final TAG concentration in the biomass was only approximately 14 wt% of the dry cell mass, and therefore its extraction may not be economically viable. The FA profiles of the TAGs produced in these batch cultures were essentially the same, whatever the initial cell density, and like the TAGs produced in cells in fermenters, comprised mainly oleic acid (C18:1; Figure 2C). This work demonstrates that if a culture that was initially grown under nitrogen sufficiency is introduced to a medium containing no nitrogen, a trade-off exists between initial cell number and final TAG concentration reached in the biomass, suggesting there is an optimal initial cell density. When considering TAG productivity,
C TAG (wt% of dry cell mass) 0 10 20 Time (days) 2.5 40 1.25 40 30 20 10 0

4 6 Time (days)

10

Figure3. Cell growth when Chlorella vulgaris cells were grown in batch culture, using culture volumes of 75ml (100ml conical flasks), in an incubator with an atmosphere consisting of 10 vol% CO2, 71 vol% N2 and 19vol% O2, at 2023C, shaken at 120 rpm and illuminated from above using fluorescent lamps at 165mol/m2/s. Varying initial cell density

illustrates the effect of varying the initial cell density in batch culture after cells were transferred to medium without nitrogen. All cultures behaved essentially similarly, in that the cells continued to multiply for the first 59 days, approximately doubling the total cell number for each initial concentration of cells (Figure4A) , whilst the total dry cell mass increased for the first 19 days (Figure 4B) . TAG levels also increased, but peaked at 5 days, falling to about 1114 wt% in all cases after 40 days (Figure 4C) . It is likely that cell growth was supported by the consumption of intracellular nitrogen pools, such as chlorophyll molecules [12] . Between 10 and 19 days, the cells had stopped multiplying and
Figure4 A Cell number (108 cells/ml) 3.0 2.5 2.0 1.5 1.0 0.5 0 0 20 Time (days) 40 Dry cell mass (g/l) B 3.0 2.5 2.0 1.5 1.0 0.5 0

20 Time (days)

40

Figure4. Growth of Chlorellavulgaris in batch culture after inoculation into medium containing no nitrogen, with varying initial cell counts of 10 107, 2.5 107 and 1.25 107 cells/ml (labeled 10, 2.5 and 1.25 respectively). (A) Cell number, (B) dry cell mass and (C)accumulation of triacylglyceride.

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Influence of nitrogen-limitation regime on lipid production by C. vulgaris

Research Article

Table1. Triacylglyceride production when growing Chlorella vulgaris in batch culture, using 75ml culture volumes with varying initial cell number, when the cells are introduced into media with no nitrogen content.
Initial cell number Parameter (cells/ml) 1.3 107 TAG content Dry cell weight TAG absolute content TAG production rate TAG content Dry cell weight TAG absolute content TAG production rate TAG content Dry cell weight TAG absolute content TAG production rate Units Time after inoculation in medium without nitrate (days) 0 1 0.1 0.10 0.005 1 0.1 1 0.1 0.19 0.01 2 0.2 1 0.1 0.78 0.04 8 0.9 05 34 4 0.14 0.008 49 6 10 1 20 2 0.48 0.03 96 11 20 2 14 2 1.67 0.09 240 28 49 6 59 24 3 0.25 0.013 60 7 32 19 2 0.52 0.03 98 12 14 14 1 1.94 0.10 271 32 8 11 919 23 2 0.34 0.018 75 9 21 17 2 0.68 0.04 118 14 22 14 1 2.4 0.13 336 39 75 1940 14 2 0.29 0.015 41 5 -2 1 11 1 0.72 0.04 76 9 -2 1 11 1 2.3 0.12 256 30 -4 2

% of dry mass g/l mg/l mg/l/day % of dry mass g/l mg/l mg/l/day % of dry mass g/l mg/l mg/l/day

2.5 107

1 108

TAG: Triacylglyceride.

the time taken to grow the initial culture in nitrogensufficient conditions must also be taken into account, as higher initial cell numbers will take longer to produce.
Nitrogenremoval regime

To try to improve the TAG productivity while ensuring high lipid content in the cells, the effect of introducing inocula into media of varying nitrate concentrations and letting the cells deplete the nitrate supply naturally was also explored. Nitrate concentrations of 10, 100, 200 and 550 mg/l were chosen, with the latter being the level in medium used for nitrogen-sufficient growth. Figure5 illustrates how cultures grown at various initial concentrations of nitrate developed after the cultures were introduced
A Cell number (108 cells/ml) 12 10 Dry cell mass (g/l) 8 6 4 2 0 0 20 Time (days) 40 N10 B 6 5 4 3 2 1 0 0

to the new medium. Cell growth continued for the first 510 days after introduction into the new medium, then leveled off. At this point there was no measurable nitrate in the media, indicating that it had been taken up by the cells. The dry cell mass (g/l) continued to increase in all cultures for the first 20 days, then was constant. Inoculation of C. vulgaris cells into medium with 10 mg/l nitrate shows that TAG accumulation (Figure5C) follows essentially the same path as that seen for inoculation into medium without nitrogen (Figure4C, squares) . In nitrogen-sufficient medium, there was a slow steady increase over the time of the experiment, reaching approximately 18 wt%. However, with both intermediate nitrate concentrations (100 and 200 mg/l),
C TAG (wt% of dry cell mass) 60 50 40 30 20 10 0 0 20 Time (days) 40

20 Time (days) N100 N200

40 N550

Figure5. Growth of Chlorella vulgaris in batch culture after inoculation into media of varying levels of nitrogen, using 75ml culture volumes with an initial cell count of 2.5 107 cells/ml. (A) Cell number, (B) dry cell mass and (C) TAG content. N10: medium containing 10mg/l nitrate; N100: medium containing 100mg/l; N200: medium containing 200mg/l; N550: medium containing 550mg/l. TAG: Triacylglyceride.

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Stephenson, Dennis, Howe, Scott & Smith The data are presented in Figure 6 ; it can be seen that there was essentially no difference between the results for the two different culture volumes. Moreover, the same profiles in cell number, dry cell mass and TAG content were seen as for the previous experiment (Figure 5) . The cell density increased before levelling off after 5 days for the algae grown in medium with 200 mg/l nitrate, and after 10 days in medium containing 550 mg/l nitrate, whereas the dry cell mass (g/l) continued to increase during the whole duration of the experiment (Figure 6AD) . For cells grown in 200 mg/l nitrate, the concentration of TAG in the biomass (wt% of dry cell mass) increased steadily to 3540 wt% after 20 days (Figure 6E) , whereas in the nitrogen-sufficient medium it reached 2025 wt%, somewhat higher than in the previous experiment, indicating the variability of biological samples. Concomitant with an increase in TAGs, there was a steady decline in both total protein (Figure 6GH) and chlorophyll (Figure 6IJ) over time. The TAG productivities calculated from these data (Table3) are similar to those in the previous experiment (Table2) when the initial concentration of nitrate in the medium was 200 mg/l, but this time the maximum rate of 138 16 mg/l/day was seen between 15 and 20 days

between 10 and 20 days after inoculation, there was a significant increase in TAG content, reaching to 39 and 46 wt%, respectively (Table2) . The TAG levels were slightly less by the 40th day, but were still over 30 wt%. Moreover, the maximum TAG productivity of 111 16 mg/l medium/day was also observed with this nitrogen regime, between 9 and 19 days after inoculation. These results indicate that both high TAG concentrations within the cells, and high TAG productivities are achievable, if cells that have initially been grown under nitrogen-sufficient conditions are introduced to a medium with an intermediate concentration of nitrate, rather than a medium containing no nitrogen.
More detailed analysis of culture parameters

In order to verify the observations shown in Figure5, the experiment was repeated, focusing on growing C. vulgaris in media with nitrate concentrations of 200 and 550 mg/l over 20 days, since the changes of interest occurred during this period. The volume of the culture was also varied (two volumes, 50 and 75 ml, were used) to investigate whether the depth of culture (0.03 and 0.043 m, respectively) and hence the availability of light had any effect.

Table2. Triacylglyceride production when growing Chlorella vulgaris in batch culture, using 75ml culture volumes, with initial cell number of 2.5 107 cells/ml, when the cells are introduced into media with varying nitrogen content.
Initial nitrate level (mg/l) 0 Parameter Units Time after inoculation into fresh medium (days) 0 1 0.1 0.19 0.01 2 0.2 05 20 2 0.48 0.03 96 11 20 2 59 19 2 0.52 0.03 98 12 14 17 2 0.58 0.03 99 12 -0.3 4 11 1 1.76 0.09 192 23 11 7 12 1 1.73 0.09 213 25 49 5 0.5 2.66 0.14 130 15 23 4 919 17 2 0.68 0.04 118 14 22 15 2 0.84 0.04 128 15 32 39 4 2.17 0.11 846 99 65 10 46 5 2.90 0.15 1320 155 111 16 6 0.7 5.21 0.28 334 39 20 4s 1940 11 1 0.72 0.04 76 9 -2 1 14 1 0.59 0.03 79 9 -2 1 33 3 2.08 0.11 677 79 -8 6 32 3 3.11 0.16 1003 118 -32 9 18 2 4.79 0.25 847 99 24 5

TAG content Dry cell weight TAG absolute content TAG production rate TAG content Dry cell weight TAG absolute content TAG production rate TAG content Dry cell weight TAG absolute content TAG production rate TAG content Dry cell weight TAG absolute content TAG production rate TAG content Dry cell weight TAG absolute content TAG production rate

% of dry mass g/l mg/l mg/l/day % of dry mass g/l mg/l mg/l/day % of dry mass g/l mg/l mg/l/day % of dry mass g/l mg/l mg/l/day % of dry mass g/l mg/l mg/l/day

10

1 0.1 22 2 0.19 0.01 0.46 0.02 2 0.2 100 12 21 3 1 0.1 15 2 0.19 0.01 1.01 0.05 2 0.2 150 17 32 4 1 0.1 14 1 0.19 0.01 1.39 0.07 2 0.2 195 23 41 5 1 0.1 2 0.2 0.19 0.01 1.75 0.09 2 0.2 38 4 81

100

200

550

TAG: Triacylglyceride.

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Influence of nitrogen-limitation regime on lipid production by C. vulgaris in cultures inoculated into medium with an initial concentration of nitrate of 550 mg/l. These results indicate that leaving cells to deplete their nitrate supply naturally leads to high TAG productivities; however, higher concentrations of TAG within the cells can be achieved if nitrate concentrations of 200 mg/l are used. Conclusion The microalga C. vulgaris has the potential to provide a renewable oil feedstock for biofuel use; however, when grown in nitrogen-sufficient conditions, only approximately 3 wt% of its lipids are triglycerides, the most suitable lipids for biodiesel production. To increase the suitability of the oil as a feedstock for biodiesel, the TAG content must be increased by, for example, removing the supply of nitrogen to the cells. In this paper, we established that maximal average TAG productivity (of 46 6 mg/l/ day over 25 days) was achieved with a two-stage method in which a culture was initially grown in nitrogen-sufficient conditions (550 mg/l) for 5 days, after which the cells were introduced to media with an intermediate nitrate concentration of 200 mg/l and left to exhaust the remaining nitrogen for a further 20 days. In comparison, previous reports in which cells were subcultured into media lacking nitrogen altogether achieved
Figure6 (right). Growth of Chlorella vulgaris in batch culture, after cultures (initial cell count of 2.5 107 cells/ml) are transferred into media with initial nitrate contents of 200mg/l and 550mg/l. (A&B)Cell number, (C&D) dry cell mass, (E&F) triacylglyceride content, (G&H) protein content and (I&J) chlorophyll content. N200: medium containing 200mg/l nitrate; N550: medium containing 550mg/l; 75ml: algae grown in 75ml batches; 50ml: algae grown in 50ml batches. dcm: Dry cell mass.

Research Article

A Cell number (10 8 cells/ml)

8 6 4 2 0 0 10 Time (days) 20

Cell number (10 8 cells/ml)

10

10 8 6 4 2 0 0 10 Time (days) 20

C Dry cell mass (g/l)

7 6 5 4 3 2 1 0 0 10 Time (days) 20

D Dry cell mass (g/l)

7 6 5 4 3 2 1 0 0 10 Time (days) 20

E TAG (wt% of dcm)

50 40 30 20 10 0 0 10 Time (days) 20

F TAG (wt% of dcm)

50 40 30 20 10 0 0 10 Time (days) 20

G Protein (wt% of dcm)

50 40 30 20 10 0 0 10 Time (days) 20

H Protein (wt% of dcm)

50 40 30 20 10 0 0 10 Time (days) 20

I Chlorophyll (wt% of dcm)

50 40 30 20 10 0 0 10 Time (days) N200 50 ml N200 75 ml 20

J Chlorophyll (wt% of dcm)

50 40 30 20 10 0 0 10 Time (days) N550 50 ml N550 75 ml 20

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Research Article

Stephenson, Dennis, Howe, Scott & Smith detailed ana lysis of the FA composition of the cellular lipids revealed that unsaturated FAs remained a high proportion under all nitrogen regimes. In particular, the level of a-linolenic acid was found to be 1518 wt%, meaning that to adhere to EN 14214, the algal TAGs must be blended with other triglycerides to reduce the level below 12 wt% [4] . If lipid productivity levels similar to those reported in this study could be achieved at a large scale, then assuming depths of algal culture of 0.043 m (as in our batch cultures), this would be equivalent to a TAG production of approximately 7.2 te/ha/y. A figure of 40 te/ ha/y has been suggested to be a reasonable target for large-scale microalgal production [11] . Nevertheless, at the TAG production rate determined in this paper, approximately 66 Mha land would be required to supply the USA with all its fuel [25] , significantly smaller than the land requirement of 3000 Mha that would be needed if corn were to be utilized for bioethanol production [25] . Future perspective In 510 years, the most promising algal species for biofuel feedstocks are likely to have been identified and extensively studied, providing general information on the pathways of lipid biosynthesis and their regulation in algae. By this means, we can expect significant advances in our ability to enhance lipid yields and increase the proportion of desired FAs within lipids by genetic or environmental manipulation. To realize the full potential of algal

productivities from 1.2 mg/l/day (with 63 wt% total lipid) [16] to 1015 mg/l/day with 4045 wt% lipid [6,18] . Ratanapoltee et al. reported a final TAG content of approximately 33 wt% when growing C. vulgaris heterotrophically for 15 days with a supplement of 10 g/l sucrose [24] . However, heterotrophic growth is difficult in a large-scale system, unless the reactor has a sterile design sufficient to exclude bacteria that thrive on the sugar. The relationships between the TAG levels in the cells (wt%) versus the average TAG productivity (mg/l/day) during the time period between introducing the cells to the new medium and reaching the final TAG concentration in our batch cultures are illustrated in Figure7. Figure7A shows that if cells are initially grown to a high density in nitrogen-sufficient conditions and then introduced to media containing no nitrogen, there is an inverse relationship between TAG productivity and TAG concentration per cell. By contrast, a positive correlation between these two parameters is seen when cells are introduced into media with nitrate concentrations of between 100550 mg/l (Figure 7B) . Nevertheless, the highest productivity is seen in the sample introduced to media containing 550 mg/l of nitrate after 19 days, but this does not have the highest wt% TAG. Instead, this is achieved with the sample introduced to media containing 200 mg/l of nitrate. Since downstream processing is likely to be facilitated by high intracellular lipid levels, there will need to be a trade-off between achieving this and the overall productivity of the cultures. Moreover,

Table3. Triacylglyceride production when growing Chlorella vulgaris in batch culture, using 50 and 75ml culture volumes, with initial cell number of 2.5 107 cells/ml, when the cells are introduced into media with varying nitrogen content.
Initial nitrate level 200mg/l 75ml Parameter Units 0 3 0.1 0.23 0.01 6 0.3 Time after NO3 removal (days) 05 14 1 1.33 0.07 183 21 37 4 510 17 2 1.81 0.10 314 37 26 8 15 2 1.99 0.11 291 34 69 15 2 2.68 0.14 412 48 66 10 10 1 3.36 0.18 339 40 34 9 1014 25 3 2.85 0.15 709 83 99 23 24 2 2.58 0.14 612 72 81 20 15 2 4.63 0.25 677 80 67 23 17 2 4.66 0.25 809 95 118 26 1420 30 3 3.32 0.18 992 116 47 24 38 4 3.08 0.16 1174 135 94 26 19 2 5.33 0.28 1019 119 86 24 26 3 6.38 0.34 1638 192 138 36

TAG content Dry cell weight TAG absolute content TAG production rate TAG content Dry cell weight TAG absolute content TAG production rate TAG content Dry cell weight TAG absolute content TAG production rate TAG content Dry cell weight TAG absolute content TAG production rate

% of dry mass g/l mg/l mg/l/day % of dry mass g/l mg/l mg/l/day % of dry mass g/l mg/l mg/l/day % of dry mass g/l mg/l mg/l/day

200mg/l 50ml

3 0.1 18 2 0.23 0.01 1.44 0.08 6 0.3 262 31 54 6 3 0.1 4 0.5 0.23 0.01 1.84 0.10 6 0.3 81 10 16 2 3 0.1 91 0.23 0.01 1.93 0.10 6 0.3 167 20 34 4

550mg/l 75ml

550mg/l 50ml

TAG: Triacylglyceride.

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Influence of nitrogen-limitation regime on lipid production by C. vulgaris

Research Article

A 90 TAG producibility (mg/l/day) 80 70 60 50 40 30 20 10 0 0 10

2.5 107 1.25 107 10 107 5 10

B 90 TAG producibility (mg/l/day) 80 70 60 50 40 30 20 10 0 0 10

100 mg/l 200 mg/l 550 mg/l 5 days 5 days 5 days 9 days 9 days 9 days 14 days 14 days 14 days 19 days 19 days 19 days

20 30 TAG (wt% of dcm)

40

50

20 30 TAG (wt% of dcm)

40

50

Figure7. Average TAG productivity versus TAG level in Chlorella vulgaris cells grown under different nitrogenlimitation regimes in batch cultures. (A) Cells were transferred into media without nitrogen and then left for 5days, initial cell counts of the cultures are shown. (B)Cultures (initial cell count of 2.5 107 cells/ml) were transferred into media with initial nitrate contents as shown. It can be observed that the 200mg/l nitrate points are further to the right on the graph as higher TAG levels are achieved, whilst the 550mg/l points are further to the left. In some cases, measurements were taken at 10 and 20days, rather than 9 and 19days. dcm: Dry cell mass; TAG: Triacylglyceride.

biodiesel production, these studies must be integrated with research into effective means of downstream processing, which will be impacted significantly by the quantity and chemical composition of the lipids within the algal cells. Furthermore, studies on the life cycle ana lysis of algal biofuel production should be reported, identifying areas of the process that need to be optimized to enhance the environmental performance of algal biofuels.
Acknowledgements
The authors are grateful to the following for the support and advice they provided: Nic Davies (Department of Chemistry, University of Cambridge, UK), Prof. Sue Harrison and Melinda Griffiths

(Department of Chemical Engineering, University of Cape Town, SA), Geraldine Heath (Department of Plant Sciences, University of Cambridge, UK), Dr. Beatrix Schlarb-Ridley and Dr. Derek Bendall (Department of Biochemistry, Universit y of Cambridge, UK).

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Executive summary When grown under nitrogen-sufficient conditions, only approximately 3wt% of the total lipids in the green microalga Chlorella vulgaris are
triacylglycerides (TAGs; the most useful lipid for biodiesel production).

Various nitrogen-limitation regimes were investigated, in which cells are transferred to medium lacking nitrogen altogether, or medium
containing different amounts of nitrate, and the cells left to exhaust nitrogen supplies naturally.

Abruptly transferring the cells to medium lacking nitrogen results in a much lower TAG productivity than if the cells are left to reduce the
nitrogen level in the medium naturally.

The most effective method of accumulating high levels of TAG was found to be transferring cells into medium containing 200mg/l nitrate,
and leaving them for a further 20days to accumulate TAG. TAG concentrations of up to 46wt% of the dry cell mass were reached using thismethod. Maximal TAG productivity of approximately 46 6mg/l/day was achieved, significantly higher than that reported previously. TAGs produced by C.vulgaris during nitrogen-deprivation contain predominantly palmitic acid (C16:0, 1621%), oleic acid (C18:1, 3237%) and a-linolenic acid (C18:3, 1518%). TAG produced by C.vulgaris during nitrogen deprivation has a significantly higher oleic acid (C18:1) content than lipids produced during growth under nitrogen-sufficient conditions, whilst the contents of the more highly unsaturated fatty acids, C18:2, C18:3 and C16:2, arelower. In order to meet EN 14214 (2008), the fuel produced from the transesterification of the TAG would have to be blended with another fuel to reduce the a-linolenic acid content to below 12wt%.

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in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert t estimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
17

Financial & competing interests disclosure

Financial support from the EPSRC is gratefully acknowledged. The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed
9

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