Research Article

Received: 25 July 2012 Revised: 9 October 2012 Accepted article published: 16 January 2013 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jctb.4027

Optimization of lipid production for algal biodiesel in nitrogen stressed cells of Dunaliella salina using FTIR analysis
Junying Liu,a Joy Mukherjee,b Jeremy J. Hawkesc and Stephen J. Wilkinsona
Abstract
BACKGROUND: Large improvements in productivity are required to make massive scale biodiesel production from microalgae an economic reality. Although the maximum neutral lipid content of microalgae has received much attention as a target for optimization, there are other factors that are equally important. These are (1) the rates of accumulation of both biomass and lipids and (2) the maximum densities of algal cells that can be sustained in continuous cultivation. The combined effect of these factors for lipid production has not been thoroughly examined in Dunaliella species. Hence this study examines the rates of growth and lipid accumulation in Dunaliella salina using Fourier transform infrared spectroscopy (FTIR) under several combinations of temperatures and cell densities. RESULTS: The FTIR signal at 2926 cm1 (rather than 1740 cm1 ) is better for measuring lipids and the PCA of the full spectrum showed a clear separation between the nitrogen replete and nitrogen depleted cells. As expected, cells subjected to nitrogen starvation (N-depleted) showed very little growth compared to the N-replete cells. N-depleted cells achieved a nal lipid content that was 78% more than the N-replete samples at 26 C, while the differential for 16 C was 28%. However, the slower growth rates caused by the stress of nitrogen starvation meant that the total lipid production over the starvation period was lower for many samples. Indeed, the only stress condition that gave signicantly higher total lipid production was the highest cell density studied at 26 C. CONCLUSION: For optimization of lipid productivity for biodiesel, the trade-off between lipid content, growth rate and cell density needs to be considered. c 2013 Society of Chemical Industry Keywords: biodiesel; Dunaliella salina19/18; lipid production; FTIR

INTRODUCTION
There is great interest in the cultivation of microalgae as a source of naturally synthesized products, particularly lipids for biodiesel, and it is called a second generation green energy source.1 Unsurprisingly, extensive research has been funded on this potential renewable fuel source due to the ability of microalgae to accumulate signicant quantities of lipids. The Dunaliella group of species is one of the most suitable microalgae for mass cultivation outdoors in open ponds.1 In this work we have used Dunaliella salina as a robust production organism in hot saline environments.2 Although D. salina can achieve a lipid content of 1020% of dry cell weight, the other key parameters effecting biodiesel productivity of microalgae are their growth rate and biomass concentration.2,3 For a continuous system operating at steady state we can dene the productivity as P = X (1)

production into different batch stages with non-stress and stress conditions since lipid production might be optimized by stress conditions that do not favour growth. In this case the equation above still applies if we consider to be the lipid content and to be the average growth rate over the total growth/stressing cycle. In this study we have investigated the three key productivity parameters given in the equation above for D. salina. Although the lipid content of this species has been measured in several studies, the overall productivity for Dunaliella has not previously been reported and published work does not agree whether or not lipid content is increased by N limitation.3 All the experiments

Correspondence to: Stephen J. Wilkinson, ChELSI Institute, Department of Chemical and Biological Engineering, University of Shefeld, Mappin St., Shefeld, UK. E-mail: s.j.wilkinson@shefeld.ac.uk

a Department of Chemical and Biological Engineering, University of Shefeld, Mappin St., Shefeld, UK b Current address: The Swire Institute of Marine Science, University of Hong Kong, Cape dAguilar road, Shek O, Hong Kong SAR c School of Chemical Engineering and Analytical Science, The University of Manchester, Oxford Road, Manchester, M13 9PL, UK

where P is lipid productivity (g L1 day1 ), is lipid content (%), is the specic growth rate (day1 ) and X is the biomass concentration (g L1 ). However, for practical applications, rather than operating at steady state, it might be better to de-couple growth and lipid
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www.soci.org carried out in this study are done in batch mode and we have therefore chosen to use the total production of lipid over a xed 29 day period to compare the different conditions. It should be noted that the factors effecting overall productivity are strongly interdependent. Most notably, the lipid content can be increased by stress conditions that also have a deleterious effect on growth rate. For example, there are many studies showing that nutrient stress conditions, such as nitrogen (N) limitation, phosphorus (P) starvation, urea limitation and low temperature can induce signicant increases in lipid content in many algal species.4 Unsurprisingly, however, it has also been observed that nutrient limitation reduces growth rate so that there is often an inverse relationship between growth rate and lipid content.1 Another key productivity parameter algal biomass concentration is also rather low, particularly in raceway ponds in which typical annual values of 515 kg m 2 have been reported.1,5 This is another parameter that is often overlooked and which also effects growth rate and lipid content. The more crowded cells become the more their growth might become compromised. However, the increased stress of overcrowding may actually help to promote lipid accumulation. Lipid production is a hot topic in algal biofuel research and more efcient and rapid methods of lipid detection are desirable. Several methods are currently in use such as gravimetric measurements, Nile red staining and FTIR. The gravimetric measurement of lipid content in algal strains includes solvent extraction and sonication fractionation6 and is therefore time-consuming. Nile-red has been used recently to image neutral lipid accumulation within cells by uorometer or to quantify lipid biosynthesis by uorescence spectroscopy. FTIR was the method chosen for this research, inspired by a previous study of freshwater algae by Dean and co-workers.7 In their work, the correlation of the FTIR signal information and an established method for the detection of lipids has been demonstrated. They found a very strong correlation between FTIR lipid/protein ratio and Nile red uorescence (up to 0.9301). More generally, FTIR has been shown to have potential as a technique for measuring the complex functional groups present in whole intact cells.8 This technology involves the measurement of infrared absorption in relation to a range of molecular vibrational modes. Specic functional groups, such as some macromolecules including proteins, lipids, carbohydrates and nucleic acids can be quantied by their specied absorption bands. This method is a rapid and non-invasive technique that requires little sample preparation. We report below the effects of biomass concentration, temperature and nitrogen starvation on lipid accumulation as well as chlorophyll concentration. At the same time we apply FTIR to demonstrate the change of algal lipids over time.

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Nitrate stress experiment D. salina was grown initially in batch mode in the medium and reactor described above until it reached stationary phase and was then run continuously using a dilution rate of 0.1 day1 . Samples from this culture were then used as the inoculum for the stress experiments. Each inoculum sample was centrifuged at 3000 g for 10 min and the supernatant discarded. The pellet was resuspended in 200 mL of new medium without nitrate. The volume of the inoculum sample was used to control the re-suspended cell density i.e. to obtain 50%, 100% and 200% of the cell concentrations of the initial algal culture in the photo-bioreactor. We centrifuged samples with volumes of 100 mL, 200 ml and 400 mL, respectively, and re-suspended each in 200 mL of nitrate free medium. This N-depleted media was fresh Dunaliella media as described above but lacking in NaNO3 . Cultures were grown in shake asks under 24 h light, at different temperatures. Two types of controls were also set-up. First, re-suspended cells in normal (i.e. nitrate-containing) medium at the same biomass concentrations (i.e. 50%, 100%, 200%) as the nitrate depleted samples. Second, un-centrifuged cells were grown as another control in order to assess whether the centrifugation process itself imposes any extra stress on the Dunaliella cells. All other conditions were operated as described above. This gave a total of 13 samples (3 N-replete labeled 200% N+ , 100% N+ and 50% N+ ; 9 N-depleted labeled 200%, 100% and 50%; and 1 uncentrifuged labeled N+ ). Relationship between cell concentration and optical density In separate experiments to those described above, cells were grown in shake asks and harvested after 15 days when optical density was equal to 0.85. At this high OD the growth rate had started to slow down. The cell density (optical density) was measured by a Thermo UV-vis spectrophotometer at a wavelength of 680 nm (OD680 ). Each sample was diluted to give an absorbance in the range 0.11.0 if optical density was greater than 1.0. The dried weight (mg mL 1 ) was determined as described in previously reported research. Briey, microalgae cells were collected by centrifuge and washed twice with distilled water at 3000 rpm for 10 min. The sample pellet was dried by freeze drier for 2 days and then the dried weight was calculated. An excellent regression relationship was obtained between algal dried weight (Y D mg mL 1 ) and OD680 value as follows: YD = 0.4552OD680 0.0371 R2 = 0.9984 (2)

METHODS AND MATERIALS

Algae, medium and pre-cultivation D. salina CCAP19/18 cells were grown in Dunaliella medium at pH 8.0 which contained: 1.5 mol L 1 NaCI, 10 m mol L 1 KCI, 20 m mol L 1 MgCI2 , 10 m mol L 1 CaCl2 , 24 m mol L 1 MgSO4 , 5 m mol L 1 NaNO3 , 24 m mol L 1 Na2 SO4 , 0.1 m mol L 1 NaH2 PO4 , 0.0015 m mol L 1 FeEDTA, 1 mL L 1 trace elements, 1 g L 1 NaHCO3 . Pre-cultures were grown under a continuous growth system in a 2 L photo-bioreactor and the light box with six bulbs of 11 W was used for illumination of the cultures. A low ambient light intensity of around 12 mol photon m2 s1 was used to simulate high density cultivation. The temperature for the continuous cultivation was 22 2 C.

Chlorophyll content measurement The chlorophyll content of each sample was determined as described by Jespersen and Christoffersen.9 2 mL samples were taken and centrifuged at 3000 g for 10 min. The pellet was re-suspended in 0.4 mL of distilled water and then 1.6 mL acetone was added. After 10 min, the sample was centrifuged again and the absorbance of the supernatant was measured at 663 nm and 645 nm. The nal chlorophyll content was calculated as the weighted average of these two absorbances using the equation YC (g chlorophy II) = 202 OD645 + 80.2 OD663 2 (3)

The nal value (Y C )) was divided by 2 to get g chlorophyll per mL (since the sample size was 2 mL).
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Optimization of lipid production for algal biodiesel using FTIR analysis FTIR spectroscopy and quantication Fourier transform infrared spectroscopy (FTIR) was conducted using the Shimadzu IRPrestige-21 Fourier Transformation Infrared Spectrophotometer (Shimadzu, U.K.) in order to characterize the various functional groups present on the whole cell surface and also the extracellular polymeric substances (EPS). At least 64 scans, with resolution of 4 cm1 , were collected for all samples. A 1.5 mL sample was taken from each replicate ask for each treatment and centrifuged before the supernatant was removed and the cells were re-suspended in 0.9% NaCl. They were washed twice to remove EPS. A portion of the sample was then applied to the FTIR plate and dried at room temperature for up to 30 min before being scanned by FTIR (Fig. 1(A)). In order to demonstrate the band corresponding to lipids in living cells, samples were treated by chloroformmethanol (2:1, v/v) to extract lipids from the live cells.6 The lipid-containing organic phase was then removed and the pellet was washed twice with 0.9% NaCI before measurement by FTIR (Fig. 1(B)). Dried lipids previously isolated from algal samples by chloroformmethanol extraction were re-suspended in methanol and measured by FTIR in the same way (Fig. 1(C)). The spectra for each sample were measured twice and then collected over the wavenumber range 4000600 cm1 . Spectral processing like baseline correction was carried out using the software within the Shimadzu FTIR instrument to minimize differences between the recorded spectra due to baseline shift. The spectral absorption bands were identied according to published information. Statistical analysis on the data was carried out using EXCEL and PASW software performing Univariate ANOVA. Principle component analysis (PCA)7 was performed using XLSTAT software to compare an entire spectral region of interest. Calculation of lipid production The total lipid production up to time t is calculated as : YL = (t Xt 0 X0 ) (4)

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a distinctive lag phase of approximately 5 days. This lag phase was extended for some conditions (e.g. 200% N+ at 20 C) which we have no obvious explanation for. All N-replete treatments entered the exponential phase at almost the same time but the N-depleted samples grow in a sub-exponential or linear manner. After day 22 most of the samples showed slower growth with a few declines. One interesting feature of the growth curves is that the growth at the lowest temperature (16 C) is still good which indicates the feasibility of growing Dunaliella in temperate climates. FTIR band assignment The bands were assigned to specic molecular groups on the basis of biochemical standards in previous studies (Table 1) and are attributed to a range of vibrational modes in residual water (3305 cm1 ); lipid CH2 (2926 cm1 ) and carbonyl (1740 cm1 ); amide I and II (1649 cm1 and 1545 cm1 , respectively); carbohydrate (1014 cm1 ); and nucleic acid (1245 cm1 ). All spectra were closely similar in this research and contained 10 clear bands over the wavenumber range 4000600 cm1 (Fig. 1(A)). Figure 1(B) spectra showed that peaks of around 1740 cm1 and 2926 cm1 disappeared after treatment by chloroformmethanol for removal of lipids and Fig. 1(C) spectra showed the total lipids from algae have much more stronger absorbance at 2926 cm1 than 1740 cm1 . The band at 2926 cm1 was therefore used to measure the relative change of lipids in this study. By analysing 160 randomly selected FTIR spectra obtained in this study it was found that the peaks of the 1649 cm1 (Amide I) band were less reliably determined than 1549 cm1 (amide II). At 1649 cm1 we observed fewer identiable peaks in the samples with more variable positions and another source of error was the close proximity of this wavelength to the 1729 cm1 (lipid) band. Because of this and the comparatively smaller variation of the 1549 cm1 (amide II) band among the different treatments, it was decided that the amide II was a suitable band for performing the normalization of FTIR spectra and ratio determination. Cells grown in different nitrogen treatments were normalized to the 1549 cm1 (amide II) band to give the lipid:amide II proles. Principle component analysis Following normalization to amide II, separate PCAs were carried out over the 1800900 cm1 wavenumber region for N-replete and N-depleted samples under different cell concentration treatments and over all time points. PCA resolved the data into two major components for all treatments (Fig. 3(A)(C)). The PC score plots of F1 against F2 were generated by all the FTIR spectra for each sample day and showed the composition of cell shift from the rst day to 29 days. For the 50% and 100% cell concentration treatments, the PC scores plots showed a very clear separation among the Nreplete and N-depleted treatments, which suggests differences in metabolism for these different conditions. F1 accounted for 74.32% and 71.35% of the variation for the 50% and 100% cell concentrations, respectively, while F2 for 13.94% and 14.41%, respectively. The 200% cell concentration treatments did not show a clear separation among the N-replete and N-depleted treatments. The PC score plots indicate which regions of the spectrum account for the main differences between the N-replete and N-depleted treatments. For all cell concentrations, these plots exhibited positive loadings for carbohydrate and lipid levels along F1. Along F2 the loadings for the lipid regions of the spectra were still positive whereas those for the carbohydrate regions

where Y L is total lipid production; t is lipid content at time t (demonstrated by FTIR spectra at 2926 cm1 ) and Xt is the dry cell weight at time t. In this case t = 29 days. Note that we do not have the true lipid content, just the relative content as determined by FTIR. We therefore express the production as relative to a control sample.

RESULTS
Cell growth under varying stress conditions The impact of nitrogen starvation on biomass concentration (i.e. dried weight) is shown in Fig. 2. As expected, it can be seen that cells lacking in nitrogen show little growth compared with those in N-replete media at all temperatures studied. Interestingly, all the N-depleted samples showed some growth which tended to be higher for the higher cell concentrations. We did nd low but detectable levels of nitrate in the N-depleted media after 22 days. We speculate that this residual nitrate is due to nitrogen release from dead cells. The growth rates of the un-centrifuged controls (which were kept in the old growth medium) were generally in between those of the N-replete and N-depleted samples. The fact that these controls also showed a long lag phase suggests that the lag phase shown by all of the samples was due to the transfer to shake ask conditions rather than due to any stress imposed by the centrifugation process. A general feature of these growth curves is
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Figure 1. Spectra from FTIR. (A) Typical infrared absorption spectra from normal live algal cells grown with nitrogen (dashed line) and without nitrate (continuous one). Band assignments are given in Table 1. (B) Absorption spectra from live algal cells treated by chloroformmethanol (2:1, v/v). (C) Absorption spectra from pure algal lipid extracted by the gravimetric method.

switched to being negative. These results indicate that variation in lipid content is the main discriminant between the N-replete and N-depleted treatments. Lipid and carbohydrate accumulation Figure 4 shows the increase in lipid content over time for stressed and unstressed cells at different temperatures and cell concentrations. All N-depleted treatments at 26 C and 20 C (50%, 100% and 200%) exhibited an increase in the lipid content as compared to the unstressed N-replete samples (50% N+ , 100% N+ , 200% N+ , N+ ) (Fig. 4(A), (B)). This demonstrates the

effectiveness of using nitrogen starvation to stress the cells and induce lipid accumulation at these temperatures. But N-depleted cells accumulated their lipids on average 78% more rapidly at 26 C compared with N-replete, while the corresponding differential at 16 C was only 28%. Conversely, at the lower temperature of 16 C the accumulation of lipids by the N-depleted cells was more modest (Fig. 4(C)). Table 2 shows the total lipid production over the duration of the experiment (29 days). The numbers are given as relative to the production for unstressed cells re-suspended at the sampled cell concentration at 26 C (100% N+ ). The total lipid production takes
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Table 1. Assignment of major macromolecular bands found in FTIR spectra of Dunaliella salina Wavenumber (cm1 ) 3277 2926 1740 1649 1539 1245 Wavenumber range(cm1 ) 3029-3639 2809-3012 1745-1734 1655-1638 1545-1540 1191-1356 Functional groups Water, protein Lipids Membrane lipids, fatty aicd Protein(Amide I) Protein(Amide II) Nucleic acids, Phosphoryl group

Band 1 2 3 4 5 6

Assignments* O-H/ N-H as CH2 C = O of esters C = O N-H, C-N as P = O C-O-C/ as P = O C-O / Si-O

Comments

Methylene groups

7 8

1147 1014

1200-900 1072-980

Polysaccharides Polysaccharides

DNA/RNA backbones, phosphoralated proteins and polyphosphate storage products Mainly polysaccharides rings Cabohydrate peaks

*Band assignments based on Murdock18 , Benning19 , Giordano20 . =symmetric stretch; as = asymmetric stretch; =symmetric deformation (bend), as = asymmetric deformation (bend).

Table 2. Total lipid accumulation at day 29 for stressed and unstressed cells at different temperatures and cell concentrations relative to that for unstressed cells re-suspended at the sampled cell concentration (100%) at 26 C. Calculated as ( t Xt 0 X0 ), where t , is lipid content (demonstrated by FTIR spectra at 2926 cm1 ) at time t (t = 29 days in this case) and Xt is the dry cell weight at time t Treatments 50% 50% N+ 100% 100% N+ 200% 200% N+ N+ 26 C 44% 84% 66% 100% 134% 92% 66% 20 C 37% 78% 72% 94% 76% 56% 61% 16 C 31% 112% 64% 92% 52% 62% 38%

The relationship between cell density and chlorophyll content The correlations between cell density and chlorophyll content for all the centrifuged samples (unstressed and stressed) were determined to show the effects of nitrogen availability on algal cell health. A quantitative equation for the relationship between biomass concentration (X g L1 ) and chlorophyll content (Y g mL1 ) under unstressed conditions was obtained as shown in the following equation: Y = 0.000020 X 2 + 0.0076X 0.0098 The equation for the stressed conditions is Y = 0.000020 X 2 + 0.0264X 0.1288 R2 = 0.6936 (6) R2 = 0.9216 (5)

account of cell growth as well as lipid content as described in the methods. Of the stressed (N-depleted) cells at 26 C only the highest cell density (200%) gave more lipid production than the unstressed control. All of the other stressed samples gave less lipid production than the unstressed control (50% N+ ). Conversely, at the lower temperature (16 C) the low cell concentration, N-replete treatment reached the second highest value for lipid production (112% relative to the control). These results show that stressing by nitrogen starvation limits growth which can offset increases in lipid content. The uncentrifuged cells displayed relatively low lipid production when compared with the centrifuged cells either in N-depleted or N-replete media. This reects their comparatively slow growth due to nutrient exhaustion combined with modest lipid accumulation with both these factors compounding each other to give low overall lipid production. A signicant difference of lipid:amide II ratio between each temperature was observed in relation to normalized band intensities (P < 0.05), but no signicant difference was found in the interaction between temperature and cell concentration (P = 0.544) or for that between the cell concentration in all treatments (P = 0.158). Figure 5 shows the change in carbohydrate over time and for stressed and unstressed cells at different temperatures and cell concentrations. All of the nitrogen stressed treatments showed an increase in the carbohydrate: amide II ratio.
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The chlorophyll content of the stressed conditions has a weak correlation with dried weight (R2 = 0.6936) as shown in Equation (6), providing direct experimental evidence that the increase of cell density does not bring a proportionate increase in chlorophyll content, i.e. nitrogen stressing negatively effects the health of the cells.

DISCUSSION
Experiments have been performed to investigate the effects of nitrogen, temperature and cell density stress on lipid production of D. salina, an important strain of algae for biodiesel production. Algal cells in nitrogen replete media grew much faster than those in new media without nitrogen. Detectable levels of nitrate in the N-depleted media were found in the late growth phase at day 22 (data not shown). The ability of algae to release nitrite or ammonium into the medium can be supported by the hypothesis that algae can assimilate and reduce nitrate in excess of nutritional demand.10 So even in N-depleted condition, the cells can grow at a reduced rate. Many microalgae exhibit an ability to enhance lipid content in cells grown in conditions of nitrogen limitation, which is most inuential on lipid storage. Lipid fractions as high as 7085% of dry weight have been reported.2 This strategy has also been applied to D. salina industrially to produce carotenoids and other pigments.11 In our work we found the low cell concentration (50%) shows greater increase in lipid/protein ratio at 26 C than the median

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Figure 3. PCA of FTIR spectra derived from different cell concentrations (50%, 100%, 200%) grown in N-depleted media (unlled) and N-replete media (lled) under different growth conditions. Two-dimensional PC plots were performed by 1800900 cm1 after normalization to amide II. Figure 2. Dried weight of algal cells grown in different temperature (A = 26 C, B = 20 C, C = 16 C). Dash lines for nitrogen depleted (50%, 100% and 200%) and solid lines for nitrogen replete (50% N+ , 100%N+ and 200% N+ ) media, 50%, 100% and 200% mean the cell concentrations of inoculum. The uncentrifuged group control (N+ ) is denoted by the thick black line. All treatments with vertical bar indicate the standard error of three replicate culture asks.

(100%) and high (200%) cell concentrations. A similar effect has been successfully applied in outdoor mass culture of Dunaliella sp. whereby the dilution of cell concentration has enhanced growth and carotenoid content.12 However, when we calculated the total lipid production using Equation (4), the maximum was obtained by the high cell concentration (200%) in N-depleted media at 26 C. In fact, this was the only sample in which nitrogen stress condition increased lipid yield. This sample was at the highest

cell density studied (200%) which shows the importance of this factor in overall productivity. It is also interesting to speculate on why the best yield was obtained at the highest temperature studied (26 C) by comparing the growth and lipid accumulation data for 26 C and 20 C for the 200% cell density. It can be seen from Fig. 2 that the unstressed cells at the 200% cell density have intrinsically faster growth at 26 C than at 20 C indicating that they are able to produce the energy and intermediates necessary for growth at a higher rate at 26 C. The nitrogen-deprived cells, however, grow at similar rates for both temperatures since growth is limited by the absence of nitrogen. However, the higher potential to produce energy and (non-nitrogen containing) intermediates at 26 C means that more of this energy/intermediates can be committed to lipid synthesis at 26 C than at 20 C if the growth
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Figure 4. Lipid:amide II ratio for different temperature (A = 26 C, B = 20 C, C = 16 C). Dashed lines for nitrogen depleted (50%, 100% and 200%) and solid lines for nitrogen replete (50% N+ , 100% N+ and 200% N+ ) media. The vertical bars indicate standard error of three FTIR spectra from pooled data. 50%, 100% and 200% mean the cell concentrations of initial inoculum. The uncentrifuged group control (N+ ) is denoted by the thick black line.

Figure 5. Carbohydrate: amide II ratio for different temperature (A = 26 C, B = 20 C, C = 16 C). Dashed lines for nitrogen depleted (50%, 100% and 200%) and solid lines for nitrogen replete (50% N+ , 100% N+ and 200% N+ ) media. The vertical bars indicate standard error of three FTIR spectra from pooled data. 50%, 100% and 200% mean the cell concentrations of initial inoculum. The uncentrifuged group control (N+ ) is denoted by the thick black line.

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www.soci.org rates at both these temperatures are constrained to similar values by nitrogen limitation. Enhancing overall productivity by adjusting cell concentration rather than using other factors is a simple and therefore potentially desirable strategy.13 Moreover, strategies to increase lipid content can be highly deleterious to cell viability and have been shown to result in signicant losses of primary product mass.1 Therefore, to optimize lipid productivity for biodiesel, the trade-off between lipid content, growth rate and cell density needs to be considered, as indicated by Equation (1). The total lipid production at a relatively low temperature (16 C) is generally lower than those at optimum temperatures (2026 C). The phenomenon may be explained by suboptimal activity of temperature-dependent enzymatic reactions involved in lipid synthesis.14,15 Finally, in relation to lipid content, it should be noted that Dunaliella strains may increase their carbohydrate rather than their lipid content under nitrogen depleted environments, and even some species respond with decreasing lipid contents.16 In this work we found that carbohydrate did increase, but generally not at the expense of lipid content. We observed orange coloring in the N-depleted samples in this study starting a few days after inoculation which is a common phenomenon and in accordance with many other studies that have observed a parallel increase of -carotene and fatty acids in N-depleted D. salina17 . This indicates profound changes brought on by N-starvation at the cellular level. Lamers, for example, used the scanning electron microscopy to analyze the induced and non-induced cells of D. salina and found that the appearance of lipid globules and the disappearance of most thylakoid membranes.11 This work shows the power of FTIR to provide an in situ, nondestructive chemical analysis of algal cells. Current FTIR allows a relatively fast spectrum acquisition time (less than 30 min per sample) for entire cells, which is necessary to monitor accurately the composition change of cells grown in heterogeneous environments. The samples were processed as rapidly as possible to minimize the cell deterioration and biochemical changes. This work, like other recent research, has faced the challenge of interpreting FTIR peaks since individual molecular groups contribute to different bands and sometimes multiple molecular groups can contribute to a single band.18 20 In this study, for example, the lipid has several bands including 2926 cm1 , 2854 cm1 and 1740 cm1 . The reliability of FTIR for quantitative measurement of lipids has already been shown in a previous study.7 This previous study strongly suggests that FTIR is an effective means of determining genuine lipid changes in cells, i.e. that the increase of lipid/protein ratio was not caused solely by a decrease of protein.

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ACKNOWLEDGEMENTS
The authors would like to thank the China Scholarship Council (CSC); the UK Carbon Trust; and MWH UK for supporting this research nancially. We also thank Dr Jim Gilmour for very useful discussions and Professor Catherine Biggs for laboratory assistance.

REFERENCES
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CONCLUSIONS
We have demonstrated the utility of FTIR for investigating the total lipid productivity for biodiesel in D. salina cells under a number of conditions. The stress factor we applied was N-depletion, which also reduces growth rate so we have established a platform to analyze the trade-off between lipid content, growth rate and cell density to give an overall measure of lipid productivity. Further work will continue this investigation for other conditions and stress factors. Ultimately, a fuller understanding will be required of the nature of lipid accumulation in D. salina cells as means of energy storage responding to unfavorable environments. Such knowledge can provide a solid basis for further continuous cultivation systems for algal biodiesel.

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J Chem Technol Biotechnol (2013)