Representative three-dimensional confocal fluorescence microscopy images of 4 days N- or S-starved CC-124 (top) and CC-125 (bottom) strains. Red, chlorophyll autofluorescence; green, Nile Red fluorescence. C, control cells; N 0 , nitrogen-starved cells; S 0 , sulfur-starved cells. [Color figure can be seen in the online version of this article, 

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... rapidly even after 1 day of nutrient starvation. Decrease in chlorophyll content was higher in N-starved cells than those of S-starved ones. The decrease in total chlorophyll content was approximately 64–60% and 76–63% on first day and lasted with 78–65% and 89–46% during 7 days of N–S starvation in CC-124 and CC-125 cells (Fig. 2c and d), respectively. Intriguingly, N starvation-based carotenoid increase was higher in CC-124 but lower in CC-125 strain than that of S starvation. Carotenoid content increased up to 634% or 427% on day 5 followed by a subsequent decline, resulting with 514% and 183% increase after 7 days of N or S starvation in CC-124. However, N or S starvation caused a gradual increase in carotenoid levels in CC-125, increasing from an initial value of 135–228% to a final value of 260– 442% after 7 days of N and S starvation (Fig. 2e and f). Restricted ability to maintain the photosynthetic functions stemming from decrease in chlorophyll content in response to N and S starvation have previously been reported (Young and Beardall, 2003; Zhang et al., 2002). Under such circumstances, some algae accumulate massive amounts of carbon in the form of carotenoids, starch, and lipids (Thompson, 1996). It has been suggested that the accumulation of carotenoid under stress conditions represents a mechanism to protect algal cells from damage by light (Ledford and Niyogi, 2005). Our results demonstrate that N and S deprivation causes a decrease in chlorophyll levels and increase of carotenoid content. However, CC-124 and CC-125 strains gave different regulation responses to N and S starvation. In particular, S starvation-based increase in carotenoid content was lower in CC-124, but higher in CC-125 than that of N starvation. This difference may be related to the mating type of CC-124 (mt À ) and CC-125 (mt þ ). Total soluble protein concentration in the algal samples decreased drastically in response to nutrient starvation. An 82% decrease in protein content was observed after 1 day of nutrient deprivation and a decrease of over 92% was observed by day 7 (Fig. 3a and b). On the other hand, total neutral lipid and starch levels increased inversely to the decrease in protein content. Maximum increase of total lipid content was reached 4 days after nutrient starvation in both strains studied. Increase in lipid content was 240% and 258% in CC-124 and 165% and 302% in CC-125 in response to N and S starvation, respectively (Fig. 3c and d). Besides fluorescence Nile Red determination, increase in neutral lipid content of 4 days N- and S-starved cells was also supported by analytical measurements. Neutral lipid content of 4 day N- and S-starved CC-124 cells showed approximately 136% and 123%, CC-125 cells showed 190% and 172% increase when compared to their respective controls. Starch levels increased rapidly after nutrient starvation. Maximum increase of starch content was reached 3 days after nutrient starvation in both strains: 706% and 713% in CC-124, 490% and 571% in CC-125 in response to N and S starvation, respectively (Fig. 3e and f). In general, decrease in protein content was followed by a simultaneous increase in lipid and starch content in both strains upon exposure to nutrient starvation. Strikingly, S deprivation caused higher lipid and starch accumulation than N deprivation. Moreover, starch content increment was faster and higher than increase in lipid content in response to nutrient starvation. Potential competition between synthesis of lipid deriva- tives and carbohydrates is an important factor when deciding optimal biofuel production strategies. If starch metabolism interferes with lipid production in N- or S- starved algae, then disabling starch synthesis may be a simple and effective method to increase net lipid production. Increased lipid synthesis on a dry weight basis has been reported in C. reinhardtii mutants with defective starch synthesis machinery (Li et al., 2010), though lipid content was not investigated on a per cell basis. Another starchless C. reinhardtii mutant was determined to display 1.5- to 2.0- fold increase in lipid content per cell, suggesting that lipid and starch syntheses do antagonize each other (Wang et al., 2009). However, high production of both starch and oils has been recently reported from C. reinhardtii and this result is inconsistent with the competition hypothesis (Work et al., 2010). Our studies show that both starch and lipid content increased greatly in response to nutrient starvation. However, maximum increase in starch content was threefold higher than lipid content increment. This may indicate that C. reinhardtii wild-type strains first accumulate starch and that lipid synthesis is induced together with carbohydrate accumulation under nutrient starvation. Ellipsoid-shaped cells (corresponding to the normal morphology of C. reinhardtii cells) were gradually replaced with larger and spherical cell shapes in the initial stages of N and S deprivation (Fig. 4), followed by cell mass reductions after longer nutrient deprivation. TAG are stored in cytosolic lipid bodies in microalgae which may account for the increase in cell volume we observed. Indeed, three dimensional pictures taken from day 4 of N- or S-starved cells show that the amount and volume of cellular cytosolic lipid bodies are higher in S-starved strains than that of N- starved ones. This suggests that increase of the density and volume of lipid bodies is directly related to increase in biovolume of the unicellular alga C. reinhardtii . Quantification of biomolecules by FTIR is a relatively new approach. Following spectrophotometric detection of neutral lipid and starch levels, we performed FTIR measurement for detection of polysaccharide, oligosaccharide, and TAG levels. Infrared spectra were recorded in transmission mode with 128 scans in the range 4,000– 600 cm À 1 . The bands were assigned to specific molecular groups on the basis of biochemical standards and published studies as previously described (Movasaghi et al., 2008). Bands were attributed to asymmetric stretching vibration of CH 2 of acyl chains (2,922 cm À 1 ); Amide I absorption (1,652 cm À 1 ); C–N stretching and CHN bending vibrations of amides from proteins (Amide II, 1,544 cm À 1 ); asymmetric CH 3 bending of the methyl groups of proteins (1,449 cm À 1 ); d CH 3 stretching of C–O groups (1,380 cm À 1 ); PO À 2 asymmetric bonds associated with phosphorus compounds (1,260 cm À 1 ) and PO À 2 symmetric stretching of phosphodiesters (1,075 cm À 1 ). Three bands were of particular interest which were attributed to ester group (C 1⁄4 O) vibration of triglycerides (1,744 cm À 1 ), membrane- bound oligosaccharide C–OH bond (1,145 cm À 1 ), and C–O stretching frequencies coupled with C–O bending frequencies of the C–OH groups of polysaccharide (1,045 cm À 1 ). According to the total soluble protein determination results, protein concentration decreased by up to 93% in response to nutrient deprivation. However, FTIR spectra levels of amide I band obtained from control cells did not at any time deviate more than 32%, therefore, we chose the amide I band for normalization of the FTIR spectra and ratio determination. Relative TAG, polysaccharide, and oligosaccharide contents were determined by calculating the ratio of TAG (1,744 cm À 1 ), polysaccharide (1,045 cm À 1 ), and oligosaccharide bands (1,145 cm À 1 ) to the amide I band (1,652 cm À 1 ). For each data set, we calculated fold-change values compared to their respective controls. Fold changes and standard errors were estimated by fitting a linear model for each time point and empirical Bayes smoothing was applied to the standard errors for all samples studied. Ratios of TAG, polysaccharide and oligosaccharides to Amide I of control samples were arbitrarily assigned a value of 1, independently for each time point. Increases in TAG, polysaccharide and oligosaccharide contents of nutrient- starved samples were defined as values displaying ‘‘—fold increase’’ in ratios of interested bands to Amide I band (Fig. 5). On the other hand, variations in the sample preparation process, such as the thickness of dried sample layers on microtiter plates, is a crucial point and can lead to nonlinear deviations. The distribution of dried cells on the microtiter plates is usually inhomogeneous. However, as a result of the drying procedure during sample preparation, all cells collapse forming a thin film of biomolecules on the sample holder (silica plate). Thus, the thickness of this layer is crucial for the absorbance through the sample, rather than the optical property of the microalgae cells itself. In the present study, we minimized deviations due to attenuation artifacts by applying limited cell numbers forming a sample film which yielded a maximum amide I absorption value of 0.2–0.3. Infrared spectroscopy results showed that relative TAG, oligosaccharide, and polysaccharide levels increased rapidly in response to nutrient starvation. Starting with an initial increment of approximately 5- or 3.1-fold in CC-124 and 2.3- or 2.1-fold in CC-125, relative TAG content reached its maximum level with 6.9- or 15.3-fold in CC-124 and 29.1- or 16.5-fold in CC-125 after 4 days of N or S starvation, respectively (Fig. 5a). However, the increase in TAG content on day 4 was followed by a subsequent decline. Clearly S starvation caused higher TAG accumulation in both strains at the end of 7 days of nutrient starvation. It is generally accepted that algae acclimate to environmental conditions by alteration of their lipid metabolism and composition (Thompson, 1996). Under nutrient deprivation, particularly N-deprivation, some algal species were reported to actively synthesize TAG as an efficient carbon sink (Guschina, 2006). It has also been shown that nitrogen limitation increases the C/N ratio, because reduced synthesis of amino acids and proteins leads to storage of an increased portion of carbon in ...