Impact of phosphorus quota and growth phase on carbon allocation in Chlamydomonas reinhardtii : an FTIR microspectroscopy study

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1 European Journal of Phycology ISSN: (Print) (Online) Journal homepage: Impact of phosphorus quota and growth phase on carbon allocation in Chlamydomonas reinhardtii : an FTIR microspectroscopy study Andrew P. Dean, James M. Nicholson & David C. Sigee To cite this article: Andrew P. Dean, James M. Nicholson & David C. Sigee (2008) Impact of phosphorus quota and growth phase on carbon allocation in Chlamydomonasreinhardtii : an FTIR microspectroscopy study, European Journal of Phycology, 43:4, , DOI: / To link to this article: Published online: 27 Nov Submit your article to this journal Article views: 911 Citing articles: 39 View citing articles Full Terms & Conditions of access and use can be found at

2 Eur. J. Phycol., (2008), 43(4): Impact of phosphorus quota and growth phase on carbon allocation in Chlamydomonas reinhardtii: an FTIR microspectroscopy study ANDREW P. DEAN 1, JAMES M. NICHOLSON 2 AND DAVID C. SIGEE 1 1 School of Biological Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK 2 Synchrotron Radiation Department, STFC Daresbury Laboratory, Warrington, Cheshire WA4 4AD, UK (Received 6 June 2007; accepted 19 December 2007) Batch cultures of Chlamydomonas reinhardtii were used to study carbon allocation in relation to growth phase and phosphorus availability. Cultures were grown at initial phosphorus (PO 4 -P) concentrations of 500 mgl 1 (high-p) and 50 mgl 1 (low-p). Cellular carbon allocation was monitored using Fourier transform infrared (FTIR) microspectroscopy with the ratio of the band intensities at 1736 cm 1 (lipid) and the cm 1 region (carbohydrate) to 1652 cm 1 (amide I) used as an index of changing carbon balance. Cellular phosphorus concentrations (P quota) were measured by energy dispersive X-ray microanalysis (EDXRMA). Both treatments entered stationary phase on day 18. Increased cell counts in the high-p treatment (max cells ml 1 at stationary phase) led to a rapid decrease in external P availability to <20 mgl 1 during early log phase, with a subsequent decrease in P quota from 0.5% to <0.1% DW. The fall of P quota to <0.1% led to an increase in the lipid/protein ratio (0.13 to 0.23) and carbohydrate/protein ratio (0.37 to 1.57), with ratios increasing further (lipid:protein 1.85; carbohydrate:protein 2.77) in late stationary phase. In the low-p treatment external P concentrations (<20 mgl 1 from day 1) restricted population growth (max cells ml 1 at stationary phase). P quotas fell to <0.1% in early log phase, with the carbohydrate/protein ratio increasing from 0.15 to 3.7 and remaining high into stationary phase while the lipid/ protein ratio increased from 0.2 to 1.2. In both treatments increasing synthesis of lipid and carbohydrate storage products resulted in an increased cell volume. Transfer of P-deficient cells (late stationary) to fresh media led to a rapid stimulation of growth, a rapid reduction in lipid/protein and carbohydrate/protein ratios, and decreased cell volumes. Key words: algae, carbon allocation, Chlamydomonas, EDXRMA, Fourier transform infrared spectroscopy, FTIR, phosphorus, phytoplankton, X-ray microanalysis Introduction In phytoplankton, the products of photosynthesis can be channelled into different biochemical pools (e.g. protein, polysaccharide, lipid, nucleic acid), resulting in a carbon balance that depends on both intrinsic (e.g. growth rate) and extrinsic (e.g. light intensity, nutrient availability) factors. Different macromolecular components are associated with different cell functions, with proteins linked to basic functions of biosynthesis and cell division, and lipids and polysaccharides serving mainly as intracellular reservoirs of carbon and energy (Geider et al., 1986). These changes in carbon allocation provide important insights into metabolic strategies adopted by phytoplankton in response to differing environmental conditions; Correspondence to: manchester.ac.uk Andrew Dean. andrew.dean@ for example under poor growth conditions (low-phosphorus) phytoplankton may preferentially synthesize storage products such as polysaccharide and/or lipid rather than protein (Healey & Hendzel, 1979; Ganff et al., 1986; Kilham et al., 1997). Fourier transform infrared (FTIR) spectroscopy is a novel method for monitoring carbon allocation in phytoplankton. This form of vibration spectroscopy can be used to collect mid-infrared absorbance spectra from air dried, intact microorganisms. It has been successfully applied to the analysis of bacteria (Naumann et al., 1996), higher plants (Wetzel et al., 1998) fungi (Fischer et al., 2006) and yeast (Galichet et al., 2001). When applied to whole organisms the resulting spectrum reflects the biochemical complexity of the cells, with absorbance bands from lipids, nucleic acids, carbohydrates and proteins. In recent years ISSN print/issn online/08/ ß 2008 British Phycological Society DOI: / Published online 27 Nov 2008

3 A. P. Dean et al. 346 FTIR spectroscopy has also been applied to the study of carbon balance in cultured algae and lake phytoplankton, although the number of environmental studies is still relatively few. Laboratorybased FTIR studies have looked at the re-allocation of carbon, particularly carbohydrate and lipid, in response to inorganic nutrient limitation (Beardall et al., 2001; Giordano et al., 2001; Heraud et al., 2005; Stehfest et al., 2005). Studies on algae sampled directly from the environment have used FTIR spectroscopy to demonstrate molecular diversity within species (Sigee et al., 2002), to discriminate between different algal species (Dean et al., 2007) and to investigate variations in carbon allocation of phytoplankton collected from different depths within a stratified lake (Dean & Sigee, 2006). FTIR spectroscopy has been used to monitor changes in carbon allocation in response to differing external phosphorus (P) concentrations, generally comparing P-replete with P-limited cells (Beardall et al., 2001; Stehfest et al., 2005). These studies looked at the effects of cellular nutrient change following the transfer of either nutrient-replete algal cells to nutrient-free media or nutrient-starved cells to nutrient-replete media. The algal spectral signature was investigated before and after transfer. The current project extends previous FTIR studies on the molecular response to P availability by investigating spectral changes at two different external P concentrations over a complete batch culture growth cycle, thus relating carbon allocation to P availability both in actively growing and nongrowing populations. In addition, since externally available P may not reflect the extent to which cells are P-deficient, due to intracellular P reserves, this investigation relates changes in carbon allocation to cellular P concentration (P quota), as determined by energy dispersive X-ray microanalysis (EDXRMA). This electron microscope technique involves the spectral analysis of the X-rays generated from micro-samples irradiated by an electron probe (Sigee, 1993) and has been used to determine the elemental composition of a range of freshwater algae (e.g. Krivtsov et al., 2000, 2005). It is used here to determine the elemental composition of cultured Chlamydomonas with particular emphasis on internal cellular quota of P, relating this to external nutrient availability and changes in carbon allocation. This paper follows on from a previous investigation that looked at the effect of phosphorus availability in batch cultures of another green alga, Scenedesmus subspicatus (Sigee et al., 2007). This showed that cellular allocation of carbon into differing macromolecules (lipids, carbohydrates) could be monitored using FTIR microspectroscopy and could be related both to nutrient availability and stage of batch culture growth. Phosphorus deficiency at low external levels limited growth and increased lipid/protein and carbohydrate/protein ratios. Evidence for P deficiency was also indicated by decreased cellular quota of phosphorus (as monitored by EDXRMA), increased cell volume and decreased cellular chlorophyll a (chl a). In this study we look at the nutrient-response of another green alga, Chlamydomonas reinhardtii, a commonly occurring member of lake phytoplankton found over a wide range of ecological conditions. The study investigates batch cultures grown at two different initial concentrations of phosphorus and seeks to link cellular growth phase, external P availability, internal P quota, and cell volume to carbon allocation by examining changes in lipid/protein and carbohydrate/protein ratios. Materials and methods An initial experiment involved the monitoring of the two cultures over a full growth phase (days 0 22) and into late stationary phase (day 35). This was followed by a short experiment, carried out to test the response of late stationary phase cells to fresh media. Growth phase experiment culture conditions Chlamydomonas reinhardtii (Dangeard, 1888) was obtained from the Culture Collection of Algae and Protozoa (CCAP 11/32A). Batch cultures of algae (200 ml of medium) were grown in 250-ml conical flasks in Jaworski s medium (UKNCC, 2001), modified to give an initial P concentration of 500 mgl 1 (High-P) and 50 mgl 1 (Low-P). Initial N and C concentrations were 20 mg l 1 and 3.8 mg l 1 respectively. Each treatment consisted of three replicate flasks. Cultures were grown under a 16 : 8-h light dark cycle, at 25 C, at a photon irradiance of 145 mmol m 2 s 1, on an orbital shaker at 120 rpm. Samples of cells (counts, chl a, cell volume, EDXRMA, FTIR spectroscopy) and culture media (nutrient concentrations) were taken at various times through the exponential and stationary phases (Fig. 1). Cell enumeration, cell volume, chl a and nutrient analysis From each culture vessel, 1 ml of medium was removed and preserved with 100 ml of Lugol s iodine. Cells were counted in a Sedgwick rafter slide using standard methodology (Woelkerling et al., 1976). Biovolume was calculated by approximating cells to a sphere (Hillebrand et al., 1999). For the determination of chl a, 5 ml of the culture medium was removed, filtered through a 0.45 mm cellulose acetate filter and the chlorophyll extracted using 96% ethanol (Jespersen & Christoffersen, 1987). The filtrate was used for the determination of nitrogen (NO 3 -N), and phosphorus (PO 4 -P), which were measured on a SKALAR SansPlus system auto-analyser, using standard methodology (Skalar Analytical, 1993).

4 FTIR spectroscopy of Chlamydomonas reinhardtii 347 Energy dispersive X-ray microanalysis At each analysis point, EDXRMA samples were obtained by combining three 0.5 ml aliquots from replicate flasks and depositing the cells on a 1 mm pore size Nucleopore ß filter membrane. Cells were washed twice with deionized water, and the excess fluid removed by purging with air. The membrane was rapidly frozen by plunging into liquid nitrogen ( 196 C), freeze dried (24 h, 60 C, 10 5 torr) then mounted on an SEM stub and carbon coated. A Cambridge 360 scanning electron microscope fitted with a Kevex detector (Kevex Ltd) and a LINK AN10000 analyser (Oxford Instruments) was used for X-ray analysis. The electron probe (20 20 mm raster) was placed over a single group (within the monolayer of cells) and a spectrum collected over a 100 s livetime with the probe current adjusted to give a count rate of approximately 10 3 c.p.s. Analyses were carried out at a magnification of 1 K, an accelerating voltage of 15 kv, a working distance of 25 mm and a take off angle of 35. Elemental quantitation was carried out using LINK ZAF/PB software. This program incorporates spectra derived from inorganic standards within a protein matrix, which were used for quantitation using the peak/continuum ratio within characteristic peak areas. The accuracy of quantitation was verified using gelatin standards containing known elemental concentrations. Elemental concentrations were determined as percentage dry weight. Five spectra were analysed from the inoculum culture (day 0) and for each of the two treatments on days 4, 8, 16, 22, 35. FTIR spectroscopy For each treatment, 0.5 ml aliquots were taken separately from replicate flasks, mixed, centrifuged and the cells re-suspended in approximately 100 ml of deionized water. Droplets of cellular suspension were immediately deposited on a Kevley ß reflectance FTIR slide, and air dried under sterile laminar flow. Preparations were stored under desiccation, and analysed within 14 days. FTIR absorption spectra were acquired on beamline 11.1 at Daresbury Laboratory, Warrington, UK, using a confocal Nicolet Continuum IR microscope coupled to a Nicolet Nexus FTIR spectrophotometer. Synchrotron radiation was piped into the spectrophotometer and used as the main source of IR radiation in lieu of the internal Globar source. The use of synchrotron radiation produces Fig. 1. Changes in batch cultures of Chlamydomonas reinhardtii grown in high-p media (initial P concentration 500 mg PO 4 -P l 1 ). (a) Changes in cell counts and chlorophyll a. Each data point is the mean (SE) of three replicate cultures. (b) Changes in nitrogen (NO 3 -N) and phosphorus concentration (PO 4 -P) within the culture media, each data point is the mean (SE) of three replicate cultures. (c) Changes in cell volume (each data point represents the mean (SE) of 20 randomly selected cells) and changes in elemental concentrations of phosphorus (P quota) as determined by energy dispersive X-ray microanalysis, where each data point represents the mean value (SE) of five spectra. (d) Changes in the lipid/amide I ratio (derived from band intensities 1736 cm 1 and 1652 cm 1 ) and carbohydrate/amide I ratio (derived from maximum band intensities within the cm 1 region and 1652 cm 1 ). Each data point is the mean (SE) of 10 FTIR spectra from pooled samples (derived from three replicate flasks) where each spectrum was collected over a randomly selected mm area of continuous cellular monolayer.

5 A. P. Dean et al. 348 spectra with a greatly improved signal-to-noise ratio, therefore providing more accurate data in a shorter time than the Globar source. A liquid nitrogen cooled, mm, single element, mercury cadmium telluride detector was used to collect data with a spectral resolution of 4 cm 1, 64 co-added scans, with a mm square aperture over the wavenumber range cm 1. The spectra were obtained in reflectance mode and displayed as absorbance spectra using the Nicolet OMNIC software. Band assignments to molecular groups were based on those previously published and described elsewhere, including general biological materials (Stuart, 1996) bacteria (Naumann, 1996) diatoms (Giordano et al., 2001); and cyanobacteria (Benning et al., 2004). Ten FTIR spectra were collected from the inoculum culture (day 0) and for each of the two treatments on days 4, 8, 16, 22 and 35. Each of the ten spectra was collected over a randomly selected mm area of continuous cellular monolayer. Analysis was carried out using the spectral region from cm 1, and a linear baseline correction applied to all spectra between these two points. Spectra were exported to Excel ß, where data analysis was carried out. Spectra were scaled to amide I and the intensity (height) of the band associated with (C ¼ O) of ester groups, primarily from lipids and fatty acids (1735 cm 1 ); and the bands associated with (C O C) stretching of polysaccharides ( cm 1 ) were recorded. For each spectrum the ratio of the lipid band and the maximum band intensity within the carbohydrate region to protein (amide I, 1652 cm 1 ) was determined and used as an index of changing carbon allocation within the cells. Investigating the response of cells to fresh media On day 35, an aliquot of cells was removed from each treatment and transferred to fresh medium with an excess phosphorus concentration of 5000 mgl 1, giving initial cell counts of cells ml 1. Cell counts and cell volume were monitored at 1, 2, 4 and 8 days following transfer, and FTIR analysis at 1, 2 and 4 days. Results Growth phase experiment: high-p cultures The initial cell count (Fig. 1a)of cells ml 1 (day 0) rapidly increased up to stationary phase on day 18, with a maximum cell count of cells ml 1. Chl a concentration showed a similar pattern, increasing from 0.1 mgml 1 to reach 3.8 mgml 1 during early stationary phase, but declined during late stationary phase (day 35) and this is reflected in the chl a per cell, which fell from 2 pg cell 1 during exponential phase to 0.7 pg cell 1 in stationary phase. The increase in cell counts correlated with a rapid decline in soluble phosphorus (PO 4 -P) concentration (Fig. 1b) within the growth medium, decreasing from 500 mgl 1 Fig. 2. Typical energy dispersive X-ray emission spectra from free-dried preparations of Chlamydomonas reinhardtii, sampled 4 days after inoculation. Each spectrum is derived from a small group of cells contained within a mm raster. (day 0) to less than 20 mgl 1 by day 4. The decline in soluble nitrogen (NO 3 -N) was less pronounced, falling from an initial value of 19.6 mg l 1 (day 0) to a minimum of 0.4 mg l 1 after the culture had entered stationary phase. Over the same period cell volume (Fig. 1c) increased from an initial value of 157 mm 3 to more than 350 mm 3. X-ray emission spectra from cells cultured in high-p medium (Fig. 2) typically showed prominent peaks of Mg, P, S, Cl, K and Ca during the early part of the growth cycle, with peaks of Na and Si also occasionally detected. Spectra obtained from the surrounding support film did not show any extraneous peaks, indicating that algal peaks were derived entirely from the specimen. The P quota of algal cells (Fig. 1c) at the initiation of the experiment (day 0) was 0.43% dry weight and after a small increase following transfer from the inoculum culture it decreased sharply, falling to below 0.1% by day 12. FTIR spectra (Fig. 3) showed nine distinct absorption bands over the wavenumber range cm 1. These bands were attributed to (C ¼ O) stretching of amides from proteins (amide I, 1652 cm 1 ); (N H) bending of amides from proteins (amide II, 1540 cm 1 ); as (CH 2 ) and as (CH 3 ) bending of methyl from

6 FTIR spectroscopy of Chlamydomonas reinhardtii 349 Fig. 3. FTIR spectra of Chlamydomonas reinhardtii over the wavenumber range cm 1 sampled 4 days after inoculation. Each spectrum is a mean of 10 replicate spectra, each collected over a randomly selected mm area of continuous cellular monolayer. Molecular assignments of the nine major absorption bands (indicated by arrows) are given in the text. proteins (1452 cm 1 ); s (CH 2 ) and s (CH 3 ) bending of methyl and s (C O) stretching of COO groups (1380 cm 1 ) and as (>P ¼ O) stretching, associated with phosphorus compounds (1245 cm 1 ). Two bands were of particular importance in reference to the P quota relationship the band at 1735 cm 1, associated with (C ¼ O) of ester groups, primarily from lipids and fatty acids, and the region from cm 1 associated with (C O C) stretching of polysaccharides. During the initial phase of algal culture the carbohydrate/protein ratio remained at 0.15 (Fig. 1d), subsequently increasing during exponential growth to 1.57 (day 16) with a greatly increased ratio of 2.77 in late stationary phase (day 35). The lipid/protein ratio also showed a marked increase, increasing from early values of 0.13 to a level of 0.23 on day 16, but with a very large increase to 1.85 occurring during stationary phase. Growth phase experiment: low-p cultures Although cell counts in the low-p cultures were generally much lower than in the high-p treatment (Fig. 4a), entry into stationary phase occurred at a similar time (day 18), with a stationary phase cell concentration of cells ml 1. This was followed by a sharp decline as cultures entered late stationary phase, with the population count falling to cells ml 1. Chl a concentration reached a maximum of 1.10 mgml 1 on day 16, then declined through stationary phase to 0.48 mgml 1 on day 35. Chl a per cell fell from values of 2 pg cell 1 during exponential phase to 1 pg cell 1 during stationary phase. Phosphorus (PO 4 -P) within the culture medium remained at mgl 1 throughout the course of the experiment (Fig. 4b) and the uptake of nitrogen (NO 3 -N) from the culture medium was much lower than that seen in the high-p culture. A large increase in cell volume was observed in the low-p cultures (Fig. 4c), increasing from 157 mm 3 (day 0) to more than 1000 mm 3 in stationary phase (days 22 and 35). During the early part of the culture period X-ray emission spectra from cells cultured in low-p medium typically had much less prominent P peaks compared to high-p treatments (Fig. 2). Low-P cells showed a marked decline in P quota (Fig. 4c) from 0.43% to 0.1% within 4 days, after which quota values decline further. FTIR absorption spectra from low-p cultures frequently had more prominent carbohydrate and lipid bands compared to high-p algal cells (Fig. 3). In the low-p treatment there was a rapid increase in the carbohydrate/protein ratio (Fig. 4d), increasing within the period of exponential growth from 0.15 (day 0) to 3.7

7 A. P. Dean et al. 350 to 1.2 during exponential phase and remained high during stationary phase. Relationship between lipid and carbohydrate ratios, P quota and cell volume Close correlation occurred between cell volume and carbon allocation in the algal cells over the major part of the growth phase experiment (days 0 to 22). This is shown by the significant linear relationship between cell volume and lipid/protein ratio (p ¼ 0.018, r ¼ 0.695, n ¼ 11), and by a similar relationship between cell volume and the carbohydrate/protein ratio (p ¼ 0.002, r ¼ 0.824, n ¼ 11). The relationship between P quota and lipid/ amide I ratio (Fig. 5a), and carbohydrate/amide I ratio (Fig. 5b) is more complex. In both cases P quota values >0.1% were associated with low molecular ratios while those <0.1% were associated with higher molecular ratios. The relationship between cell volume and P Quota (Fig. 5c) shows a substantial increase in cell volume when quota values were <0.1%. Fig. 4. Low-P treatment. Changes in batch cultures of Chlamydomonas reinhardtii grown in low-p media (initial concentration 50 mg PO 4 -P l 1 ). Remaining legend as Figure 1. (day 8), and remaining at a high level into early stationary phase but with a decrease towards the end of the culture period. The lipid/protein ratio (Fig. 4d) increased from an initial value of 0.13 Response of cells to fresh media In both high-p and low-p cultures, the volume of inoculum transferred to fresh medium on day 35 was adjusted to give an initial population count of cells ml 1 (Fig. 6a). In cultures derived from the original high-p treatment, cell counts rapidly increased to reach cells ml 1 within 2 days of transfer. Cultures derived from the original low-p treatment also showed a rapid population increase, reaching cells ml 1 over the same period. Both populations then remained static for the rest of the experiment. Cell volumes decreased markedly during the 24 h following transfer (Fig. 6b), falling from 460 mm 3 to 140 mm 3 in the high-p culture and 1130 mm 3 to 210 mm 3 in the low-p culture. After 48 h, cell volumes in both cultures were 100 mm 3. Cell transfer to fresh medium also resulted in a reversal of the FTIR spectral changes previously observed over the course of the growth phase experiment. Within 48 h of transfer, original spectral differences between the two cultures (Fig. 7a) were almost indistinguishable (Fig. 7b). The majority of the spectral changes occurred over the first 24 hrs, with both lipid/protein (Fig. 8a) and carbohydrate/protein (Fig. 8b) ratios showing marked reductions over this period. Discussion This study used batch cultures to study carbon allocation in relation to growth phase and

8 FTIR spectroscopy of Chlamydomonas reinhardtii 351 Fig. 6. Changes in (a) cell count before (day 35) and after transfer to fresh (5000 mg PO 4 -P l 1 ) medium. For each treatment one flask was used. For each data point a minimum of 200 cells was counted in a Sedgwick rafter slide following appropriate dilution of the culture. (b) Changes in cell volume before and after transfer to fresh high-p medium. Each data point is the mean (SE) of 20 cells. Fig. 5. Relationship between (a) lipid/amide I ratio and P quota (b) carbohydrate/amide I ratio and P quota and (c) cell size and P quota over the major part of the growth phase experiment (days 0 to 22). phosphorus availability in C. reinhardtii. Initial phosphorus concentrations in the two treatments were ecologically relevant, spanning differences in P availability between oligotrophic and hypertrophic lakes (Sigee, 2004). Both treatments exhibited the logarithmic and stationary phases typical of batch culture (Subba Rao, 2006) but lacked an initial lag phase. This can be attributed to the fact that the inoculum cells (log phase cells cultured in high-p media) were already acclimatized to the general laboratory growth conditions subsequently employed. Although changes in external and internal P concentrations varied markedly between the two treatments, both high-p and low-p cultures entered stationary phase at the same time, suggesting that P availability may not be the primary cause of phase transition in these experiments. P availability was important, however, in determining the maximum cell count, which differed markedly in the two situations. As discussed below, both treatments exhibited characteristics previously observed in P-deficient cells, including decreased cellular P quota, increased lipid/protein and carbohydrate/protein ratios, and increased cell volume. In both treatments a reduction in external P availability led to a decline in cellular P quota, suggesting that P storage ( luxury consumption ) was minimal under these conditions. In the low-p culture, which had low external P-availability from the start of the experiment, P quotas rapidly fell to

9 A. P. Dean et al. 352 Fig. 7. FTIR spectra showing the spectra (a) before (day 35) and (b) 48 h after cells were placed in fresh high-p (5000 mg PO 4 -Pl 1 ) media. Each spectrum is a mean of 10 replicate spectra, each collected over a randomly selected mm area of continuous cellular monolayer. 0.1% while in the high-p treatment the fall in external P availability to low levels had occurred by day 4 and a fall in cellular P quota (to <0.1% dry weight) followed soon after. Studies on other algae suggest that the P quota values of <0.1% measured in this study are consistent with severe P-deficiency. An investigation on cultured Scenedesmus (Sterner & Robinson, 1994), for example, showed that the P content of the cells varied between 0.9% DW in nutrient replete conditions to 0.08% DW in P-limited cells. The internal P quota values observed in this study would suggest that the low-p culture was P-deficient from the start of the experiment, while the high-p treatment was P-deficient from mid-log phase. The relationship between lipid and carbohydrate ratios and internal P availability (P quota) shows that changes in carbon allocation are correlated more with changes in P quota rather than external P concentrations, with increases in lipid/protein and carbohydrate/protein ratios occurring when the P quota falls below 0.1%. This result is in agreement with recent studies on Fig. 8. Changes in the (a) lipid/amide I ratio and (b) carbohydrate/amide I ratio before (day 35) and after transfer to fresh (5000 mg PO 4 -P l 1 ) media. Each point is the mean (SE) of 10 FTIR spectra, each collected over a randomly selected mm area of continuous cellular monolayer. S. subspicatus (Sigee et al., 2007), where changes in carbon allocation also occurred when P quota fell to less than 0.1%. The increased carbohydrate/protein and lipid/ protein ratios seen in this study are most likely due to increased synthesis of carbohydrates and lipids rather than to a decrease in proteins. This is evidenced by the increased cell volume, which is consistent with increased storage products, and by the fact that within treatments changes in the lipid/ protein and carbohydrate/protein ratios occurred both at different times, and to differing extents. If protein was the main determinant of these changes the changes in both ratios would be expected to correlate. Thus although changes to protein will have occurred during the experiment (e.g. declining cellular chl a during stationary phase will have resulted in a decrease in chlorophyll associated protein) it is likely that the observed changes in the molecular ratios are due primarily to changes in lipid and carbohydrate synthesis.

10 FTIR spectroscopy of Chlamydomonas reinhardtii 353 The increased synthesis of carbohydrates and lipids seen in this study reflect those seen under P-deficiency in other algae, including the green alga Ankistrodesmus (Kilham et al., 1997) and the diatom Stephanodiscus (Lynn et al., 2000). Changes in carbon allocation during exponential phase suggest that increases in cellular storage products, particularly carbohydrate, occurred when the cells were still undergoing division. This suggests that under good growth conditions not all carbon was allocated to cell division, and a progressive increase in lipids and carbohydrate synthesis occurred as growth conditions deteriorated and cells entered stationary phase. In stationary phase (poor growth conditions) there was a cessation of cell division but continued synthesis of carbohydrates and lipids. This synthesis and intracellular storage of lipids and carbohydrates resulted in large increases in cell volume, as has been observed in a number of P-stressed algae. (Mitchell et al., 1992; Sterner et al., 1993; Kilham et al., 1997; van Donk et al., 1997). The changes due to P-deficiency seen in the growth phase experiment were reversed when stationary phase cells were placed in fresh medium. On entry to fresh medium there was a rapid increase in cell division over a 48 h period, during which reserves of lipids and carbohydrates were rapidly depleted, with cell division ceasing when reserves were exhausted. Other studies have also shown a reduction in cellular storage products during active cell division, however the reduction in storage products observed in Chlamydomonas was more rapid than that seen, for example, in the diatom Phaeodactylum (Stehfest et al., 2005) and may reflect the r-selected nature of Chlamydomonas. The results presented here make an interesting comparison to those observed in a parallel experiment on another green alga, S. subpicatus (Sigee et al., 2007). In the 50 mgl 1 treatment a decline in the P quota of Scenedesmus to <0.1% also resulted in increased lipid and carbohydrate ratios, with the increase occurring throughout the log and stationary phases. However, the 200% increases in the lipid/protein and carbohydrate/ protein ratios were much lower than in Chlamydomonas, where the percentage increase in the molecular ratios was 800% and 2000% respectively. Scenedesmus also showed much lower increase in cell volume, only rising by 50%, compared with 600% in Chlamydomonas. In the 500 mgl 1 treatment Scenedesmus displayed increases in lipid/protein and carbohydrate/protein ratio of 100 and 50% respectively. However, this only occurred when the cells were already in stationary phase, and no increase in cell volume was observed, with cellular volume decreasing by 30%. These two species of green algae therefore showed a clear difference in the magnitude of their response to P limitation, particularly in relation to cell volume. In Chlamydomonas the close relationship between cell volume and increasing lipid and carbohydrate synthesis is clear in both treatments, and in agreement with the findings of a number of studies using conventional methodology (Mitchell et al., 1992; Sterner et al., 1993; Kilham et al., 1997; van Donk et al., 1997), whereas the relationship seen in Scenedesmus may be atypical. This study has shown that cellular carbon allocation can show rapid and distinctive changes with both growth stage and availability of an essential macronutrient, and builds upon a previous body of work using bulk analysis and FTIR spectroscopy to relate cellular carbon allocation to P availability. The novel combination of FTIR and EDXRMA spectroscopy brings together two high-resolution techniques that have the capacity to analyse single cells and individual colonies within algal populations (Sigee et al., 2002, Dean et al., 2007). These techniques have particular potential for studying the carbon allocation-p quota relationship in single species within mixed environmental populations, and for extrapolating the information obtained in this laboratory study to an environmental context. Acknowledgements The authors gratefully acknowledge the Leverhulme Trust (Grant Number F/00 120/AO) for funding the work carried out in this study. The authors also thank CCLRC funding (Beamtime Grant Number 45109) for the use of the synchrotron based FTIR facilities at Daresbury Laboratory, UK and the Electron Microscope Unit at Manchester University for the EDXRMA facilities. 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