Biomass production from the microalga Phaeodactylum tricornutum: Nutrient stress and chemical composition in exponential fed-batch cultures

b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 3 ) 1 e8

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Biomass production from the microalga Phaeodactylum tricornutum: Nutrient stress and chemical composition in exponential fed-batch cultures Matilde S. Chauton a,*, Yngvar Olsen b, Olav Vadstein a a b

Dept. Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway Dept. Biology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway

article info

abstract

Article history:

Microalgae are a resource for production of renewable energy, but there are challenges that

Received 16 May 2012

must be met to optimize the biomass production at a reasonable price. Diatoms are rich in

Received in revised form

carbon storage compounds such as carbohydrates and lipids, and studies have shown that

2 November 2012

nitrogen or phosphorus limitation may increase the cellular carbon storage. To understand

Accepted 4 October 2013

better the physiological effects of resource limitation we need a controlled steady-state

Available online xxx

environment, and this can be achieved in chemostats where growth is regulated by the availability of a limiting substrate through regulation of the dilution rate. We present data

Keywords:

from exponential fed batch-cultures of the model diatom Phaeodactylum tricornutum, to

Biomass

which either nitrogen or phosphorus limiting medium was added in small doses every

Fed-batch cultivation

minute over the whole cultivation period. P limitation led to a higher carbon content per

Nutrient limitation

cell than N limitation. A large fraction of the carbon was stored in carbohydrates in N

Lipid

limited cells, and this biomass may be a suitable raw material for fermentation. Inde-

Carbohydrates

pendent of treatment, a lipid content of around 10% of dry weight was found in this study.

Phaeodactylum tricornutum

In our data the biomass production expressed in terms of cell numbers was highest at intermediate dilution rates, and the chemical composition of the cells was stable over several cycles of exponential increase in culture volume and following harvests. Higher yield of storage carbon can be achieved by increasing the total biomass production instead of maximizing the per cell content of lipids or carbohydrates. ª 2013 Elsevier Ltd. All rights reserved.

1.

Introduction

The search for carbon neutral and renewable complements to the present use of fossil fuels has resulted in increased interest in using microalgae as producers of carbon-rich compounds for bioenergy production. There are many thousand different species of microalgae, many of these are marine

species that grow without competing for arable land and freshwater. However, there are challenges related to microalgal biomass production, and a fundamental issue is how to achieve maximum biomass or carbon production with the desired quality at a reasonable price [1]. Diatoms store energy and reduced carbon as the water-soluble polysaccharide chrysolaminaran (1,3-b-glucan) [2] and neutral lipids in the

* Corresponding author. Tel.: þ47 73597867. E-mail address: [email protected] (M.S. Chauton). 0961-9534/$ e see front matter ª 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biombioe.2013.10.004

Please cite this article in press as: Chauton MS, et al., Biomass production from the microalga Phaeodactylum tricornutum: Nutrient stress and chemical composition in exponential fed-batch cultures, Biomass and Bioenergy (2013), http://dx.doi.org/ 10.1016/j.biombioe.2013.10.004

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form of triacylglycerides (TGAs) [3,4]. Many diatoms are easy to cultivate and yield considerable biomass densities, and studies have shown that nutrient stress such as nitrogen (N) or phosphorus (P) limitation increases the cellular content of lipids and/or carbohydrates. Phytoplankton growing under nutrient replete conditions are characterized by relatively stable C:N:P ratios, and deviation from these ratios may indicate carbon storage or nutrient depletion. A median C:N value (weight) of around 7 is observed in marine microalgae growing under replete conditions, but the ratio may vary from 3 to 17 [5]. Many studies have been performed on the effect of N or P limitation in microalgae, but these studies are not directly applicable for the discussion of microalgae as providers of raw material for biofuel. Studies of the nutritional value of algae used as a feed in aquaculture will normally focus on the contents of long-chained, polyunsaturated fatty acids [6,7] while for biofuel production main focus is on short-chained fatty acids with few double bonds. Studies on nutrient deprivation are often performed in batch cultures and not in a controlled environment with a steady state relation between available nutrients and cell components such as in balanced, exponential growth [25]. Chemostats on the other hand, contain cells in steady state conditions and growth is governed by the supply rate of a limiting resource [8]. When the system is in steady state the growth rate (m) equals the dilution rate (D), and the relationship between growth rate and cell quota for the limiting nutrient (Q, i.e. ratio of internal concentration to biomass) is expressed by:   Q0 m ¼ m0max 1  Q

(1)

where m0max is the apparent theoretical (i.e. asymptotic) maximum growth rate when Q is infinite and Q0 is the subsistence cell quota when m ¼ 0 [9]. Cellular uptake and handling of N and P are different in the cells so the functional relationships of N and P quota vs. growth rate may therefore be different in practice: inorganic P is stored in the cells in amounts that may support cell division when the external levels are very low, whereas N is acquired and incorporated quickly into organic compounds such as amino acids, proteins, nucleic acids and pigments. Biomass (B) production is limited by either nutrient or light, and in the case of nutrient limitation the biomass yield is related to the cell quota by rearranging Equation (1): Y¼

  1 1 m ¼ 1 0 Q Q0 mmax

(2)

Hence, one way to modulate biomass production is to vary the dilution rates of the cultivation system, because the dilution rate D is inversely related to the biomass B and lower dilution rates yield higher biomass. On the other hand, production of carbon-containing biomass constituents (PC, mg C L1 day1) is expressed as a function biomass on a carbon basis (BC) and the net growth rate (m): PC ¼ m  BC

(3)

Maximum production is theoretically achieved at a dilution rate that corresponds to 1/2mmax [10]. Cells in steady state may be achieved in a fed-batch culture system, where

medium is added in small doses on a frequent basis (e.g. every minute) to minimize perturbations of the growth environment. In an exponential fed-batch system, medium is added to a batch culture in an exponentially increasing manner, and the ratio of medium added (DV) per culture volume (VC) is constant and considered as analogous to the dilution rate D: D¼

DV VC

(4)

Exponential fed-batch cultures may be used for repeated harvests of culture containing cells in the same metabolic state. To study the effects of nutrient stress on cellular carbon and energy storage, the marine diatom Phaeodactylum tricornutum was grown in exponential fed-batch cultures at five different dilution rates under N or P limitation. In general, N limitation affects protein synthesis and cell cycle progress at an early stage [27] while P limitation leads to cell cycle arrest in the G2 þ M phase when the cells are large and preparing to divide [28]. It is therefore expected that cells produced under N limitation will be chemically different from cells produced under P limitation, and this can be used to “design” the optimal raw material in e.g. bioenergy production. Our data fit the predictions from chemostat theory: there is a hyperbolic or linear relation between intracellular nutrient concentration and growth rate, and the biomass yield is inversely related to the growth rate. Our data also indicate that fed-batch cultures can be used to provide steady-state cells: cultures of varying growth rates could be harvested at several time points and when the culture volume was replenished the cells continued to be physiologically the same as in previous samplings. In terms of resource production, the fed-batch cultivation system may provide a steady delivery of biomass with controlled quality over time.

2.

Material and methods

The diatom P. tricornutum Bohlin (originating from the CCMP 2561 strain) was grown at 20  C and illuminated with 100 mmol photons m2 s1 in 16:8 (light:dark) cycles. Growth medium (Guillard’s f/2) was prepared [11] from filtered and autoclaved seawater and macro and micronutrients/vitamins, but N:P was adjusted to 3 (molar ratio) in the N reduced medium and 195 in the P reduced medium by reducing the concentration of the limiting nutrient. Cultures were grown in BD Falcon optically clear flasks, and aerated with air/CO2 (1e2% vol:vol) to prevent settling of cells and C limitation. The flasks were mounted in plexiglass holders and biomass changes were monitored by measurements of transmittance: Siemens GaAIA Infrared emitters (880 nm) were mounted on one side of the holder, and the transmitted radiance was recorded by matching phototransistors on the opposite side [12,13]. Fresh medium was added to the cultures every minute by micropumps (Bio-Chem Valve Inc.) and medium doses increased exponentially based on information on the actual start volume and the desired dilution rate (¼growth rate): Dilution rates of 0.33, 0.45, 0.65, 0.90 and 1.10 day1 was chosen corresponding to approximately 24, 32, 46, 65 and 80% of mmax determined from in vivo fluorescence measurements on N and P limited batch cultures prior to the fed-batch study.

Please cite this article in press as: Chauton MS, et al., Biomass production from the microalga Phaeodactylum tricornutum: Nutrient stress and chemical composition in exponential fed-batch cultures, Biomass and Bioenergy (2013), http://dx.doi.org/ 10.1016/j.biombioe.2013.10.004

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Samples for gravimetric determination of total neutral (chloroform extractable) lipids (TNL) were collected by centrifugation (5000 rpm/3  10 min at 5  C) and lyophilized before they were blown with N2 and stored at 80  C until analysis. Lipid extraction was performed with chloroform/ methanol (2:1), and a known volume was taken from the chloroform phase and the solvent was evaporated before weighing the remaining neutral lipids [19]. Two technical replicates were measured from each sample, and up to 5% difference between replicates was accepted. Chlorophyll a (Chl a) was analysed in a HewlettePackard HPLC 1100 Series system, with extraction and solvent compositions according to Rodriguez et al. [20]. Protein was estimated from N cell1 using a conversion factor of 4.87 [21] and dry weight (DW) was estimated from carbon using ash free dry weight (AFDW) ¼ C/0.46 and AFDW/DW 0.86 [22,23]. Phaeodactylum is an atypical diatom and fusiform cells contain very little silica, sometimes as little as 0.4% [24]. The dry weight is therefore not influenced by the presence of silica such as in other diatoms with proper silica frustules.

3.

Results

Turbidity was logged every minute and showed a circadian rhythm of increasing turbidity during the light period and decreasing turbidity during dark phases. As an example, Fig. 1 shows a period of 9 light:dark phases and two cycles of culture harvest and exponential increase(s). Turbidity remained relatively stable over several days (average 783 r.u.  4.2%). Harvest of culture gave a small increase in the average

1000 900 800

Turbidity (r.u.)/volume (ml)

All cultures were filled and harvested several times in the exponential fed-batch modus. Sampling was initiated when transmittance and optical density was stable for several consecutive days and varied less than 10% from one harvest to another. Optical density at 750 nm (OD750) and Nile Red-induced fluorescence (NRF) served as proxies of biomass and neutral lipids, respectively, and were measured every day or every other day during the experimental periods. Analyses of dissolved nutrients, cell numbers, particulate carbon (C), nitrogen (N), phosphorus (P), lipids and chlorophyll were performed on samples from early in the steady-state period and then again at the end of the experimental period when the exponential fed-batch cycles had been repeated up to 10 times, the actual number of cycles depending on the dilution rate for each culture. Aliquots of 2.5 mL were sampled regularly from each culture and OD750 was measured from 2 or 3 replicates in a Pharmacia Biotech Ultraspec 2000 UV/VIS spectrophotometer. The same samples were stained with Nile red (25 mg NR in 100 mL acetone, final concentration 1.5 mM), incubated for 15 min at room temperature and transferred to a Perkin Elmer LS 50B spectrofluorometer for measurement of NRF (ex. 530  5 nm, em. 575  10 nm) [14]. Cells were counted in a Becton Dickinson FACScan flow cytometer, equipped with an argon-ion laser that provides excitation light of 488 nm. Subsamples of cells fixed with glutaraldehyde (2% final concentration) were analysed by collecting signals of side scatter and autofluorescence (detector FL3 with a 650 nm/LP filter), and Fluoresbrite Carboxylate YG 1 mm Microspheres (Polysciences, Inc.) were used as an internal reference. Bacteria numbers were monitored in selected samples during the experimental period using SYBR Green I nucleic acid stain (Invitrogen) staining (detector FL1 with a 530 nm/30 filter). Samples for analysis of particulate C and N were collected on precombusted Whatman GF/F filters (47 mm in diameter) and stored at 23  C until analysis. Three (or sometimes two) replicates and blank filter pieces were bored out from the large filters, and the filter pieces were treated with fuming HCl (37%) to remove inorganic C before they were packed into tin capsules and dried at 60  C for 48 h. The samples were analysed on an ECS 4010 Costech Instruments element analyser. Particulate P was determined by spectrophotometric readings of absorbance after persulfate oxidation of three replicate filter pieces bored out from a larger GF/F filter [15]. Samples for analysis of dissolved NO3/NO2 and PO4 were collected using GF/F filters to remove particulate material, and stored at 20  C until analysis. Duplicate culture filtrate samples were analysed in an auto analyser according to standard methods [16]. Samples for analysis of carbohydrates were bored out from the same precombusted Whatman GF/F filters that were used for CN analysis for dilution rates of 0.33, 0.65 and 1.1 day1 and were analysed using a modified version of the phenolesulphuric method [17] using glucose as a standard. The reported values are the mean of two replicates with C.V. less than 15% glucans extracted with warm water/weak acid have been shown to be intracellularly stored chrysolaminarans, often dominated by glucose [18].

700 600 500 400 300 200 100 0 Days

Fig. 1 e Turbidity (relative units, upper graph) and fedbatch culture volume (ml, lower graph) logged over 9 consecutive days. Cultures were harvested on day 1 and 5, and medium was added in exponential fed-batch modus between harvests.

Please cite this article in press as: Chauton MS, et al., Biomass production from the microalga Phaeodactylum tricornutum: Nutrient stress and chemical composition in exponential fed-batch cultures, Biomass and Bioenergy (2013), http://dx.doi.org/ 10.1016/j.biombioe.2013.10.004

b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 3 ) 1 e8

0.12

A

N P

OD 750 nm

0.10 0.08 0.06 0.04 0.02 0.00 0.0

0.5

1.0

1.5

6

10 cells ml

2.0

B

3.0

2.5

-1

N P

2.0 1.5

6

10 cells ml

-1

2.5

1.0 0.5 0.0 0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

-1

Dilution rate, day

C

N P

1.0

l-1

day

-1

1.2

0.8 0.6 0.4

6

turbidity, but this difference was less than 5% of the overall mean for this period. Results from analyses of dissolved nutrients, cell numbers, carbon, nitrogen, phosphorus, carbohydrates, lipids and chlorophyll and calculated protein contents from two time points (sampled with approximately two weeks between them) were analysed using Pearson’s correlation coefficient (variable pairs where one of the measurements is missing was omitted from the correlation analysis, providing in total 17 correlations of 3e5 data points, r ¼ 0.8842). Hence, the reproduceability of the measurements was good and in support of prolonged steady-state. Optical density is often used to monitor biomass changes, and the results here show that OD is linked to biovolume and not directly to cell number or carbon: Analysis of the relation between OD750 vs. C ml1 showed no difference between the treatments (not shown), but a difference in cell volume is seen from the relation of OD750 vs. cell numbers (Fig. 2A). Diatoms in general contain less carbon per cell volume compared to other groups due to the presence of vacuoles of varying sizes [26] and silicified cell frustules instead of e.g. the cellulose theca of dinoflagellates. The frustule is very small (or absent) in P. tricornutum, so the analyses are little influenced by Si here. Cell numbers and carbon biomass decreased as a function of dilution rate, in accordance with chemostat theory [25]. Cell numbers varied a factor 3 and 2 over the range of dilution rates for N and P limited cultures, respectively (Fig. 2B). In the Nlimited cultures, cell biomass production was maximum at dilution rate 0.65 day1 (or approximately 50% of the estimated umax) and decreased at the highest rate studied here. In the P-limited cultures the cell biomass production increased with dilution rate up to 0.65 day1 and was unchanged at higher dilution rates (Fig. 2C). Bacteria numbers were 3e4  106 ml1 in the N limited cultures at different points during the experiment, and 6e10  106 ml1 in the P limited cultures. Assuming up to 30 fg C per bacteria [40,41] bacteria C biomass is not more than 1e3% compared to the algae C biomass. Such bacteria numbers are comparable to normal seawater, were algae and bacteria co-exist but do not outcompete each other. The minimum cellular concentrations of N, P or C that will support growth (Q0, pg cell1) and maximum theoretical growth rate (m0max , day1) were found by linear regression of cellular quota of N, P or C (Q, pg cell1) vs. dilution rate (D, day1) as presented in Fig. 3AeC; in the case of N and C quota in P limited cells, the fit was linear while in the other cases a non-linear fit (known as the Droop model) was made using Eq. (1) as model (Table 1). Cellular N varied from 0.57 to 2.20 pg cell1 in N limited cells, and from 1.52 to 2.39 pg cell1 in P limited cells (Fig. 3A). Cellular P varied between 0.44 and 1.01 pg cell1 in N limited cells, and between 0.17 and 0.60 pg cell1 in P limited cells (Fig. 3B). Cellular C varied between N and P limited cells, and the N limited cells contained less than 11 pg C cell1 at all dilution rates except the highest; all other treatments resulted in cells with 17.5e19.5 pg C cell1 (Fig. 3C). C:N (mol:mol) decreased linearly with increasing dilution rates in both treatments and with similar slopes (6.446x/p ¼ 0.0048 and 6.0170x/p ¼ 0.001), and C:N varied from 8.0 to 18.8 in N limited cells, and from 8.7 to 14.9 in P limited cells (Table 2).

10 cells prod. m

4

0.2 0.0 0.0

0.2

0.4

0.6

0.8

1.0

Dilution rate, day

1.2

1.4

-1

Fig. 2 e Biomass measurements and productivity in N limited (filled circle) and P limited (open circle) fed-batch cultures of Phaeodactylum tricornutum. (A) Cells (3106 mlL1) vs. optical density (750 nm), trend lines are shown (regression statistics: y [ 0.0409*x/r2 0.9162 in N limited cells and y [ 0.0792*x/r2 0.9495 in P limited cells). (B) Cells (3106 mlL1) as a function of dilution rates (dayL1), (regression statistics: y [ L1.937*x D 2.818/r2 0.9537 in N limited cells and y [ L0. 782*x D 1.444/r2 0.82 in P limited cells). (C) Cells produced (106 mlL1 dayL1) as a function of dilution rates (dayL1).

Please cite this article in press as: Chauton MS, et al., Biomass production from the microalga Phaeodactylum tricornutum: Nutrient stress and chemical composition in exponential fed-batch cultures, Biomass and Bioenergy (2013), http://dx.doi.org/ 10.1016/j.biombioe.2013.10.004

b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 3 ) 1 e8

Algal biomass is usually categorized into the major metabolite fractions lipids, carbohydrates and proteins, and “ash” or “other”. In N limited cells neutral lipids varied from 7 to 12%, while in the P limited cells the lipid content varied from 9 to 17% (Fig. 4/Table 2). N limited cells contained almost 60% carbohydrates and 16e26% proteins, while P limited cells contained 29e39% carbohydrates and 19e23% proteins. The chlorophyll content varied from 0.055 to 0.227 pg cell1 in N limited cells, and from 0.151 to 0.384 pg cell1 in P limited cells, corresponding to 0.2e0.5% of dry weight (Table 2).

3.0

2.5

A

2.0

pg N cell-1

5

1.5

1.0

4.

N P

0.5

0.0 0.00

0.25

0.50

0.75

1.00

1.25

Dilution rate (day-1) 1.2

1.0

B

pg P cell-1

0.8

0.6

0.4

N P

0.2

0.0 0.00

0.25

0.50

0.75

1.00

1.25

Dilution rate (day-1) 25

C

pg C cell -1

20

15

10

5

0 0.00

N P 0.25

0.50

0.75

1.00

1.25

Dilution rate (day-1) Fig. 3 e Cell quota (pg cellL1) as a function of dilution rates (dayL1) in N limited (filled circle) and P limited (open circle) fed-batch cultures of Phaeodactylum tricornutum. Subsistence cell quota (Q0) and maximum theoretical growth rate ðm0max Þ for (A) nitrogen, (B) phosphorus and (C) carbon. See Table 1 for regression statistics.

Discussion

Fed-batch cultivation of microalgae is a way to produce biomass of a relatively constant quality over a period of time, and variations in dilution rates and nutrient availability determine the biomass amounts and the content e.g. lipids or carbohydrates. Carbon accumulation and/or N limitation is reflected in high C:N ratios, and in all the samples here the C:N was higher than the median value of 7 that is observed in microalgae growing under unlimited conditions [5]. A coupling of N and C metabolism has been shown in algae, and lack of N in microalgae affects amino acid synthesis and protein metabolism. Protein content is reported to be 30e65% of the cell mass in unlimited cells [5,32] but in our nutrient limited samples the protein content was lower than 26%. N limitation also affects C accumulation and synthesis of chlorophylls [28,34], and a reduction in photosynthetic carbon assimilation may be observed if N limitation restricts synthesis of the protein Rubisco [35]. Such coupling of N and C metabolism and possibly adverse effect on photosynthetic carbon assimilation can be seen also in our data, where N limited cells contained approximately half as much C as the P limited cells. Diatoms may display a low affinity for P [29] and P. tricornutum has been shown to stop dividing when the intracellular P concentration was 2 fmol cell1 [30]. In our study, P limited cells were fewer in terms of numbers but contained more C than N limited cells, and supports the view that P limitation leads to cell cycle arrest at a point close to cell division. However, the minimum cell quota (Q0) of P limited cells in our study was more than twice as high, so under the experimental conditions applied here the P availability does not appear to have been limiting to such an extent that normal cell metabolism ceases. Increased cell size is related to decreased affinity for phosphate uptake in many phytoplankton [31] but the vacuole may counter-effect this by increasing the surface-to-volume ratio of the cell. A clear difference in absorbance characteristics between N and P limited cells was observed (Fig. 2A) and is interpreted as a difference in cell size or ultimately vacuolar size between the N and the P limited cells, which in turn may affect the nutrient uptake. Bacteria also add to the turbidity in the cultures, but the bacterial biomass was low (1e3% bacteria C compared to microalga C) and can therefore not explain the observed difference in absorption per biomass in the two treatments. Despite the observations of high C:N ratios and low protein content, the increase in cellular content of C, N and P at higher dilution rates here seem to be related to metabolic activity that

Please cite this article in press as: Chauton MS, et al., Biomass production from the microalga Phaeodactylum tricornutum: Nutrient stress and chemical composition in exponential fed-batch cultures, Biomass and Bioenergy (2013), http://dx.doi.org/ 10.1016/j.biombioe.2013.10.004

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Table 1 e Regression statistics from linear regression of cell quota Q vs. dilution rate D in Fig. 3AeC; in the case of N and C quota in P limited cells, the fit was linear (lines 2 and 6 in the table) while in the other cases a non-linear fit (the Droop model) was applied to the data (Eq. (1)). Cell quota N P C

Treatment

m0max (day1)

Q0 (pg cell1)

N lim. P lim. N lim. P lim. N lim. P lim.

1.354  0.128

0.476  0.043

1.552  0.186 1.274  0.243 1.627  0.299

0.300  0.028 0.137  0.027 6.214  1.035

Const.

Slope

n

1.048  0.374

0.888  0.194

13

18.286  0.390

1.722  1.261

10

Table 2 e C:N (mol:mol), proteins, chlorophyll a, and carbohydrates (pg cellL1), and total chloroform-extractable lipids (% of dry weight, lyophilized) in P. tricornutum grown under N or P limitation at five different dilution rates. Roman numbers I and II indicate samplings at two different time points during the steady-state cultivation period. Sampling

I

II

I

0.33

II

I

II

0.45

I

0.65

II

I

0.9

II 1.1

Dilution N C:N (mol:mol) Proteins Chl a Carbohydrates Total lipids P C:N (mol:mol) Proteins Chl a Carbohydrates Total lipids

14.9 3.29 0.073 11.6 10.0 14.3 7.94 0.209 16.9 20.6

18.8 3.01

2.53 0.087

3.36 0.055

15.9 12.9 17.9 6.83 17.5 12.8

12.4 8.14 0.151

8.05 0.148 12.1

Fig. 4 e Ratios of metabolites in N and P limited cultures grown at different dilution rates from 0.33 to 1.1 dayL1: Lipids (grey), carbohydrates (dark grey), proteins (white), and “other” (shaded).

14.7 3.87 0.077 13.4 11.3

14.0 3.92

8.8 11.08 0.187 9.2 11.9

9.0 10.16

13.7 3.79 0.201

14.0 8.9

13.7 12.1

8.0 5.87 0.144 13.7

7.6 7.26 0.384

9.39

10.3

9.5 9.57 0.227 24.6 7.8

10.8 9.08 21.5 6.2

8.7 10.65 0.220 14.4 9.0

9.5 9.82 0.249 17.1 8.7

is not targeted at intracellular accumulation of neutral lipids because these samples also contained the lowest amounts of neutral lipids. A study of P. tricornutum showed that in actively growing cells around 10% of the dry weight was chloroform extractable lipids [36], a number very similar to the findings in this study (Fig. 4). We also found that the carbohydrate content was higher in cells that grew under N limitation compared to P limitation, while lipid and protein content was similar. Carbohydrate content increases with longer light periods or continuous light since the synthesis period is longer, and the period of darkness when carbohydrates are catabolized is shorter (or absent). Chrysolaminaran increase was also observed in N limited cells of the diatom Skeletonema costatum, but when the cells were resupplied with N the chrysolaminaran pool was used to supply carbon skeletons for amino acid synthesis [37,38]. The sum of lipids, carbohydrates and proteins in the biomass we analysed was sometimes low (<50% of the dry weight in some instances). The fraction that is not protein, carbohydrate or protein is sometimes named ‘ash’ (that is, inorganic carbon) or ‘miscellaneous’ (usually with a description of solubility), and typical reported values of ash content is 8e12% and for ‘miscellaneous’ 10e12% [24,33]. Dependent on extraction procedures, these categories may contain many different metabolites: inorganic carbon, nucleic acids, polar membrane lipids/phospholipids, pigments, osmolytes and cell

Please cite this article in press as: Chauton MS, et al., Biomass production from the microalga Phaeodactylum tricornutum: Nutrient stress and chemical composition in exponential fed-batch cultures, Biomass and Bioenergy (2013), http://dx.doi.org/ 10.1016/j.biombioe.2013.10.004

b i o m a s s a n d b i o e n e r g y x x x ( 2 0 1 3 ) 1 e8

wall polysaccharides (that are not water-extractable), to mention some. In summary, N limitation of P. tricornutum leads to a reduction in cell size and less carbon per cell, and more carbon was stored in carbohydrates. Biomass from N limited cells may be a suitable raw material for fermentation and gas production due to the elevated carbohydrate content. P limitation leads to larger cell with more carbon per cell, however, a larger fraction of this carbon was associated with constituents other than neutral lipids, carbohydrates and proteins. A closer inspection of this category of compounds may reveal highvalue compounds that can be exploited to improve the budget balance in microalgae production. Overall, the cellular content of lipids was not as high as that reported elsewhere [39] instead we found levels of cellular total lipids that reflect the lipid content of actively growing cells rather than cells that have passed into stationary culture phase. Considering the lipid production potential, a higher biomass yield is a way to increase e.g. the lipid output and the fed-batch cultivation system is a way to provide a continuous production of biochemically optimized, high-value biomass.

Acknowledgement Funding was provided by the Nordic Energy Research/NINNER and the Norwegian Research Council through the LIPIDO project (NFR 187161), and the funding source was not involved any further in the development of the experimental work or the writing of the report. All authors contributed to the study design, interpretation of data, in the writing of the report and in the decision to submit the paper for publication. MSC and OV were responsible for the collection of data and the analytical work. Ole Støren wrote the software for the automatic data logging system that was used in the cultivation work.

references

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Please cite this article in press as: Chauton MS, et al., Biomass production from the microalga Phaeodactylum tricornutum: Nutrient stress and chemical composition in exponential fed-batch cultures, Biomass and Bioenergy (2013), http://dx.doi.org/ 10.1016/j.biombioe.2013.10.004

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Please cite this article in press as: Chauton MS, et al., Biomass production from the microalga Phaeodactylum tricornutum: Nutrient stress and chemical composition in exponential fed-batch cultures, Biomass and Bioenergy (2013), http://dx.doi.org/ 10.1016/j.biombioe.2013.10.004