Process Biochemistry 40 (2005) 297–305

Mixotrophic growth of the microalga Phaeodactylum tricornutum Influence of different nitrogen and organic carbon sources on productivity and biomass composition
M.C. Cerón Garc´a, A. Sánchez Mirón, J.M. Fernández Sevilla, ı E. Molina Grima, F. Garc´a Camacho∗ ı
Departamento de Ingenier´a Qu´mica, Universidad de Almer´a, E-04071 Almer´a, Spain ı ı ı ı Received 30 July 2003; accepted 2 January 2004

Abstract The mixotrophic growth of the diatom Phaeodactylum tricornutum UTEX-640 was studied using diverse substrates at different concentrations in discontinuous and fed-batch modes. The nutrients used were acetate (0.005–0.1 M), starch (0.5–5 g l−1 ), lactic acid (0.005–0.1 M), glycine (0.005–0.02 M), glucose (0.5–5 g l−1 ) and glycerol (0.005–0.1 M). Biomass concentration and biochemical profile were monitored. The capacity of the different nutrients to promote mixotrophic growth varied not only with its nature, but also with the concentration used for the experiment, showing how comparisons at the same concentration may be misleading. Subsequent fed-batch cultures using glycerol (0.1 M), and supplemented urea (0.01 M) and sodium nitrate (1 g l−1 ) as nitrogen sources, showed that repeated additions of organic substrate can sustain mixotrophic growth at very high density cultures. The best results were obtained using with urea (0.01 M), which resulted in maximum biomass and eicosapentaenoic acid productivities that were, respectively, 1.52 g l−1 per day and 43.13 mg l−1 per day, significantly higher than those obtained for the photoautotrophic control. Although the results reported here were obtained in flask cultures of only 1 l working volume and under low irradiance (165 Em−2 s−1 ), similar data were reported for photoautotrophic growth on glycerol of this same strain in outdoor pilot-scale tubular photobioreactors (tube diameter 3 and 6 cm and to 50 and 200 l working volume, respectively), which suggest the possibility of using mixotrophy for the mass production of microalgae. © 2004 Elsevier Ltd. All rights reserved.
Keywords: Microalga; Biochemical composition; Mixotrophic growth; Phaeodactlum tricornutum

1. Introduction Photosynthetic algae are a feed component for aquaculture and produce numerous valuable compounds including pigments (e.g., -carotene, phycobiliproteins), oils (e.g., eicosapentaenoic acid, docosahexaenoic acid), and stable isotope-labelled biochemicals (e.g.,[13 C] glucose); they also have potential in the discovery of new pharmaceuticals [1]. The commercial exploitation of photosynthetic microalgae is based on typical large, outdoor open ponds and closed tubular photobioreactors occupying vast land extensions. These systems although make use of natural sunlight for the production of energy fixed chemically [1,2], they present a great disadvantage, however, cellular self-shading hinders

Corresponding author. Fax: +349-5001-5484. E-mail address: fgarcia@ual.es (F. Garc´a Camacho). ı

light availability, severely limiting biomass production and the low biomass concentrations reached decrease efficient harvesting of the cells. Strategies to improve the efficient use of light or eliminate its requirement by cells and so reduce the cost of microalgal biomass production, involve mixotrophic, photoheterotrophic or heterotrophic growth of algae. The election of one or another depends on the bioproducts of interest and the strains. For example, heterotrophic production of PUFAs from microalgae is possible [3–5], and has recently been achieved by Martek Biosciences [6]. However, heterotrophic growth is not suitable for pigment production (e.g., phycocyanin, astaxanthin, etc.) since many of these are photosynthetic accessory pigments [7]. Otherwise, mixotrophy and photoheterotrophy allow microalgal cells to synthesise simultaneously compounds characteristic of both heterotrophic and photosynthetic metabolisms at high production rates [5,8].

0032-9592/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2004.01.016

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On the other hand, most microalgae are obligate photoautrophs and are unable to use organic carbon sources [9]. This imposes the necessity of screening for species endowed with this capacity. One microalga with this possibility is Phaeodactylum tricornutum UTEX 640 [10]. This species has been broadly studied as a potential source of PUFAs (mainly EPA) and of a great diversity of pigments [1,11,12]. So far, a limited number of preliminary studies have reported that P. tricornutum cells can develop mixotrophic activity in the presence of some organic carbon sources such as alcohols, sugars, diverse organic compounds, growth promoters, soluble fractions of cereals and potato fermented flours, rye and wheat flours [10,13,14,15,16,17,18,19]. Nonetheless, most of them were carried out with the only intention of demonstrating the use of these nutrients by the cells, without considering practical aspects derived of growth kinetics and biomass/bioproduct yield data. In view of the limited amount of information on the potential of mixo- versus photoautotrophic growth, this research was aimed to test different organic nutrients and to analyse the effect of concentration on growth and biochemical composition (focused on fatty acid profile and pigments) of the marine microalga P. tricornutum UTEX 640 grown in laboratory-scale batch cultures.

ditions of glycerol 0.1 M and urea (at optimal concentration previously determined). Additions were done when a steady state biomass concentration had been reached. The initial pH was fixed at 8.0. The temperature was 20 ± 0.5 ◦ C. All experiments were performed in duplicate (the mean values were presented) and they included a control culture (without organic nutrient). Biomass concentration was estimated by absorbance at 625 nm [21] and periodically checked by dry-weight determinations. Freeze-dried biomass was used for fatty acid analysis by gas chromatography, according to the method described by Rodriguez et al. [22]. Carotenoids were quantified according to the method described by Whyte [23] and chlorophylls content were determinated by Hansman’s spectrometric method [24] and using Parsons and Strickland’s equations [25] for the quantification. The following sigmoidal equation was used as suggested by Cerón Garc´a et al. [10] to smooth the growth data by ı nonlinear regression. ln Cb Cbo = Yo + a (1 + exp(−t − to /b))c (1)

2. Materials and methods The alga P. tricornutum UTEX-640 was obtained from the collection of University of Texas, Austin. The inocula were grown in 100 ml conical flasks with 50 ml of culture under aseptic conditions, on an orbital shaker and without aeration. The culture medium of Garc´a Sánchez et al. [20] ı was used for maintenance. One-litre flat-bottom spherical flasks were used as growth vessels. The culture medium was enriched seawater prepared as indicated elsewhere [20]. Inoculation was done using the above described inoculum in linear growth phase and adding sufficient quantity to start with 80 mg l−1 . Growth vessels were sparged with filter-sterilised air at 0.1 min (v/v) for mixing and aeration. The cultures were continuously illuminated by one Philips TLD 36W/54 fluorescent lamp at a light intensity 165 E m−2 s−1 measured at the surface of the culture. The culture medium, except the organic substrates, was sterilised in an autoclave at 126 ◦ C for 20 min. The organic nutrients (lactate, acetate, starch, glycine, glycerol and glucose) were separately sterilised by filtration through 0.2 m pore membranes. Several experimental runs, one for each organic nutrient, were initiated and conducted in discontinuous mode ranged between 0.005 and 0.1 M (0.5, 1, 2 and 5 g l−1 for starch), including a photoautotrophic control. Urea was used as supplementary nitrogen source, and optimised for concentration in the experiments with glycerol (glycerol at 0.1 M was found to be optimal in a previous study [10]). Finally, a fed-batch culture was carried out with successive ad-

This procedure allows the elimination of the influence of experimental error in the evaluation of instantaneous values of growth kinetics parameters. Thus, Eq. (1) was used to obtain the values of the specific growth rate, µ, and the theoretical biomass productivity, Pb, at any time during the experiment and from there the maximum values of those parameters.

3. Results The experimental results of concentration (C) versus time (t) obtained for the discontinuous experiments are represented in Fig. 1 as points of ln(C/C0 ) versus t accompanied by lines that represent the corresponding regressions of the experimental data to Eq. (1). More than 90% of the experimental data were fitted within a ±15% margin using Eq. (1). The results presented in Fig. 1 show that the capacity to promote and sustain mixotrophic growth varies widely among the different substrates depending on the nature and initial concentration utilised. Several circumstances can hinder the appreciation of differences among experiments. Among these are the occurrence of lag phases at the beginning of the experiments and the fact that the organic nutrient can be consumed during the experiment causing a decrease in the initial concentration of substrate. Bearing this in mind it is easy to see how important it is to allow sufficient time to ensure biomass development so that nutrient that initially causes lag phase but has a great capability to promote mixotrophic growth is not deemed as bad. It is desirable to use for comparison a procedure that allows the separation of the effect of the different factors, nutrient type, initial concentration and time. These differences are

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Fig. 1. Variation of ln(C/C0 ) vs. culture time. Regression (lines) were obtained from Eq. (1) [10] for the different initial concentrations tested.

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Fig. 2. Variation of biomass concentration means for each organic nutrient concentration and the intervals around based on Fisher’s least significant difference (LSD) procedure.

more clearly and concisely shown by a two-way analysis of variance (multifactor ANOVA). As expected in batch cultures, time has always had a statistically significant effect on biomass concentration and its contribution to variance was also always high. However, interesting differences were observed for the different organic nutrient concentrations. Fig. 2 shows the mean values of biomass concentration reached for each organic nutrient concentration and the intervals around each mean value. The method used to discriminate among the means values was the Fisher’s least significant difference (LSD) procedure.

In experiments with acetate (Fig. 2a), six pairs of means showed statistically significant differences and growth inhibition was established above 0.005 M. When starch was used as organic nutrient (Fig. 2b), five pairs of means showed statistically significant differences. A stimulated growth compared to control was clearly identified for all starch concentrations assayed, even though a slight inhibitory effect was observed above 2 g l−1 of starch. Glycerol also influenced growth favourably, as can be seen in Fig. 2c, where seven pairs of means showed statistically significant differences. For the concentration 0.05 M,

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Table 1 Dimensionless maximum specific growth rate and maximum biomass productivity in relation to phototrophic control for all the organic sources assayed [Nutrient] Lactate µD [1] [2] [3] [4] 1.58 1.54 1.37 1.37 PD 1.56 1.41 1.15 0.73 Acetate µD 1.00 0.68 0.86 0.71 PD 0.82 1.16 1.21 1.16 Starch µD 1.88 1.22 1.79 1.56 PD 1.58 2.08 1.91 1.52 Glycine µD 0.83 1.03 0.72 PD 1.01 1.11 0.98 Glycerol µD 1.00 1.13 0.44 0.73 PD 1.35 1.51 1.74 2.33 Glucose µD 1.10 1.34 1.33 0.96 PD 0.36 0.50 0.72 1.43 Glycerol + urea µD 1.16 1.50 1.34 1.84 PD 0.88 0.94 0.66 0.77

Data are theoretical values estimated from sigmoidal equation fitted to experimental primary data. [number]: the numbers in upward order correspond with the upward order of the concentrations assayed for each nutrient.

a decrease of the maximum biomass concentration was observed due, possibly, to a marked lag phase (see Fig. 1b). For glycine (Fig. 2d), no statistically significant differences between control mean and others were observed. Glucose also enhanced growth and five pairs of means showed statistically significant differences (Fig. 2e). For sodium lactate (Fig. 2f), eight pairs of means showed statistically significant differences at the 95.0% confidence level. This graph suggests that lactate, in relation to the control, stimulated the growth for concentrations below 0.05 M; although, excluding the control, its effect was inhibitory above the minimum concentration of lactate (0.005 M). Experiments with glycerol + urea (Fig. 2g) suggest that urea stimulates slightly the growth at concentration of 0.01 M, but a contrary effect is exhibited above 0.02 M. Table 1 summarises the most relevant dimensionless kinetic parameters for comparison. Basically, it can be observed the same above described pattern, matched with that shown by dimensionless specific growth rate (µD ) and biomass productivity (PD ) data estimated from the five parameters sigmoidal equation (Eq. (1)) (see Table 1). Only the PD from experiment with acetate and µD from the experiment with glycerol + urea did not follow the expected tendency. This can be justified, partly, by the fact comparing with a photoautotrophic control culture, a nutrient growth-inhibited culture may show a low exponential specific growth rate, but a large linear growth phase associated

with biomass concentrations more high (i.e., maximum biomass productivity more elevated). In general, the µD values reported in Table 1 did not correspond to the same growth phase, although PD did in linear growth phase. 3.1. Pigments Pigments were quantified from harvested biomass once the culture finished. As example, in Table 2 maximum values of pigments content at best nutrient concentrations are shown. The effect of organic substrate on total pigments content and its distribution in carotenoids and chlorophylls was varied. Whereas starch, glycerol, glycine and lactate stimulated the biosynthesis of total pigments (19.2, 24.7, 8.8 and 32.8%, respectively, in relation to control culture), glucose and urea had a marked opposite effect (−47.6 and −40% on control, respectively). The chorophyll contents followed the same tendency than total pigment content. However, the fraction of carotenoids had three exceptions, originated in cultures with glycine, glucose and glycerol. The most significative one was the extraordinary low carotenoids content in culture conducted with glycine. The influence of organic nutrient concentration was not considered in Table 2 because of the best results showed in Table 2 matched with those obtained of statistical analysis applied to kinetics data in Fig. 2.

Table 2 Final carotenoids and chlorophylls content on dry weight for the best concentration of each nutrient Nutrient Starch Control Glycerol Control Glycine Control Glucose Control Lactate Control Glycerol (0.1 M) + urea Control Concentration 2 g l−1 0.1 M 0.01 M 5 g l−1 0.005 M 0.01 M With glycerol (0.1 M) Carotenoid (% dry weight) 1.04 1.14 0.59 0.34 0.02 0.38 0.49 0.35 0.64 0.21 0.49 0.33 Chlorophyll (% dry weight) 3.32 2.29 2.83 2.31 2.97 2.37 0.71 1.95 3.79 3.13 1.09 2.40 Total pigment (% dry weight) 4.36 3.43 3.43 2.75 2.99 2.75 1.20 2.29 4.45 3.35 1.63 2.72

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Table 3 Final EPA content on biomass dry weight and maximum EPA yield productivity for the best concentration of each organic nutrient Nutrient Starch Control Glycerol Control Glycine Control Glucose Control Lactate Control Glycerol (0.1 M) + urea Control Glycerol (0.1 M) + urea implementations Concentration 1 g l−1 0.1 M 0.01 M 5 g l−1 0.005 M 0.01 M With glycerol (0.1 M) 0.01 M Dry weight (%) 1.84 2.24 2.04 1.90 1.76 1.78 2.62 2.24 2.20 1.73 2.91 2.10 2.83 EPA yield (mg l−1 per day) 4.98 2.92 8.56 3.35 4.99 2.52 6.55 4.07 4.09 2.05 11.53 6.47 43.13

same concentration (0.01 M). Ukeles and Rose [16] reported growth stimulatory effect for the three substrates, whereas Hayward [26] observed this behaviour only for glycerol. Additionally, in accordance with Hayward [26], Cooksey [15] also reported that P. tricornutum Böhlin incorporated acetate in the light but the microorganism grew poorly. Growth variability among different species has also been widely reported. Two of the substrates most used in the last decades, not the only, in the culture of economically interesting microalgae have been acetate and glucose. Among others, noteworthy studies detailing growth stimulatory effects of acetate uptake are those carried out with Haematococcus lacustris (astanxanthin producers) [27], Navicula saprophila, Rhodomonas salina and Nitzschia sp. (EPA potential producers) [28], Haematococcus pluvialis (astanxanthin) [29], and Brachiomonas submarina [30]. Contrarily, Moya et al. [31] reported growth inhibitory effects by acetate in batch cultures of Haematcocus lacustris specific; growth rate was affected by a variable inhibition term depending on irradiance level and acetate concentration. 4.1. Growth kinetics In order to establish accurate comparisons, primary growth data were consulted from literature and treated as described here. Only Ukeles and Rose [16] presented primary growth data graphically for acetate, lactate, and glycerol at different concentrations. Tables 1 and 4 display the results of such calculations. The Ukeles’ µD data tendency and scale matched with our results. The sodium acetate had a negative effect in P. tricornutum in all cases, slowing down the growth as much as greater was concentration assayed, with the consequent reduction in biomass concentration as well as in biomass productivity (Table 1). These results are coherent with those obtained by Ukeles [16], who also found a growth reduction for P. tricornutum with the use of acetate, although it is possible to emphasise that these authors registered an increase in the final biomass concentration when prolonging the culture life over 240 h. In this sense, the possibility that P. tricornutum is able to use acetate for mixotrophic growth cannot be
Table 4 Maximum dimensionless specific growth rate and biomass productivity in relation to phototrophic control for all batch experiments in Ukeles’ assay [16] Concentration Nutrient Acetate µD 0.05 0.01 0.1 0.2 1 0.64 0.85 0.67 PD 0.51 0.99 0.77 Lactate µD 0.49 0.47 PD 0.66 0.64 Glycerol µD 0.66 0.56 0.28 PD 0.90 1.01 0.84

3.2. EPA content The type of nutrient influenced markedly the EPA synthesis. Urea and lactate notably increased the EPA content, 38.6 and 27.2%, respectively, on that reached with control culture. On the contrary, cellular EPA content decreased in relation control with the use of starch. No significant effect was observed with glycerol and glycine. Among all the EPA percentages displayed in Table 3, the value corresponding to the culture conducted with glycerol and urea (2.9%) highlights on the other ones.

4. Discussion The ability of obligate photoautrophy microalgae to grow mixotrophycally (or photoheterotrophically) is a phenomenon which appears to exist in a number of genera and species distributed throughout the major taxonomic divisions [16]. From the little literature published about the subject, it is not possible to extract conclusions on the reasons for the lack of ability of microalgae to use reduced carbon sources. These causes probably vary with the species, the strains and culture conditions. They are associated to permeability of the cell, membrane diffusion, active transport and enzymatic processes. Therefore, it implies that any reported study on stimulation of growth by a organic substrate under illumination may not be extrapolable even to the same species. For instance, Hayward [26] and Ukeles and Rose [16] studied the effect of a wide range of externally supplied carbon compounds on the growth of P. tricornutum Böhlin in mixotrophic conditions. In both studies, glycerol, sodium acetate and sodium lactate, among others, were tested at

Data are theoretical values estimated from sigmoidal equation fitted to experimental primary data.

M.C. Cer´ n Garc´a et al. / Process Biochemistry 40 (2005) 297–305 o ı Table 5 Best biomass productivity and biomass concentration values for different nutrients Nutrient Concentration Maximum biomass productivity (mg l−1 h−1 ) 13.2 10.90 11.30 5.44 17.5 7.49 11 9.89 10.70 7.47 7.73 4.95 16.51 17.50 63.5 Maximum biomass concentration (g l−1 ) 1.15 1.10 1.79 0.57 2.99 1.48 2.46 2.10 2.01 1.35 2.18 1.40 2.87 2.70 15.4

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Acetate Control Starch Control Glycerol Control Glycine Control Glucose Control Lactate Control Glycerol (0.1 M) + urea Control Glycerol (0.1 M) + urea successive implementations

0.05 M 1 g l−1 0.1 M 0.01 M 5 g l−1 0.005 M 0.01 M With glycerol (0.1 M) 0.01 M

rejected, but in any case requires a long adaptation time or is able to assimilate acetate only under conditions of strong light deprivation. In relation to starch (Table 1), this nutrient stimulated cells growth for all the concentrations tested, although a slight inhibitory effect was observed above 1 g l−1 acetate. The maximum biomass concentration attained was 1.79 g l−1 for the culture with an initial starch concentration of 1 g l−1 (Table 5). This value exceeds by 175% the value achieved for the control culture. Fábregas et al. [18] also demonstrated, how potato starch was uptaken mixotrophically by P. tricornutum Böhlin reaching an enhanced productivity around 2.4-fold higher than that obtained in photoautotrophic control. In our assays, an analogous improvement (2.1-fold) was observed. However, the present result is more valuable if one keeps in mind that the volumetric scale of our cultures was bigger than those used by Fábregas et al. [18]. The trialcohol glycerol was used as substrate at concentrations of 0.005, 0.01, 0.05 and 0.1 M (0.46, 0.92, 4.6 and 9.2 g l−1 , respectively). For all concentrations a slight enhanced maximum specific growth rate was observed up to 0.01 M glycerol concentration (Table 1). Maximum biomass productivity was clearly in all case above the photoautotrophic control. It is necessary to emphasise that the highest glycerol concentration gave rise to a phase of initial adaptation that decreased the growth in the first hours of culture, giving place to a growth lower than the control. The maximum biomass concentration 2.59 g l−1 was obtained at 0.01 M glycerol concentration, two- fold the value attained in the corresponding phototrophic control (Table 5).

Glycine moderately affected the µD (Table 1), however, its presence was unnoticed in the PD which was practically constant. The maximum concentration (2.46 g l−1 ) was reached at 0.01 M (Table 5), exceeding the control value by 18.95%. This improvement is notably inferior to the one reported by Hayward (152% on the control culture) with a high concentration of glycine (1 M). Glucose is the final product of the photosynthesis, thus allowing the assumption that any photosynthetic microorganism must be able to incorporate it to its metabolism. It is reasonable to expect that its incorporation to the metabolism is straightforward. For that reason, glucose, stimulated growth compared with the control for all concentrations used although not very intense (Table 1). The maximum biomass concentration corresponded to the culture with the highest glucose concentration, being 2.01 g l−1 and exceeding the control in a 148% (Table 5). This result is similar to that obtained by Hayward [26] with the same microalga but a different strain. In experiments with lactate, the nutrient consistently promoted growth in most of experiments (Table 1). The most favourable result was obtained for the concentration of 0.005 M, which was the lowest tried, resulting in a biomass concentration of 2.18 g l−1 which supposed an increase of 40% compared to the photoautotrophic control (Table 5). Increasing lactate concentrations, 0.01 and 0.05 M, gave rise to improvements over the control but always less intense than the 0.005 M concentration. The highest substrate concentration originated a reduction of the final biomass concentration which caused an inferior result than the control, indicating a possible inhibition by substrate. Lactate was therefore a nutrient appropriate for mixotrophic growth, obtaining in the best of cases a biomass productivity of 7.74 mg l−1 h−1 that means an improvement of 56% on the photoautotrophic control. These results also mean an improvement in productivity of 136% compared to the results published by Ukeles and Rose [16] for the same lactate concentration (0.005 M), and 122% when compared with the results for the lactate concentration of 0.01 M. These improvements are more significant when the scales are compared; the work volume in our experiments was 100 times greater (1 l as opposed to 10 ml) than the other works cited above. As shown in Table 1, in the cultures supplemented with added urea at different concentrations the maximum biomass productivity was reached for 0.01 M urea concentration but never exceeding the control. On the contrary, the maximum specific growth rate always exceed the control. On the other hand, the maximum biomass concentration was slightly higher to the control with glycerol 0.1 M (Table 5). Previous studies carried out with P. tricornutum adding nitrates and urea as nitrogen sources demonstrated that the consumption rate of nitrates and urea is always lower when two forms are added together than when they are added separately, although the total nitrogen assimilation rate is higher [32]. Nevertheless, in our experiments these results

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did not reproduce, instead the two forms added together, giving better productivities than separately (see Table 1, glycerol + urea). Since the highest maximum biomass productivity relative to control with glycerol was attained in the culture supplemented with 0.1 M glycerol and urea 0.01 M, these conditions were assayed in fed-batch mode. Glycerol and urea were added at the beginning; three more additions were made once steady state had been reached. This same treatment was applied to the control culture, but using only glycerol. A maximum productivity of 63.5 mg l−1 h−1 was reached after the third addition and was eight-fold the control culture. This value is close to 81 mg l−1 h−1 , obtained with the same specie but operated in continuous mode in photoautotrophically conditions and during the summer, when irradiance is high [33]. By the contrary, it is greater than 44.08 mg l−1 h−1 , obtained by Wen and Chen [34] under mixotrophic conditions with a different strain. The maximum biomass concentration, 15.4 g l−1 , was also higher than that reported by Zhang et al. [35], 10.2 g l−1 , working with Spirulina at irradiance range similar to fixed in our experiments. Ogbonna and Tanaka [5] reported a greater maximum biomass concentration in fed-batch mode of 60 g l−1 . However, the volumetric scale was markedly lower (80 ml) and a different light availability (200 E m−2 s−1 at the surface of 100 ml flask). Yongmanitchai and Ward [36] carried out experiments using nitrate, ammonium and urea as nitrogen sources, finding that the growth was satisfactory improved with nitrate as well as with urea, while the culture supplemented with ammonium did not grow. Similar results were obtained for the combinations nitrate and urea and nitrate and ammonium. 4.2. Pigments In mixotrophic culture, cellular photosynthetic components concentration depends on the relation of the time that cells remain in dark and illuminated zones, that is to say, the relative weight of the heterotrophic/autotrophic metabolisms. At high cellular concentrations, light begins to be limiting and the interval of autotrophic growth is very low compared with the heterotrophic one. Under these conditions, the cellular chlorophyll content is much higher than in the autotrophic cultures [37]. Similar results were observed in the results described in this work; the mixotrophic cultures experience an increase in pigments that is dependent on the increase in biomass concentration and that agrees more with an antenna pigment function than with a photoprotector function, since they are accumulated due to the low intensity of available light [38]. This is because light attenuation increases as cells number increases, and therefore, the average irradiance inside the reactor diminishes. This process implies that cells must synthesise more radiation receiving pigments, in order to maintain growth.

4.3. EPA yield The highest EPA productivity of 43.13 mg l−1 per day was obtained in the culture carried out with 0.1 M glycerol and supplemented periodically with urea 0.01 M. This yield was 13-fold higher than the maximum EPA productivity obtained in the photoautotrophically grown control culture. If we compare results obtained by Wen and Chen [34], 6.37 mg l−1 per day, the present results exceed theirs in 570.3%. Yongmanitchai and Ward [36] reported an EPA productivity of 18.9 mg l−1 per day for P. tricornutum grown in laboratory batch cultures using nitrates as nitrogen source. In outdoor culture, Molina Grima et al. [33] achieved similar yields (47.8 mg l−1 per day) in a pilot-scale outdoor tubular reactor, under photoautotrophic conditions. Clearly, P. tricornutum use all these organic nutrients efficiently in mixotrophic growth. The biomass productivity and EPA yield are notably enhanced in comparison with photoautotrophic culture (Tables 3 and 5). 5. Conclusions In conclusion, P. tricornutum UTEX-640 is able to utilise a variety of organic nutrients assayed in this experiment efficiently under mixotrophic growth, enhancing notably the biomass and EPA production, in comparison with photoautotrophic culture. Mixotrophic growth requires relatively low light intensities and consequently lower energy costs. At high cellular concentration, light becomes to be limiting and the grade of autotrophic growth is very low compared with the heterotrophic one. Under these conditions, the pigments and fatty acids content of cells are significantly higher than in autotrophic cultures. The high irradiance requirements to support growth and enhanced productivity at high cell density can be partially covered by the addition of a suitable organic nutrient in adequate concentration. In consequence these results show the possibility to substitute the photoautotrophic growth by the mixotrophic growth. The repercussions in the photobioreactors design is also clear: lower height/diameter aspect ratio. Therefore, irradiance, land surface occupied and refrigeration may be reduced significantly. Acknowledgements This work was supported by the CICYT (BIO-98-0522), Spain. We wish to express our gratitude to our late co-worker and friend Cristobal Sánchez Mart´n for all the help during ı these years. References
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