2 Mixotrophic Growth of the Microalga Phaeodactylum Tricornutum

7/27/2019 2 Mixotrophic Growth of the Microalga Phaeodactylum Tricornutum

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Process Biochemistry 40 (2005) 297305

Mixotrophic growth of the microalga Phaeodactylum tricornutumInfluence of different nitrogen and organic carbon sources on

productivity and biomass composition

M.C. Cern Garca, A. Snchez Mirn, J.M. Fernndez Sevilla,E. Molina Grima, F. Garca Camacho

Departamento de Ingeniera Qumica, Universidad d e Almera, E-04071 Almera, Spain

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 concentra-tions in discontinuous and fed-batch modes. The nutrients used were acetate (0.0050.1 M), starch (0.55g l1), lactic acid (0.0050.1 M),glycine (0.0050.02M), glucose (0.55 g l1) and glycerol (0.0050.1 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 usedfor the experiment, showing how comparisons at the same concentration may be misleading. Subsequent fed-batch cultures using glycerol(0.1 M), and supplemented urea (0.01M) and sodium nitrate (1 g l1) as nitrogen sources, showed that repeated additions of organic substratecan sustain mixotrophic growth at very high density cultures. The best results were obtained using with urea (0.01 M), which resulted inmaximum biomass and eicosapentaenoic acid productivities that were, respectively, 1.52 g l1 per day and 43.13 mg l1 per day, significantlyhigher than those obtained for the photoautotrophic control. Although the results reported here were obtained in flask cultures of only 1 lworking volume and under low irradiance (165Em2 s1), 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), whichsuggest 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 aquacul-ture and produce numerous valuable compounds includ-ing pigments (e.g., -carotene, phycobiliproteins), oils (e.g.,

eicosapentaenoic acid, docosahexaenoic acid), and stableisotope-labelled biochemicals (e.g.,[13C] glucose); they alsohave potential in the discovery of new pharmaceuticals [1].

The commercial exploitation of photosynthetic microal-gae is based on typical large, outdoor open ponds and closedtubular photobioreactors occupying vast land extensions.These systems although make use of natural sunlight for theproduction of energy fixed chemically [1,2], they presenta great disadvantage, however, cellular self-shading hinders

Corresponding author. Fax: +349-5001-5484.E-mail address: [email protected] (F. Garca Camacho).

light availability, severely limiting biomass production andthe low biomass concentrations reached decrease efficientharvesting of the cells. Strategies to improve the efficientuse of light or eliminate its requirement by cells and soreduce the cost of microalgal biomass production, involvemixotrophic, photoheterotrophic or heterotrophic growth ofalgae. The election of one or another depends on the bioprod-ucts of interest and the strains. For example, heterotrophicproduction of PUFAs from microalgae is possible [35],and has recently been achieved by Martek Biosciences [6].However, heterotrophic growth is not suitable for pigmentproduction (e.g., phycocyanin, astaxanthin, etc.) since manyof these are photosynthetic accessory pigments [7]. Other-wise, mixotrophy and photoheterotrophy allow microalgalcells to synthesise simultaneously compounds characteris-tic of both heterotrophic and photosynthetic metabolisms athigh 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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298 M.C. Ceron Garca et a l. / Process Biochemistry 40 (2005) 297305

On the other hand, most microalgae are obligate pho-toautrophs and are unable to use organic carbon sources [9].This imposes the necessity of screening for species endowedwith this capacity. One microalga with this possibility isPhaeodactylum tricornutum UTEX 640 [10]. This specieshas 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 re-ported that P. tricornutum cells can develop mixotrophicactivity in the presence of some organic carbon sources suchas alcohols, sugars, diverse organic compounds, growthpromoters, soluble fractions of cereals and potato fermentedflours, rye and wheat flours [10,13,14,15,16,17,18,19].Nonetheless, most of them were carried out with the onlyintention of demonstrating the use of these nutrients bythe cells, without considering practical aspects derived ofgrowth kinetics and biomass/bioproduct yield data. In viewof the limited amount of information on the potential ofmixo- versus photoautotrophic growth, this research was

aimed to test different organic nutrients and to analyse theeffect of concentration on growth and biochemical com-position (focused on fatty acid profile and pigments) ofthe marine microalga P. tricornutum UTEX 640 grown inlaboratory-scale batch cultures.

2. Materials and methods

The alga P. tricornutum UTEX-640 was obtained fromthe collection of University of Texas, Austin. The inoculawere grown in 100 ml conical flasks with 50 ml of culture

under aseptic conditions, on an orbital shaker and withoutaeration. The culture medium of Garca Snchez et al. [20]was used for maintenance.

One-litre flat-bottom spherical flasks were used as growthvessels. The culture medium was enriched seawater preparedas indicated elsewhere [20]. Inoculation was done using theabove described inoculum in linear growth phase and addingsufficient quantity to start with 80 mg l1. Growth vesselswere sparged with filter-sterilised air at 0.1 min (v/v) formixing and aeration. The cultures were continuously illu-minated by one Philips TLD 36W/54 fluorescent lamp at alight intensity 165E m2 s1 measured at the surface ofthe culture.

The culture medium, except the organic substrates, wassterilised in an autoclave at 126 C for 20 min. The organicnutrients (lactate, acetate, starch, glycine, glycerol and glu-cose) were separately sterilised by filtration through 0.2mpore membranes. Several experimental runs, one for each or-ganic nutrient, were initiated and conducted in discontinuousmode ranged between 0.005 and 0.1 M (0.5, 1, 2 and 5 g l1

for starch), including a photoautotrophic control. Urea wasused as supplementary nitrogen source, and optimised forconcentration in the experiments with glycerol (glycerol at0.1 M was found to be optimal in a previous study [10]). Fi-nally, a fed-batch culture was carried out with successive ad-

ditions of glycerol 0.1 M and urea (at optimal concentrationpreviously determined). Additions were done when a steadystate biomass concentration had been reached. The initialpH was fixed at 8.0. The temperature was 20 0.5 C. Allexperiments were performed in duplicate (the mean valueswere presented) and they included a control culture (without

organic nutrient).Biomass concentration was estimated by absorbance at625nm [21] and periodically checked by dry-weight de-terminations. Freeze-dried biomass was used for fatty acidanalysis by gas chromatography, according to the methoddescribed by Rodriguez et al. [22]. Carotenoids were quan-tified according to the method described by Whyte [23] andchlorophylls content were determinated by Hansmans spec-trometric method [24] and using Parsons and Stricklandsequations [25] for the quantification.

The following sigmoidal equation was used as suggestedby Cern Garca et al. [10] to smooth the growth data bynonlinear regression.

ln

Cb

Cbo

= Yo +

a

(1+ exp(t to/b))c(1)

This procedure allows the elimination of the influence ofexperimental error in the evaluation of instantaneous valuesof growth kinetics parameters. Thus, Eq. (1) was used toobtain the values of the specific growth rate, , and thetheoretical biomass productivity, Pb, at any time during theexperiment and from there the maximum values of thoseparameters.

3. Results

The experimental results of concentration (C) versus time(t) obtained for the discontinuous experiments are repre-sented in Fig. 1 as points of ln(C/C0) versus t accompaniedby lines that represent the corresponding regressions of theexperimental data to Eq. (1).

More than 90% of the experimental data were fitted withina 15% margin using Eq. (1).

The results presented in Fig. 1 show that the capacityto promote and sustain mixotrophic growth varies widelyamong the different substrates depending on the nature andinitial concentration utilised. Several circumstances canhinder the appreciation of differences among experiments.Among these are the occurrence of lag phases at the be-ginning of the experiments and the fact that the organicnutrient can be consumed during the experiment causing adecrease in the initial concentration of substrate. Bearingthis in mind it is easy to see how important it is to allowsufficient time to ensure biomass development so that nutri-ent that initially causes lag phase but has a great capabilityto promote mixotrophic growth is not deemed as bad. Itis desirable to use for comparison a procedure that allowsthe separation of the effect of the different factors, nutrienttype, initial concentration and time. These differences are


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M.C. Ceron Garca et al. / Process Biochemistry 40 (2005) 297305 299

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 Fishers least significantdifference (LSD) procedure.

more clearly and concisely shown by a two-way analysis ofvariance (multifactor ANOVA). As expected in batch cul-tures, time has always had a statistically significant effecton biomass concentration and its contribution to variancewas also always high. However, interesting differences wereobserved for the different organic nutrient concentrations.Fig. 2 shows the mean values of biomass concentrationreached for each organic nutrient concentration and theintervals around each mean value. The method used to dis-criminate among the means values was the Fishers leastsignificant difference (LSD) procedure.

In experiments with acetate (Fig. 2a), six pairs of meansshowed statistically significant differences and growth inhi-bition was established above 0.005 M. When starch was usedas organic nutrient (Fig. 2b), five pairs of means showed sta-tistically significant differences. A stimulated growth com-pared to control was clearly identified for all starch concen-trations assayed, even though a slight inhibitory effect wasobserved above 2 g l1 of starch.

Glycerol also influenced growth favourably, as can beseen in Fig. 2c, where seven pairs of means showed statis-tically significant differences. For the concentration 0.05 M,


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Table 1Dimensionless maximum specific growth rate and maximum biomass productivity in relation to phototrophic control for all the organic sources assayed

[Nutrient] Lactate Acetate Starch Glycine Glycerol Glucose Glycerol + urea

D PD D PD D PD D PD D PD D PD D PD

[1] 1.58 1.56 1.00 0.82 1.88 1.58 0.83 1.01 1.00 1.35 1.10 0.36 1.16 0.88[2] 1.54 1.41 0.68 1.16 1.22 2.08 1.03 1.11 1.13 1.51 1.34 0.50 1.50 0.94

[3] 1.37 1.15 0.86 1.21 1.79 1.91 0.72 0.98 0.44 1.74 1.33 0.72 1.34 0.66[4] 1.37 0.73 0.71 1.16 1.56 1.52 0.73 2.33 0.96 1.43 1.84 0.77

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

a decrease of the maximum biomass concentration was ob-served due, possibly, to a marked lag phase (see Fig. 1b).For glycine (Fig. 2d), no statistically significant differencesbetween control mean and others were observed.

Glucose also enhanced growth and five pairs of meansshowed statistically significant differences (Fig. 2e).

For sodium lactate (Fig. 2f), eight pairs of means showed

statistically significant differences at the 95.0% confidencelevel. This graph suggests that lactate, in relation to the con-trol, stimulated the growth for concentrations below 0.05 M;although, excluding the control, its effect was inhibitoryabove the minimum concentration of lactate (0.005 M).

Experiments with glycerol + urea (Fig. 2g) suggest thaturea stimulates slightly the growth at concentration of0.01M, but a contrary effect is exhibited above 0.02 M.

Table 1 summarises the most relevant dimensionlesskinetic parameters for comparison. Basically, it can beobserved the same above described pattern, matched withthat shown by dimensionless specific growth rate (D) andbiomass productivity (PD) data estimated from the five pa-

rameters sigmoidal equation (Eq. (1)) (see Table 1). Onlythe PD from experiment with acetate and D from the ex-periment with glycerol + urea did not follow the expectedtendency. This can be justified, partly, by the fact com-paring with a photoautotrophic control culture, a nutrientgrowth-inhibited culture may show a low exponential spe-cific growth rate, but a large linear growth phase associated

Table 2Final carotenoids and chlorophylls content on dry weight for the best concentration of each nutrient

Nutrient Concentration Carotenoid (%dry weight)

Chlorophyll (%dry weight)

Total pigment(% dry weight)

Starch 2 g l1

1.04 3.32 4.36Control 1.14 2.29 3.43

Glycerol 0.1 M 0.59 2.83 3.43Control 0.34 2.31 2.75

Glycine 0.01 M 0.02 2.97 2.99Control 0.38 2.37 2.75

Glucose 5 g l1 0.49 0.71 1.20Control 0.35 1.95 2.29

Lactate 0.005 M 0.64 3.79 4.45Control 0.21 3.13 3.35

Glycerol (0.1 M) + urea 0.01 M 0.49 1.09 1.63Control With glycerol (0.1 M) 0.33 2.40 2.72

with biomass concentrations more high (i.e., maximumbiomass productivity more elevated). In general, the Dvalues reported in Table 1 did not correspond to the samegrowth phase, although PD did in linear growth phase.

3.1. Pigments

Pigments were quantified from harvested biomass oncethe culture finished. As example, in Table 2 maximum val-ues of pigments content at best nutrient concentrations areshown. The effect of organic substrate on total pigments con-tent and its distribution in carotenoids and chlorophylls wasvaried. Whereas starch, glycerol, glycine and lactate stimu-lated the biosynthesis of total pigments (19.2, 24.7, 8.8 and32.8%, respectively, in relation to control culture), glucoseand urea had a marked opposite effect (47.6 and 40%on control, respectively).

The chorophyll contents followed the same tendency thantotal pigment content. However, the fraction of carotenoidshad three exceptions, originated in cultures with glycine,

glucose and glycerol. The most significative one was theextraordinary low carotenoids content in culture conductedwith glycine.

The influence of organic nutrient concentration was notconsidered in Table 2 because of the best results showed inTable 2 matched with those obtained of statistical analysisapplied to kinetics data in Fig. 2.


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Table 3Final EPA content on biomass dry weight and maximum EPA yieldproductivity for the best concentration of each organic nutrient

Nutrient Concentration Dryweight (%)

EPA yield(mgl1 per day)

Starch 1 g l1 1.84 4.98Control 2.24 2.92

Glycerol 0.1 M 2.04 8.56Control 1.90 3.35

Glycine 0.01 M 1.76 4.99Control 1.78 2.52

Glucose 5 g l1 2.62 6.55Control 2.24 4.07

Lactate 0.005 M 2.20 4.09Control 1.73 2.05

Glycerol (0.1 M)+ urea

0.01 M 2.91 11.53

Control With glycerol(0.1M)

2.10 6.47

Glycerol (0.1 M)+ urea imple-mentations

0.01 M 2.83 43.13

3.2. EPA content

The type of nutrient influenced markedly the EPA syn-thesis. Urea and lactate notably increased the EPA content,38.6 and 27.2%, respectively, on that reached with controlculture. On the contrary, cellular EPA content decreased inrelation control with the use of starch. No significant effectwas observed with glycerol and glycine. Among all the EPA

percentages displayed in Table 3, the value corresponding tothe culture conducted with glycerol and urea (2.9%) high-lights on the other ones.

4. Discussion

The ability of obligate photoautrophy microalgae togrow mixotrophycally (or photoheterotrophically) is a phe-nomenon which appears to exist in a number of genera andspecies distributed throughout the major taxonomic divi-sions [16]. From the little literature published about the sub-

ject, it is not possible to extract conclusions on the reasonsfor the lack of ability of microalgae to use reduced carbonsources. These causes probably vary with the species, thestrains and culture conditions. They are associated to perme-ability of the cell, membrane diffusion, active transport andenzymatic processes. Therefore, it implies that any reportedstudy on stimulation of growth by a organic substrate un-der illumination may not be extrapolable even to the samespecies. For instance, Hayward [26] and Ukeles and Rose[16] studied the effect of a wide range of externally suppliedcarbon compounds on the growth of P. tricornutum Bhlinin mixotrophic conditions. In both studies, glycerol, sodiumacetate and sodium lactate, among others, were tested at

same concentration (0.01M). Ukeles and Rose [16] reportedgrowth stimulatory effect for the three substrates, whereasHayward [26] observed this behaviour only for glycerol.Additionally, in accordance with Hayward [26], Cooksey[15] also reported that P. tricornutum Bhlin incorporatedacetate in the light but the microorganism grew poorly.

Growth variability among different species has also beenwidely reported. Two of the substrates most used in the lastdecades, not the only, in the culture of economically inter-esting microalgae have been acetate and glucose. Amongothers, noteworthy studies detailing growth stimulatory ef-fects of acetate uptake are those carried out with Haema-tococcus lacustris (astanxanthin producers) [27], Naviculasaprophila, Rhodomonas salina and Nitzschia sp. (EPA po-tential producers) [28], Haematococcus pluvialis (astanxan-thin) [29], and Brachiomonas submarina [30]. Contrarily,Moya et al. [31] reported growth inhibitory effects by acetatein batch cultures ofHaematcocus lacustris specific; growthrate was affected by a variable inhibition term depending on

irradiance level and acetate concentration.

4.1. Growth kinetics

In order to establish accurate comparisons, primarygrowth data were consulted from literature and treated asdescribed here. Only Ukeles and Rose [16] presented pri-mary growth data graphically for acetate, lactate, and glyc-erol at different concentrations. Tables 1 and 4 display theresults of such calculations. The Ukeles D data tendencyand 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 greaterwas concentration assayed, with the consequent reductionin biomass concentration as well as in biomass productivity(Table 1). These results are coherent with those obtained byUkeles [16], who also found a growth reduction for P. tri-cornutum with the use of acetate, although it is possible toemphasise that these authors registered an increase in the fi-nal biomass concentration when prolonging the culture lifeover 240h. In this sense, the possibility that P. tricornu-tum is able to use acetate for mixotrophic growth cannot be

Table 4Maximum dimensionless specific growth rate and biomass productivity

in relation to phototrophic control for all batch experiments in Ukelesassay [16]

Concentration Nutrient

Acetate Lactate Glycerol

D PD D PD D PD

0.05 0.49 0.660.01 0.64 0.51 0.47 0.64 0.66 0.900.1 0.85 0.99 0.56 1.010.2 0.67 0.771 0.28 0.84

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


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Table 5Best biomass productivity and biomass concentration values for differentnutrients

Nutrient Concentration Maximumbiomassproductivity(mgl1 h1)

Maximumbiomassconcentra-tion (g l1)

Acetate 0.05 M 13.2 1.15Control 10.90 1.10

Starch 1 g l1 11.30 1.79Control 5.44 0.57

Glycerol 0.1 M 17.5 2.99Control 7.49 1.48

Glycine 0.01 M 11 2.46Control 9.89 2.10

Glucose 5 g l1 10.70 2.01Control 7.47 1.35

Lactate 0.005 M 7.73 2.18Control 4.95 1.40

Glycerol (0.1 M)+ urea

0.01 M 16.51 2.87

Control With glycerol(0.1M)

17.50 2.70

Glycerol (0.1 M)+ urea successiveimplementations

0.01 M 63.5 15.4

rejected, but in any case requires a long adaptation time oris able to assimilate acetate only under conditions of stronglight deprivation.

In relation to starch (Table 1), this nutrient stimulated cellsgrowth for all the concentrations tested, although a slight

inhibitory effect was observed above 1gl1

acetate. Themaximum biomass concentration attained was 1.79 g l1 forthe culture with an initial starch concentration of 1 g l1

(Table 5). This value exceeds by 175% the value achieved forthe control culture. Fbregas et al. [18] also demonstrated,how potato starch was uptaken mixotrophically by P. tri-cornutum Bhlin reaching an enhanced productivity around2.4-fold higher than that obtained in photoautotrophic con-trol. In our assays, an analogous improvement (2.1-fold) wasobserved. However, the present result is more valuable ifone keeps in mind that the volumetric scale of our cultureswas bigger than those used by Fbregas et al. [18].

The trialcohol glycerol was used as substrate at con-centrations of 0.005, 0.01, 0.05 and 0.1 M (0.46, 0.92, 4.6and 9.2gl1, respectively). For all concentrations a slightenhanced maximum specific growth rate was observedup to 0.01 M glycerol concentration (Table 1). Maximumbiomass productivity was clearly in all case above the pho-toautotrophic control. It is necessary to emphasise that thehighest glycerol concentration gave rise to a phase of initialadaptation that decreased the growth in the first hours ofculture, giving place to a growth lower than the control. Themaximum biomass concentration 2.59 g l1 was obtained at0.01M glycerol concentration, two- fold the value attainedin the corresponding phototrophic control (Table 5).

Glycine moderately affected theD (Table 1), however, itspresence was unnoticed in the PD which was practically con-stant. The maximum concentration (2.46 g l1) was reachedat 0.01 M (Table 5), exceeding the control value by 18.95%.This improvement is notably inferior to the one reported byHayward (152% on the control culture) with a high concen-

tration of glycine (1 M).Glucose is the final product of the photosynthesis, thus al-lowing the assumption that any photosynthetic microorgan-ism must be able to incorporate it to its metabolism. It is rea-sonable to expect that its incorporation to the metabolism isstraightforward. For that reason, glucose, stimulated growthcompared with the control for all concentrations used al-though not very intense (Table 1). The maximum biomassconcentration corresponded to the culture with the highestglucose concentration, being 2.01 g l1 and exceeding thecontrol in a 148% (Table 5). This result is similar to thatobtained by Hayward [26] with the same microalga but adifferent strain.

In experiments with lactate, the nutrient consistently pro-moted growth in most of experiments (Table 1). The mostfavourable result was obtained for the concentration of0.005M, which was the lowest tried, resulting in a biomassconcentration of 2.18 g l1 which supposed an increase of40% compared to the photoautotrophic control (Table 5).Increasing lactate concentrations, 0.01 and 0.05M, gaverise to improvements over the control but always less in-tense than the 0.005M concentration. The highest substrateconcentration originated a reduction of the final biomassconcentration which caused an inferior result than the con-trol, indicating a possible inhibition by substrate. Lactate

was therefore a nutrient appropriate for mixotrophic growth,obtaining in the best of cases a biomass productivity of7.74mgl1 h1 that means an improvement of 56% on thephotoautotrophic control. These results also mean an im-provement in productivity of 136% compared to the resultspublished by Ukeles and Rose [16] for the same lactateconcentration (0.005 M), and 122% when compared withthe results for the lactate concentration of 0.01 M. Theseimprovements are more significant when the scales arecompared; the work volume in our experiments was 100times greater (1 l as opposed to 10 ml) than the other workscited above.

As shown in Table 1, in the cultures supplemented withadded urea at different concentrations the maximum biomassproductivity was reached for 0.01 M urea concentration butnever exceeding the control. On the contrary, the maxi-mum specific growth rate always exceed the control. Onthe other hand, the maximum biomass concentration wasslightly higher to the control with glycerol 0.1 M (Table 5).Previous studies carried out with P. tricornutum adding ni-trates and urea as nitrogen sources demonstrated that theconsumption rate of nitrates and urea is always lower whentwo forms are added together than when they are addedseparately, although the total nitrogen assimilation rate ishigher [32]. Nevertheless, in our experiments these results


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did not reproduce, instead the two forms added together, giv-ing better productivities than separately (see Table 1, glyc-erol + urea).

Since the highest maximum biomass productivity relativeto control with glycerol was attained in the culture supple-mented with 0.1 M glycerol and urea 0.01 M, these condi-

tions were assayed in fed-batch mode. Glycerol and ureawere added at the beginning; three more additions were madeonce steady state had been reached. This same treatmentwas applied to the control culture, but using only glycerol.A maximum productivity of 63.5mgl1 h1 was reachedafter the third addition and was eight-fold the control cul-ture. This value is close to 81mgl1 h1, obtained withthe same specie but operated in continuous mode in pho-toautotrophically conditions and during the summer, whenirradiance is high [33]. By the contrary, it is greater than44.08mgl1 h1, obtained by Wen and Chen [34] undermixotrophic conditions with a different strain. The maxi-mum biomass concentration, 15.4 g l1, was also higher than

that reported by Zhang et al. [35], 10.2gl1, working withSpirulina at irradiance range similar to fixed in our experi-ments. Ogbonna and Tanaka [5] reported a greater maximumbiomass concentration in fed-batch mode of 60 g l1. How-ever, the volumetric scale was markedly lower (80 ml) anda different light availability (200E m2 s1 at the surfaceof 100 ml flask).

Yongmanitchai and Ward [36] carried out experimentsusing nitrate, ammonium and urea as nitrogen sources,finding that the growth was satisfactory improved with ni-trate as well as with urea, while the culture supplementedwith ammonium did not grow. Similar results were ob-

tained for the combinations nitrate and urea and nitrate andammonium.

4.2. Pigments

In mixotrophic culture, cellular photosynthetic compo-nents concentration depends on the relation of the timethat cells remain in dark and illuminated zones, that is tosay, the relative weight of the heterotrophic/autotrophicmetabolisms. At high cellular concentrations, light be-gins to be limiting and the interval of autotrophic growthis very low compared with the heterotrophic one. Underthese conditions, the cellular chlorophyll content is muchhigher than in the autotrophic cultures [37]. Similar resultswere observed in the results described in this work; themixotrophic cultures experience an increase in pigmentsthat is dependent on the increase in biomass concentrationand that agrees more with an antenna pigment function thanwith a photoprotector function, since they are accumulateddue to the low intensity of available light [38]. This is be-cause light attenuation increases as cells number increases,and therefore, the average irradiance inside the reactor di-minishes. This process implies that cells must synthesisemore radiation receiving pigments, in order to maintaingrowth.

4.3. EPA yield

The highest EPA productivity of 43.13 mg l1 per daywas obtained in the culture carried out with 0.1M glyc-erol and supplemented periodically with urea 0.01 M. Thisyield was 13-fold higher than the maximum EPA produc-

tivity obtained in the photoautotrophically grown controlculture. If we compare results obtained by Wen and Chen[34], 6.37mgl1 per day, the present results exceed theirsin 570.3%. Yongmanitchai and Ward [36] reported an EPAproductivity of 18.9 mg l1 per day for P. tricornutum grownin laboratory batch cultures using nitrates as nitrogen source.In outdoor culture, Molina Grima et al. [33] achieved similaryields (47.8mg l1 per day) in a pilot-scale outdoor tubularreactor, under photoautotrophic conditions.

Clearly, P. tricornutum use all these organic nutrients ef-ficiently in mixotrophic growth. The biomass productivityand EPA yield are notably enhanced in comparison withphotoautotrophic culture (Tables 3 and 5).

5. Conclusions

In conclusion, P. tricornutum UTEX-640 is able to utilisea variety of organic nutrients assayed in this experiment ef-ficiently under mixotrophic growth, enhancing notably thebiomass and EPA production, in comparison with photoau-totrophic culture. Mixotrophic growth requires relatively lowlight intensities and consequently lower energy costs.

At high cellular concentration, light becomes to be lim-iting and the grade of autotrophic growth is very low com-pared with the heterotrophic one. Under these conditions,

the pigments and fatty acids content of cells are significantlyhigher than in autotrophic cultures. The high irradiance re-quirements to support growth and enhanced productivity athigh cell density can be partially covered by the addition ofa suitable organic nutrient in adequate concentration.

In consequence these results show the possibility to substi-tute the photoautotrophic growth by the mixotrophic growth.The repercussions in the photobioreactors design is alsoclear: lower height/diameter aspect ratio. Therefore, irradi-ance, land surface occupied and refrigeration may be re-duced significantly.

Acknowledgements

This work was supported by the CICYT (BIO-98-0522),Spain. We wish to express our gratitude to our late co-workerand friend Cristobal Snchez Martn for all the help duringthese years.

References

[1] Apt KE, Behrens PW. Commercial developments in microalgalbiotechnology. J Phycol 1999;35:21526.

[2] Borowitzka MA. Commercial production of microalgae: ponds, tanks,tubes, and fermentors. J Biotech 1999;70:31321.


9/9


[3] Harrington GW, Beach DH, Dunham JE, Holz Jr GG. Thepolyunsaturated fatty acids of marine dinoflagellates. J Proto-zool1970;17:2139.

[4] Henderson RJ, Leftley JW, Sargent JR. Lipid composition and biosyn-thesis in the marine dinoflagellate Crypthecodinium cohnii. Phyto-chemistry 1988;27:167983.

[5] Ogbonna JC, Tanaka H. Production of pure photosynthetic cellbiomass for environmental biosensors. Mater Sci Eng C 2000;12:915.

[6] Chen F. High cell density culture of microalgae in heterotrophicgrowth. Technol Biotechnol 1996;14:4216.

[7] Chen F, Zhang Y. High cell density mixotrophic culture of Spirulinaplatensis on glucose for phycocyanin production using a fed-batchsystem. Enzyme Microb Technol 1997;20:2214.

[8] Ogbonna JC, Toshihiko S, Hideo T. An integrated solar and arti-ficial light system for internal illumination of photobioreactors. JBiotechnol 1999;70:28997.

[9] Droop MR. Heterotrophy of carbon. In: Steward WDP, editor. Al-gal physiology and biochemistry. Berkeley: University of CaliforniaPress; 1974. p. 53059.

[10] Cern Garca MC, Fernndez Sevilla JM, Acin Fernndez FG,Molina Grima E, Garca Camacho F. Mixotrophic growth of Phaeo-dactylum tricornutum on glycerol: growth rate and fatty acid profile.J Appl Phycol 2000;12:23948.

[11] Borowitzka MA. Vitamins and fine chemicals from microalgae. In:Borowitzka, Borowitzka, editors. Microalgal biotechnology. Cam-bridge: Cambridge University Press; 1988. p. 15396.

[12] Bermejo R, Talavera M, Eva del Valle C, lvarez-Pez Jos M.C-phycocyanin incorporated into reverse micelles: a fluorescencestudy. Colloids Surf B: Biointerfaces 2000;18:519.

[13] Cheng J, Antia N. Enhancement by glycerol of photoheterotrophicgrowth of marine planktonic algae and its significance to the ecol-ogy of glycerol pollution. J Fisheries Res Board Can 1970;27:33546.

[14] Lewin J, Lewin R. Auxotrophy and heterotrophy in marine littoraldiatoms. Can J Microbiol 1960;6:12731.

[15] Cooksey K. Acetate metabolism by whole cells of Phaeodactylum

tricornutum Bhlin. J Phycol 1974;10:2537.[16] Ukeles R, Rose WE. Observations on organic carbon utilisationby photosynthetic marine microalgae. Marine Biol 1976;37:1128.

[17] Komoda Y, Isogai Y, Satoh K. Isolation from humus and identifica-tion of two growth promoters: adenosine and 2 -deoxyadenosine inculturing the diatom Phaeodactylum tricornutum. Chem Pharm Bull1983;31:37714.

[18] Fbregas J, Morales ED, Polanco N, Patio M, Otero Tobar JF.Use of agricultural surpluses for production of biomass by marinemicroalgae. World J Microbiol Biotechnol 1996;12.

[19] Fabregas J, Morales ED, Lamela T, Cabezas Otero. Short communi-cation: Mixotrophic productivity of the marine diatom Phaeodacty-lum tricornutum cultured with soluble fractions of rye, wheat andpotato. World J Microbiol Biotechnol 1997;13:34951.

[20] Garca Snchez JL, Molina Grima E, Garca Camacho F, SnchezPrez JA, Lpez Alonso D. Estudio de macronutrientes para laproduccin de PUFAs a partir de la microalga marina Isochrysisgalbana. Grasas y aceites 1994;45(5):32332.

[21] Molina Grima E, Snchez Prez JA, Garca Camacho F, GarcaSnchez JL, Lpez Alonso D. n-3 PUFA productivity in chemostatcultures of microalgae. Appl Microbiol Biotechnol 1993;38:599605.

[22] Rodrguez Ruiz J, Belarbi E-H, Garca Snchez JL, Lpez Alonso D.Rapid simultaneous lipid extraction and transesterification for fattyacid analyses. Biotechnol Tech 1998;12(9):68991.

[23] Whyte JN. Biochemical composition and energy content of sixspecies of phytoplankton used in mariculture of bivalves. Aquacul-ture 1987;60:23141.

[24] Hansmann E. Pigment analysis. In: Stein JR, editor. Handbook ofphycological methods, culture methods and growth measurements.London: Cambridge University Press; 1973. p. 35968.

[25] Strickland JGH, Parsons TR. A practical handbook of seawater anal-ysis. Bull Fish Res Bd Can 1972;167:15363.

[26] Hayward J. Studies on the growth ofPhaeodactylum tricornutum. II.The effect of organic substances on growth. Physiologia Plantarum1968;21:1008.

[27] Chen F, Chen H, Gong XD. Mixotrophic and heterotrophicgrowth of Haematococcus-lacustris and rheological behavior of thecell-suspensions. Bioresour Technol 1997;62(12):1924.

[28] Kitano M, Matsukawa R, Karube I. Changes in eicosapentaenoic acidcontent of Navicula saprophila, Rhodomonas salina and Nitzschiasp under mixotrophic conditions. J Appl Phycol 1997;9(6):55963.

[29] Tripathi U, Sarada R, Rao SR, Ravishankar GA. Production ofAstaxanthin in Haematococcus-Pluvialis cultured in various media.Bioresour Technol 1999;68(2):1979.

[30] Tsavalos Alexander J, Day John G. Development of media forthe mixotrophic/heterotrophic culture of Brachiomonas submarina. JAppl Phycol 1994;6:4313.

[31] Moya MJ, Snchez Guardamino ML, Vilavella A, Barbera E. Growthof Haematococcus-Lacustrisa contribution to kinetic modeling. JChem Technol Biotechnol 1997;68(3):3039.

[32] Molloy CJ, Syrett PJ. Effect of light and N deprivation on inhibi-tion of nitrate uptake by urea in microalgae. J Exp Mar Biol Ecol1988;118:97101.

[33] Molina Grima E, Garca Camacho F, Snchez Prez JA, Urda Car-dona J, Acin Fernndez FG, Fernndez Sevilla JM. Outdoor chemo-

stat culture of Phaeodactylum tricornutum UTEX 640 in a tubularphotobioreactor for the production of eicosapentaenoic acid. Biotech-nol Appl Biochem 1994;20:27990.

[34] Wen Z, Chen F. Production potential of eicosapentaenoic acid bythe diatom Nitzschia laevi. Biotechnol Lett 2000;22:72733.

[35] Zhang KW, Gong XD, Chen F. Kinetic models for astaxanthinproduction by high cell density mixotrophic culture of the microalgaSpirulina platensis. J Ind Microbiol Biotechnol 1998;21:2838.

[36] Yongmanitchai W, Ward OP. Growth of and Omega-3 fatty acidproduction by Phaeodactylum tricornutum under different cultureconditions. Appl Environ Microbiol 1991;57:41925.

[37] Ogbonna JC, Masui H, Tanaka H. Sequential heterotrophic/autotrophic cultivationan efficient method of producing Chlorellabiomass for health food and animal feed. J Appl Phycol 1997;9:35966.

[38] Garca Camacho F, Contreras Gmez A, Acin Fernndez FG, Fer-nndez Sevilla J, Molina Grima E. Use of concentric-tube airliftphotobioreactors for microalgal outdoor mass cultures. Enzyme Mi-crob Technol 1999;24:16472.

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2 Mixotrophic Growth of the Microalga Phaeodactylum Tricornutum