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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 Garcı́a, A. Sánchez Mirón, J.M. Fernández Sevilla,
E. Molina Grima, F. Garcı́a Camacho∗
Departamento de Ingenierı́a Quı́mica, Universidad de Almerı́a, E-04071 Almerı́a, 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 (0.005–0.1 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 light availability, severely limiting biomass production and


the low biomass concentrations reached decrease efficient
Photosynthetic algae are a feed component for aquacul- harvesting of the cells. Strategies to improve the efficient
ture and produce numerous valuable compounds includ- use of light or eliminate its requirement by cells and so
ing pigments (e.g., ␤-carotene, phycobiliproteins), oils (e.g., reduce the cost of microalgal biomass production, involve
eicosapentaenoic acid, docosahexaenoic acid), and stable mixotrophic, photoheterotrophic or heterotrophic growth of
isotope-labelled biochemicals (e.g.,[13 C] glucose); they also algae. The election of one or another depends on the bioprod-
have potential in the discovery of new pharmaceuticals [1]. ucts of interest and the strains. For example, heterotrophic
The commercial exploitation of photosynthetic microal- production of PUFAs from microalgae is possible [3–5],
gae is based on typical large, outdoor open ponds and closed and has recently been achieved by Martek Biosciences [6].
tubular photobioreactors occupying vast land extensions. However, heterotrophic growth is not suitable for pigment
These systems although make use of natural sunlight for the production (e.g., phycocyanin, astaxanthin, etc.) since many
production of energy fixed chemically [1,2], they present of these are photosynthetic accessory pigments [7]. Other-
a great disadvantage, however, cellular self-shading hinders wise, mixotrophy and photoheterotrophy allow microalgal
cells to synthesise simultaneously compounds characteris-
∗ Corresponding author. Fax: +349-5001-5484. tic of both heterotrophic and photosynthetic metabolisms at
E-mail address: fgarcia@ual.es (F. Garcı́a Camacho). high production rates [5,8].

0032-9592/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2004.01.016
298 M.C. Cerón Garcı́a et al. / Process Biochemistry 40 (2005) 297–305

On the other hand, most microalgae are obligate pho- ditions of glycerol 0.1 M and urea (at optimal concentration
toautrophs and are unable to use organic carbon sources [9]. previously determined). Additions were done when a steady
This imposes the necessity of screening for species endowed state biomass concentration had been reached. The initial
with this capacity. One microalga with this possibility is pH was fixed at 8.0. The temperature was 20 ± 0.5 ◦ C. All
Phaeodactylum tricornutum UTEX 640 [10]. This species experiments were performed in duplicate (the mean values
has been broadly studied as a potential source of PUFAs were presented) and they included a control culture (without
(mainly EPA) and of a great diversity of pigments [1,11,12]. organic nutrient).
So far, a limited number of preliminary studies have re- Biomass concentration was estimated by absorbance at
ported that P. tricornutum cells can develop mixotrophic 625 nm [21] and periodically checked by dry-weight de-
activity in the presence of some organic carbon sources such terminations. Freeze-dried biomass was used for fatty acid
as alcohols, sugars, diverse organic compounds, growth analysis by gas chromatography, according to the method
promoters, soluble fractions of cereals and potato fermented described by Rodriguez et al. [22]. Carotenoids were quan-
flours, rye and wheat flours [10,13,14,15,16,17,18,19]. tified according to the method described by Whyte [23] and
Nonetheless, most of them were carried out with the only chlorophylls content were determinated by Hansman’s spec-
intention of demonstrating the use of these nutrients by trometric method [24] and using Parsons and Strickland’s
the cells, without considering practical aspects derived of equations [25] for the quantification.
growth kinetics and biomass/bioproduct yield data. In view The following sigmoidal equation was used as suggested
of the limited amount of information on the potential of by Cerón Garcı́a et al. [10] to smooth the growth data by
mixo- versus photoautotrophic growth, this research was nonlinear regression.
aimed to test different organic nutrients and to analyse the  
Cb a
effect of concentration on growth and biochemical com- ln = Yo + (1)
Cbo (1 + exp(−t − to /b))c
position (focused on fatty acid profile and pigments) of
the marine microalga P. tricornutum UTEX 640 grown in This procedure allows the elimination of the influence of
laboratory-scale batch cultures. 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
2. Materials and methods theoretical biomass productivity, Pb, at any time during the
experiment and from there the maximum values of those
The alga P. tricornutum UTEX-640 was obtained from parameters.
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 3. Results
aeration. The culture medium of Garcı́a Sánchez et al. [20]
was used for maintenance. The experimental results of concentration (C) versus time
One-litre flat-bottom spherical flasks were used as growth (t) obtained for the discontinuous experiments are repre-
vessels. The culture medium was enriched seawater prepared sented in Fig. 1 as points of ln(C/C0 ) versus t accompanied
as indicated elsewhere [20]. Inoculation was done using the by lines that represent the corresponding regressions of the
above described inoculum in linear growth phase and adding experimental data to Eq. (1).
sufficient quantity to start with 80 mg l−1 . Growth vessels More than 90% of the experimental data were fitted within
were sparged with filter-sterilised air at 0.1 min (v/v) for a ±15% margin using Eq. (1).
mixing and aeration. The cultures were continuously illu- The results presented in Fig. 1 show that the capacity
minated by one Philips TLD 36W/54 fluorescent lamp at a to promote and sustain mixotrophic growth varies widely
light intensity 165 ␮E m−2 s−1 measured at the surface of among the different substrates depending on the nature and
the culture. initial concentration utilised. Several circumstances can
The culture medium, except the organic substrates, was hinder the appreciation of differences among experiments.
sterilised in an autoclave at 126 ◦ C for 20 min. The organic Among these are the occurrence of lag phases at the be-
nutrients (lactate, acetate, starch, glycine, glycerol and glu- ginning of the experiments and the fact that the organic
cose) were separately sterilised by filtration through 0.2 ␮m nutrient can be consumed during the experiment causing a
pore membranes. Several experimental runs, one for each or- decrease in the initial concentration of substrate. Bearing
ganic nutrient, were initiated and conducted in discontinuous this in mind it is easy to see how important it is to allow
mode ranged between 0.005 and 0.1 M (0.5, 1, 2 and 5 g l−1 sufficient time to ensure biomass development so that nutri-
for starch), including a photoautotrophic control. Urea was ent that initially causes lag phase but has a great capability
used as supplementary nitrogen source, and optimised for to promote mixotrophic growth is not deemed as bad. It
concentration in the experiments with glycerol (glycerol at is desirable to use for comparison a procedure that allows
0.1 M was found to be optimal in a previous study [10]). Fi- the separation of the effect of the different factors, nutrient
nally, a fed-batch culture was carried out with successive ad- type, initial concentration and time. These differences are
M.C. Cerón Garcı́a et al. / Process Biochemistry 40 (2005) 297–305 299

Fig. 1. Variation of ln(C/C0 ) vs. culture time. Regression (lines) were obtained from Eq. (1) [10] for the different initial concentrations tested.
300 M.C. Cerón Garcı́a et al. / Process Biochemistry 40 (2005) 297–305

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 In experiments with acetate (Fig. 2a), six pairs of means
variance (multifactor ANOVA). As expected in batch cul- showed statistically significant differences and growth inhi-
tures, time has always had a statistically significant effect bition was established above 0.005 M. When starch was used
on biomass concentration and its contribution to variance as organic nutrient (Fig. 2b), five pairs of means showed sta-
was also always high. However, interesting differences were tistically significant differences. A stimulated growth com-
observed for the different organic nutrient concentrations. pared to control was clearly identified for all starch concen-
Fig. 2 shows the mean values of biomass concentration trations assayed, even though a slight inhibitory effect was
reached for each organic nutrient concentration and the observed above 2 g l−1 of starch.
intervals around each mean value. The method used to dis- Glycerol also influenced growth favourably, as can be
criminate among the means values was the Fisher’s least seen in Fig. 2c, where seven pairs of means showed statis-
significant difference (LSD) procedure. tically significant differences. For the concentration 0.05 M,
M.C. Cerón Garcı́a et al. / Process Biochemistry 40 (2005) 297–305 301

Table 1
Dimensionless 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

µD PD µD PD µD PD µD PD µD PD µD PD µD 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 correspond
with the upward order of the concentrations assayed for each nutrient.

a decrease of the maximum biomass concentration was ob- with biomass concentrations more high (i.e., maximum
served due, possibly, to a marked lag phase (see Fig. 1b). biomass productivity more elevated). In general, the µD
For glycine (Fig. 2d), no statistically significant differences values reported in Table 1 did not correspond to the same
between control mean and others were observed. growth phase, although PD did in linear growth phase.
Glucose also enhanced growth and five pairs of means
showed statistically significant differences (Fig. 2e). 3.1. Pigments
For sodium lactate (Fig. 2f), eight pairs of means showed
statistically significant differences at the 95.0% confidence Pigments were quantified from harvested biomass once
level. This graph suggests that lactate, in relation to the con- the culture finished. As example, in Table 2 maximum val-
trol, stimulated the growth for concentrations below 0.05 M; ues of pigments content at best nutrient concentrations are
although, excluding the control, its effect was inhibitory shown. The effect of organic substrate on total pigments con-
above the minimum concentration of lactate (0.005 M). tent and its distribution in carotenoids and chlorophylls was
Experiments with glycerol + urea (Fig. 2g) suggest that varied. Whereas starch, glycerol, glycine and lactate stimu-
urea stimulates slightly the growth at concentration of lated the biosynthesis of total pigments (19.2, 24.7, 8.8 and
0.01 M, but a contrary effect is exhibited above 0.02 M. 32.8%, respectively, in relation to control culture), glucose
Table 1 summarises the most relevant dimensionless and urea had a marked opposite effect (−47.6 and −40%
kinetic parameters for comparison. Basically, it can be on control, respectively).
observed the same above described pattern, matched with The chorophyll contents followed the same tendency than
that shown by dimensionless specific growth rate (µD ) and total pigment content. However, the fraction of carotenoids
biomass productivity (PD ) data estimated from the five pa- had three exceptions, originated in cultures with glycine,
rameters sigmoidal equation (Eq. (1)) (see Table 1). Only glucose and glycerol. The most significative one was the
the PD from experiment with acetate and µD from the ex- extraordinary low carotenoids content in culture conducted
periment with glycerol + urea did not follow the expected with glycine.
tendency. This can be justified, partly, by the fact com- The influence of organic nutrient concentration was not
paring with a photoautotrophic control culture, a nutrient considered in Table 2 because of the best results showed in
growth-inhibited culture may show a low exponential spe- Table 2 matched with those obtained of statistical analysis
cific growth rate, but a large linear growth phase associated 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 Concentration Carotenoid (% Chlorophyll (% Total pigment
dry weight) dry weight) (% dry weight)
Starch 2 g l−1 1.04 3.32 4.36
Control 1.14 2.29 3.43
Glycerol 0.1 M 0.59 2.83 3.43
Control 0.34 2.31 2.75
Glycine 0.01 M 0.02 2.97 2.99
Control 0.38 2.37 2.75
Glucose 5 g l−1 0.49 0.71 1.20
Control 0.35 1.95 2.29
Lactate 0.005 M 0.64 3.79 4.45
Control 0.21 3.13 3.35
Glycerol (0.1 M) + urea 0.01 M 0.49 1.09 1.63
Control With glycerol (0.1 M) 0.33 2.40 2.72
302 M.C. Cerón Garcı́a et al. / Process Biochemistry 40 (2005) 297–305

Table 3 same concentration (0.01 M). Ukeles and Rose [16] reported
Final EPA content on biomass dry weight and maximum EPA yield growth stimulatory effect for the three substrates, whereas
productivity for the best concentration of each organic nutrient
Hayward [26] observed this behaviour only for glycerol.
Nutrient Concentration Dry EPA yield Additionally, in accordance with Hayward [26], Cooksey
weight (%) (mg l−1 per day) [15] also reported that P. tricornutum Böhlin incorporated
Starch 1 g l−1 1.84 4.98 acetate in the light but the microorganism grew poorly.
Control 2.24 2.92
Growth variability among different species has also been
Glycerol 0.1 M 2.04 8.56 widely reported. Two of the substrates most used in the last
Control 1.90 3.35 decades, not the only, in the culture of economically inter-
Glycine 0.01 M 1.76 4.99 esting microalgae have been acetate and glucose. Among
Control 1.78 2.52 others, noteworthy studies detailing growth stimulatory ef-
Glucose 5 g l−1 2.62 6.55 fects of acetate uptake are those carried out with Haema-
Control 2.24 4.07 tococcus lacustris (astanxanthin producers) [27], Navicula
Lactate 0.005 M 2.20 4.09 saprophila, Rhodomonas salina and Nitzschia sp. (EPA po-
Control 1.73 2.05 tential producers) [28], Haematococcus pluvialis (astanxan-
Glycerol (0.1 M) 0.01 M 2.91 11.53 thin) [29], and Brachiomonas submarina [30]. Contrarily,
+ urea Moya et al. [31] reported growth inhibitory effects by acetate
Control With glycerol 2.10 6.47 in batch cultures of Haematcocus lacustris specific; growth
(0.1 M) rate was affected by a variable inhibition term depending on
Glycerol (0.1 M) 0.01 M 2.83 43.13 irradiance level and acetate concentration.
+ urea imple-
mentations
4.1. Growth kinetics

3.2. EPA content In order to establish accurate comparisons, primary


growth data were consulted from literature and treated as
The type of nutrient influenced markedly the EPA syn- described here. Only Ukeles and Rose [16] presented pri-
thesis. Urea and lactate notably increased the EPA content, mary growth data graphically for acetate, lactate, and glyc-
38.6 and 27.2%, respectively, on that reached with control erol at different concentrations. Tables 1 and 4 display the
culture. On the contrary, cellular EPA content decreased in results of such calculations. The Ukeles’ µD data tendency
relation control with the use of starch. No significant effect and scale matched with our results.
was observed with glycerol and glycine. Among all the EPA The sodium acetate had a negative effect in P. tricornutum
percentages displayed in Table 3, the value corresponding to in all cases, slowing down the growth as much as greater
the culture conducted with glycerol and urea (2.9%) high- was concentration assayed, with the consequent reduction
lights on the other ones. 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. tri-
4. Discussion cornutum with the use of acetate, although it is possible to
emphasise that these authors registered an increase in the fi-
The ability of obligate photoautrophy microalgae to nal biomass concentration when prolonging the culture life
grow mixotrophycally (or photoheterotrophically) is a phe- over 240 h. In this sense, the possibility that P. tricornu-
nomenon which appears to exist in a number of genera and tum is able to use acetate for mixotrophic growth cannot be
species distributed throughout the major taxonomic divi-
sions [16]. From the little literature published about the sub- Table 4
Maximum dimensionless specific growth rate and biomass productivity
ject, it is not possible to extract conclusions on the reasons in relation to phototrophic control for all batch experiments in Ukeles’
for the lack of ability of microalgae to use reduced carbon assay [16]
sources. These causes probably vary with the species, the
Concentration Nutrient
strains and culture conditions. They are associated to perme-
ability of the cell, membrane diffusion, active transport and Acetate Lactate Glycerol
enzymatic processes. Therefore, it implies that any reported µD PD µD PD µD PD
study on stimulation of growth by a organic substrate un-
0.05 0.49 0.66
der illumination may not be extrapolable even to the same 0.01 0.64 0.51 0.47 0.64 0.66 0.90
species. For instance, Hayward [26] and Ukeles and Rose 0.1 0.85 0.99 0.56 1.01
[16] studied the effect of a wide range of externally supplied 0.2 0.67 0.77
carbon compounds on the growth of P. tricornutum Böhlin 1 0.28 0.84
in mixotrophic conditions. In both studies, glycerol, sodium Data are theoretical values estimated from sigmoidal equation fitted to
acetate and sodium lactate, among others, were tested at experimental primary data.
M.C. Cerón Garcı́a et al. / Process Biochemistry 40 (2005) 297–305 303

Table 5 Glycine moderately affected the µD (Table 1), however, its


Best biomass productivity and biomass concentration values for different presence was unnoticed in the PD which was practically con-
stant. The maximum concentration (2.46 g l−1 ) was reached
nutrients
Nutrient Concentration Maximum Maximum
at 0.01 M (Table 5), exceeding the control value by 18.95%.
biomass biomass
productivity concentra- This improvement is notably inferior to the one reported by
(mg l−1 h−1 ) tion (g l−1 ) Hayward (152% on the control culture) with a high concen-
Acetate 0.05 M 13.2 1.15 tration of glycine (1 M).
Control 10.90 1.10 Glucose is the final product of the photosynthesis, thus al-
Starch 1 g l−1 11.30 1.79 lowing the assumption that any photosynthetic microorgan-
Control 5.44 0.57 ism must be able to incorporate it to its metabolism. It is rea-
Glycerol 0.1 M 17.5 2.99 sonable to expect that its incorporation to the metabolism is
Control 7.49 1.48 straightforward. For that reason, glucose, stimulated growth
Glycine 0.01 M 11 2.46 compared with the control for all concentrations used al-
Control 9.89 2.10 though not very intense (Table 1). The maximum biomass
Glucose 5 g l−1 10.70 2.01 concentration corresponded to the culture with the highest
Control 7.47 1.35 glucose concentration, being 2.01 g l−1 and exceeding the
Lactate 0.005 M 7.73 2.18 control in a 148% (Table 5). This result is similar to that
Control 4.95 1.40 obtained by Hayward [26] with the same microalga but a
Glycerol (0.1 M) 0.01 M 16.51 2.87 different strain.
+ urea In experiments with lactate, the nutrient consistently pro-
Control With glycerol 17.50 2.70 moted growth in most of experiments (Table 1). The most
(0.1 M)
favourable result was obtained for the concentration of
Glycerol (0.1 M) 0.01 M 63.5 15.4 0.005 M, which was the lowest tried, resulting in a biomass
+ urea successive
concentration of 2.18 g l−1 which supposed an increase of
implementations
40% compared to the photoautotrophic control (Table 5).
Increasing lactate concentrations, 0.01 and 0.05 M, gave
rejected, but in any case requires a long adaptation time or rise to improvements over the control but always less in-
is able to assimilate acetate only under conditions of strong tense than the 0.005 M concentration. The highest substrate
light deprivation. concentration originated a reduction of the final biomass
In relation to starch (Table 1), this nutrient stimulated cells concentration which caused an inferior result than the con-
growth for all the concentrations tested, although a slight trol, indicating a possible inhibition by substrate. Lactate
inhibitory effect was observed above 1 g l−1 acetate. The was therefore a nutrient appropriate for mixotrophic growth,
maximum biomass concentration attained was 1.79 g l−1 for obtaining in the best of cases a biomass productivity of
the culture with an initial starch concentration of 1 g l−1 7.74 mg l−1 h−1 that means an improvement of 56% on the
(Table 5). This value exceeds by 175% the value achieved for photoautotrophic control. These results also mean an im-
the control culture. Fábregas et al. [18] also demonstrated, provement in productivity of 136% compared to the results
how potato starch was uptaken mixotrophically by P. tri- published by Ukeles and Rose [16] for the same lactate
cornutum Böhlin reaching an enhanced productivity around concentration (0.005 M), and 122% when compared with
2.4-fold higher than that obtained in photoautotrophic con- the results for the lactate concentration of 0.01 M. These
trol. In our assays, an analogous improvement (2.1-fold) was improvements are more significant when the scales are
observed. However, the present result is more valuable if compared; the work volume in our experiments was 100
one keeps in mind that the volumetric scale of our cultures times greater (1 l as opposed to 10 ml) than the other works
was bigger than those used by Fábregas et al. [18]. cited above.
The trialcohol glycerol was used as substrate at con- As shown in Table 1, in the cultures supplemented with
centrations of 0.005, 0.01, 0.05 and 0.1 M (0.46, 0.92, 4.6 added urea at different concentrations the maximum biomass
and 9.2 g l−1 , respectively). For all concentrations a slight productivity was reached for 0.01 M urea concentration but
enhanced maximum specific growth rate was observed never exceeding the control. On the contrary, the maxi-
up to 0.01 M glycerol concentration (Table 1). Maximum mum specific growth rate always exceed the control. On
biomass productivity was clearly in all case above the pho- the other hand, the maximum biomass concentration was
toautotrophic control. It is necessary to emphasise that the slightly higher to the control with glycerol 0.1 M (Table 5).
highest glycerol concentration gave rise to a phase of initial Previous studies carried out with P. tricornutum adding ni-
adaptation that decreased the growth in the first hours of trates and urea as nitrogen sources demonstrated that the
culture, giving place to a growth lower than the control. The consumption rate of nitrates and urea is always lower when
maximum biomass concentration 2.59 g l−1 was obtained at two forms are added together than when they are added
0.01 M glycerol concentration, two- fold the value attained separately, although the total nitrogen assimilation rate is
in the corresponding phototrophic control (Table 5). higher [32]. Nevertheless, in our experiments these results
304 M.C. Cerón Garcı́a et al. / Process Biochemistry 40 (2005) 297–305

did not reproduce, instead the two forms added together, giv- 4.3. EPA yield
ing better productivities than separately (see Table 1, glyc-
erol + urea). The highest EPA productivity of 43.13 mg l−1 per day
Since the highest maximum biomass productivity relative was obtained in the culture carried out with 0.1 M glyc-
to control with glycerol was attained in the culture supple- erol and supplemented periodically with urea 0.01 M. This
mented with 0.1 M glycerol and urea 0.01 M, these condi- yield was 13-fold higher than the maximum EPA produc-
tions were assayed in fed-batch mode. Glycerol and urea tivity obtained in the photoautotrophically grown control
were added at the beginning; three more additions were made culture. If we compare results obtained by Wen and Chen
once steady state had been reached. This same treatment [34], 6.37 mg l−1 per day, the present results exceed theirs
was applied to the control culture, but using only glycerol. in 570.3%. Yongmanitchai and Ward [36] reported an EPA
A maximum productivity of 63.5 mg l−1 h−1 was reached productivity of 18.9 mg l−1 per day for P. tricornutum grown
after the third addition and was eight-fold the control cul- in laboratory batch cultures using nitrates as nitrogen source.
ture. This value is close to 81 mg l−1 h−1 , obtained with In outdoor culture, Molina Grima et al. [33] achieved similar
the same specie but operated in continuous mode in pho- yields (47.8 mg l−1 per day) in a pilot-scale outdoor tubular
toautotrophically conditions and during the summer, when reactor, under photoautotrophic conditions.
irradiance is high [33]. By the contrary, it is greater than Clearly, P. tricornutum use all these organic nutrients ef-
44.08 mg l−1 h−1 , obtained by Wen and Chen [34] under ficiently in mixotrophic growth. The biomass productivity
mixotrophic conditions with a different strain. The maxi- and EPA yield are notably enhanced in comparison with
mum biomass concentration, 15.4 g l−1 , was also higher than photoautotrophic culture (Tables 3 and 5).
that reported by Zhang et al. [35], 10.2 g l−1 , working with
Spirulina at irradiance range similar to fixed in our experi- 5. Conclusions
ments. Ogbonna and Tanaka [5] reported a greater maximum
biomass concentration in fed-batch mode of 60 g l−1 . How- In conclusion, P. tricornutum UTEX-640 is able to utilise
ever, the volumetric scale was markedly lower (80 ml) and a variety of organic nutrients assayed in this experiment ef-
a different light availability (200 ␮E m−2 s−1 at the surface ficiently under mixotrophic growth, enhancing notably the
of 100 ml flask). biomass and EPA production, in comparison with photoau-
Yongmanitchai and Ward [36] carried out experiments totrophic culture. Mixotrophic growth requires relatively low
using nitrate, ammonium and urea as nitrogen sources, light intensities and consequently lower energy costs.
finding that the growth was satisfactory improved with ni- At high cellular concentration, light becomes to be lim-
trate as well as with urea, while the culture supplemented iting and the grade of autotrophic growth is very low com-
with ammonium did not grow. Similar results were ob- pared with the heterotrophic one. Under these conditions,
tained for the combinations nitrate and urea and nitrate and the pigments and fatty acids content of cells are significantly
ammonium. higher than in autotrophic cultures. The high irradiance re-
quirements to support growth and enhanced productivity at
4.2. Pigments high cell density can be partially covered by the addition of
a suitable organic nutrient in adequate concentration.
In mixotrophic culture, cellular photosynthetic compo- In consequence these results show the possibility to substi-
nents concentration depends on the relation of the time tute the photoautotrophic growth by the mixotrophic growth.
that cells remain in dark and illuminated zones, that is to The repercussions in the photobioreactors design is also
say, the relative weight of the heterotrophic/autotrophic clear: lower height/diameter aspect ratio. Therefore, irradi-
metabolisms. At high cellular concentrations, light be- ance, land surface occupied and refrigeration may be re-
gins to be limiting and the interval of autotrophic growth duced significantly.
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 Acknowledgements
were observed in the results described in this work; the
This work was supported by the CICYT (BIO-98-0522),
mixotrophic cultures experience an increase in pigments
Spain. We wish to express our gratitude to our late co-worker
that is dependent on the increase in biomass concentration
and friend Cristobal Sánchez Martı́n for all the help during
and that agrees more with an antenna pigment function than
these years.
with a photoprotector function, since they are accumulated
due to the low intensity of available light [38]. This is be-
cause light attenuation increases as cells number increases, References
and therefore, the average irradiance inside the reactor di-
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