Bioresource Technology 55 (1996) 83-88

0 1996 Elsevier Science Limited

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ELSEVIER

EICOSAPENTAENOIC ACID-RICH BIOMASS PRODUCTION

BY THE MICROALGA PHAEODACTYLUM TRI%ORNUTUM IN
A CONTINUOUS-FLOW REACTOR

Albert0 Reis, * Luisa Gouveia,a Vera Veloso,b Helena L. Fernandes,b Josh A. Empis
& Julio M. NovaiC

Institute National de Engenharia e Tecnologia Industrial, DER Estrada do PaGodo Lumiar; 1699 Lisboa Codex, Portugal
bLaborat6rio de Engenharia Bioquimica, Institute Superior Tkcnico, Avenida Rovisco Pais, 1099 Lisboa Codex, Portugal

(Received 15 December 1994; revised version received 4 October 1995; accepted 10 October 1995)

Abstract PC phosphatidylcholine
The marine diatom Phaeodactylum tricornutum Boh- PE phosphatidylethanolamine
lin is a potential source of the pharmaceutically PG phosphatidylglyceride
valuable w3 polyunsaturated fatty acid eicosapentae- PI phosphatidylinositol
noic acid (EPA). The results of indoor continuous PS phosphatidylserine
growth of Phaeodactylum tricornutum Bohlin are PUFA polyunsaturated fatty acid
reported. r volumetric output rate (productivity/reac-
The relationships between dilution rate (D), nitrate tor volume)
concentration and chemical composition were studied. SFA saturated fatty acid
Higher biomass and lipid productivities were obtained SQ sulfoquinovosyldiglyceride
at low D values. EPA was found to be an intermediate TG triglyceride
metabolite and the best productivity (6 mg 1-r day-r) X biomass on ash-free dry-weight basis
was achieved for D values ranging from 0.32 to 0.50 WDW
day-. Under optimum conditions, 84 and 1170, x:ywz fatty acid with x carbon atoms, y double
respectively, of total recovered EPA were present in bonds, where z is the distance between
monogalactosyldia~lglycerol (MGDG) and in triacyl- the last double bond and the methyl end
glycerol (TG) moieties, respectively.Recorded EPAI! group
and EPA/20,4 03 ratios for all tested dilution rates IJ specific growth rate (dxixdt)
were among the highest values ever reported, showing
EPA purification to be easier to perform from this
INTRODUCTION
starting material than from many others commonly in
use. Copyright 0 1996 Elsevier Science Ltd.
The Omega-3 (~3) polyunsaturated fatty acids mar-
ket (EPA and DHA) dates from 1982 and has an
Key words: Microalga, diatom, Phaeodactylum tricor-
annual estimated value in excess of 25 million US
nutum, fatty acids, continuous reactor, lipids, EPA.
dollars (CQVB, 1988). The therapeutic value of
these compounds has been shown in the reduction
NOMENCLATURE of blood cholesterol (Bonaa et al., 1990), in the pre-
vention of blood-platelet aggregation, and as a
arachidonic acid (20 : 4 w6) protection against cardiovascular and coronary heart
chain with n carbon atoms diseases, atherosclerosis, hyperlipidemy, hypercho-
dilution rate (volumetric flow/reactor lesterolemy and hypertriglyceridemy (Simopoulos,
volume) 1986). Other known applications include the therapy
DGDG digalactosyldiglyceride of chronic inflammation processes (Vitale, 1988;
DHA docosahexaenoic acid (22 : 6 w3) Goetzl et al., 1986) and improvement of vision
EPA eicosapentaenoic acid (20 : 5 03) (Dratz & Deese, 1986). Encouraged by recent multi-
MGDG monogalactosyldiglyceride disciplinary studies about beneficial effects upon
MUFA monounsaturated fatty acid human health, mainly in the prophilaxis and therapy
P productivity/reactor surface of chronic and degenerative diseases (obesity, dia-
PA phosphatidic acid betes, hypertension, cardiovascular and
brain-vascular diseases, digestive and metabolic dis-
*Author to whom correspondence should be addressed. eases, as well as cancer), a wide range of functional
83
84 A. Reis, L. Gouveia, K Woso, H. L. Fernandes,J. A. Empis, J. M. Novak
food products enriched with marine 03 fatty acids
has penetrated the market (Lauritzen, 1994). A fast
growth of this market is expected, to the 200 million
US dollars mark before the end of the century
(CQVB, 1988), mainly directed towards the health-
food industry and aquaculture. Marine fish products,
mainly menhaden oil, have been the traditional
sources of 03 polyunsaturated fatty acids. Several
publications have pointed out the feasibility of EPA
and DHA production from microbial sources, with
special emphasis on fungi (Yongmanitchai & Ward,
1989; Kennedy et al., 1993) and microalgae (Yong-
manitchai & Ward, 1989; Kennedy et al., 1993;
Bajpai & Bajpai, 1993). Microalgae, despite their
high production costs, show several advantages over
fish oil, as reviewed by Karuna-Karan (1986) and by
Reis (1993).
Mass production of the Bacillarophyceae Phaeo-
dactylurn tricomutum as a source of lipids (Dubinsky
et al, 1978; SERI, 1986) and for w3 PUFA produc-
tion (Moreno et aZ., 1979) has already been
reported, though under relatively low temperatures
and at low light intensities (Ansell et al, 1963; Reis
et al., 1990; Veloso et aZ., 1991); conditions which
are markedly different from those registered in this
work.
METHODS
Organism and growth media
Phaeodactylum tricomutum Bohlin SiPHAEO-1
(TFX-1) was obtained from the Solar Energy
Research Institute (SERI) Culture Collection
(Golden, Colorado, USA) and was cultivated in fil-
tered sea water enriched with components of the
MN medium (Borowitzka, 1988), but with some
modifications, as previously described (Reis, 1993).
Growth conditions
The non-sterile reactor used (Fig. 1) was a poly-
ethylene bag placed in a water bath at 24 +05C
(Reis et al, 1994). Dimensions were: volume, l-4 1;
height, 36 cm; diameter, 7 cm. A conical bottom was
shaped on to it, using a sealing device, in order to
minimize dead volumes and biomass deposition. A
14 1 h- air flow was provided through a ceramic
porous plate at the bottom. Fresh medium was
pumped by means of a peristaltic pump (Pharmacia,
model Pl) through the bottom. Medium and bio-
mass overflowed through a side tube (Fig. 1).
Continuous illumination was obtained by six verti-
cally placed 36 W fluorescent lamps, giving a total
light intensity of 250 PE m-* s-l.
Growth parameters (absorbance, ash-free dry
weight, chlorophyll and nutrient concentrations), as
well as algal chemical composition (protein, carbo-
hydrates, lipids and fatty acids) were measured at
each dilution rate value, immediately after stationary
state was attained. A steady-state was defined when-
r inlet
niRlct
s
i
7
2
Ic
1 I I
Fig. 1. Schematic diagram of the continuous-flow reac-
tor. 1 - Magnetic stirrer; 2 - stirring bar; 3 - nutrient
vessel; 4 - water bath, 5 - polyethylene bag; 6 - liquid
circulation peristaltic pump; 7 - side tube; 8 - ceramic
porous plate; 9 - gas valve; 10 - fluorescent lamps (light
source).
ever constant absorbance, as measured by five
consecutive absorbance readings (sampling interval:
2 h) within an interval range below 2% deviation,
was encountered (Reis et al., 1994). This situation
always materialized no later than four residence
times after setting new and different conditions.
Analytical methods
Measurements of algal growth were performed as
described in previous work (Reis et al, 1990; Veloso
et aZ., 1991). Nitrogen concentration was measured
using the Cawse method (Cawse, 1967) and by
means of a specific nitrate electrode (Ingold Mes-
stechni KAG type 15222300) (APHA, 1976). Fatty
acid methyl esters were prepared by transesterifica-
tion of freeze-dried samples according to Cohen et
al. (1988a). Fatty acid analyses were performed in a
Varian 3300 gas-liquid chromatograph equipped
with FID. Separation was carried out with a O-32
mm x 30 m fused silica capillary column, with (film:
0.32pm) Supelcowax 10 (Supelco) and He as carrier
at a flow rate of 1.5 ml.min-l. The column tempera-
ture was programmed at an initial temperature of
175C for 5 min, then increased at 2*5C.min-1 to
235C and held there for 20 min. Injector tempera-
ture, detector temperature and split ratio were,
respectively, 280, 300C and 1OO:l. Heptadecanoic
acid (Merck) was used as internal standard. Lipid
extraction and separation of its individual compo-
nents by thin-layer chromatography has been
described in previous publications (Reis et al, 1990;
Veloso et aZ., 1991). Other lipidic standards were
supplied by Sigma. Individual bands were scraped
off and transesterified as stated above.
RESULTS AND DISCUSSION
The biomass concentration and measured absor-
bance of the outflow, as well as the nitrate uptake,
are presented in Fig. 2, for all tested dilution rates.
 Continuous EPA production from Phaeodactylum tricornutum
:540 nm)
2 I'2
.
.
1.5
1 -1
.
. .
0.5
.
.
* * v .
* * I
* I
,t ,* ,
C n
0 0.2 0.4 0.6 0.6
D (day-)
Fig. 2. Biomass concentration (x) on ash-free dry-weight
basis (AFDW), culture absorbance (A) at 540 nm and
nitrate uptake evolution with dilution rate for continuous
growth of Phueodactylum tricomutum. ??Absorbance (540
nm); v biomass concentration (x) on ash-free dry-weight
basis (AFDW); * NO3 uptake. All values are averages of
duplicates.
The cell density curve exhibited a strong decrease
with the increase of D showing a hyperbolic pattern.
This decrease was similar to that obtained by Marsot
et al. (1991) for a continuous growth of Phaeoducty-
lum tricomutum in a dialysis system. The inverse
relationship between p (or D) and x appears to be a
normal algal response to conditions in the medium
which are affected by cell density (nutrient avail-
ability). The nitrate uptake can be considered
negligible for cultures with D 2 O-68 day-, showing
enhanced N-conversion efficiency in terms of bio-
mass production. The data suggest that N was a
non-limiting nutrient for all assayed conditions.
The chemical composition of the biomass has
been studied under continuous conditions at dif-
ferent dilution rates (Reis et al, 1994). At lower D
values, higher lipid and fatty acid concentrations,
ranging from 19.9% of lipids and 57% of fatty acids
(on AFDW of biomass basis) to 43.7% of lipids and
24.8% of fatty acids, were obtained. This increase in
total lipid contribution with slower-growing cultures
appears to be a normal behaviour for Eukaryotic
cells and was reported for Phaeodactylum tricomu-
turn by Kaixian and Borowitzka (1993). Clearly,
protein and carbohydrate concentrations did not
exhibit any significant variation, contributing 20 and
10% of AFDW biomass.
Figure 3 shows that there was a direct correlation
between consumed N per biomass unit and dilution
rate. Assuming that nearly all consumed N had been
used in protein synthesis, and since the protein con-
tent did not significantly change with D, a rough
calculation could be performed to determine N used
for maintenance. The higher the dilution rate of the
culture the lower was the N uptake for maintenance
and the higher the efficiency of N assimilation. This
conclusion agrees with data from Marsot et al.
85
NOjuptake
.
- 1.5
A
- 0.5 0 0.2 0.4 0.6 0.8 1
D (day')
Fig. 3. Nitrate uptake per biomass unit weight versus
dilution rate.
1 1.2 1.d
(1991), and may be a consequence of the ability for
nutrient adaptation to oligotrophic conditions, as
has been pointed out in other publications (Raim-
bault & Gentilhomme, 1990; Raimbault et al., 1990).
Chlorophyll-a and EPA concentrations as a func-
tion of dilution rate were studied by Reis et al.
(1994). For D<O*6 day- (higher biomass concen-
tration) the chlorophyll-a plot showed a wide
plateau (1% of AFDW), probably caused by light
limitation. Decrease of chlorophyll concentration at
D > 0.6 day- probably meant that the photosyn-
thetic locus was damaged by the higher light
intensity per cell at lower biomass concentrations.
The fatty-acid profile versus dilution rate is shown
in Table 1 with emphasis on EPA/AA and EPA/
20:4w3 ratios. The increase in D values produced an
increase in 14:0, 16:3, 20503 and 22:6w3, while 16:0
and 16:lco7 decreased sharply. As a rule, monoun-
saturated fatty acids may be seen to have been
replaced by polyunsaturated ones, especially those
belonging to the w3 family. For cultures at D ~0.32
day- the biomass showed optimal nutritional value
for aquaculture purposes in terms of the 03/06
ratio, according to the classification of Weeb and
Chu (1983). For higher D values, the biomass
showed moderate values.
Biomass, lipid and EPA volumetric formation
rates were presented by Reis et al. (1994). The
higher the D, the lower the biomass and lipid pro-
ductivities. Maximum productivities were obtained
at D = 0.14 day-: 0.23 g 1-l day- and 0.10 g 1-l
day-, respectively, showing the ability of this alga
to grow under conditions of high cell density. Maxi-
mum productivity for EPA (rEPA= 6 mg l- day- )
was obtained at higher D values (O-32<D ~0.50
day-).
In the determination of fatty acid distribution and
EPA distribution among lipid classes in the harves-
ted biomass at D = 0.32 day-, which gave
maximum EPA productivity, the percentages of
recovered fatty acids (Fig. 4) and recovered EPA
(Fig. 5) were 80 and 89.9%, respectively. Based
upon this, it can be said that 62% of recovered fatty
acids were bonded to the glycolipid MGDG fraction
and 21% to TG (Fig. 4).
86 A. Reis, L. Gouveia, K I/eoso, H. L. Fernandes, J. A. Empis, .I. M. Novais

The situation was different when the distribution
of recovered EPA throughout the lipid classes was
described (Fig. 5). It may be seen that this valuable
component was to be found preferably in the
MGDG (84% of EPA) and TG (11% of EPA) pha-
ses. Similar results were found by Arao et al. (1987)
who, nevertheless, had reported that only a negli-
gible percentage of EPA was to be found in the TG
fraction.
The presence of 95% of total EPA in the least
polar lipidic fractions (TG and MGDG), suggests an
easy way to separate, concentrate and purify this
product, and this may be of commercial significance.
CONCLUSIONS
An inexpensive continuous-flow apparatus worked
successfully for 6 months without any kind of con-
tamination, showing it to be suitable for inoculum
production for outdoor, large-scale reactors, the per-
formance of which has already been studied (Reis et
al., 1990).
The stationary-state hypothesis (p = D) seems to
be supported by stability of growth parameters,
absence of cell deposition on the reactor bottom and
no cell lysis.
An extrapolation of these small-scale biomass and
EPA productivities, with outdoor experiments,
assuming rough calculations in terms of the illumi-
nated surface was made, and the extrapolation gave
optimum biomass productivities which exceeded 23 g
m - day- (PEPA - 0.6 g mm2 day-), more than
four-fold higher when compared with our previous
reports (Reis et aZ., 1990; Veloso et al., 1991) and
higher than data published for Porphiridium cruen-
turn (Cohen et al., 1988b).
Biomass of uniform and controlled quality in
terms of fatty acid composition, was produced.
Therefore, this continuous-production system may
be recommended as adequate to provide a suitable
and stable diet for hatchery production (larval mol-
lusts and crustacea), when operating at low D.
Operation for EPA, which is based on the value of
MGDG+TG content in harvested biomass, will
depend upon the further development of down-
stream processing operations.
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Table 1. Relative distribution of fatty acids (FA) of Phueoabctylum t&corn&m grown in a continuous reactor versus
dilution rate. All values are averages of duplicates

rate (day-)
FA 0.14 0.32 Dilu:;; 0.59 0.68 0.85 l-22

14:o ;:; ;:; 5.0 5.0 5.8 5.0 5.1

15:o o-3 0.2 o-2 0.2 o-3
16:0 22.5 25.6 13.8 13.8 13.3 14.4 14.8
16:lw9 0 0 0.6 0.7 1.0 0.8 0.3
16:107 43.3 39-7 25.5 27.9 22.0 20.4 21.0
16:105 0 0.1 0.5 0.6 1.0 1.3
16:206 1.0 ;:7
0:: ;:; ;:; ;:; ;:s
16:204
16:3w4,3 2.5 ;:; 6.6 8-5 6-7 5.0
0.7 8!5
16:404,1 0.2 0.5 o-7 0.5 0.9
18:0 0.4 0.5 o-3 ;:; 0.4 O-6 O-6
18:lw9 ::: 1.3 o-5 1-o 0.6 l-2 1.3
18:lw7
18:2w6 ::y l-6
1-l 1.2
1.2 1.7
1.3 o-9
2-o ;:f ;:;

18:306
18:303 0.1 1.3
0.1 0.7
o-1 0.1
8:: 0.7
0.3 ;:; ;:;
18:4w3 o-5 0.9 0.5
20:3w6 lV4 0 ;:; :5 ;:; 0.4 ;:;
20:4w6 l-2 0.4 1.1 0.5 0.4
20:303 0.8 0
201403 ;:; ;:; ;:; ;:; 0:;
20:503 10.2 12.8 25.8 20.4 27.3 2:*; 2:.33
22:l 0 8:; 0.4 0.3 ;:; 0.5
22:4w6 1-o ;:; 0 1-o ;:;
22:50t3 1.3 1.1 2-3 2-3 2.9 2.0
22~603 2z 3Y.F I;.: 1.3 1.9 2.8 2.5
SFA 19.4 19.7 20-3 20.8
MUFA 46.8 43.3 28.7 32-2 25.9 25.4 26.1
PUFA 23.2 25.4 463 41.7 47.5 46.0 46.7
03/w6 1.8 2.8 4.7 5.1 5.3
EPA/AA 7.3 10.7 64.5 13366 24.8 540.54 63.3
EPA/20:4w3 25.5 16-O 43.0 51-o 136.5 126.0 84.3
 Continuous EPA production from Phaeodactylum tricornutum 87

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