1 eicosapentaenoic acid-rich biomass production
TRANSCRIPT
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ELSEVIER0960-8524(95)00157-3
Bioresource Technology 55 (1996) 83-88
0 1996 Elsevier Science Limited
Printed in Great Britain. All rights reserved
0960-8524196 $15.00
EICOSAPENTAENOIC ACID-RICH BIOMASS PRODUCTION
BY THE MICROALGA PHAEODACTYLUM TRI%ORNUTUM IN
A CONTINUOUS-FLOW REACTOR
Albert0 Reis,” * Luisa Gouveia,a Vera V eloso,b H elena L. Fernandes,b Josh A. Emp is”
& Julio M . NovaiC
“ I ns t i t u t e N a t i ona l de Engenha r i a e Tecno log ia ndus t r i a l , D ER Est r ada do PaGo o Lum ia r ; 1699 L i sboa Codex , Po r t uga l
bLabo ra t 6 r i o de Engenha r i a B ioqu i m i ca , I ns t i t u t e Super i o r Tkcn i co , Av en ida Rov i sco Pa i s , 1099 L i sboa Codex , Po r t uga l
(Received 15 December 1994; revised version received 4 October 1995; accepted 10 October 1995)
AbstractThe mari ne diat om Phaeodactylum tricornutum Boh-
l i n i s a po ten t i a l sou r ce o f t he pha rm aceu t i ca l l y
v a l u a b l e w3 po l y u n sa t u r a t e d fatty acid eicosapentae-
noic acid (EPA). The result s of i ndoor conti nuous
grow th of Phaeodactylum tricornutum Bohlin are
reported.
The relat ionships betw een dil uti on rat e (D), ni t ra te
concentr ati on and chemi cal composi ti on w ere studied.
Hi gher biomass and li pid producti vi t ies w ere obtai ned
at ow D val ues. EPA w as ound t o be an i ntermediat e
metabol it e and t he best producti vi ty (6 mg 1-r day-r )
w as achieved for D val ues ranging rom 0.32 to 0.50
day-‘. Under optimum conditions, 84 and 1170,
respectively, o f t ot al recovered EPA w ere present i n
monogalactosyldi a~lglycerol (MGDG) and in tri acyl -
gly cerol (TG) moi et i es, respecti vely .Recorded EPAI !
and EPA/20,4 03 rat ios or al l test ed dil uti on rat es
w ere among t he hi ghest va lues ever report ed, show ing
EPA puri fi cati on to be easier to perform from thi s
starti ng materi al than rom many others commonly in
use. Copy ri ght 0 1996 Elsevi er Science Ltd.
Key w ords: Microalga, diatom, Phaeodacty lum tr icor-
nutum, fatty acids, continuous reactor, lipids, EPA .
NOMENCLATURE
arachidonic acid (20 :4 w6)
chain with n carbon atoms
dilution rate (volumetric flow/reactor
volume)
DGDG digalactosyldiglyceride
DHA docosahexaenoic acid (22 : 6 w3)
EPA eicosapentaenoic acid (20 : 5 03 )
MGDG monogalactosyldiglyceride
MU FA monounsaturated fatty acid
P productivity/reactor surface
PA phosphatidic acid
*Author to whom correspondence should be addressed.
83
PCPE
PG
PI
PS
PUFA
r
SF A
SQ
TG
X
x:ywz
IJ
phosphatidylcholinephosphatidylethanolamine
phosphatidylglyceride
phosphatidylinositol
phosphatidylserine
polyunsaturated fatty acid
volumetric output rate (productivity/reac-
tor volume)
saturated fatty acid
sulfoquinovosyldiglyceride
triglyceride
biom ass on ash-free dry-weight basis
W DW
fatty acid with x carbon atoms, y doublebonds , w here z is the distance between
the last double bond and the methyl end
groupspecific growth rate (dxixdt)
INTRODUCTION
The Om ega-3 (~3) polyunsaturated fatty acids mar-
ket (EPA and DHA) dates from 1982 and has an
annual estimated value in excess of 25 million US
dollars (CQV B, 1988). The therapeutic value of
these compo unds has been sho wn in the reductionof blood cholesterol (Bonaa et al., 1990), in the pre-
vention of blood-platelet aggregation, and as a
protection against cardiovascular and coronary heart
diseases, atherosclerosis, hyperlipidemy, hypercho-
lesterolemy an d hypertriglyceridemy (Simopoulos,
1986). Other k nown applications include the therapy
of chronic inflammation processes (Vitale, 1988;
Goetzl et a l . , 1986) and improvement of vision
(Dratz & Deese, 1986). Encouraged by recent mu lti-
disciplinary studies about beneficial effects upon
huma n health, mainly in the prophilaxis and therapy
of chronic and degenerative diseases (obesity, dia-
betes, hypertension, cardiovascular an d
brain-vascular diseases, digestive and metabolic dis-
eases, as well as cancer), a wide range of functional
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84 A. Reis, . Gouveia, K Woso, H. L . Fernandes, . A . Empis, . M . Nov ak
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 traditionalsources 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).
r inlet
niRlct
s
27
1 I
Ic
I
i
Fig. 1. Schematic diagram of the continuous-flow reac-tor. 1 - Magnetic stirrer; 2 - stirring bar; 3 - nutrientvessel; 4 - water bath, 5 - polyethylene bag; 6 - liquidcirculation peristaltic pump; 7 - side tube; 8 - ceramicporous plate; 9 - gas valve; 10 - fluorescent lamps (light
Mass production of the Bacillarophyceae Phaeo-
dacty lurn ri comutum 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.
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
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
Th e non-sterile reactor used (Fig. 1) was a poly-
ethylene bag placed in a water bath at 24 +05”C
(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 tominimize 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.
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
175°C for 5 min, then increased at 2*5”C.min-1 to
235°C and held there for 20 min. Injector tempera-
ture, detector temperature and split ratio were,
respectively, 280, 300°C 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.
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 ateach dilution rate value, immediately after stationary
state was attained. A steady-state was defined when-
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.
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Con t i n u ou s EPA p r o du c t i o n f r om Phaeodactylum tricornutum 85
2
1.5
1
0.5
C t0
:540 nm )
NOjuptake
I ' 2.
.
- 1.5
- 1
.
. .
.- 0.5
.
* * v .* * ‘I
* I,* ,
n
0.2 0.4 0.6 0.6 1 1.2 1.d
D (day-‘)
Fig. 2. Biom ass concentration (x) on ash-free dry-weightbasis (AFDW ), culture absorbance (A ) at 540 nm andnitrate uptake evolution with dilution rate for continuousgrowth of Phueoda ct y l um t r i c omu t um . ??Absorbance (540nm); v bioma ss concentration (x) on ash-free dry-w eightbasis (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 a l . (1991) for a continuous growth of Phaeoducty-
l um t r i c omu t um 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 considerednegligible 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 a l , 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 Eukaryoticcells and was reported for Phaeoda c t y l um t r i c omu -
t u r n 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 e t a l .
.
A
0 0.2 0.4 0.6 0.8 1
D (day' )
Fig. 3. Nitrate uptake p er biomass unit weight versusdilution rate.
(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 a l . , 1990).
Chlorophyll-a and EPA concentrations as a func-tion of dilution rate were studied by Reis e t a l .
(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 16:0and 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 e t a l . (1994). The
higher the D, the lower the biomass and lipid pro-
ductivities. Maximum productivities were obtainedat 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).
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86 A . R ei s , L . Gouv eia , K I / eoso , H . L . Fernandes, J. A . Em pis , . I . M . No va is
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 a l . (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.
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 previousreports (Reis et aZ. , 1990; Veloso et a l . , 1991) and
higher than data published for Porphiridium cruen-
turn (Cohen et a l . , 1988b).
CONCLUSIONS
An inexpensive continuous-flow apparatus worked
successfully for 6 months without any kind of con-
tamination, showing it to be suitable for inoculumproduction for outdoor, large-scale reactors, the per-
formance of which has already been studied (Reis et
a l . , 1990).
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.
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.
REFERENCES
APHA (1976). St a nda r d M et h od s f o r t h e E xam i n a t i o n o f
W a t e r and W as t ew a t er .American Public Health Associa-tion, Springfield, pp. 393-4.
Table 1. Relative distribution of fatty acids (FA) of P h u eo a bc t y l um t & c or n & m g row n in a continuous reactor versus
dilution rate. Al l values are averages of duplicates
FA
14:o
15:o
16:0
16:lw9
16:107
16:105
16:206
16:2046:3w4,3
16:404,1
18:0
18:lw9
18:lw718:2w6
18:30618:303
18:4w320:3w6
20:4w6
20:303
201403
0.14
;:;
22.5
0
43.3
0
0”::
2.5
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::‘:
::y
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o-5lV4
;:;
0.32
;:;
25.6
0
39-7
0.1
;:;
;:;
0.5
1.3
l-61-l
1.30.1
0.90
l-2
;:;
Dilu:;;
rate (day-‘)
0.59 0.68 0.85 l-22
5.0 5.0 5.8 5.0 5.1
o-3 0.2 o-2 0.2 o-3
13.8 13.8 13.3 14.4 14.8
0.6 0.7 1.0 0.8 0.3
25.5 27.9 22.0 20.4 21.0
0.5 0.6 1.0 1.3
;:; ;:; ;:‘s1.0 ;:“7
0.7.6 8-5 6-7 5.0 8!5
0.5
;:;
o-7 0.5 0.9
o-3 0.4 O-6 O-6
o-5 1-o 0.6 l-2 1.3
1.2 1.7 o-91.2 1.3 2-o ;:f ;:;
0.7 0.1 0.7o-1
;:;
8:: 0.3 ;:; ;:;
:5 ;:;0.50.4 ;:;
0.4 1.1 0.5 0.4
;:; ;:; 0”:;0.8 0
20:503 10.2 12.8 25.8 20.4 27.3 2:*; 2:.33
22:l 0 0.4 0.3 0.5
22:4w6 1-o
8:;
0
;:;
1-o ;:;
22:50t3 1.3 1.1
;:;
2-3 2-3 2.9 2.0
22~603 1.3 1.9 2.8 2.5
SFA
2z 3Y.F I ;.:
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 5.3EPA/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
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Conti nuous EPA producti on f rom Phaeodactylum tricornutum 87
PE
PG
PA
OTHER DGDG
16.7
Pc+scl
‘,.
PI
TG
Fig. 4. Recovered fatty-acid distribution among lipids
from harvested biomass of Phaeodactylum tricomutum at
D = 0.32 day- ‘. In the determination of fatty-acid dis-
tribution among lipid classes, the percentage of recovered
fatty acids was 80% o f total fatty acids; 62% of the recov-
ered fatty acids were bonded to the glycolipid MGD G and
21% to TG, the most important lipids. Distribution offatty acids among other lipid classes is shown in the
expanded bar chart on the right.
PG
PE
MGDG
64.3
PC+SQ
DGDG
PA
Fig. 5. Recovered EPA distribution among lipids from
harvested biomass of Phaeodactylum tricomutum at
D = 0.32 day--‘. In the determination of recovered EPA
distribution among lipid classes, the percentage of recov-
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