1 Eicosapentaenoic Acid-rich Biomass Production

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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



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.



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).



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

0.2

0.4

::‘:

::y

0.1

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



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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