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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 Veloso,b Helena L. Fernandes,b & Julio M. NovaiC Josh A. Empis

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 The marine diatom Phaeodactylum

tricornutum

Boh-

lin is a potential source of the pharmaceutically valuable w3 polyunsaturated fatty acid eicosapentae-

noic acid (EPA). The results of indoor continuous growth of Phaeodactylum tricornutum Bohlin are reported. The relationships between dilution rate (D), nitrate concentration and chemical composition were studied. Higher biomass and lipid productivities were obtained at low D values. EPA was found to be an intermediate metabolite and the best productivity (6 mg 1-r day-r) was achieved for D values ranging from 0.32 to 0.50 day- . Under optimum conditions, 84 and 1170, respectively, of total recovered EPA were present in monogalactosyldia~lglycerol (MGDG) and in triacylglycerol (TG) moieties, respectively.Recorded EPAI! and EPA/20,4 03 ratios for all tested dilution rates were among the highest values ever reported, showing EPA purification to be easier to perform from this starting material than from many others commonly in use. Copyright 0 1996 Elsevier Science Ltd. Key words: Microalga, diatom, Phaeodactylum tricornutum, fatty acids, continuous reactor, lipids, EPA. NOMENCLATURE arachidonic acid (20 : 4 w6) chain with n carbon atoms dilution rate (volumetric flow/reactor volume) digalactosyldiglyceride docosahexaenoic acid (22 : 6 w3) eicosapentaenoic acid (20 : 5 03) monogalactosyldiglyceride monounsaturated fatty acid productivity/reactor surface phosphatidic acid
should be addressed.

PC PE PG PI PS PUFA r SFA SQ TG X x:ywz

phosphatidylcholine phosphatidylethanolamine phosphatidylglyceride phosphatidylinositol phosphatidylserine polyunsaturated fatty acid volumetric output rate (productivity/reactor volume) saturated fatty acid sulfoquinovosyldiglyceride triglyceride biomass on ash-free dry-weight basis WDW fatty acid with x carbon atoms, y double bonds, where z is the distance between the last double bond and the methyl end group specific growth rate (dxixdt)

IJ

INTRODUCTION The Omega-3 (~3) polyunsaturated fatty acids market (EPA and DHA) dates from 1982 and has an annual estimated value in excess of 25 million US dollars (CQVB, 1988). The therapeutic value of these compounds has been shown in the reduction of blood cholesterol (Bonaa et al., 1990), in the prevention of blood-platelet aggregation, and as a protection against cardiovascular and coronary heart diseases, atherosclerosis, hyperlipidemy, hypercholesterolemy and hypertriglyceridemy (Simopoulos, 1986). Other known applications include the therapy of chronic inflammation processes (Vitale, 1988; Goetzl et al., 1986) and improvement of vision (Dratz & Deese, 1986). Encouraged by recent multidisciplinary studies about beneficial effects upon human health, mainly in the prophilaxis and therapy of chronic and degenerative diseases (obesity, diabetes, hypertension, cardiovascular and brain-vascular diseases, digestive and metabolic diseases, as well as cancer), a wide range of functional 83

DGDG DHA EPA MGDG MUFA P PA

*Author to whom correspondence

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 healthfood 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 (Yongmanitchai & 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 Phaeodactylurn tricomutum as a source of lipids (Dubinsky et al, 1978; SERI, 1986) and for w3 PUFA production (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

r inlet niRlct s

2 1 I

7 Ic I

Fig. 1. Schematic diagram of the continuous-flow reactor. 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

Bohlin SiPHAEO-1 (TFX-1) was obtained from the Solar Energy Research Institute (SERI) Culture Collection (Golden, Colorado, USA) and was cultivated in filtered 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 biomass overflowed through a side tube (Fig. 1). Continuous illumination was obtained by six vertically 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, carbohydrates, lipids and fatty acids) were measured at each dilution rate value, immediately after stationary state was attained. A steady-state was defined when-

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 Messtechni KAG type 15222300) (APHA, 1976). Fatty acid methyl esters were prepared by transesterification 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 temperature 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 temperature, 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 components 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 absorbance 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

85

NOjuptake
2 :540 nm) . I'2

.
- 1.5

. 1.5

1 . . 0.5 . . .

-1

- 0.5

0.2

0.4
D (day')

0.6

0.8

*
C

*
0.2 0.4

v . * * I
,*
0.6

,t
0

*
1

I n
1.2 1.d

Fig.

3.

0.6

Nitrate uptake per biomass unit weight versus dilution rate.

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 Phaeoductylum 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 availability). The nitrate uptake can be considered negligible for cultures with D 2 O-68 day- , showing enhanced N-conversion efficiency in terms of biomass 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 different 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 tricomuturn 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 content 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.

(1991), and may be a consequence of the ability for nutrient adaptation to oligotrophic conditions, as has been pointed out in other publications (Raimbault & Gentilhomme, 1990; Raimbault et al., 1990). Chlorophyll-a and EPA concentrations as a function of dilution rate were studied by Reis et al. (1994). For D<O*6 day- (higher biomass concentration) 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 photosynthetic 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, monounsaturated 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 productivities. 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. Maximum 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 harvested 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) phases. Similar results were found by Arao et al. (1987) who, nevertheless, had reported that only a negligible 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 contamination, showing it to be suitable for inoculum production for outdoor, large-scale reactors, the performance 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.
Table 1. Relative distribution

An extrapolation of these small-scale biomass and EPA productivities, with outdoor experiments, assuming rough calculations in terms of the illuminated 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 cruenturn (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 mollusts 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 downstream processing operations.

REFERENCES
APHA (1976). Standard Methods for the Examination of Water and Wastewater.American Public Health Associa-

tion, Springfield,pp. 393-4.

of fatty acids (FA) of Phueoabctylum t&corn&m grown in a continuous reactor versus dilution rate. All values are averages of duplicates 0.32 ;:; 25.6 0 39-7 0.1 ;:; ;:; 0.5 1.3 l-6 1-l 1.3 0.1 0.9 0 l-2 ;:; 12.8 8:; 1.1 3Y.F 43.3 25.4 2.8 10.7 16-O Dilu:;; 5.0 o-3 13.8 0.6 25.5 0.5 ;:; 6.6 0.5 o-3 o-5 1.2 1.2 0.7 o-1 ;:; 0.4 ;:; 25.8 0.4 ;:; I;.: 28.7 463 4.7 64.5 43.0 ;:; 20.4 0.3 0 2-3 1.3 19.4 32-2 41.7 13366 51-o rate (day- ) 0.59 5.0 0.2 13.8 0.7 27.9 0.6 ;:; 8-5 ;:; 1-o 1.7 1.3 0.1 8:: :5 0.68 5.8 o-2 13.3 1.0 22.0 1.0 ;: s 6-7 o-7 0.4 0.6 o-9 2-o 0.7 0.3 ;:; 1.1 0 :; 27.3 ;:; 2-3 1.9 19.7 25.9 47.5 5.1 24.8 136.5 0.85 5.0 0.2 14.4 0.8 20.4 1.3 1.0 5.0 0.7 0.5 O-6 l-2 ;:f ;:; 0.5 0.4 0.5 0.8 2:*; 0.5 1-o 2.9 2.8 20-3 25.4 46.0 540.54 126.0 l-22 5.1 o-3 14.8 0.3 21.0 ;: 7 8!5 0.9 O-6 1.3 ;:; ;:; ;:; 0.4 0 2:.33 ;:; 2.0 2.5 20.8 26.1 46.7 5.3 63.3 84.3

FA 14:o 15:o 16:0 16:lw9 16:107 16:105 16:206 16:204 16:3w4,3 16:404,1 18:0 18:lw9 18:lw7 18:2w6 18:306 18:303 18:4w3 20:3w6 20:4w6 20:303 201403 20:503 22:l 22:4w6 22:50t3 22~603 SFA MUFA PUFA 03/w6 EPA/AA EPA/20:4w3

0.14 ;:; 22.5 0 43.3 0 0 :: 2.5 0.2 0.4 :: : ::y 0.1 o-5 lV4 ;:; 10.2 0 1-o 1.3 2z 46.8 23.2 1.8 7.3 25.5

Continuous EPA production from Phaeodactylum

tricornutum

87

PE PG PA

OTHER 16.7

DGDG

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 distribution among lipid classes, the percentage of recovered fatty acids was 80% of total fatty acids; 62% of the recovered fatty acids were bonded to the glycolipid MGDG and 21% to TG, the most important lipids. Distribution of fatty acids among other lipid classes is shown in the expanded bar chart on the right.

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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 recovered EPA was 89.8% of total EPA; 84% of total EPA was esterified to MGDG and 11% to TG. Distribution of recovered EPA among other lipid classes is shown in the expanded bar chart on the right. Ansell, A. D., Raymont, J. E. G., Lander, K. F. & Shackley, P. (1963). Studies on the mass culture of Phaeodactylum. II. The growth of Phaeodactylum and other species in outdoor tanks. Limnol. Oceanogr, 8,
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