Appl Microbiol Biotechnol (1994) 41:23-27

Applied Microbiology Biotechnology

Springer-Verlag 1994

Effect of growth rate on the eicosapentaenoic acid and docosahexaenoic acid content of Isochrysis galbana in chemostat culture
E. Molina Grima, J. A. Sfinchez P~rez, F. Garcia Camacho, J. M. Ferndndez Sevilla, F. G. Aci~n Ferndndez
Departamento de Ingenierfa Quimica, Facultad de Ciencias Experimentales, Universidad de Almerfa, E-04071 Almerfa, Spain Received: 1 June 1993/Received revision: 27 September 1993/Accepted: 30 September 1993

Abstract. An isolate of Isochrysis galbana rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) has been grown as a chemostat culture at 20 C and pH 8.00 controlled by CO2 injection. From a low dilution rate (D) of 0.0024 h -1 to 0.0377 h - l , close to maximum growth, a decrease in E P A content from 5.21% dry weight (d.w.) to 2.80% d.w. was observed, although the percentage of E P A in the total fatty acids increased. Lipids were fractionated, E P A being the major fatty acid found in the glycolipid fraction, whereas in the neutral lipid fraction were mainly palmitic and palmitoleic acids. At the same time, the biomass concentration also decreased from 1015 mg-1-1 to 202 mg. 1-1 over the range of Ds mentioned. Nonetheless, E P A productivity had a maximum value of 15.26 m g ' l - l . d a y -1 at D =0.0208 h -1.

have been studied, with improved productivity of EPA (L6pez Alonso et al. 1992a, b; Molina Grima et al. 1993). In the present paper, data from experiments aimed at increasing P U F A productivity are given and the biochemical changes related to growth rate are discussed.

Materials and methods

Organism. The microalga used was an isolate (labelled II-4) selected from among 42 isolates of a single strain of L galbana in a phenotypic selection programme carried out in our laboratory to select an EPA-rich strain of microalgae (L6pez Alonso et al. 1992a, b). Growth conditions. Cultures were carried out in a 5-1 fermentor (New Brunswick Scientific Bioflo III) that was computer controlled. The culture vessel, head plate and components of the head plate could be sterilized by autoclaving. The culture medium and sterilization processes were as described by Molina et al. (1991). The cultures were constantly illuminated with four Osram Dulux EL (20 W) fluorescent lamps arranged around the culture vessel. The incident light intensity on the culture surface was measured with a Mavolux digital meter (Gossen) in W . m -2 (Photosynthetically active radiation 1 W . m - 2 = 4 . 6 6 ~ E . m - 2 . s - 1 ) . Cell concentration was determined by absorbance at 530 nm. Experiments were carried out at a temperature of 20 C, CO2-injection-controlled pH at 8.00, air supply at 1.5 1.min - I sterilized with 0.22-t~m Millipore filters, and 150 rpm agitation. Analytical methods. Chlorophylls were measured according to the method of Hansmann (1973). Carotenoid determination was the same as that used by Whyte (1987). Lipids were extracted by the method of Kochert (1978). Fatty acid methylation was done by direct transesterification with acetyl chloride: methanol (1 : 20) following the method o f Lepage and Roy (1984). The analysis of methyl esters was carried out by gas chromatography using a 30m capillary column of fused silica (SP2330, Supelco, Bellefonte, Pa., USA), internal diameter of 0.25 mm, 0.20 txm standard film, split ratio 100:1, and a flame ionization detector. Sigma Lipid Standard 189-15, Supelco rapeseed oil mixture and Supelco PUFA-1 patterns were used for the determination. Nonadecanoic acid was used as an internal standard to quantify the fatty acid content in biomass dry weight (d.w.). Proteins were assayed as described by Lowry et al. (1951).

Introduction Polyunsaturated fatty acids (PUFAs) play an important role in human health, from the prevention of heart and circulatory diseases to brain development in infancy (Kyle 1992). Some strains of microalgae have recently been proposed as an alternative to fish oil as a source of these products (Radwan 1991). The main fatty acids studied were the n-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In the production of E P A and D H A in microalgae, several factors affecting the growth and biochemical composition of the cells must be balanced. Choice of species, growth conditions and age of the culture determine the productivity of fatty acids as expressed in terms of culture volume and time. In this context, a strain selection programme has been carried out with the marine microalga Isochrysis galbana and growth conditions that enhance the E P A and D H A contents

Correspondence to: E. Molina Grima

24 Lipids were fractioned in a chromatography column using 250-400 mesh silica gel and eluted with chloroform, acetone and methanol at 10, 40 and 10 times the volume of bed of each solvent, respectively (Kates 1986). The volmne of each solvent was then collected and analysed by gas chromatography as indicated above. e~ 2;0 -

1.5

1.0

Results and discussion

Growth rate

0.5

The dilution rate (D) ranged between 0.0024 h -~ and 0.0377 h-~, giving rise to a decrease in biomass concentration from 1024 mg.1 -a to 202mg-1-1. On the other hand, biomass productivity increased with D to a maximum plateau of 13.05mg.l-~-h -1 between D=0.0208 h -~ and D = 0 . 0 2 9 h -a (see Fig. 1). These variations of biomass and productivity with D are characteristic of light-limited continuous cultures, where steady-state growth is determined by the availability of light, that is to say the average light intensity inside the culture, which is dependent on the cell concentration (self-shadingeffect). Thus, as the biomass concentration increases, the mean light intensity decreases and so does the growth rate.

0 40

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Biochemical composition

The relationship of biomass concentration, light and growth described above has a paramount influence on cellular biochemical composition. As can be seen in Fig. 2a, the pigment content tends to decrease as D increases and there is more light available for photosynthesis, so the need for chlorophyll and carotenoids is lower. This is generally observed in chemostat cultures of photosynthetic micro-organisms due to the inverse variation between pigment and light intensity (Herzig and Falkowski 1989). Over the dilution range assayed,

4
2
X

0.002

0 . 0 0 7 0.012

0 . 0 1 7 0 . 0 2 2 0 . 0 2 7 0 . 0 3 2 0.037 Dilution rate, h"1

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Fig. 2a-c. Biochemical composition changes at different dilution rates, a Pigments: x , chlorophyll a; @, chlorophyll c; 2x, carotenoids, b Total lipids and proteins: A, lipids; O, proteins, c Main fatty acids: A, 1 4 : 0 ; . , 16:0; , 16:1; x , 18:4; 7?, 20:5; ~ , 22:6
12

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~! 400;

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2

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r 0.025

i 0.03

0.035

0.04

Dilation rate, h4 Fig, 1. Effect of dilution rate on biomass concentration () and productivity (R)

chlorophyll a decreased from 1.59% d.w. to 0.65% d.w. and chlorophyll c from 0.39% d.w. to 0.11% d.w. Carotenoids also dropped from 1.07% d.w. to 0.29% d.w. In the synthesis of biochemical components, cells that have a short duplication period contain more proteins and less lipids in the biomass than when the duplication period is longer. This is shown in Fig. 2b, in which the protein content increases markedly with D accompanied by a significant decrease in lipid content. This variation is related to the structural function of proteins as well as to the role played by some kinds of, mainly neutral, lipids in storage (Whyte 1988).

25 As old cells synthesize threefold more lipids than young cells, the lipid content may vary from 33% d.w. at D =0.0024 h - l i to 11% d.w. at D =0.0377 h-1. This change also affects the fatty acid composition of lipids. Taking into account only the main fatty acids found in the total saponificable fraction of lipids, in Fig. 2c saturated and mono-unsaturated acids, palmitic (16:0) and palmitoleic (16:1), (above 7% d.w.) are observed to be the major components of lipids in cells grown at low Ds. The polyunsaturated 20:5 content Was lower, around 5% d.w. However, as D increased, this fatty acid profile changed, and at D=0.0208 h -1 the major fatty acid was E P A (5%), while the 16:0 and 16:1 contents dropped to 2.74% and 3.36%, respectively. At higher Ds, all the fatty acids decreased, E P A remaining the major fatty acid. At the lowest D tested, 16:0 and 16:1 represented 21.67% and 22.17% of the total fatty acids, respectively, E P A 15.76% and D H A 7.82%. At D =0.0048 h - l , cell growth was also strongly limited by light, and a similar fatty acid profile was found, with a slight increase in 16: 0, 16:1 and EPA, although the dry weight of total fatty acid decreased. The most interesting feature appears when the fatty acid profile at D = 0 . 0 0 4 8 h -1 is compared to D = 0 . 0 2 0 8 h -1. The percentage of 14:0 increased slightly, but the 16:0 and 16:1 percentages dropped to 15.49% and 19.04%, respectively, according to the decrease observed in their dry weights. On the other hand, E P A increased to 27.9% of the total fatty adds, as its dry weight content remained approximately constant. At the higher Ds of 0.029 h -1 and 0.0377 h -1 the fatty acid profile was found to be the same in spite of the lower total lipid content of the biomass. The variation described is related to the abovementioned change in proteins. At low growth rates, storage lipids (16:0, 16:1) served as a sink for photosynthetically fixed carbon as the need for structural biomolecules was reduced. This was reflected in the low amount of proteins synthesized. Nonetheless, as growth was light-limited the cells had to maintain a substantial photosynthetic apparatus (chloroplasts) in order to collect the scarce light available. Thus, structural lipids, mainly those allocated to chloroplast membranes, which are highly unsaturated (Kates and Volcani 1966), were still found in high proportion. This could be the reason why the E P A content was approximately constant from D =0.0024 h -1 to 0.0208 h -1 The total lipids in the biomass obtained at D=0.0048 h -1 and D =0.029 h -1 were separated into three main fractions: neutral lipids, glycolipids and phospholipids. In Fig. 3, the variations in classes of lipids are presented in percentages of total saponifiable lipids and percentages of dry weight. The proportion of neutral lipids in biomass grown at 0.0048h -1 was 67.5%, which corresponds to 19.1% d.w. This is approximately three times higher than glycolipid fraction and five times higher than phospholipids. On the other hand, in biomass grown at 0.029 h - l , the neutral lipid proportion decreased down to half of that obtained at 0.0048 h -1, with a consequent increase in glycolipids
70 60 50 ~i40 30 20 .....

10
0.005 Dilution rate, h 1 0.029

Fig. 3. Distribution of lipid classes of cells grown at low and high dilution rate. Contents are given as percentages of total fatty acids and as percentages of dry weight: [],neutral lipids; rN, glycolipids; B, phospholipids

and phospholipids. The remarkable fact is that the decrease in total lipids shown in Fig. 2b was mainly due to the reduction in neutral lipids, as the glycolipids remained constant, although the phospholipids also decreased, but to a much lower extent. The fatty acid composition of the separated lipid fractions is given in Fig. 4. The main fatty acids found in the neutral lipids were 16:0 and 16:1. Polyunsaturated fatty acids were also present and E P A was the third, in order of percentage. At 0.029 h - l , unsaturation was somewhat higher than at 0.0048 h -1 with a significant increase in D H A content (from 3% to 10%). E P A constituted more than 30% of the glycolipids, followed by 16:0, 16:1 and 18:4 in much lower proportions. The overall profile did not change between the two Ds. On the contrary, the phos'pholipids became more saturated in fast-growing cells, 16:0 being the major constituent. In general, 14:0 was found in the three fractions as a minor component, in percentages ranging from 5% to 12%. Both 16:0 and E P A were the principal fatty acids in the phospholipid fraction. At the same time, E P A was found mainly in glycolipids and ] 6 : 1 in neutral lipids. From the above data, it may be concluded that when the target using a microalgal culture system is to obtain PUFAs, it is not worth growing cells under strongly limiting conditions for high lipid content in biomass, because increased lipid comes from the synthesis of storage lipids, which are saturated fats. On the other hand, there is an inverse relationship between lipid content and growth (Fig. 2b and c), the best growth rate not being the highest. Thus, a balanced growth must be obtained. When the fatty acid contents presented here are compared with those corresponding to the same strain and culture system described previously (Molina Grima et al. 1993), an inverse variation of fatty acid profile vet-

26
35 3O Neutral lipids

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20

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Glycolipids
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whereas in the present experiments, carbon was supplied by CO2 injection into the medium to keep the pH constant at 8.00. In practice, in the first set of experiments, the cells were CO2-1imited. This was evident in the increase in pH up to 8.65, and in the latter this limitation was prevented. Therefore, at low Ds, non-carbon-limited cells are capable of synthesizing higher amounts of storage lipids than carbon-limited cells. Furthermore, the EPA content under both growing conditions at low Ds are analogous, around 5% d.w., due to the effect of light on PUFA synthesis. Finally, the aim was to obtain high fatty acid productivity. EPA productivity varied with D, reaching a maximum of 15.26 mg .l - l . day -1 at D =0.0208 h-~. At this rate, neither EPA content (4.93% d.w.) nor biomass concentration (620 mg.1-1) were the highest obtained, but as the time factor is considered in productivity, the net EPA synthesis rate was maximal. Nevertheless, at D = 0.029 h-1, the biomass productivity obtained was the same as at D=0.0208 h -~, although the biomass concentration was lower (450mg-1-1) as was the EPA content (3.19% d.w.). Thus, at D = 0.029 h - ~ effluent from the culture vessel was less concentrated, with a lower fatty acid content, making this D unfavorable in comparison to D =0.0208 h -1. This is a clear example of how conditions optimizing a bioproduct are generally different from those that optimize: bi0mass productivity.

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Acknowledgement. This research was supported by a grant from

Phospholipids

the Comisi6n Interministerial de Ciencia y Tecnologfa (CICYT), (BIO 91-0652).

References

".'.'.'.',

iii!iiiil
0.005

0.029 Dilution rate, h "I

Fig. 4a-c. Fatty acid profiles of each lipid fraction: I1, 14:0; [],

16:0; [], 16:1; N, 18:4; N, 20:5; Nil,22:6. a Neutral lipids, b Glycolipids, c Phospholipids

sus growth rate is found. It was reported that the percentage of 16:0 and 16:1 over total fatty acids increased with D, from 10.12% and 10.65% at D=0.006h -1 to 17.64% and 16.07% at D=0.0377h -1, respectively, and the percentage of EPA decreased from 25.10% to 22.87% for the same Ds. What makes these sets of experiments different is that in Molina Grima et al. (1993), the culture received CO2 only by air bubbling,

Hansmann E (1973) Pigment analysis. In: Stein JR (ed) Handbook of phycological methods, culture methods and growth measurements. Cambridge University Press, Cambridge, p 359 Herzig R, Falkowski PG (1989) Nitrogen limitation in Isochrysis galbana (Haptophyceae). I. Photosynthetic energy conversion and growth efficiencies. J Phycol 25:462-471 Kates M (1986) Techniques of lipidology: isolation, analysis and identification of lipids. 2nd revised edn. In: Burdon RH, Knippenberg PH van (eds) Laboratory techniques in biochemistry and molecular biology, vol 3, part 2. Elsevier, Amsterdam, pp 190-195 Kates M, Volcani BE (1966) Lipid components of diatoms. Biochem Biophys Acta 116:264-278 Kyle D (1992) Production and use of lipids from microalgae. Lipid Technol May-June: 59-64 Kochert G (1978) Quantitation of the macromolecular components of microalgae. In: Hellebust J, Cragie S (eds) Handbook of phycological methods. Physiological and biochemical methods. Cambridge University Press, Cambridge, p 189 Lepage G, Roy C (1984) Improved recovery of fatty acid through direct transesterification without prior extraction or purification. J Lipid Res 25:1391-1396 Ldpez Alonso D, Molina Grima E, S~inchez P6rez JA, Garcfa S4nchez JL, Oarcia Camacho F (1992a) Isolation of clones of Isochrysis galbana rich in eicosapentaenoic acid. Aquaculture 102: 363-371 Ldpez Alonso D, Molina Grima E, S4nchez P6rez JA, Oarcfa S4nchez JL, Garcfa Camacho F (1992b) Fatty acid variation

27 among different isolates of a single strain of Isochrysis galbana. Phytochemistry 31 : 3901-3904 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275 Molina Grima E, Martfnez ME, S~inchez S, Garcfa F, Contreras A (1991) Growth and biochemical composition with amphasis on the fatty acids of Tetraselmis sp. Appl Microbiol Biotechnol 36 : 21-25 Molina Grima E, Sfinchez P4rez JA, Garcfa Camacho F, Garc~a S~inchez JL, L6pez Alonso D (1993) n-3 PUFA productivity in chemostat cultures of microalgae. Appl Microbiol Biotechnol 38: 599-605 Radwan SS (1991) Sources of C20-polyunsaturated fatty acids for biotechnological use. Appl Microbiol Biotechnol 35:421--430 Whyte JN (1987) Biochemical composition and energy content of six species of phytoplankton used in mariculture of bivalues. Aquaculture 75:193-203 Whyte JN (1988) Fatty acid profiles from direct methanolysis of lipids in tissue of cultured species. Aquaculture 75:193-203