Outdoor Turbidostat Culture of the Marine Microalga

Aquaculture and Fisheries Management 1994, 25, 547-555

Outdoor turbidostat culture of the marine microalgaTetraselmis sp.

E. MOLINA GRIMA, F. GARCf A CAMACHO, J. A. S A N C H E Z PEREZ,A. CONTRERAS G O M E Z & F. VALD^S SANZ Dpto. Ingenien'a Quimica,Universidad de Almeria, Almeria, Spain

Abstract. Biomass productivity, photosynthetic efficiency and fatty acid profiles of the marinemicroalga Tetraselmis sp. in a turbidostat culture system using an outdoor photobioreactor aredescribed. The maximum productivity depended on the units in which it was expressed; itreached 5-41 x 10"' g/l/h in volumetric units, and 7-26g/m^/day in surface units, the former atthe smallest culture depth (0-12m) and the latter at the greatest culture depth (0-25 m). Datafrom the experiments were adjusted to an equation for light limitation. The comparison of fattyacid composition at both depths showed that biosynthesis of polyunsaturated fatty acids(PUFAs), including eicosapentaenoic acid (EPA), was favoured when available light was low(P'17m). Moreover, fatty acid 16:0 seemed to be an indicator of the type of limitation in theculture, i.e. nutrient or light limitation.

Introduction

The use of chemostat and turbidostat continuous culture techniques, for the study of thegrowth and behaviour of microorganisms, is widespread (Myers & Clark 1944). Comparedwith batch culture, their advantages are that steady states are reproducible and uninfluencedby pretreatment of the inoculum. This is of particular interest when the produced biomass isto be used in aquaculture, because these techniques can ensure a particular fatty acid profile.Specially, PUFAs are essential for the development of early larval stages of many fish andshellfish (Watanabe, Kitajima & Fujita 1983; Randall, Bolis & Agradi 1990).

In a previous study (Camacho, Molina, Martinez, Sinchez & Garcia 1990), perform-ance of an outdoor continuous culture facility operating as a chemostat at constant dilutionrates was described. The steady state was difficult to reach in this device. Variations in lightintensity cause continuous changes in the specific growth rate of the algae during a given solarcycle. Furthermore, interpretation and modelling of the experimental results, a necessarytool for the comparison of productivities obtained from different laboratories, is alsoparticularly difficult.

In view of the difficulties described above, we decided to use our facility as a turbidostat.Thus the influence of operating variables (average dilution rate, D, concentration of biomassin steady state, C, and the depth of the culture, d) on biomass productivity and fatty acidprofile of Tetraselmis sp. were studied.

Correspondence: Dr E. Molina Grima, Dpto. Ingenieria Quimica, Universidad de Almerfa, 04071 Almerfa,Spain.

547

548 E. Molina Grima et al.

Materials and methods

The microalga used was Tetraselmis sp. from the Torre de la Sal Aquaculture Institute inCastell5n (Spain). The culture pond was a rectangular glass container with maximum depthof 0-3 m. More specifications in relation to experimental setup and culture medium aredescribed in a previous paper (Camacho et al. 1990). The operating variables under studywere modified within the following ranges: dilution rate, D, between 0-111 per day and 0-464per day; steady-state biomass concentration, C, between 0-060 and 0-250g/l; and culturedepth, d, 0-12m (corresponding to 441), 0-17m (681) and 0-25m (1001). The average dailyintensity of solar irradiation ranged from 352 to 530 W/m^. The air flow was 2v/v per min.

Each experiment began with a discontinuous period of growth until the desired cellconcentration was achieved. Continuous operation began early in the morning. Biomassconcentration was maintained constant by adjusting the dilution rate. The experiment wascontinued under these conditions until the end of the afternoon.

Fatty acid methylation was done by direct transesterification with acetyl chloride:methanol (1:20) following the Lepage & Roy method (1984). The analysis of methyl esterswas carried out by gas chromatography using a 30 m capillary column of fused silica (SP2330,Supelco, Bellefonte, PA, USA), internal diameter 0-25 mm, 0-20 jj,m standard film, split ratio100:1, and a flame ionization detector. SIGMA Lipid Standard 189-15, Supelco Rapeseed oilmixture and Supelco PUFA-1 patterns were used for the determination. Pigment content wassubtracted from total lipids to calculate the fatty acid content of dry weight (L6pez, Molina,Sanchez, Garcia & Garcia 1992; Molina, Sanchez, Garcia, Garcia & L6pez 1992).

The biomass extinction coefficient, Ka, was numerically determined using the variation inthe average biomass production rate (g/h), Gm, with average incident light intensity, IQ,published in Camacho et al. (1990). The procedure is based on the use of the law ofLambert-Beer to describe the attenuation of light through a culture of concentration C anddepth x:

I(x) = /o.e-'^''-^' (1)

The average light intensity in the inside of the culture can be calculated by integrating theabove expression for the total culture depth.

= — rdidi ' ^ Ka.C.d

By substituting /„ for /o in the expression which relates Gm to /o (Camacho et al. 1990), thefollowing is obtained:

where /^ is average light intensity in the inside of the culture corresponding to saturation.The experimental results were adjusted to Equation 3 by non-linear regression, providing

the following parameters: 4 = 35-5 W/m^, Ka = 0-035 m /̂g and Cmax = 2-62 x 10"^ g/h (r^ =0-998).

Tetraselmis culture 549

Table 1. Influence of culture depth and steady state biomass concentration on biomass productivity andphotosynthetic effiiciency. For terminology see text

Depth(m)

0-12

0-17

0-25

0-17

Exp.no.

12345

6789101112131415161718

1920212223

24

C(g/1)

0-0530-0980-1420-1480-150

0-0320-0600-0830-1370-1480-1670-1810-1920-2060-2070-2080-2180-237

0-0960-0980-1490-1760-180

0-250

D(per day)

0-4640-3920-3420-2720-163

0-2790-2500-2400-2210-2170-1400-2030-1660-1470-1430-1430-1360-111

0-2550-2350-1850-1650-132

0-225

/o(W/m^)

352371391410410

478432520525530476519508495519407503554

458469410432510

431

P'lO-'(g/l/h)

2-774-265-414-472-72

1-001-652-203-353-563-894-063-533-373-293-233-302-90

2-712-553-063-222-65

6-25

P'(g/m^/day)

2-74-15-34-32-3

1-22-53-75-55-65-66-55-25-55-44-55-44-7

6-25-66-97-65-4

7-0

(%)

0-781-141-381-080-66

0-310-560-620-940-991-201-151-021-000-931-170-960-77

1-281-081-611-611-12

1-59

Results and discussion

Biomass and productivity

Productivities obtained by the system, P (g/l/h) = D.C and P' (g/m^/day) = P.d, arepresented in Table 1 as a function ofthe operating variables in the 24 experiments performed.

With sunlight as the input, productivity and reactor scale are expressed in terms of area{P'), not volumetric units, as in fermenters, and both of them are limited by the intensity ofthe sunlight and photosynthetic conversion efficiencies. P was included in Table 1 becauseproductivity in volumetric units was thought to be a characteristic intensive parameter ofculture system and growth conditions which allows comparisons between totally differentculture systems. Thus, for example, of two given rectangular photobioreactors with the samevolume of culture but different surface, and therefore depth, the shallower attains the greaterproductivity P (g/l/h), because the effect of light limitation is lower. However, productivity P'(g/m^/day) is greater in the deeper until the increase in depth, d, no longer compensates thedecrease in P'.

Thus in our cultures, P maximum is found in the shallowest depth tested (0-12 m) and P' inthe deepest (0-25m). P' at a depth of 0-17m is observed to be almost linearly dependent onbiomass concentration up to approximately 0-160g/l (Fig. 1). This, if there is no growthlimitation by nutrients, is due to the microorganism's growing at the maximum possible


10

a -

2 -

-

//fo/%

a

,

D

0

oi

^ ^o

0 2 5

,__ 0.17

\ . 0.12

100 200 300 400

O 0.17 m a 0.12 m 0 0.25 m

500

Figure 1. Variation of the biomass productivity, P' (g/m^/day) with biomass concentration, C (g/m').

growth rate under the given operating conditions. This linear increase continues until thespecific growth rate is affected by one or more of the following factors: (a) the average lightintensity inside the tank may be below saturation due to cell density, even though incidentillumination may be high enough; (b) there may be a mortality term which includes theprocesses leading to cellular decay, and it is directly proportional to their concentration; (c) athigh biomass concentrations the amounts of one or several nutrients may be too small tomaintain the maximum growth rate, so that these nutrients become the limiters of growth.

Only the first two, (a) and (b), are possible because there could not be limitation bynutrients in view of the results under the same experimental conditions in a discontinuousculture in which a specific growth rate of around 0-07 per hour and a biomass concentration ofapproximately 0-3g/1 at the end of the exponential phase were obtained (Garcia 1991;Molina, Martinez, Sanchez, Garcia & Contreras 1991). These data would give rise to aproductivity at this point of 0-021 g/l/h or 32g/m^/day in a 0-17m deep tank, almost five timesgreater than the maximum values obtained.

For mass cultures, probably the most extensive analyses of various light curves are thoseby Van Oorschot (1955) and Shelef, Oswald & Golveke (1968). Using the simplestapproximations of Van Oorschot (first order-zero order model):

|JL = M-m /o S /s (4)

and considering the Lambert-Beer equation (Equation 1) for describing light dissipationthrough depth {d), the final integrated relationship for productivity P' as a function ofincident light intensity, /o was:

— In • e '} - Kd.C.d(5)


where Eq is thermodynamic efflciency (0-2), assuming a quantum requirement of 10einstein/mol CO2 reduced to glucose, A is saturation light intensity, J is heat of combustion ofalgae, assuming that the heat content of algae is 501-6kJ/mol (Goldman 1979), Ka is theextinction coefflcient obtained in Equation 3, C is biomass concentration, d is culture depth,Kd is a cell decay coefficient and jim is maximum speciflc growth rate.

Equation 5 is useful in demonstrating the importance of growth rate on algal biomassconcentration and resulting yield in a particular geographical region. The coefficient ofextinction obtained (Ka = 0-1035m^/g) was used to apply Equation 5 to these results, asmentioned above.

The adjustment was made by multiple regression of P' vs /o and C data (Table 1) fromwhich Is, Eq and Kd values presented in Table 2 were obtained. The continuous lines in Fig. 1represent the theoretical values calculated with Equation 5 for each depth.

Thermodynamic efficiency (Eq), as a parameter not depending on the depth of theculture, is observed to be practicaliy constant at around 0-15. This value is very near to thatobtained by Shelef et al. (1968), 0-2, although the latter is for Chlorella.

Keeping in mind that the incidence of cellular decay processes could vary with depth,differences obtained in Kd are logical. Thus, the lower depth and the higher Kd value are notsurprising. This may be because photorespiration and photooxidation are greater at smallestdepth in comparison with respiration at highest depth.

Photosynthetic efficiency

The efficiency of solar energy assimilation, Eo, is another parameter of great importance inthe evaluation of an outdoor culture facility. £0 is generally regarded as the fraction ofavailable light energy in the visible spectrum range converted to fixed chemical energy in theform of algal biomass. An empirical equation for Eo is given below:

cellular chemical energyt-o {/o) = —:—— r—r lUO , , .

incident light energy (6a)

_ Productivity (g/l/h). culture volume (1). heat of biomass combustion (J/g)visible fraction of total sunlight (0-43). Irradiance (J/h) (6b)

and by making the necessary unit conversions the following is obtained:

Eo(%) = m-3^h (6)

Table 2. Parameters of Equation 5determination coefficient)

Depth (m) Eq

0-12 0-170-17 0-140-25 0-12

obtained by multiple regression

A (W/m^)

334161322

of data (P', Jo, C) for each culture depth (r̂ is

Kd (per day)

0-2140-0540-023

r"

0-8230-9110-918


The obtained values of Eo are shown in Table 1. Equation 6 may obviously be used tocalculate the productivity of a Tetraselmis sp. culture with known system efficiency for oneday and given IQ. In this sense. Equation 6 is very similar to the model proposed by Oswald(1988) for the prediction of productivity in g/m /̂day of a 2700 m^-surface culture pool locatedat latitude 37°N in California:

P' = 4-02-10-3 . £^ (o/̂ ) . i^ (7)

The differences between Equations 6 and 7 arise because in Equation 7 Oswald uses anaverage of 23 kJ/g for the heat of biomass combustion. In this study, the average of 13-4kJ/gfor a strain of Tetraselmis suecica determined previously by Whyte (1987) was taken as theexperimental value. If in Equation 6 the average of 5-5kcal/g used by Oswald is substituted,we obtain an equation:

P' = 4-28-10-3 - Eo (%) - /o (8)

analogous to Oswald's model (Equation 7). The slight difference between them is probablydue to differences in the spectral distribution of the solar radiation in the two sites. Ourinstallation is located at latitude 36°50'N, very nearly that of Oswald. However, theenvironmental conditions of relative atmospheric humidity, air speed etc., which affect thespectral distribution, are probably different. Besides, the response of a particular algal straintoward light varies in the different parts of the visible spectrum. In this regard, the strainsused by Oswald, and the culture media, in no case agree with those used in this study.

Experiment 24 was performed with a larger illuminated surface area (5 = 0-536m^) thanthe others and at a depth 0-17m, doubling the £o in experiment 14 (the operating conditionsof which were the most similar to this one, but with 5 = 0-400m^). This demonstrates that inexperiment 24 the light reached places within the culture where it could be used moreefficiently. The same effect was obtained by Winokur (Lee, Erickson & Yang 1984) withChlorella cultures, but with agitation: the agitated cultures doubled the photosyntheticefficiency of those that were not agitated because in the latter the frequency of exposure tolight was decreased.

Fatty acid profile

No significant influence of biomass concentration on the fatty acid profile was observed in thestationary stage. Therefore, Table 3 shows the averages of the most representative fatty acidsat 0-12 and 0-17 m.

Although the change in the amount of essential fatty acids with growth irradiance appearsto be species specific, some similarities can be found between them. Thus, the ratio ofomega-3 polyunsaturated fatty acid to the sum of saturated and monoenoic fatty aciddecreased slightly with decreasing culture depth, as occurred with Isochrysis aff. galbana(clone T-ISO) grown in turbidostats at various levels of irradiance (Sukenik & Wahnow1991).

The percentage of fatty acid 16:0 declined with increasing culture depth, i.e. as availablelight intensity decreased and growth rate diminished, which is in agreement with the results ofthe experiments of other authors for several species (Thompson, Harrison & Whyte 1990;


Table 3. Variation of fatty acid profiles (as percentage of total fatty acids) with culture depth and culture conditions

Fatty acids

16:016:116:218:018:118:218:320:118:420:420:5SaturatedMonoenoicPUFAsPUFAs/(Sat. + Monoen.)

0-12

20-31-91-91-5

22-84-2

23-91-33-80-63-5

21-826-231-10-65

Depth (m)

0-17

11-32-22-11-2

22-35-3

25-51-27-21-06-7

12-526-939-4

1-0

Batch cultures

18-31-62-60-6

26-99-8

17-71-23-6-

6-818-931-428-10-56

'D.C, data from batch cultures of Tetraselmis sp. (Molina etal. 1991).

Renaud, Parry, Think, Padovan & Sammy 1991). Keeping in mind that a substantial amountof 16:0 is normally located in triacylglycerides (Fisher & Schwarzenbach 1978), it would seemreasonable to consider this fatty acid largely a storage product for excess energy, aninterpretation corroborated by Roessler (1988) and Calderwood (1989) in diatoms.

Thus, from these results, it may be stated that the lower the growth rate, the lower the16:0 content. Nevertheless, in the literature, 16:0 content is found to rise as growth rate slows(Cohen, Vonshak & Richmond 1988; Hodgson, Henderson, Sargent & Leftley 1991). Bothstatements are true. The difference between them is in the origin of the limiting factors whichcause the growth rate to decline. In the experiments presented here, and in those ofThompson et al. (1990) and Renaud etal. (1991), cultures were light limited, so that at higherintensities, when photosynthetic activity is high, 16:0 synthesis is enhanced as a primary stepin the fatty acid metabolic pathway. On the other hand, when growth limitation is due tonutrient limitation (N, P, Si), the percentage of 16:0 increases sharply because of decreases instructural fatty acid synthesis (mainly PUFAs) and carbon is fixed as storage Hpids (Cohen etal. 1988; Roessler 1988; Calderwood 1989; Hodgson etal. 1991). In conclusion, a percentageincrease in 16:0 is a sign that the culture is nutrient-deprived when light imposes no limit, orlight-saturated when nutrients are in excess as in the experiments carried out at 0-12m.

The relative amount of 20:5/i3, very important in the nutrition of marine animals (DePauw, Morales & Persoone 1984), diminished with decreasing culture depth. The samevariation occurred in four species grown under laboratory conditions at continuousillumination and in the red alga Porphyridium cruentum in light-limited chemostat cultures.This variation supports the contention that this fatty acid represents an important componentof chloroplast Hpids for some marine phytoplankton (Thompson etal. 1990). Lastly, the high18:3/i3 content is not strange because it is typical of green algae.

Although culture conditions and harvested biomass from outdoor continuous culturesand indoor discontinuous cultures are not comparable at all, Tetraselmis sp. PUFA contentswere higher in the former than in the latter. The average n3 PUFA content was 31-1% at


0-12m and 39-4% at 0-17m, two and ten points above the content obtained in the indoordiscontinuous experiments carried out at the same temperature and with the same mediumcomposition (Table 3). On the other hand, the average monoenoic fatty acid content inoutdoor culture is lower. These variations confirm that most unfavourable culture conditionshelp the biosynthesis of high-energetic-content molecules such as PUFAs (the outdoorcultures accomplish this on a larger scale, with less agitation, more dead zones, and lowerratio of surface to volume than in the discontinuous cultures).

Therefore, the quality and richness of microalgae as food for larvae which dependprimarily on polyunsaturated fatty acid composition may be managed by acting on depth ofculture. Moreover, the use of a turbidostat may secure a fatty acid profile of the biomass overa long period of time. This could be of interest, because the irregular coverage of PUFAneeds of both breeders and larvae explains the variation in survival rate and qualityfrequently observed in shellfish hatcheries.

Acknowledgments

This research was supported by a grant from the Direcci6n General de Universidades eInvestigacidn de la Junta de Andalucia, Spain.

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Documents

Outdoor Turbidostat Culture of the Marine Microalga