Productivity and photosynthetic efficiency of outdoor cultures of Tetraselmis suecica in annular columns

Aquaculture 261 (2006) 932 – 943 www.elsevier.com/locate/aqua-online

Productivity and photosynthetic efficiency of outdoor cultures of Tetraselmis suecica in annular columns Graziella Chini Zittelli a , Liliana Rodolfi b , Natascia Biondi b , Mario R. Tredici b,⁎ b

a Istituto per lo Studio degli Ecosistemi, CNR, Via Madonna del Piano 10, 50019 Sesto Fiorentino, Firenze, Italy Dipartimento di Biotecnologie Agrarie, Università degli Studi di Firenze, P.le delle Cascine 24, 50144 Firenze, Italy

Received 9 June 2006; received in revised form 4 August 2006; accepted 7 August 2006

Abstract In this study, 120-l annular columns were used to cultivate Tetraselmis suecica outdoors. The mass transfer at different aeration rates and the influence of the harvest rate on productivity and biochemical composition were investigated. The potential of the system was evaluated by estimating productivity at full-scale. Two different arrangements to simulate a full-scale plant and determine the “overall areal productivity” (OAP) were experimented with. In August 2003, one experimental column (full-scale column) was placed between seven dummy columns. All the reactors were positioned at a distance of 0.8 m wall to wall and centred at the vertices of equilateral triangles. A second experimental column (isolated column) was placed in a separate area under full sunlight. In August 2004, the columns were placed side by side in an east–west oriented row at a distance of 0.24 m wall to wall. In the first experiment, the mean volumetric productivity of the full-scale column was not significantly lower than that achieved by the isolated column (0.46 against 0.49 g l− 1 day− 1) in spite of the shading by the dummy units. The average OAP and efficiency of conversion of visible solar radiation (PE) were 36.3 g m− 2 day− 1 and 9.4%, respectively. In the second experiment, the full-scale column attained a mean volumetric productivity of 0.42 g l− 1 day− 1. The OAP and the PE were 38.2 g m− 2 day− 1 and 9.3%, respectively. © 2006 Elsevier B.V. All rights reserved. Keywords: Annular column; Mass culture; Full-scale simulation; Overall areal productivity (OAP); Photosynthetic efficiency; Tetraselmis suecica

1. Introduction Tetraselmis is a marine green flagellate widely used in aquaculture facilities as feed for bivalve molluscs, penaeid shrimp larvae and rotifers (Muller-Feuga et al., 2003). This marine genus has been found to have a large spectrum of antimicrobial activity (Austin and Day, 1990; Austin et al., 1992) and its members have shown a high potential as probiotics (Irianto and Austin, 2002). Because ⁎ Corresponding author. Tel.: +39 055 3288306; fax: +39 055 3288272. E-mail address: [email protected] (M.R. Tredici). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.08.011

of its high content of vitamin E, Tetraselmis has also been proposed as a source of this vitamin for human and animal consumption (Carballo-Cárdenas et al., 2003). Mass cultivation of Tetraselmis has been carried out in different kinds of open ponds (Materassi et al., 1983; Pedroni et al., 2004). The main problem in outdoor cultivation in open systems is contamination of the culture by other algal species (Materassi et al., 1983). At present, culture methods used in hatcheries for Tetraselmis production rely mainly on polyethylene bags and transparent glass-fibre cylinders (up to 500 l) usually kept indoors with artificial light (Fulks and Main, 1991). These systems are inefficient, leading to

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low productivities and little reliable cultures (Tredici, 1999). In the last decade, research efforts have been directed towards the development of more efficient, high surface-to-volume ratio photobioreactors for microalgae cultivation (Tredici, 2004). Some of these systems have been used, in laboratory or at small scale level outdoors, to cultivate Tetraselmis spp. (Tredici et al., 1996; Borowitzka, 1997; Pedroni et al., 2004). Tetraselmis has been also grown under heterotrophic conditions with yields in excess of 100 g l− 1 day− 1 (Day et al., 1991). It is generally thought that photobioreactors are more productive than ponds, but in reality, the higher performance of photobioreactors has never been clearly proved. One of the reasons for this is that a correct evaluation of photobioreactors in productivity terms is difficult, especially if the photobioreactors are of the vertical type. As recently proposed (Tredici, 2004), a correct evaluation of vertical reactors can be done in terms of overall areal productivity (OAP). To calculate the OAP the units must be arranged in the experimental set-up so as to simulate full-scale arrangement and carefully avoid peripheral effects. Both the type of arrangement and the distance between the units have a profound influence on reactor productivity, and must be carefully planned. Unless the reactors are placed at great distance, it is expected that, as a consequence of self-shading among the units, the productivity of the reactor in full-scale arrangement will be significantly decreased in comparison with isolated un-shaded reactors. The closer the reactors, the higher will be the decrease of productivity. The extent of this decrease has never been determined. The annular column photobioreactor has been developed at the Dipartimento di Biotecnologie Agrarie of the University of Florence and used to cultivate bioactive Nostoc strains (Rodolfi et al., 2002), Nannochloropsis sp. (Chini Zittelli et al., 2003; Rodolfi et al., 2003) and Isochrysis sp. T-ISO (Chini Zittelli et al., 2004) under artificial, natural and combined illumination. In the present work, annular columns have been used to cultivate Tetraselmis suecica outdoors and the potential of the system was estimated by determining productivity at fullscale in two different arrangements: 1) with columns placed at the vertices of equilateral triangles or 2) in parallel rows. Having optimised O2 mass transfer and temperature control, outdoor experiments were carried out to investigate the influence of the harvest rate on productivity, cell weight and biochemical composition of the biomass, and to determine volumetric productivity at full-scale. From the latter, the OAP and the photosynthetic efficiency (PE), necessary to correctly evaluate the potential of a culture system, were calculated.

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2. Materials and methods 2.1. The annular column The annular column was devised at the Dipartimento di Biotecnologie Agrarie of the University of Florence (Florence, Italy) in 1997. It consists of two 2m-high Plexiglas® cylinders of 40 and 50 cm in diameter placed one inside the other so as to form an annular culture chamber 4.5-cm-thick, 1.9-m-high and 120 l in volume (Chini Zittelli et al., 2003). The illuminated surface area and the surface-to-volume ratio of the reactor are 5.3 m2 and 44 m− 1, respectively. Compressed air is bubbled at the bottom of the annular chamber through a perforated plastic tube, for mixing and gas exchange. CO2 from cylinders is injected into the culture through a gas diffuser placed in a un-aerated zone of the annular chamber, as carbon source and for pH regulation. 2.2. Experimental plan The experiments were carried out during the summer, in 2003 and 2004, at our experimental stations of San Casciano Val di Pesa (latitude: 43°39′0″N; longitude: 11°11′0″E, 310 m above sea level) and Scandicci (latitude: 43°46′1″N; longitude: 11°9′20″E, 47 m above sea level), near Florence (Italy). In 2003, from July to August, the influence of the harvest rate on productivity and biochemical composition of the biomass was investigated using five 120-l annular columns placed at a distance of 1.0 m wall to wall in an east–west oriented row. The experimental columns were positioned in the middle of the row and operated in parallel at three different daily harvest rates (30, 40 and 50%). In the same year, a simulation of a full-scale arrangement with columns placed at the vertices of equilateral triangles was set up and the performance of the fullscale experimental reactor was compared with that of an isolated reactor during a quasi-steady state period of 16 days (from 21st August to 5th September). A second full-scale experiment, with columns placed in an east– west oriented row, was carried out in 2004. The experimental column was positioned in the middle of the row and operated during a quasi-steady state period of 23 days (from 9th to 31st August). 2.3. Full-scale annular column arrangements During the second half of August 2003, an experiment was carried out to determine the OAP (the productivity obtained from the overall – including

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Fig. 1. Plan view of the full-scale arrangement of annular columns at the vertices of equilateral triangles (•, experimental column; , dummy column).

empty spaces – ground area occupied by the reactors constituting the plant, as defined by Tredici, 2004) of annular columns placed at the vertices of equilateral triangles at a distance of 0.8 m wall to wall (Fig. 1). This distance was chosen as the minimum allowing operators' work between the reactors. One experimental column (full-scale column), which was filled with the culture, was surrounded from the south side by seven empty columns wrapped in green plastic (dummy columns) (Fig. 1). The number and position of the dummy columns were chosen considering that only these seven reactors shade the experimental one during August at the latitude of Florence. This arrangement allowed to accommodate 0.68 columns per square meter. The ground was white-washed to increase its reflectivity. The second full-scale simulation was carried out in the summer 2004, with columns placed on a concrete floor at a distance of 0.24 m wall to wall in an east–west oriented row (Fig. 2). The OAP was calculated by considering that, in the period of the experiment, parallel east–west oriented rows of 2.0-m-high annular columns spaced of at least 1.66 m apart centre to centre do not shade each other. The hourly change of the length and of the azimuth angle of the shadow made by a 2.0-m-high column on the 18th of August in Florence is shown in Fig. 3. In this type of arrangement there were 0.81 columns per square meter.

Fig. 2. T. suecica OR cultures in annular columns arranged in an east– west oriented row.

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Fig. 3. Length and azimuth angle of the shadow made by a 2.0-m-high annular column from 7 a.m. to 5 p.m. on the 18th August at the latitude of Florence (Italy).

2.4. Organism and culture conditions The marine prasinophyte T. suecica strain OR from the culture collection of the Dipartimento di Biotecnologie Agrarie of the University of Florence (Italy), was grown in f medium (Guillard and Ryther, 1962) made with artificial seawater (Adriatic Sea Equipment, Forlí, Italy) at 30 g l− 1 salinity and enriched with NaHCO3 (0.5 g l− 1). The artificial seawater was filtered through 10- and 1.0μm polypropylene filters (Domnick Hunter, UK) and then added with the sterile nutrient solutions. NaNO3 and NaH2PO4 were added to the culture when required to prevent nutrient limitation. The air injected into the reactors was filtered through 1.0-μm Polycap HD encapsulated filters (Arbor Tech, USA). The total volume of the aerated culture (liquid plus gas phase) was 117.6 l. The air-flow rate was maintained at 0.23 l l− 1 min− 1 and the gas hold-up was about 0.022. Pure CO2 from cylinders was injected during daylight hours to provide carbon to the culture and regulate its pH at 8.0 ± 0.2. A control unit including a thermostat provided temperature regulation by automatically activating tap water circulation through a 0.6-cm diameter stainless steel tube immersed into the culture, when the temperature exceeded the preset value of 27 °C. During the night, the culture temperature was allowed to equilibrate with ambient. A semi-continuous daily harvesting regimen was adopted in all the experiments. Each day, in the early morning a predetermined culture fraction (30, 40 or 50%) was withdrawn and replaced with the same volume of

fresh medium. In the full-scale experiments, a 40% daily harvest rate was adopted. All the experiments started when quasi-steady state conditions were achieved. 2.5. Analytical procedures Culture growth was estimated by measurement of cell number, using a Bürker haemocytometer, and dry biomass concentration. Dry weight was determined on a daily basis before culture harvesting according to Chini Zittelli et al. (2000). The mean doubling time (td) was calculated as: td = ln2 / μ, where μ is the specific growth rate calculated according to Hu et al. (1996). Biochemical analysis of T. suecica biomass was made on culture samples collected at the end of the dark period. The samples were centrifuged, washed and lyophilised. The lyophilised biomass was analysed for carbohydrate (Dubois et al., 1956), protein (Lowry et al., 1951) and lipid (Marsh and Weinstein, 1966). The vitamin E content was determined according to Carballo-Cárdenas et al. (2003). The dissolved oxygen concentration was measured by means of an OXY 323 oxygen meter equipped with a CellOX 325 polarographic Clark-type electrode (WTW, Germany). The mass transfer coefficient for oxygen, KLa(O2), in air-bubbled annular columns was determined in tap water at a constant temperature of 25 °C, according to Babcock et al. (2002). The photosynthetic photon flux density (PPFD) at the reactor surface was measured by a LI-190SB cosine quantum sensor connected to a LI-185B quantum/

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radiometer/photometer (Li-Cor, Inc., Lincoln, USA). The PPFD was measured at 20 different points on the column circumference and at a distance of 0.85 m from the reactor base. At each hour, the PPFD values measured on the column frontal directly illuminated surface were averaged, as well as the values measured on the back surface, reached only by dispersed radiation. The photosynthetic photon flux (PPF) intercepted by the frontal and the back surfaces of the column was estimated by integrating the curves representing the diurnal variation of the PPFD impinging on each surface and multiplying these values for half column surface area (1.45 m2). The total PPF intercepted by the annular column was obtained by summing the PPF intercepted by the frontal and back surfaces of the reactor. The PPF impinging on a horizontal surface of 0.2 m2 (i.e., equal to the cross-sectional area of the annular column) was added to take into account the irradiance impinging on the column top. The daily global solar radiation on the horizontal and the air temperature values were obtained from LaMMA Agrometeorological Station (CNR-IBIMET, Florence, Italy). The caloric content of T. suecica OR biomass was calculated from its biochemical composition, assuming a caloric content of 38.9 kJ g− 1 for lipid, 17.6 kJ g− 1 for carbohydrate, and 23.8 kJ g− 1 for protein (Milner, 1953). To calculate the photosynthetic efficiency (PE) achieved by the cultures in the annular column, the reactor productivity (g day− 1) was multiplied by the mean caloric content of T. suecica OR biomass (24.2 kJ g− 1) and divided by the solar energy input (PAR) on the reactor surface (kJ day− 1). The impinging PPF was converted from mol photons s− 1 to kJ s− 1 using 217 as conversion factor. The overall areal productivity (OAP) (g m− 2 day− 1) in the full-scale experiments was calculated multiplying the reactor productivity (g day− 1) by the number of reactors per square meter. The PE of the full-scale arrangement was calculated from the OAP multiplied by the mean caloric content of T. suecica OR and divided by the visible solar energy input per unit ground area (kJ m− 2 day− 1). The visible fraction of global solar radiation was assumed to be 45%.

3. Results 3.1. Oxygen mass transfer and temperature control The mass transfer coefficient for oxygen (KLa(O2)) and the gas hold-up in the annular column were measured at three different aeration rates (0.11, 0.23 and 0.49 l l− 1 min− 1), corresponding to superficial gas velocities (Ug) of 0.003, 0.007 and 0.015 m s− 1. When the Ug increased from 0.003 to 0.015 m s− 1, the KLa (O2) and the gas hold-up increased linearly from 14.1 to 74.9 h− 1 and from 0.010 to 0.043, respectively (Fig. 4). A mass transfer coefficient of 36.4 h− 1 (attained at a Ug of 0.007 m s− 1) was sufficient to keep the dissolved oxygen concentration little above air saturation values at noontime on sunny days (data not shown), when usually the oxygen evolution rate reaches the maximum. A superficial gas velocity of 0.007 m s− 1 was thus adopted in all the following experiments. In sunny days, the total solar energy impinging on an isolated annular column at midday often exceeded 3 MJ h− 1. Under this radiation, and with air temperatures of 35–36 °C, a culture without temperature control reached and maintained for 3–4 h temperatures of 41–42 °C (data not shown), well above the optimum for T. suecica (about 27 °C). By circulating tap water at a flow rate of 120 l h− 1, the cooling system was able to remove more than 2.5 MJ h− 1 and maintain the culture temperature below 33 °C, even in very hot summer days (Fig. 5). During the hottest hours of the day, the water temperature at the inlet of the cooling tube varied between 24 and 26 °C, and increased of 4–5 °C during its passage through the tube. The diurnal temperature

2.6. Statistical analysis Growth and biochemical data that compared two groups were analysed by paired t-test. Growth data that compared more than two groups were analysed by oneway ANOVA. Differences between treatments were compared by Tukey's multiple comparison test, using the software Graph Pad 4.00 (Graph Pad Software, San Diego, USA). The significance level was P b 0.05.

Fig. 4. Oxygen mass transfer coefficient (KLa(O2)) (●) and gas holdup (■) at 25 °C in an annular column as function of the superficial gas velocity (Ug). Error bars represent the standard deviation from three replicate experiments. (●) n = 3, r = 0.99 (P b 0.05); (■) n = 3, r = 0.99 (P b 0.05).

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3.3. Light distribution on isolated and full-scale arranged columns

Fig. 5. Diurnal variation of the temperature in a T. suecica OR culture grown in the isolated annular column (●), compared to ambient temperature (○) on the 21st August 2003.

pattern in the full-scale arrangement did not vary significantly compared with the isolated column. 3.2. Influence of the harvest rate on productivity and biochemical composition

Fig. 6 shows the diurnal variation of the average PPFD on the frontal (directly illuminated) and back (receiving only dispersed radiation) surfaces of the isolated and the full-scale (in the triangular arrangement) column on the 21st August 2003. Solar irradiance on the horizontal is also shown for comparison. The frontal surface of both columns was averagely exposed to a lower PPFD as compared to that impinging on the horizontal. Moreover, while the PPFD on the horizontal draws a bell-shaped curve centred at midday, the average PPFD on the frontal surface of the reactors, is nearly constant after reaching its maximum value. In particular, the isolated column received a maximum average PPFD of about 1000 μmol photons m− 2 s− 1 from 8:20 to 16:20, and the full-scale column intercepted a lower PPFD (about 900 μmol photons m− 2 s− 1 ) for a shorter period (from 10:20 to 15:20). Diversely, on the back surface, no significant differences were found between the isolated and the full-scale

The harvest rate affected the main growth parameters of T. suecica cultures (Table 1). The highest volumetric productivity (0.56 g l− 1 day− 1, corresponding to 64.4 g reactor− 1 day− 1) was attained at 50% harvest rate. Productivity decreased to about 0.50 g l− 1 day− 1 at 40% and 30% harvest rate. The increase of the harvest rate from 30 to 50% resulted in a halving of the doubling time and in a 25% reduction of the cell weight (Table 1). The biochemical composition was less affected, protein being always the major component (41–44%) of the dry biomass, followed by lipid (30–32%) and carbohydrate (10–13%). Ashes were about 17.5% of dry biomass at all the harvest rates tested. The average vitamin E content of the biomass from cultures kept at 50% harvest rate was 125 μg g− 1 dry weight. Table 1 Influence of the harvest rate on growth parameters of T. suecica OR cultivated in annular columns Harvest rate (%) 30 0.48 ± 0.08a Volumetric productivity −1 −1 (g l day ) Biomass concentration at 1.71 ± 0.13 harvesting (g l− 1) Cell weight (pg cell− 1) 212.3 ± 34.1 Doubling time (day) 2.13 ± 0.35

40

50

0.51 ± 0.05ab

0.56 ± 0.08b

1.25 ± 0.06a

1.16 ± 0.11a

nd 1.33 ± 0.16

157.8 ± 20.8 1.07 ± 0.13

Mean values ± SD (n = 16) are reported. Values across the same row sharing a common superscript letter are not significantly different (P N 0.05). nd = not determined.

Fig. 6. Diurnal variation of the PPFD impinging on the horizontal (A) and on the frontal (●, ■) and back (○, □) surface of annular columns (B) on the 21st August 2003. ■, □: isolated column; ●, ○: full-scale column in the triangular arrangement. The PPFD values reported for the columns are averages of the PPFD measured once per hour at different points along the column circumference at 0.85 m from the bottom.

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reactor. It is to point out that the PPFD values shown in Fig. 6 are averages of the irradiance values measured every hour at twenty different points along the reactor circumference and do not illustrate the distribution of the irradiance at the column surface. The actual irradiance values measured hourly along the circumference of the isolated column on the 21st August are shown in Fig. 7. During the central daylight hours (from 10:20 to 14:20) the maximum irradiance values on the column surface were about 20% lower than the corresponding irradiances on the horizontal. In the early morning and in the evening, the opposite was true, with about 30% higher maximum irradiances on the column than on the horizontal. On the 21st August, the isolated column intercepted a total PPF (see Section 2.5) of 82 mol photons, while the estimated PPF on the full-scale column in the triangular arrangement was 68 mol photons. The latter value was overestimated as it can be inferred from the drawings

Fig. 8. Shading pattern on the experimental column in the full-scale triangular arrangement during the first half of the day on the 21st August 2003.

Fig. 7. Diurnal variation of the light distribution along the circumference of the isolated column.

(Fig. 8), showing the shading pattern in the full-scale triangular arrangement for the first half of the day (the other half being its mirror image). The PPF received by the full-scale column from 9:20 to 12:20 (and hence from 12:20 to 15:20) was overestimated because, when this column was shaded, the shadow was below the measurement height (0.85 m from the column bottom). During the remaining hours of the day, the PPF was, instead, underestimated, since the measurement level was in the shadow, but the column top was illuminated. However, considering that from 9:20 to 15:20 the columns received the bulk of the daily PPF, the PPF intercepted by the experimental column during the daylight period was, on the whole, overestimated.

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3.4. Productivity and photosynthetic efficiency of isolated vs full-scale annular columns The daily volumetric productivities attained by the isolated column and the full-scale column in the triangular arrangement are shown in Fig. 9, together with the global solar radiation. The average volumetric productivity achieved in the experimental period by the full-scale column (0.46 g l− 1 day− 1) was not significantly different (P N 0.05) from that attained by the isolated column (0.49 g l− 1 day− 1). The simulation of a full-scale plant, with columns placed at the vertices of equilateral triangles, gave an OAP of 36.3 g m− 2 day− 1. From this value, given the caloric content of the biomass (24.2 kJ g− 1) and the average visible solar radiation on the horizontal (9.6 MJ m− 2 day− 1), an efficiency of conversion of solar energy into biomass of 9.4% was calculated for the whole experimental period. It is worth noting that in sunny days (average solar radiation of 23 MJ m− 2 day− 1) the mean OAP and PE were 38.7 g m− 2 day− 1 and 9.1%, respectively, while in cloudy days (with less than 12 MJ m− 2 day− 1) the OAP decreased to 19.7 g m− 2 day− 1 and the PE increased to 11.4%. On the 21st August the PPF intercepted by the columns was measured and, by taking into account the reactor productivity and the biomass caloric content, a PEs of 10.0 and of 10.6% were calculated for the

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isolated and the full-scale annular column, respectively. It is worth noting that the PE achieved by the culture in the full-scale column was underestimated, since the PPF intercepted by this reactor was overestimated for the reasons reported in Section 3.3. The average volumetric productivity obtained in the row-arrangement (Fig. 2) during a quasi-steady state period of 23 days was 0.42 g l− 1 day− 1. Considering a full-scale plant made by parallel column rows placed at a distance of 1.66 m centre to centre to avoid shading (see Section 2.3), an OAP of 38.2 g m− 2 day− 1 and a PE of 9.3% (with an average visible solar radiation on the horizontal of 10.0 MJ m− 2 day− 1) were calculated. The sole factor that might reduce productivity at real fullscale, and not considered in this simulation, is that in a real situation the ground between column rows would be shaded and thus would reflect less light compared to the un-shaded ground. This fact, however, has not had a significant influence in our work given that the columns were placed on a low reflectivity concrete floor. 4. Discussion Vertical annular columns were devised in 1997 at the Dipartimento di Biotecnologie Agrarie of the University of Florence (Italy) and have been used since then to cultivate various cyanobacteria and microalgae, among

Fig. 9. Volumetric productivity of T. suecica OR grown outdoors in the isolated and in the full-scale (triangular arrangement) annular columns. Daily solar radiation on the horizontal is also shown. Data were collected during a quasi-steady state period of 16 days. Average irradiance was 21.3 MJ m− 2 day− 1.

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which bioactive Nostoc strains (Rodolfi et al., 2002), Nannochloropsis sp. (Chini Zittelli et al., 2003; Rodolfi et al., 2003) and Isocrhysis galbana T-ISO (Chini Zittelli et al., 2004) under artificial, natural or combined illumination. The annular columns have been shown to be capable of producing high quality algae biomass on a regular basis and attain much higher productivities than traditional systems used in hatcheries, where a high productivity is desirable since it reduces the need for space and labour, and running costs. Under artificial illumination, provided by a set of fluorescent tubes placed inside the inner cylinder of the column, a very high efficiency of light utilisation was achieved (Chini Zittelli et al., 2003). These systems are at present industrially produced and commercialised by two Italian companies (Fotosintetica & Microbiologica s.r.l. and Exenia s.r.l.) and used by several fish and mollusc hatcheries for on-site production of marine microalgae. In this work, the marine T. suecica strain OR was cultivated in annular columns outdoors during two consecutive summers. Productivity was high and stable, showing only variations clearly dependent on climatic conditions (e.g., productivity decreases in cloudy days). A 120-l annular column typically produces more than 60 g of dry biomass per day and, due to its high surfaceto-volume ratio, achieves a cell concentration at harvesting and a volumetric productivity (1.7 g l− 1 and 0.56 g l− 1 day− 1, respectively) about three times higher than those attained with Tetraselmis spp. in polyethylene bags and transparent cylinders (Fulks and Main, 1991). Oxygen accumulation up to inhibitory levels and overheating of the culture strongly limit the performance of closed reactors in sunny weather (Tredici, 2004). For this reason, efficient systems for temperature control and oxygen degassing are a necessity in closed reactors kept outdoors. In the annular columns temperature increase above damaging values was prevented by circulating tap water through a metal pipe immersed into the culture. T. suecica OR cultures attained good performances in spite of the temperature exceeding the optimum of 2– 6 °C for about 8 h per day in very hot days. The oxygen removal capacity in a photobioreactor is governed by the magnitude of the gas–liquid mass transfer coefficient, which in bubble columns is in general higher than in tubular reactors mixed with pumps or airlifts (Sánchez Mirón et al., 1999). Significantly higher KLa(O2) values (up to 75 h− 1) were obtained in the annular columns compared to those measured in a nearhorizontal tubular reactor (Tredici-design) experimented with in the past (Chini Zittelli et al., 1999), even if this latter was operated at greater superficial gas velocities (Babcock et al., 2002). In high surface-to-volume ratio

reactors, high superficial gas velocities and mass transfer rates usually lead to higher productivities (Barbosa et al., 2003; Zhang et al., 2002). Very likely, higher superficial gas velocities than that adopted in this work (0.007 m s− 1) would have increased productivity due to the beneficial effects of high mixing in general, and of short light–dark cycles in particular (Janssen et al., 2003). However, economical considerations and the risk of cell damage discourage the use of very intense mixing. Very high bubbling and mass transfer rates were not necessary in the annular columns, at least as far as oxygen removal is concerned. A superficial gas velocity of 0.007 m s− 1 and a KLa(O2) of 36.4 h− 1 were, in fact, suitable to avoid cell sedimentation and maintain the dissolved oxygen concentration below toxic values, even at noontime on sunny summer days. A fundamental parameter governing growth rate, productivity and biochemical composition in algae mass cultures is the population density (Richmond, 2004), which in turn depends on the harvest rate. In our work with outdoor T. suecica cultures in annular columns, a harvest rate of 50% of the culture volume per day stabilized the culture at a mean cell density of 0.88 g l− 1 and gave an average productivity of 0.56 g l− 1 day− 1. At this harvest rate, a growth rate about half of that reported by Carballo-Cárdenas et al. (2003) in exponential-phase cultures of another strain of the same species, was attained. At 50% harvest rate, cell weight was the lowest, confirming the findings of Fábregas et al. (1997) that T. suecica cell weight decreases with the increase of the harvest rate (i.e., with the increase of the growth rate). It is known that a small cell size positively affects the ingestibility of the alga by microfeeders (MullerFeuga et al., 2003). Differently from previous studies carried out with Tetraselmis spp. in the laboratory, which found large variations in cell composition with culture conditions (Otero and Fábregas, 1997), in our outdoor experiments the biochemical composition was little affected by the harvest rate, with protein always the major constituent (41–44% of dry biomass), followed by lipid (30–32%) and carbohydrate (10–13%). According to Wikfors et al. (1996), who found that high-lipid strains of Tetraselmis supported faster oyster growth, and considering that in general, mollusc larvae require 30 to 60% protein in the diet for an adequate growth (Brown et al., 1989), the above composition seems appropriate for biomass to be used as feed in aquaculture. A lower content of vitamin E was found in T. suecica biomass produced outdoors in annular columns compared to that observed in laboratory grown cultures of the same species (CarballoCárdenas et al., 2003).

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Numerous Tetraselmis species have been cultivated outdoors, both in open ponds (Materassi et al., 1983; Laws et al., 1986; Pedroni et al., 2004) and pilot-scale bioreactors (Borowitzka, 1997; Pedroni et al., 2004). In the latter systems higher volumetric productivities (up to 1.2 g l− 1 day− 1) have been recorded. This is a positive feature, but it is important to point out that not necessarily a high volumetric productivity translates into high areal productivity and PE. Although, very few direct side-by-side comparisons (Weissmann and Benemann, 2003; Pedroni et al., 2004) of open ponds and photobioreactors have been carried out, the general perception is that closed photobioreactors are more productive (on an areal basis) and efficient than open ponds. Indeed, it has been proved that with some algae, e.g. those that have high temperature optima, productivity and PE are enhanced in photobioreactors (Tredici and Materassi, 1992), but these findings can not be generalized. Recently, Pedroni et al. (2004) compared, over a period of several months, the performance of T. suecica strain OR cultivated in near-horizontal tubular photobioreactors and raceway ponds, using flue gas as carbon source. The two culture systems exhibited similar areal productivities (with maximum values averaging 26 g m− 2 day− 1). The comparison of open ponds or horizontal photobioreactors with elevated (vertical) culture systems has been rarely attempted (Tredici and Chini Zittelli, 1998). One of the reasons is that elevated culture systems are difficult to evaluate in terms of areal productivity. As recently proposed (Tredici, 2004), vertical photobioreactors can be evaluated, and thus compared with ponds or other systems, only in terms of OAP (overall areal productivity). In plants based on vertical culture systems, both the arrangement and the distance between the reactors have profound influence on productivity. Thus, in general, the OAP potentially achievable by a plant made of vertical reactors can not be extrapolated from data obtained with a single unit, but an adequate number of units must be set up and carefully operated avoiding peripheral effects (Tredici, 2004). The measure of PE is also useful for comparison of performances of different photobioreactors outdoors, although it should not be generalized because high efficiencies might be simply the result of low irradiance conditions (Richmond, 2004). With T. tetrathele grown outdoors in mixing-board ponds in Southern Italy, Materassi et al. (1983) achieved a maximum areal productivity of 32 g m− 2 day− 1 in July and an areal productivity of 26 g m− 2 day− 1 in August with PEs of 5.6 and 5.1% (PAR), respectively. In the present work, the areal productivity (OAP) and PE

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measured during August with T. suecica were 40–47 and 82–84% higher, respectively, than those obtained by Materassi et al. (1983). The fact that different species have been used in the two studies, does not allow to draw conclusions. Comparison with the work by Pedroni et al. (2004), who cultivated the same strain used in this study, and in the same period, is more straightforward. The annular columns achieved about 80% higher productivity and were about two times more efficient in converting light energy into biomass than the ponds and the horizontal reactors experimented with by Pedroni et al. (2004). Since any negative influence on growth and productivity of the flue gas used in the latter experiments has to be excluded (Capuano F., personal communication), the better performance of the annular columns should be ascribed to the vertical nature of this system. We think that the superiority of the annular column over horizontal reactors and open ponds, in terms of areal productivity and PE, is related to its capacity to dilute the direct solar radiation impinging on its vertical, curved surface. As demonstrated, light dilution reduces the negative effects of photosaturation and photoinhibition, leading to significant increases of PE and productivity (Tredici and Chini Zittelli, 1998). Besides, the rear surface of a vertical reactor receives only dispersed (diffuse and reflected) radiation of low intensity, which is known to be used with high efficiency (Hu et al., 1996). The mean PPFD values measured on the annular columns during a typical summer day showed that, compared to a horizontal surface, the frontal directly illuminated surface of the column is exposed to lower irradiances. In particular during the central daylight hours, when the negative effect of excessive light upon outdoor cultures is maximal, the mean radiation incident on the annular columns undergoes a ca twofold dilution compared with that impinging on the horizontal. Besides, while the irradiance on the horizontal draws the well known bell-shaped curve centred at midday, the culture in the column is exposed to a more homogeneous light environment (on average 900– 1000 μmol photons m− 2 s− 1) for most of the day. However, the main advantage of this system (i.e., that around solar noon a large part of the direct radiation is reflected given that it strikes the column surface with a large incidence angle) can be seen also as a limitation, since it leads to significant losses of useful light. These losses can be reduced in a full-scale plant if the units are closely arranged so that most of the photons reflected by the reactors walls (and by the ground) are intercepted again by the reactors in the vicinity. The performance of vertical reactors in any kind of full-scale plant will depend on the number of units

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deployed on a given area and the type of arrangement. Both these factors influence light distribution on the reactor surface and thus reactor productivity. Obviously, reactors can not be placed at a great distance from each other so as to avoid self-shading because, even if this arrangement maximises productivity per reactor, it will lead to negligible areal yields. When the reactors are placed as close as possible to maximise areal productivity yet allowing operations, it is expected that, as consequence of self-shading, reactor productivity will be significantly reduced in comparison with that obtained by an isolated un-shaded unit. The closer the reactors, the higher will be the decrease in productivity per reactor. It was thus unexpected the observation made in this work, with the columns placed at the vertices of equilateral triangles, that discontinuous, short-lasting shading of the culture in the full-scale column did not decrease its productivity, and, even if the PE of the full-scale column was underestimated, the culture in this reactor attained a 6% higher PE compared to that in the isolated one (10.6 and 10.0%, respectively). This is an aspect worth of further study. It is also interesting that the PE of the whole plant (9.4%) did not show a large decrease in comparison with that attained by the single full-scale column, suggesting that part of the light that reached the ground was reflected and intercepted again. Our results indicate that in a full-scale plant made of vertical reactors it is not profitable spacing the units by at least the maximum extent of the shadow in winter to assure that the reactors are never mutually shaded and the maximum productivity is obtained year round by each unit, as previously suggested by Sánchez Mirón et al. (1999). This type of arrangement will necessarily lead to low overall areal productivity and inefficient use of land. A large number of columns must be placed on a given ground area if we want to improve the efficiency of land use. Moreover, having a large number of reactors offers advantages, such as flexibility, possibility to grow different strains and vary culture conditions to optimise growth, and capacity to improve product quality by disposing of the cultures in worst conditions. These advantages may somewhat compensate the much higher complexity and cost. The small size and a relatively high energy requirement for mixing discourage the use of the annular column in large scale production of algae biomass. These limitations are, however, of minor importance in fish and mollusc hatcheries given their absolute need for a sustained production of high quality biomass and the very high cost of microalgae aquaculture feeds (Benemann, 1992).

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