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Journal of Applied Phycology 12: 521526, 2000. 2000Kluwer Academic Publishers. Printed in the Netherlands.

521

A Modular Flat Panel Photobioreactor (MFPP) for indoor masscultivation ofNannochloropsissp. under artificial illumination

Graziella Chini Zittelli, Roberta Pastorelli & Mario R. Tredici

Dipartimento di Biotecnologie Agrarie, Universita di Firenze and Centro di Studio dei Microrganismi Autotrofi,

CNR, P.le delle Cascine 24, 50144 Firenze, Italy

(Author for correspondence; e-mail [email protected])

Received 1 February 2000; revised 20 May 2000; accepted 20 May 2000

Key words:eicosapentaenoic acid, fatty acid profile, Modular Flat Panel Photobioreactor, Nannochloropsissp.

Abstract

Nannochloropsissp. was grown in a Modular Flat Panel Photobioreactor (MFPP) consisting of six alveolar panelseach with 20.5 L culture volume and 3.4 m2 illuminated surface area. The panels formed a closely-packed unit

with illumination provided by banks of fluorescent tubes placed between the panels. The whole unit was contained

in a thermoregulated cabinet. Continuous illumination of one side of the panels with 115 mol photon m2 s1

attained a mean volumetric productivity of 0.61 g (d. wt) L1 24 h1, increasing to 0.97 g (d. wt) L1 24 h1

when the same irradiance was provided on both sides of the panels. With 230 mol photon m2 s1 on one side

of the panel, a mean productivity of 0.85 g (d. wt) L1 24 h1 was achieved, which reached 1.45 g (d. wt) L1 24

h1 when both sides were illuminated. Increasing the amount of light provided to the culture (either by increasing

irradiance or the illuminated surface area) decreased pigment and enhanced the total fatty acid content, but did

not change significantly the content of eicosapentaenoic acid. A MFPP of the present dimensions could produce

sufficient microalgae to support a hatchery producing 6 million sea bream fingerlings annually.

Introduction

Nannochloropsis is a marine microalga cultivated in

many fish hatcheries as the basis of an artificial food

chain system (Zmora et al., 1993; Lubzens et al.,

1995). The nutritional value ofNannochloropsis for

rotifers and fish larvae is related to its biochemical

composition, especially its lipid and fatty acid content.

Due to its high content of eicosapentaenoic acid (EPA,

20:5n3), Nannochloropsis has also been proposed as

a potential source of this important polyunsaturated

fatty acid for human consumption (Sukenik, 1998;Chini Zittelli et al., 1999).

Mass cultivation ofNannochloropsisis at present

carried out in fish hatcheries in 100 to 150-L poly-

ethylene bags or 200 to 500-L transparent glass-fibre

cylinders, generally used indoors with artificial light,

and different kinds of large open ponds and tanks,

usually kept outdoors under sunlight (Fulks & Main,

1991; Hoffman, 1999). These culture systems suffer

from several limitations, among which the lack of con-

trol of culture parameters, in particular irradiance and

temperature, the low cellular concentration attainable

and the difficulty of preventing contamination are the

most important (Tredici, 1999).

Recently, Zou & Richmond (1999) and Chini Zit-

telli et al. (1999) have demonstrated the feasibility of

outdoor cultivation ofNannochloropsis sp. in closed

reactors, obtaining much higher volumetric productiv-

ities and cell densities than those achieved by the

traditional systems. Outdoor culture, however, is not

always feasible, particularly in temperate climates

during the winter, due to low solar irradiances and

temperatures. Yet, it is during the winter that require-

ments forNannochloropsisbiomass by fish hatcheries

are highest.

Few data are available on the cultivation ofNan-

nochloropsis under artificial light in controlled cul-


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Figure 1. Schematic drawing of the Modular Flat Panel Photobioreactor (MFPP). (a) removable alveolar panel; (b) fluorescent tubes; (c)cabinet; (d) air and CO2 injection point.

ture systems of sufficiently large volumes to provide

hatcheries with the needed amount of biomass. The

present paper reports the ability of a newly designed

Modular Flat Panel Photobioreactor (MFPP), op-

erated under artificial illumination, to provide large

amounts of good qualityNannochloropsisbiomass.

Materials and methods

The Modular Flat Panel Photobioreactor

The Modular Flat Panel Photobioreactor (MFPP) con-

sisted of six removable alveolar panels placed vertic-

ally back to front and 24 cm apart, forming a closely-packed unit. The alveolar panels were 108 cm wide

165 cm high, with an internal thickness of 1.2 cm.

The total working volume of the MFPP was 123 L

and the total illuminated surface area 20.4 m2. Mixing

and degassing of the culture suspension were achieved

by bubbling compressed air at the base of each panel

through a perforated plastic tube. Alveolar panels are

described in detail in Tredici et al. (1991), Tredici

& Materassi (1992) and Tredici (1999). Illumination

was provided by banks of twelve day-light fluores-

cent tubes (Osram L 58 W/11-860) placed between

the panels. The lighting device allowed illumination

of the reactors from one or both sides at different ir-

radiance levels depending on the number of lamps in

operation. The six panels and the banks of fluores-

cent tubes were contained in a thermoregulated white

painted cabinet. A schematic drawing of the MFPP is

shown in Figure 1.

Organism and culture conditions

The marine eustigmatophyte Nannochloropsis sp.used in the experiments was obtained from Dr. Zmora

of the National Center for Mariculture, Israel Ocean-

ographic and Limnological Research (Eilat, Israel).

Nannochloropsiscultures were grown in artificial sea-

water at 33 g L1 salinity (Adriatic Sea Equipment,

Forl, Italy), enriched with F/2 medium nutrients

(Guillard & Ryther, 1962). NaNO3 and NaH2PO4


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were added to the cultures when required in order

to prevent nutrient limitation. The artificial seawa-

ter was filtered through 1.5-m polypropylene filters

(Domnick Hunter, UK) and then integrated with sterile

nutrient solutions.

The air injected into the reactors was filtered

through 0.2-m filters (Sartorius, Goettingen, Ger-many). Pure CO2 from cylinders was added continu-

ously to the air stream (3% v/v) to provide carbon

to the culture and regulate pH at 7.5 0.2. Air-flow

rate was maintained at 0.5 L L1 min1; gas hold-up

was about 3.3%. The cultures were kept at the optimal

growth temperature of 25 1 C by an air condi-

tioner that automatically introduced cooled air into

the cabinet. Two different irradiance levels (115 and

230 mol photon m2 s1) and both single-side and

two-side illumination were tested. A semicontinuous

harvesting regimen was adopted to maintain cell con-

centration in the optimal range, which was: 2.63.2 g

(d. wt) L1 for single-side illumination with 115molphoton m2 s1, 3.14.0 g (d. wt) L1 for single-

side illumination with 230 mol photon m2 s1 and

two-side illumination with 115 mol photon m2 s1,

and 3.85.0 g (d. wt) L1 for two-side illumination

with 230mol photon m2 s1. Culture samples were

collected weekly and lyophilised for the biochemical

analysis. Each trial lasted one month after steady-state

conditions had been achieved.

Analytical procedures

Growth of the cultures was estimated by measure-ment of the dry biomass concentration. For dry

weight determination, triplicate culture samples (2

5 mL) were diluted (1:10) with distilled water and

filtered through pre-weighed 1.2-m glass-fibre fil-

ters (Whatman GF/C; 47 mm). The filtered cells were

washed with distilled water (25 mL) and dried at

105 C to constant weight. Irradiance (PAR) was

measured using a Li-Cor quantum sensor (Model LI-

190 SB, Li-Cor, Inc., Lincoln, NE) connected to a

quantum/radiometer/ photometer (Model LI-185 B).

The efficiency of light conversion was calculated as

the ratio between the mean reactor productivity (g d.wt reactor1 24 h1) and the mean photosynthetic

photon flux intercepted by the reactor (mol photon

reactor1 24 h1), corrected for the absorbance by

the reactors walls. It has the unit of gram of bio-

mass per mole of photons (g mol photon1). The Chl

aand total carotenoid content was estimated spectro-

photometrically following the procedure described by

Parson and Strickland (1963). For fatty acid determ-

inations, the cells were harvested by centrifugation

and aliquots of the paste obtained were lyophilised

and analysed. Fatty acid analyses were performed as

previously described (Chini Zittelli et al., 1999).

Results

The productivities attained by Nannochloropsis sp.

grown in the MFPP under the different light condi-

tions are reported in Table 1, together with the light

conversion efficiencies and the pigment content of the

biomass. When one side of the panels was illuminated

with 115 mol photon m2 s1, the mean volumet-

ric productivity was 0.61 g (d. wt) L124 h1 and

increased to 0.85 g (d. wt) L124 h1 when the irradi-

ance was doubled. When illumination was provided on

both sides, the culture productivity rose to 0.97 g (d.

wt) L1 24 h1 at 115 mol photon m2 s1 and to

1.45 g (d. wt) L1 24 h1 at 230 mol photon m2

s1. The efficiency of light conversion was 0.80 g

(d. wt) mol photon1 when the panel was illuminated

from one side with the lower irradiance and decreased

at the higher irradiance and with two-side illumina-

tion (Table 1). Although the cultures illuminated with

115 mol photon m2 s1 from both sides and those

illuminated with 230 mol photon m2 s1 from one

side received the same amount of photons per unit

time, the former showed a somewhat (14%) higher

productivity, and hence higher light conversion ef-

ficiency, and a greatly reduced pigment content (by31.6% for Chl a and by 12.6% for carotenoids). Pig-

ments decreased markedly also when two-side instead

of one-side illumination was provided at both irra-

diances, and when the irradiance was doubled with

two-side illumination. The pigment decrease was less

marked when the irradiance was doubled with one side

illumination (Table 1).

The fatty acid content and profile of Nanno-

chloropsis sp. was also greatly influenced by irradi-

ance levels and type of illumination (Table 2). The in-

crease of irradiancefrom 115 to 230 molphoton m2

s

1

caused an increase of total fatty acids (TFA) byone third: from 14.7 to 19.6% of d. wt when illu-

mination was provided from one side, and from 24.3

to 32.5% of d. wt when illumination was provided

from both sides. An increase of one fifth (from 19.6 to

24.3% of d. wt) was observed when, without varying

the total amount of photons provided to the culture,

illumination was changed from 230 mol photon m2


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Table 1 . Volumetric productivity, efficiency of light conversion and pigment content ofNannochloropsissp.cultivated in the MFPP under different light conditions. One-side and two-side illumination was providedat 115 or 230 mol photon m2 s1. Mean values ( s.d.; n = 30 for volumetric productivity; n = 8 forpigment content) are shown

Irradiance and type of illumination

115 mol photon m2 s1 230 mol photon m2 s1

From one side From two sides From one side From two sides

Volumetric productivity

(g L1 24 h1) 0.61 0.13 0.97 0.13 0.85 0.19 1.45 0.27

Efficiency of light conversion

(g mol photon1) 0.80 0.17 0.64 0.10 0.56 0.13 0.48 0.10

Pigment content

(% d. wt)

Chla 3.54 0.86 2.01 0.37 2.94 0.76 1.37 0.22

Carotenoids 0.96 0.24 0.76 0.05 0.87 0.28 0.48 0.07

Table 2. Fatty acid content (% d.wt) of Nannochloropsis sp. grown in the MFPP under different lightconditions. One-side and two-side illumination was provided at 115 or 230 mol photon m2 s1. Mean

values ( s.d.; n = 4) are shown. Only major fatty acids ( 0.3%) are reported

Fatty acid Irradiance and type of illumination

115 mol photon m2 s1 230 mol photon m2 s1

From one side From two sides From one side From two sides

14:0 0.83 0.21 1.67 0.11 1.14 0.25 1.95 0.33

16:0 4.74 1.25 10.6 0.63 7.45 0.92 15.2 0.52

16:1n7 4.02 0.49 6.45 0.59 5.71 0.45 9.01 0.75

18:1n9 0.71 0.32 1.60 0.17 1.09 0.19 2.04 0.64

18:2n6 0.48 0.05 0.38 0.04 0.35 0.03 0.37 0.04

20:4n6 0.78 0.17 0.85 0.11 0.67 0.06 0.86 0.05

20:5n3 2.72 0.44 2.03 0.18 2.64 0.15 2.28 0.33

TFA 14.7 2.16 24.3 1.27 19.6 1.75 32.5 1.26

s1on one side to 115 mol photon m2 s1 on

two sides. Increasing illumination from 115 mol

photon m2 s1 on one side to 230 mol photon m2

s1 on two sides caused a doubling of the TFA content.

These increases in TFA were due to the increases of

saturated and monounsaturated fatty acids (14:0, 16:0,

16:1n7 and 18:1n9) that are mainly associated with

storage lipids (triacylglycerols). The content of 20:4n6

and of EPA changed less significantly with light con-

ditions, EPA content varying in the range 2.02.7% ofthe d. wt.

Discussion

The MFPP has been successfully used to cultivate

Nannochloropsis sp. with artificial light, and has

shown the potential to overcome the limitations en-

countered in traditional culture systems. A single

123-L MFPP, like the present one, can produce about

180 g d. wt of high-qualityNannochloropsisbiomass

per day, the requirements of a hatchery producing 6

million sea bream fingerlings annually (G. Cassizzi,

pers. comm.). Due to the high surface-to-volume ratio

of the single panels, the cell concentration at harvest-

ing and the volumetric productivities (as high as 7 g

(d. wt) L1 and 1.45 g (d. wt) L1 24 h1 withtwo-side illumination at 230 mol photon m2 s1)

achievable in the MFPP are 15 and 30 times higher

than those attained with Nannochloropsis in 200 L

fiber glass cylinders under artificial light (James &

Al-Khars, 1990). This means a substantial saving in

terms of culture operation, culture medium and space.


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Besides, when two-side illumination is applied, the

MFPP can take advantage of the spatial light dilution

effect (Tredici & Chini Zittelli, 1998). In fact, two-

side illumination compared with one-side illumination

of double irradiance reduces mutual shading and in-

creases the efficiency of light conversion. That spatial

light dilution causes more light to be available to thesingle cell is demonstrated by the higher productivity

and lower pigment content that characterise cultures

illuminated from both sides. The effect of increasing

illumination from either one or both sides, decreases,

as expected, light conversion efficiency, but is made

up by increasing productivity.

Sukenik et al. (1989) have shown that Nanno-

chloropsis sp. grown in the laboratory under saturating

light conditions is characterised by a high content of

lipids and fatty acids as compared to cells grown in

light limiting conditions. The increase in lipid content

coincided with a reduction in the percentage of EPA

(mainly associated with galactolipids), and with an in-crease in the relative abundance of 16:0 and 16:1n7

that are associated with triacylglycerols (Sukenik et

al., 1993). Similarly, Renaud et al. (1991) showed

an increasing saturation of the fatty acids ofN. ocu-

lata with increasing irradiance in large scale outdoor

cultures. On the contrary, Seto et al. (1992) reported

that high light intensity has no effect on the EPA dis-

tribution and content. The experiments carried out in

the MFPP confirm that increasing the amount of light

provided to the culture, by increasing irradiance level

or the illuminated surface area, causes an increase of

the TFA content (that accumulate up to 33% of the d.wt) due to the increase of saturated and monounsat-

urated short chain fatty acids. However, in agreement

with Seto et al. (1992), we have not found a signi-

ficant influence of the irradiance level on the content

of polyunsaturated fatty acids (20:4n6 and EPA). Thus

an energy-rich biomass (33% TFA) and with a good

EPA content (about 2.3%) can be produced under

conditions that also lead to high productivity.

Compared to other systems for microalgae cul-

ture (e.g. that operated by James and Al-Khars), the

MFPP provides a very efficient utilisation of light

since photons do not escape from the system and are

very efficiently conveyed to the culture. Assuming an

electricity cost of 0.08 US$ per kWh, the cost of arti-

ficial illumination can be estimated at 3045 US$ per

kg ofNannochloropsissp. dry biomass. This figure is

relatively low if we consider the total production cost

of algal biomass in hatcheries (Benemann, 1992).

Acknowledgements

We are indebted to Francesco Favilli for his excellent

technical help in the construction of the Modular Flat

Panel Photobioreactor. This work was partly supported

by Exenia Group S.r.l. (Milano, Italy).

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a modular flat panel photobioreactor (mfpp) for indoor mass culti

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