a modular flat panel photobioreactor (mfpp) for indoor mass culti
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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 mario.tredici@unifi.it)
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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