fermentation

Article
Optimization of Arthrospira platensis (Spirulina)
Growth: From Laboratory Scale to Pilot Scale
Florian Delrue 1, * ID , Emilie Alaux 1 , Lagia Moudjaoui 1 , Clément Gaignard 1 , Gatien Fleury 1 ,
Amaury Perilhou 2 , Pierre Richaud 3 , Martin Petitjean 4 and Jean-François Sassi 1
1 Commissariat à l’Energie Atomique et aux Energies Alternatives (CEA Cadarache), Groupe Biomasse 3G,
F-13108 Saint-Paul-lez-Durance, France; E.alaux@hotmail.fr (E.A.); L.moudjaoui@laposte.net (L.M.);
Clement.gaignard@hotmail.com (C.G.); Gatien.fleury@cea.fr (G.F.); Jean-Francois.SASSI@cea.fr (J.-F.S.)
2 Microphyt, 713 Route de Mudaison, F-34670 Baillargues, France; Amaury.perilhou@microphyt.eu
3 Commissariat à l’Energie Atomique et aux Energies Alternatives (CEA Cadarache), Centre National de la
Recherche Scientifique, Aix Marseille Université, UMR7265, Institut de Biosciences et Biotechnologies
Aix Marseille, F-13108 Saint-Paul-lez-Durance, France; Pierre.richaud@cea.fr
4 Spiruline de la côte bleue, Les fortunés, Route des Bassins, F-13180 Gignac-la-Nerthe, France;
Spirulinedelacotebleue@gmail.com
* Correspondence: florian.delrue@cea.fr; Tel.: +33-4-42-25-72-22

Received: 12 October 2017; Accepted: 2 November 2017; Published: 7 November 2017

Abstract: Arthrospira platensis (Spirulina) is the most cultivated microalga worldwide. Improving its
cultivation in terms of biomass productivity, quality, or production cost could significantly
impact the Spirulina industry. The objectives of this paper were defined as to contribute to this
goal. Spirulina biomass productivity was investigated through medium choice. A modified
Zarrouk’s medium was selected as it gave higher final dry weights and longer sustained growth
than Hiri’s and Jourdan’s media. Then, in order to reduce Spirulina production cost, modified
Zarrouk’s medium was rationalized by testing different dilutions. It was found that modified
Zarrouk’s medium could be diluted up to five times without impacting the growth rates in
a 28-days batch cultivation. Higher dry weights were even observed after 21 days of batch
cultivation (1.21 g/L for 20%-modified Zarrouk’s medium in comparison to 0.84 g/L for modified
Zarrouk’s medium). Iron uptake was then investigated as one of the major contributors to Spirulina
nutritional quality. An increase in iron content was obtained by replacing iron sulfate by iron EDTA
at a concentration of 10 mgFe /L (2.11 ± 0.13 mgFe /gbiomass for EDTA-FeNa,3H2 O at 10 mgFe /L
compared to 0.18 ± 0.13 for FeSO4 ,6H2 O at 2 mgFe /L). Impact of light intensity on Spirulina
biomass productivity was also investigated in a 2 L Photobioreactor (PBR). Specific growth rates
were calculated for Photosynthetically Photon Flux Densities (PPFD) from 85 to 430 µmol/m2 /s.
At 430 µmol/m2 /s, photoinhibition was not observed and the specific growth rate was maximum
(0.12/day). Finally, a 40-day cultivation experiment was conducted in a 1000 L PBR giving a maximum
daily areal productivity of 58.4 g/m2 /day. A techno-economic analysis gave production cost two
to 20 times higher for PBR (from 18.71 to 74.29 €/kg) than for open ponds (from 3.86 to 9.59 €/kg)
depending on Spirulina productivity.

Keywords: Spirulina cultivation; iron content; light intensity; medium rationalization

1. Introduction
Arthrospira platensis is an aquatic, filamentous cyanobacterium which is often classified as a
blue/green microalga. The common name of its commercialized biomass is “Spirulina” (the name used
in this paper) which production makes this microorganism to be the most cultivated worldwide [1].
The Spirulina production has been estimated to be between 3000 and 20,000 tons/year.

Fermentation 2017, 3, 59; doi:10.3390/fermentation3040059 www.mdpi.com/journal/fermentation

Fermentation 2017, 3, 59 2 of 14

In 2014, in France, 105 Spirulina farmers were registered in the association “Fédération des
Spiruliniers de France”. Spirulina is considered to be a nutraceutical due to its high nutritional quality
(proteins, essential amino acids and fatty acids, polysaccharides, carotenoids, vitamins, and iron) [2].
Spirulina is almost exclusively produced in open ponds which are low-cost, and easy to build and
operate. However, the difficulty of these systems are the low biomass productivity [3,4], less than
15 g/m2 /day, the difficultly to maintain optimal cultivation parameters, their high evaporation rates
and their weakness towards contamination [5]. However, Arthrospira platensis is a cyanobacterium
growing at elevated pH (9.5 to 11.0 with an optimum at 10.5 [6]) and is, therefore, less subject to
contaminations [7].
Cultivation medium has a great impact on the productivity of biomass and other compounds
of interest. For example, nitrogen concentration in the medium [8] (optimum at 2.5 g/L) and also
nitrogen source (urea better than ammonium or nitrate) [9] has a great effect on Spirulina productivity.
Additionally, a phosphate concentration of 250 mg/L in the form of K2 HPO4 was found to optimize
biomass production [10]. A study showed that Zarrouk’s medium (ZM) was the best medium in terms
of biomass productivity, while modified Blue-Green 11 medium (BG11) gave the highest chlorophyll,
carotenoid, phycocyanin, and allophycocyanin contents [11]. The authors also observed the maximum
content of phycoerythrin in synthetic human urine (SHU) medium. However the differences observed
by the authors were not significant (less than 10% for most of the results for experiments not performed
in triplicates). ZM was also selected over 5 other media (Rao’s, CFTRI, OFERR, revised media,
and Bangladesh medium No. (3) for its higher Spirulina biomass productivity [12]).
Three media from the literature were selected in this study: Zarrouk’s [13], Hiri’s [14],
and Jourdan’s [15] media (ZM, HM, and JM, respectively). They were compared based on their
biomass productivity. Spirulina media contain generally high concentrations of nutrients which impact
their cost. Media with reduced cost can be as effective as ZM in terms of final biomass concentration,
chlorophyll and protein content [16]. Reduction of the medium cost by dilution with ultrapure water
was then tested.
Initial biomass concentration was previously described to have an effect on biomass productivity
for the microalga Haematococcus pluvialis [17]. An initial biomass concentration of 0.5 g/L was found
to optimize asthaxanthin productivity. The influence of the initial biomass concentration in a batch
cultivation on the growth of Spirulina was also studied.
Spirulina media have generally low iron concentration (2 mgFe /L for ZM and HM, and 0.2 mgFe /L
for JM). Using typical iron content value of 1 mgFe /g [18], maximal theoretical biomass concentrations
are only 0.2, 2, and 2 g/L for JM, ZM, and HM, respectively (assuming that iron is always bio-available
for Spirulina). Iron is a very important element in human nutrition since anemia is the most common
food deficiency concerning two billions people worldwide [19]. Despite low iron concentrations
in its cultivation media, Spirulina contains high amount of iron [20] (0.58–1.8 g/kgbiomass ).
Therefore, an experiment was designed to increase the Spirulina’s iron content by using higher
iron concentrations in the medium and by using two different sources of Fe-EDTA.
Light intensity has a great impact on Spirulina productivity. Two independent studies found that
Spirulina final biomass concentration productivity was the highest at the highest photosynthetically
photon flux density (PPFD) they used (around 60 µmol/m2 /s) [10,21]. Light saturation was not
reached; an increase in biomass productivity could certainly be achievable with PPFD higher than
60 µmol/m2 /s. The effect of PPFD) from 85 to 430 µmol/m2 /s on the growth of Spirulina was,
therefore, studied.
Finally, a 40-day cultivation run was performed in a 1000 L-photobioreactor (PBR) during
spring 2015 for evaluating the possibility of growing Spirulina in PBRs in order to improve Spirulina
biomass productivity.
Fermentation 2017, 3, 59 3 of 14

2. Materials and Methods

2.1. Strain
Arthrospira platensis from Paracas (Peru, strain no. 14067 from Limnologie, Rennes, France) was
used in this study. It was selected for its higher growth rates (data not shown) in comparison to
another Arthrospira platensis strain (Arthrospira platensis from Lonar, India, strain no. 14039, Limnologie,
Rennes, France).
Modified versions of three media designed for Spirulina growth were used in this study: JM,
HM, and ZM. Their composition is shown in Table 1. A modification of these media were done for
comparison purposes and practical reasons. The same trace elements solution was used for these three
media: the Hutner’s solution without iron [22]. Its composition is: 50 mg/L of EDTA; 11.4 mg/L of
H3 BO3 ; 22 mg/L of ZnSO4 ,7H2 O; 5.06 mg/L of MnCl2 ,4H2 O, 1.61 mg/L of CoCl2 ,6H2 O; 1.57 mg/L of
CuSO4 ,5H2 O; and 1.1 mg/L of Mo7 O24 (NH4 )6 ,4H2 O. These minerals were diluted in ultrapure water
(Purelab Ultra, Veolia Water STI, Le Plessis Robinson, France).
Fe-EDTA solutions were obtained from Akzo Nobel (Dissolvine® E-Fe-13 with 13% of
EDTA-FeNa,3H2 O, Amsterdam, The Netherlands) and Plantin (Ferro 8 with 8% of EDTA-FeNH4 ,
Courtezon, France).

Table 1. Modified ZM, HM, and JM composition.

Chemicals Modified ZM [13] (g/L) Modified HM [14] (g/L) Modified JM [15] (g/L)
NaHCO3 16.8 16 8
NaCl 1 0 1
(NH4 )3 PO4 0 0.1 0.2
MgSO4 ,6H2 O 0.2 0.1 0.2
FeSO4 ,6H2 O 0.01 0.01 0.001
K2 SO4 1 0.5 1
CaCl2 ,2H2 O 0.04 0.1 0.1
CH4 N2 O 0 0.1 0.009
KNO3 0 0 1
NaNO3 2.5 0 0
K2 HPO4 0.5 0 0
Hutner’s solution without iron 1 mL 1 mL 1 mL
Major Elements
Modified ZM [13] (mg/L) Modified HM [14] (mg/L) Modified JM [15] (mg/L)
Concentrations
Carbonate CO3 2− 12,000 11,430 5710
Nitrogen N 412 47 143
Phosphorus P 89 23 46
Sulfur S 238 120 237

2.2. Culture Conditions

Spirulina cultures were grown in incubation shakers (HT Multitron Pro from Infors, Baar,
Switzerland) which were set to 120 RPM, 32 ◦ C, and 11 µmol/m2 /s as PPFD.
Lab-scale 2 L-photobioreactors from Diachrom Biotechnology (Bottmingen, Switzerland) were
used for assessing the effect of light intensity on the growth of Spirulina. Temperature was controlled
at 32 ◦ C and agitation was set to 50 RPM. Light was delivered by 4000 K white LEDs (LCW 45M
from Osram, Munich, Germany). Light intensities were measured using a light meter ULM-500 and a
Spherical Micro Quantum Sensor US-SQS/L both from Walz (Effeltrich, Germany). PPFD varied from
85 to 430 µmol/m2 /s (volumetric average value on the whole reactor filled with ultrapure water).
Spirulina was also cultivated in a 1000 L tubular Camargue PBR from Microphyt [23] located
in a greenhouse. The PBR, oriented north-south, consists of a 240 m piping serpentine glass circuit,
with a 76 mm inside diameter and 4.5 mm wall thickness, folded horizontally in 24 straight runs with
a vertical height of 3 m and a width of 0.3 m, forming a 10 m long tubular fence. The 1000 L Camargue
PBR has a specific area of 8.84 m2 /m3 .
Fermentation 2017, 3, 59 4 of 14

2.3. Growth Data

The dry weight was measured after desiccation on pre-weighed filters with a porosity of 0.7 µm
(Sartorius Stedim, Göttingen, Germany): 10 mL of Spirulina cultures were filtered and rinsed twice
with the same volume of ultrapure water. The prefilters were maintained at 80 ◦ C overnight in a
ventilated oven.
Optical density was measured at 880 nm (OD880 ) in order to reduce the influence of the pigments
absorption (mainly chlorophylls and phycocyanin) with an Epoch 2 Microplate Spectrophotometer
from BioTek Instruments, Inc. (Winooski, VT, USA).
Productivities and nutrient consumption rates were calculated using the following formula, with t
being the time and At being the dry weight or the nutrient concentration:

A t2 − A t1
Rate of At1 →t2 =
t2 − t1

2.4. Biomass Analyses

Iron content was measured using inductively-coupled plasma (ICP) coupled with atomic emission
spectrometry (AES) using an ICP-AES Vista MPX (Agilent Technologies, Santa Clara, CA, USA).
Fifty milliliters of Spirulina culture were centrifuged at 4750 rpm, 4 ◦ C for 10 min. The iron content
in the supernatant was measured. Then, the pellet was washed twice with 15 mL of a 10 mM EDTA
solution. The resulting 2 × 15 mL (separated by centrifugation) was analyzed for its iron content
from which the adsorbed iron content of the biomass could be deducted. Finally, the washed pellet
was hydrolyzed for 24 h at 80 ◦ C in 1 mL of 70% nitric acid (ICP grade, JT Baker) and then diluted
to 6 mL with ultrapure water. The hydrolysate was analyzed for its iron content from which the
internalized iron content of the biomass could be deducted. The iron concentrations were determined
using standard curve obtained by analyzing ICP grade iron standard solutions.
The C/N ratio was determined using an organic elemental analyzer (Thermo Scientific FLASH™
2000 CHNS/O, Thermo Fisher Scientific, Waltham, MA, USA).

3. Results and Discussion

3.1. Culture Media Comparison

Media conventionally used in lab-scale experiments and by Spirulina producers were compared
in triplicates, i.e., modified ZM, modified HM and modified JM. Pre-cultures were done in each
medium for acclimation. Growth curves are shown in Figure 1. For 10 days, modified ZM and
HM showed similar growth curves with higher optical density than modified JM. This difference
could be a consequence of the higher sodium bicarbonate concentrations in those two media in
comparison to modified JM (twice less sodium bicarbonate). Then, after 10 days, growth continued
for modified ZM but not for modified HM. The higher N, P, and S content in modified ZM could
explain this more sustained growth. Modified ZM also showed a higher biomass productivity
(91.5 ± 4.0 mg/L/day from day 0 to day 13) than modified HM (80.5 ± 1.6 mg/L/day) and
modified JM (77.9 ± 3.4 mg/L/day). Modified ZM was then selected for the cultivation experiments.
These results were in accordance to another study where the use of ZM gave a higher final dry weight
than SHU and modified BG11 media [11].
Fermentation 2017, 3, 59 5 of 14
Fermentation 2017, 3, 59 5 of 14

Figure 1. OD880 of Spirulina for three different media: modified ZM (blue diamonds), modified HM
Figure 1. OD880 of Spirulina for three different media: modified ZM (blue diamonds), modified HM
(red squares) and modified JM (purple triangles), in triplicate. Error bars are the standard deviations of
(red squares) and modified JM (purple triangles), in triplicate. Error bars are the standard deviations
the biological triplicates.
of the biological triplicates.

3.2. Effect of the Initial Biomass Concentration on Spirulina Growth Using Modified Zarrouk’s Medium
3.2. Effect of the Initial Biomass Concentration on Spirulina Growth Using Modified Zarrouk’s Medium
The influence of the initial biomass concentration was tested with modified ZM (no replicate).
The influence of the initial biomass concentration was tested with modified ZM (no replicate).
As shown in Figure 2, there is no significant difference in the final OD880 for all experiments. All growth
As shown in Figure 2, there is no significant difference in the final OD880 for all experiments. All
curves reached their stationary phase after 20 days with similar OD . This was possibly due to
growth curves reached their stationary phase after 20 days with similar880 OD880. This was possibly due
higher growth rates measured for culture experiment started at lower initial concentration (Table 2) as
to higher growth rates measured for culture experiment started at lower initial concentration (Table
this could imply more light availability per cell leading to higher growth rates. In a similar study it
2) as this could imply more light availability per cell leading to higher growth rates. In a similar
was found that higher initial biomass concentrations led to lower growth rates due to the shadowing
study it was found that higher initial biomass concentrations led to lower growth rates due to the
effect [24].
shadowing effect [24].
However, the final biomass concentrations were slightly higher when the initial biomass
However, the final biomass concentrations were slightly higher when the initial biomass
concentration was also higher (Table 2). Similar optical density measurements and different biomass
concentration was also higher (Table 2). Similar optical density measurements and different biomass
concentrations meant that a change occurred in the biomass optical properties due to different
concentrations meant that a change occurred in the biomass optical properties due to different
biomass composition. This should be further investigated as a mean to advantageously modify
biomass composition. This should be further investigated as a mean to advantageously modify the
the biomass composition.
biomass composition.
Table 2. Final dry weights (day 27) and productivity between day 7 and day 15 for different initial dry
Table 2. Final dry weights (day 27) and productivity between day 7 and day 15 for different initial
weights in modified ZM.
dry weights in modified ZM.
Initial
Initial DryDry Weight
Weight (g/L) Final
(g/L) Final DryWeight
Dry Weight (g/L)
(g/L) Productivity between
Productivity between Day
Day7 7and
andDay
Day15 15
(in(in
mg/L/day)
mg/L/day)
0.350.35 2.76
2.76 67.7
67.7
0.350.35 2.93
2.93 67.5
67.5
0.5 2.99 63.5
0.5 2.99 63.5
0.7 3.07 62.5
0.7 1 3.07
3.40 62.5
53.2
1 3.40 53.2
Fermentation 2017, 3, 59 6 of 14
Fermentation 2017, 3, 59 6 of 14

Figure 2. OD880 measurements of Spirulina for four different initial dry weights in modified ZM: 0.35 g/L
Figure 2. OD880 measurements of Spirulina for four different initial dry weights in modified ZM: 0.35
(orange diamonds and red squares, duplicate), 0.5 g/L (green triangles), 0.7 g/L (purple diamonds),
g/L (orange diamonds and red squares, duplicate), 0.5 g/L (green triangles), 0.7 g/L (purple
and 1 g/L (blue circles). No replicates.
diamonds), and 1 g/L (blue circles). No replicates.

3.3. Cost
3.3. Cost Optimization
Optimization of
of Modified
Modified Zarrouk’s
Zarrouk’s Medium
Medium Composition
Composition
Modified ZM
Modified ZMgavegavebetter
bettergrowth
growth than the other
than two media
the other two mediatested tested
but it contains higher amounts
but it contains higher
of various chemicals leading to significant increased costs. The nitrogen
amounts of various chemicals leading to significant increased costs. The nitrogen content content of modified ZM can of
theoretically lead to a maximal biomass concentration of 4.6 g/L for
modified ZM can theoretically lead to a maximal biomass concentration of 4.6 g/L for Spirulina (ifSpirulina (if nitrogen was the
only limiting
nitrogen was theelement). This would
only limiting be 74,
element). 31, would
This and 395 beg/L for and
74, 31, phosphorus,
395 g/L for sulfur, and potassium,
phosphorus, sulfur,
respectively.
and potassium, These calculationsThese
respectively. were based on the elemental
calculations were based analyses
on theofelemental
three Spirulinaanalysesstrains
of [25].
three
Therefore, dilution
Spirulina strains [25]. of modified ZM was investigated in order to reduce the cost of the cultivation.
A growth experiment
Therefore, dilutionwas of conducted
modified ZM using was 100%-, 50%-, and
investigated in 20%-modified
order to reduce ZMthe (diluted
cost ofusing
the
ultrapure water), in triplicate. Pre-cultures were done in each medium
cultivation. A growth experiment was conducted using 100%-, 50%-, and 20%-modified ZM (diluted for acclimation. It was found
that the
using 20%-modified
ultrapure water),ZM in showed
triplicate.thePre-cultures
best growthwere (OD880 readings),
done in eachquitemedium similar
forto the 50%-modified
acclimation. It was
found that the 20%-modified ZM showed the best growth (OD880 readings), quiteexcept
ZM (Figure 3). The same behavior was found for the dry weight curves (Figure 4), similarthetodrop
the
observed with the 20%-modified ZM at the end of the cultivation. The
50%-modified ZM (Figure 3). The same behavior was found for the dry weight curves (Figure 4), decrease of the dry weight was
found to
except theoccur
dropearlier
observed (around
withdaythe 20 in comparison
20%-modified ZMtoatday
the23endfor of
optical densities) and
the cultivation. Thetodecrease
be steeper.
of
The dry
the decline
weightphasewas is found
indeedtodetected earlier(around
occur earlier in dry weight
day 20than in optical density
in comparison to day measurements.
23 for optical
Cell fragments
densities) and to may still be accounted
be steeper. The decline in optical
phase isdensity
indeedmeasurements
detected earlier since theyweight
in dry still have
than absorbance,
in optical
but will pass through the 0.7 µm glass-fiber prefilters and not be accounted
density measurements. Cell fragments may still be accounted in optical density measurements for in the dry weight.
since
they The
still average of daily growth
have absorbance, ratespass
but will fromthrough
day 0 to theday 0.7
15 was
µm higher for 20%
glass-fiber and 50%-modified
prefilters and not be
ZM (0.145 and
accounted 0.142/day,
for in respectively) than for 100%-modified ZM (0.094/day). Similarly, the average
the dry weight.
biomass productivity from
The average of daily growth day 0 to dayfrom
rates 21 wasday found to be15higher
0 to day for 20%
was higher forZM20% thanandin50%-modified
the two other
media
ZM (Tableand
(0.145 3). However,
0.142/day,when calculatedthan
respectively) fromfor day100%-modified
0 to day 24, the ZM average biomass productivity
(0.094/day). Similarly, the is
lower for the 20%-modified ZM than for the 50%- and 100%-modified
average biomass productivity from day 0 to day 21 was found to be higher for 20% ZM than in the ZM. In continuous cultivation
experiments
two other media (concentration of 0.4 g/L,when
(Table 3). However, media renewal rate
calculated frombetween
day 0 to40% dayand 24, 60% and duration
the average biomass of
productivity is lower for the 20%-modified ZM than for the 50%- and 100%-modified ZM. Ina
60 days), Radmann et al. also found higher growth rates for a 20% ZM (0.138/day) in comparison to
undiluted ZM
continuous (0.134/day)
cultivation [26].
experiments (concentration of 0.4 g/L, media renewal rate between 40% and
60% and duration of 60 days), Radmann et al. also found higher growth rates for a 20% ZM
(0.138/day) in comparison to a undiluted ZM (0.134/day) [26].
Fermentation 2017,3,3,59
Fermentation2017, 59 77 of
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14
Fermentation 2017, 3, 59 7 of 14

Figure 3. OD880 measurements of Spirulina grown in 100% (blue diamonds), 50% (red squares), and
Figure 3. OD
OD880
880 measurements
measurements ofSpirulina
of Spirulinagrown
growninin 100%
100% (blue
(blue diamonds),
50%50%
(red(red squares), and
Figure 3.
20% (green triangles) modified ZM, triplicates. Error bars arediamonds),
standard deviations squares), and 20%
of the biological
20% (green
(green triangles)
triangles) modified
modified ZM, triplicates.
ZM, triplicates. Error
Error bars are bars are standard
standard deviationsdeviations of the triplicates.
of the biological biological
triplicates.
triplicates.

Figure 4.4. Dry

Figure Dry weights
weights of
of Spirulina
Spirulina grown
grown in in 100%
100% (blue
(blue diamonds),
diamonds), 50%
50% (red
(red squares),
squares), and
and 20%
20%
Figure 4. Dry weights of Spirulina grown in 100% (blue diamonds), 50% (red squares), and 20%
(greentriangles)
(green triangles) modified
modified ZM,ZM, triplicates.
triplicates. ErrorError bars
bars are are standard
standard deviations
deviations of the triplicates.
of the biological biological
(green triangles) modified ZM, triplicates. Error bars are standard deviations of the biological
triplicates.
triplicates.
Nitrate was found to be the limiting nutrient in the experiments leading to the decline phase
aroundNitrate
day was found
20 for the to be the limiting
20%-modified ZMnutrient
(Figurein5).theTheexperiments leading to
average nutrient the decline phase
consumption rates
Nitrate was found to be the limiting nutrient in the experiments leading to the decline phase
around day
(nitrate, 20 for the
phosphate, and20%-modified
sulfate) wereZM (Figure(Table
calculated 5). The3).
average nutrient consumption
These consumption rates tendrates (nitrate,
to decrease
around day 20 for the 20%-modified ZM (Figure 5). The average nutrient consumption rates (nitrate,
phosphate,
with and dilution
increasing sulfate) were
rate. calculated
This tendency(Tableis3).less
These consumption
pronounced rates tend
for nitrates buttovery
decrease
rapidwith
for
phosphate, and sulfate) were calculated (Table 3). These consumption rates tend to decrease with
increasing dilution rate. This tendency is less pronounced for nitrates but very rapid
phosphates (10 times lower consumption rates for 20% and 50% diluted modified ZM in comparison to for phosphates
increasing dilution rate. This tendency is less pronounced for nitrates but very rapid for phosphates
(10 times modified
undiluted lower consumption
ZM). This could ratesmean
for that
20%Spirulina
and 50%growth diluted modified
is more ZMinindiluted
effective comparison
medium, to
(10 times lower consumption rates for 20% and 50% diluted modified ZM in comparison to
undiluted the
enhancing modified
interestZM). This20%-
for using couldandmean that Spirulina
50%-modified ZM. Thegrowth is more
next step wouldeffective
be to useina diluted
design
undiluted modified ZM). This could mean that Spirulina growth is more effective in diluted
medium,
of enhancing
experiment the interest
to optimize forphosphate
nitrate, using 20%- andand 50%-modified
sulfate ZM. The
concentrations. next step results
Impressive would were
be to
medium, enhancing the interest for using 20%- and 50%-modified ZM. The next step would be to
use a design
obtained of experiment
on other microalgaetostrains
optimize
(such nitrate, phosphate
as Chlorella and sulfate
protothecoides withconcentrations.
40% increase of Impressive
biomass
use a design of experiment to optimize nitrate, phosphate and sulfate concentrations. Impressive
results were obtained
concentration and 85%onofother
lipid microalgae
concentration strains
using (such as Chlorella
a response protothecoides
surface with which
method [27]) 40% increase
could
results were obtained on other microalgae strains (such as Chlorella protothecoides with 40% increase
of biomass
inspire concentration
Spirulina cultivationand 85% of lipid concentration using a response surface method [27])
optimization.
of biomass concentration and 85% of lipid concentration using a response surface method [27])
which could inspire Spirulina cultivation optimization.
which could inspire Spirulina cultivation optimization.
Fermentation 2017, 3, 59 8 of 14
Fermentation 2017, 3, 59 8 of 14

Figure5.5.Nitrate
Nitrateconcentrations
concentrations
in in mgN-NO
mgN-NO − 3−/Lin100%
100% (blue diamonds),
50%50%
(red(red squares), and
Figure 3 /Lin (blue diamonds), squares), and 20%
20% (green
(green triangles)
triangles) modified
modified ZM growing
ZM growing Spirulina,
Spirulina, triplicates.
triplicates.

Table 3.
Table 3. Average
Averagenutrient
nutrientconsumption
consumptionrate
ratefor
for100%,
100%,50%,
50%,and
and20%-modified
20%-modifiedZM.
ZM.

Average Consumption Rate Average Productivity (mg/L/day)

ZM Average Consumption Rate Average Productivity (mg/L/day)
ZM Nitrate Phosphate Sulfate From Day 0 to From Day 0 to
Proportion Nitrate Phosphate Sulfate From Day 0 to From Day 0 to
Proportion (mgN/L/day) (mgP/L/day) (mgS/L/day) Day 21 Day 24
(mgN /L/day) (mgP /L/day) (mgS /L/day) Day 21 Day 24
100% 7.25 ± 0.10 2.38 ± 0.06 1.39 ± 0.11 36.9 ± 3.2 34.4 ± 1.5
100% 7.25 ± 0.10 2.38 ± 0.06 1.39 ± 0.11 36.9 ± 3.2 34.4 ± 1.5
50% 8.13 ± 0.10 0.26 ± 0.24 1.06 ± 0.24 47.8 ± 2.5 34.6 ± 2.4
50% 8.13 ± 0.10 0.26 ± 0.24 1.06 ± 0.24 47.8 ± 2.5 34.6 ± 2.4
20%20% 6.42 ± 0.91
6.42 ± 0.91 0.38 ± 0.12
0.38 ± 0.12 0.60±± 0.71
0.60 0.71 53.5 ± 5.4
53.5 ± 5.4 23.5
23.5 ± 7.5
± 7.5

3.4. Iron Source and Iron Content in Spirulina

3.4. Iron Source and Iron Content in Spirulina
Spirulina is a source of highly available iron with a 6.5 times more available iron than beef meat
Spirulina is a source of highly available iron with a 6.5 times more available iron than beef
[28]. Increasing iron content in Spirulina could lead to a very beneficial increase of its nutritional
meat [28]. Increasing iron content in Spirulina could lead to a very beneficial increase of its
value.
nutritional value.
Modified ZM contains iron in the form of FeSO4,6H2O (2 mgFe/L) stabilized with EDTA (50
Modified ZM contains iron in the form of FeSO4 ,6H2 O (2 mgFe /L) stabilized with EDTA
mg/L). The opportunity to use commercial formulations of Fe-EDTA was tested in Spirulina
(50 mg/L). The opportunity to use commercial formulations of Fe-EDTA was tested in Spirulina
cultivation experiments using Fe-EDTA from Plantin and Akzo Nobel at two different
cultivation experiments using Fe-EDTA from Plantin and Akzo Nobel at two different concentrations
concentrations (3 and 10 mgFe/L) in duplicates. Figures 6 and 7 show the growth curves in OD880 for
(3 and 10 mgFe /L) in duplicates. Figures 6 and 7 show the growth curves in OD880 for these
these experiments. Concentration of 3 mgFe/L of Fe-EDTA did not impact significantly the growth of
experiments. Concentration of 3 mgFe /L of Fe-EDTA did not impact significantly the growth of
Spirulina (Figure 6). However, at 10 mgFe/L, Spirulina growth was slightly inhibited, especially after
Spirulina (Figure 6). However, at 10 mgFe /L, Spirulina growth was slightly inhibited, especially
10 days (Figure 7). Nevertheless, the Spirulina iron content was significantly increased when
after 10 days (Figure 7). Nevertheless, the Spirulina iron content was significantly increased
Fe-EDTA concentration in the medium was increased from 3 to 10 mgFe/L for the Plantin solution
when Fe-EDTA concentration in the medium was increased from 3 to 10 mgFe /L for the Plantin
(Table 4). Iron mass balances were not always coherent with large disparities over the duplicates.
solution (Table 4). Iron mass balances were not always coherent with large disparities over the
However, the Spirulina iron content (internalized iron) showed three consistent behaviors. First,
duplicates. However, the Spirulina iron content (internalized iron) showed three consistent behaviors.
Fe-EDTA increased the iron content. Then, higher Fe-EDTA concentrations in the medium increased
First, Fe-EDTA increased the iron content. Then, higher Fe-EDTA concentrations in the medium
the iron content. And, Fe-EDTA solution from Plantin led to higher iron content in comparison to the
increased the iron content. And, Fe-EDTA solution from Plantin led to higher iron content in
solution from Akzo-Nobel at 10 mgFe/L. This difference could be explained by Fe-EDTA counter-ion
comparison to the solution from Akzo-Nobel at 10 mgFe /L. This difference could be explained by
which is NH4+ for the Plantin solution and Na+ for the Akzo-Nobel solution. Further studies should
Fe-EDTA counter-ion which is NH4 + for the Plantin solution and Na+ for the Akzo-Nobel solution.
be conducted on the counter-ion effect on iron accumulation, as well as on the chelating agent
Further studies should be conducted on the counter-ion effect on iron accumulation, as well as on the
(EDDHA, DTPA for example).
chelating agent (EDDHA, DTPA for example).
 Fermentation2017,
Fermentation 2017,3,3,59
59 99of
of14
14
Fermentation 2017, 3, 59 9 of 14

Figure 6.
Figure 6. OD
OD880 measurements
measurementsofof Spirulina
Spirulina grown
grownusing Fe-EDTA
using Fe-EDTAfrom Plantin
from at 3atmg
Plantin /L Fe
3 Femg (blue
/L
Figure 6. OD880880measurements of Spirulina grown using Fe-EDTA from Plantin at 3 mgFe/L (blue
diamonds),
(blue Fe-EDTA
diamonds), from Akzo-Nobel
Fe-EDTA at 3 mgFe/Lat(red
from Akzo-Nobel squares)
3 mg Fe /L and
(red modified
squares)ZM and(control,
modified2 mgZM
Fe/L
diamonds), Fe-EDTA from Akzo-Nobel at 3 mgFe/L (red squares) and modified ZM (control, 2 mgFe/L
in the form
(control, 2 mgof FeSO
Fe /L ,6Hform
in 4the 2O with 50 mg/L
of FeSO 4 ,6H2 of EDTA,
O with green of
50 mg/L triangles), in duplicate.
EDTA, green Error
triangles), bars are
in duplicate.
in the form of FeSO4,6H2O with 50 mg/L of EDTA, green triangles), in duplicate. Error bars are
standard
Error bars deviations of deviations
are standard the biological duplicates.
of the biological duplicates.
standard deviations of the biological duplicates.

Figure 7. OD880 measurements of Spirulina grown using Fe-EDTA from Plantin at 10 mgFe /L
Figure 7. OD880 measurements
(blue of Spirulina grown using Fe-EDTA from Plantin at 10 mgFe/L (blue
Figurediamonds), Fe-EDTA from
7. OD880 measurements Akzo-Nobel
of Spirulina grownat using
10 mg Fe /L (red
Fe-EDTA fromsquares)
Plantinand
at 10modified ZM
mgFe/L (blue
diamonds),
(control, 2 Fe-EDTA
mg /L of from
iron inAkzo-Nobel
the form at
of 10
FeSO mg Fe/L (red squares) and modified ZM (control, 2
,6H O with 50 mg/L of EDTA, green triangles),
diamonds), Fe-EDTA
Fe from Akzo-Nobel at 10 mg 4 Fe/L 2 (red squares) and modified ZM (control, 2
mgFe/L of iron
in in the form
are of FeSO4,6H 2O with 50 mg/L of EDTA, green triangles), in duplicate. Error
mgduplicate. Error bars standard deviations of the biological triplicates.
Fe/L of iron in the form of FeSO4,6H2O with 50 mg/L of EDTA, green triangles), in duplicate. Error
bars are standard deviations of the biological triplicates.
bars are standard deviations of the biological triplicates.
Table 4. Iron mass balance for the Spirulina cultivation experiments using Fe-EDTA formulations
Table 4. Iron mass balance for the Spirulina cultivation experiments using Fe-EDTA formulations
from Plantin and Akzo Nobel at 3 and 10 mgFe/L of iron in comparison to the control, modified ZM.
from Plantin and Akzo Nobel at 3 and 10 mgFe/L of iron in comparison to the control, modified ZM.
Residual in Adsorbed Internalized Sum Adsorbed Internalized
Iron mass balance in Fe/L) Adsorbed
Residual(mg
Iron mass balance Supernatant (mgFe/L) Internalized
(mgFe/L) Sum
(mg Fe/L)
Adsorbed
(mgFe/g) Internalized
(mgFe/g)
Supernatant (mgFe/L) (mgFe/L) (mgFe/L) (mgFe/L) (mgFe/g) (mgFe/g)
Initial iron concentration of 3 mgFe/L
Initial iron concentration of 3 mgFe/L
Fe-EDTA Plantin 1 0.124 2.324 0.503 2.95 2.501 0.32
Fe-EDTA
Fe-EDTA Plantin
Plantin 12 0.124
0.081 2.324
2.353 0.503
0.532 2.95
2.966 2.501
2.721 0.32
0.541
Fe-EDTA Plantin
Fe-EDTA Akzo Nobel 2 1 0.081
0.062 2.353
2.359 0.532
0.424 2.966
2.845 2.721
2.507 0.541
0.451
Fe-EDTA
Fe-EDTA Akzo
Akzo Nobel
Nobel 12 0.062
0.074 2.359
2.349 0.424
0.53 2.845
2.954 2.507
2.925 0.451
0.67
Fe-EDTA Akzo Nobel 2 0.074 2.349 0.53 2.954 2.925 0.67
Fermentation 2017, 3, 59 10 of 14

Table 4. Iron mass balance for the Spirulina cultivation experiments using Fe-EDTA formulations from
Plantin and Akzo Nobel at 3 and 10 mgFe /L of iron in comparison to the control, modified ZM.

Residual in Adsorbed Internalized Sum Adsorbed Internalized

Iron Mass Balance
Supernatant (mgFe /L) (mgFe /L) (mgFe /L) (mgFe /L) (mgFe /g) (mgFe /g)
Initial iron concentration of 3 mgFe /L
Fe-EDTA Plantin 1 0.124 2.324 0.503 2.95 2.501 0.32
Fermentation 2017, 3, 59 10 of 14
Fe-EDTA Plantin 2 0.081 2.353 0.532 2.966 2.721 0.541
Fe-EDTA Akzo Nobel 1 0.062 2.359 0.424 2.845 2.507 0.451
Fe-EDTA1Akzo
Control Nobel
(2 mg Fe/L)2 0.074
0.096 2.349
0.628 0.53
0.12 2.954
0.844 2.925
0.492 0.67
0.094
Control 1 (2 mgFe /L) 0.096 0.628 0.12 0.844 0.492 0.094
Control 2 (2 mgFe/L) 0.045 0.342 0.207 0.594 0.453 0.273
Control 2 (2 mgFe /L) 0.045 0.342 0.207 0.594 0.453 0.273
Initial iron concentration of 10 mgFe/L
Initial iron concentration of 10 mgFe /L
Fe-EDTA Plantin 1 8.482 N.C. 0.690 N.C. N.C. 2.015
Fe-EDTA Plantin 1 8.482 N.C. 0.690 N.C. N.C. 2.015
Fe-EDTA Plantin 2 5.357 1.029 0.743 7.130 3.049 2.204
Fe-EDTA Plantin 2 5.357 1.029 0.743 7.130 3.049 2.204
Fe-EDTA Akzo Nobel
Fe-EDTA Akzo Nobel 1 1 1.557
1.557 6.011
6.011 0.461
0.461 8.030
8.030 11.702
11.702 0.898
0.898
Fe-EDTA
Fe-EDTAAkzo Nobel22
Akzo Nobel 7.544
7.544 0.443
0.443 0.296
0.296 8.282
8.282 0.889
0.889 0.593
0.593
Control 11 (2
Control (2 mg
mgFeFe/L)
/L) 0.002
0.002 0.403
0.403 0.238
0.238 0.644
0.644 0.626
0.626 0.369
0.369
Control 2 (2 mg /L) N.C. N.C. 0.233 N.C. N.C. 0.357
Control 2 (2 mgFeFe/L) N.C. N.C. 0.233 N.C. N.C. 0.357
N.C.: Not consistent, values out-of-range which were not reflecting real values.
N.C.: Not consistent, values out-of-range which were not reflecting real values.

3.5.
3.5. Effect
Effect of
of Light
Light Intensity
Intensity on
on Spirulina
Spirulina Growth
Growth
The
The effect
effect of
of PPFD
PPFD on on the
the growth
growth of of Spirulina
Spirulina was
was investigated
investigated inin 22 LL PBRs
PBRs running
running in in batch
batch
mode. Five PPFDs from 85 to 430 µmol/m 2 /s were tested. As predicted, the higher the light
mode. Five PPFDs from 85 to 430 µmol/m /s were tested. As predicted, the higher the light intensity,
2
intensity,
the faster the
thefaster
growth. the Agrowth. A linear relationship
linear relationship was found was found growth
between betweenratesgrowth
andrates
lightand light
intensity
intensity (Figure 8), except for the first point at 85 µmol/m 2 /s. Light saturation could not be
(Figure 8), except for the first point at 85 µmol/m2/s. Light saturation could not be reached at 430
reached 2 /s, while some studies found photoinhibition around 300 µmol/m2 /s [29]
µmol/m2at /s,430 µmol/m
while some studies found photoinhibition around 300 µmol/m2/s [29] or 432 µmol/m2/s
or 432 2 /s [30]. These differences
µmol/m
[30]. These differences might be explainedmight be explained
by different by different
Spirulina strain and Spirulina
differentstrain and
lighting
different lighting conditions. However, Figure 8 shows a curving of the specific
conditions. However, Figure 8 shows a curving of the specific growth rate line near 400 µmol/m2/sgrowth rate line near
400 2
µmol/m
meaning that/s meaning
it is reaching that it is reaching photoinhibition.
photoinhibition.

Figure 8. Spirulina growth rates with respect to PPFD. The correlation (blue diamonds) does not
Figure 8. Spirulina growth rates with respect to PPFD. The correlation (blue diamonds) does not
include the
include the first
first point
point at
at 85
85 µmol/m
µmol/m22/s
/s(red
(redsquare).
square).No
Noreplicate.
replicate.

3.6. Spirulina Cultivation Run in a 1000 L Camargue PBR

3.6. Spirulina Cultivation Run in a 1000 L Camargue PBR
During 40 days, between the 11 March 2015 and 15 April 2015, a Spirulina cultivation was
During 40 days, between the 11 March 2015 and 15 April 2015, a Spirulina cultivation was
conducted in a 1000 L tubular Camargue PBR located in a 60 m22 greenhouse (Figure 9). A fed-batch
conducted in a 1000 L tubular Camargue PBR located in a 60 m greenhouse (Figure 9). A fed-batch
mode was used with harvesting and medium supplementation during days 12, 21, and 27 in order to
maintain sufficient nutrients for the growth.
The daily average temperature varied slightly between 19.7 °C and 27.6 °C (daily average
diurnal temperature between 20.7 °C and 33.3 °C and daily average nocturnal temperature between
15.3 °C and 22.6 °C).
Fermentation 2017, 3, 59 11 of 14

mode was used with harvesting and medium supplementation during days 12, 21, and 27 in order to
maintain sufficient nutrients for the growth.
The daily average temperature varied slightly between 19.7 ◦ C and 27.6 ◦ C (daily average diurnal
temperature between 20.7 ◦ C and 33.3 ◦ C and daily average nocturnal temperature between 15.3 ◦ C
and 22.6 ◦ C).
Fermentation 2017, 3, 59 11 of 14

Figure 9.9.Daily
Figure Dailyaverage
average PPFD
PPFD (measured
(measured inside
inside the greenhouse)
the greenhouse) andweights
and dry dry weights for Spirulina
for Spirulina grown
grown in a 1000 L Camargue PBR. No replicates.
in a 1000 L Camargue PBR. No replicates.

Averagevolumetric
Average volumetricand
andareal
areal productivities
productivities varied
varied during
during the experiment
the experiment with with a minimum
a minimum from
from5 to
day day
10 5(20
tomg/L/day
10 (20 mg/L/day 2 /day)
or 2.26
or 2.26 g/m g/mand
2/day) and a maximum between day 10 and 11 (520
a maximum between day 10 and 11 (520 mg/L/day
or 58.4 g/mor2 /day),
mg/L/day 58.4 g/m
with averages of 55 mg/L/day and 6.19 g/m2 /day
2/day), with averages of 55 mg/L/day and 6.19 g/mfrom
2/day from day 5 to 40.
day 5 to 40.

3.7.
3.7. Cost Comparison:
Comparison: PBR and Open Ponds
The
The model
modeldescribed
described in in
a previous
a previousworkwork
[31] was
[31] used
was to compare
used the production
to compare cost of Spirulina
the production cost of
in open ponds
Spirulina andponds
in open in PBRs.andTypical
in PBRs.productivities in open ponds
Typical productivities cultivating
in open pondsSpirulina
cultivating areSpirulina
in the range
are
of
in 4the g/m2of
to 7range /day
4 to(weekly production
7 g/m2/day (weeklyfrom 46.5 kg to
production 77 kg
from in akg
46.5 1750 m2kg
to 77 open
in apond
1750during
m2 open thepond
year
2015).
duringThree productivities
the year 2015). Three were tested in the
productivities model:
were 4, 7,
tested inand
the 10 g/m24,/day
model: for10
7, and open
g/mponds,
2/day forand 10,
open
25, and 40
ponds, and g/m 2 /day
10, 25, for40
and PBRs.
g/m2Capital
/day forcosts (CAPEX)
PBRs. Capitalwere
costsestimated
(CAPEX)atwere 50 €/m 2 for open
estimated ponds
at 50 €/m2and
for
open€/m
1000 ponds 2 for PBR. These 2more realistic values are, therefore, quite different from the initial model
and 1000 €/m for PBR. These more realistic values are, therefore, quite different from
which wasmodel
the initial designed for was
which extrapolation
designed for purposes. Open ponds
extrapolation purposes. haveOpen
a depth of 25
ponds cm aand
have are agitated
depth of 25 cm
by
andpaddlewheels
are agitated byatpaddlewheels
a velocity of 25 at acm/s.
velocityAnofevaporation rate of 2.2 m/year
25 cm/s. An evaporation wasm/year
rate of 2.2 also assumed.
was also
Residence
assumed. time for both
Residence openfor
time ponds
bothandopenPBRs was set
ponds andto seven
PBRs days.was setAir was supplemented
to seven days. Airbywas2%
CO 3 3
2 at a rate ofby
supplemented 7 L/min/m
2% CO2 at for PBRof[23]
a rate and 2.7 L/min/m
7 L/min/m 3 for PBR [23] for and
open2.7ponds.
L/min/mAdditional
3 pumping
for open ponds.
for liquid circulation
Additional pumping was also implemented
for liquid circulation was for also
PBR implemented
(0.3 m/s) [23].for The
PBRmodel does [23].
(0.3 m/s) not account
The modelfor
further
does not processing
account for (harvesting by filtration
further processing and/or drying)
(harvesting since the
by filtration processes
and/or willsince
drying) be similar for both
the processes
PBRs
will be and open ponds.
similar for bothResults
PBRs and are shown in TableResults
open ponds. 5. Production
are shown costsinare significantly
Table higher
5. Production in PBR
costs are
due to their higher CAPEX. However, the model does not account for the
significantly higher in PBR due to their higher CAPEX. However, the model does not account for the higher production quality
in PBR. The
higher productivity
production qualityhasina higher
PBR. Theimpact on specific energy
productivity consumption
has a higher impactinon PBRs than inenergy
specific open
ponds.
consumption However,in PBRsthese energy
than in open specific
ponds. consumptions
However, these are energy
similar specific
to the caloric value ofare
consumptions Spirulina
similar
(5.67
to thekWh/kg)
caloric value[32]. ofThis means(5.67
Spirulina that kWh/kg)
if high productivities
[32]. This means can bethatobtained, energy applications
if high productivities can be
(where
obtained, energy
energyreturn on investment,
applications (whereEROI,
energyhasreturn
to be above 1) could be
on investment, considered.
EROI, has to be above 1) could
be considered.
Fermentation 2017, 3, 59 12 of 14

Table 5. Production cost and specific energy consumption for Spirulina cultivation in open ponds and
PBR for different productivities.

Techno-Economic Productivity Production Energy Consumption

Analysis (g/m2 /day) Cost (€/kg) (kWh/kg)
4 9.59 4.26
Open ponds 7 5.49 3.51
10 3.86 3.20
10 74.29 6.33
PBR 25 29.82 3.00
40 18.71 2.18

4. Conclusions
Spirulina cultivation can be improved though three criteria: productivity, quality, and cost.
Spirulina biomass productivity can be significantly improved by using the appropriate medium.
Indeed, modified ZM was found to provide higher biomass productivity than the two other media
tested (modified HM and modified JM). To reduce Spirulina production cost, dilutions of the modified
ZM were tested and it was shown that modified ZM could be diluted up to five times without
impacting the biomass productivity up to 21 days after inoculation. These results also suggest that
improvements are still possible and that a design of experiments approach for optimizing Spirulina
medium would be highly beneficial for productivity but also production cost. Spirulina biomass
productivity could not be increased by increasing the initial biomass concentration in a batch culture.
However, higher light intensity was found to increase Spirulina growth rate for PPFD up to, at least,
430 µmol/m2 /s.
Spirulina biomass nutritional quality can be improved by increasing its iron content.
Using Fe-EDTA at a concentration of 10 mgFe /L allows an increase of the Spirulina iron content
from around 0.4 mgFe /g to more than 2 mgFe /g. Improvements could also be expected by using
different chelating agents since Fe-EDTA stability ranged from pH 4.0 to 6.3 and Spirulina cultures
have pH ranging from 9.5 to 11.0.
Cultivation of Spirulina in a 1000 L pilot-scale tubular PBR was investigated as it could improve
biomass productivity. Indeed, values up to 58.4 g/m2 /day were observed during the 40-day cultivation.
However, a cost analysis resulted in higher production cost for PBR (from 18.71 to 75.29 €/kg) than for
open pond cultivation (from 3.86 to 9.59 €/kg). Low production cost, high biomass productivity and
high biomass quality are difficult to combine. Equilibrium has to be found which will be driven by the
market that is addressed (i.e., human nutrition, cosmetic, therapeutic, etc.).

Acknowledgments: The authors are very grateful to the editor for offering the opportunity to publish a paper in
Special Issue entitled “Cultivation and Downstream Processing of Microalgal Biomass”.
Author Contributions: Florian Delrue, Emilie Alaux, Lagia Moudjaoui, Gatien Fleury, Martin Petitjean and
Jean-François Sassi conceived and designed the experiments; Florian Delrue, Emilie Alaux, Lagia Moudjaoui,
Clément Gaignard, Gatien Fleury, Amaury Perilhou, Pierre Richaud performed the experiments; Florian Delrue
and Emilie Alaux analyzed the data; Pierre Richaud performed the ICP analyses; Florian Delrue, Emilie Alaux,
Pierre Richaud and Jean-François Sassi wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.

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