AbstractArthrospira 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, 3 H2O at 10 mgFe/L compared to 0.18 ± 0.13 for FeSO4,6H2O 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. View Full-Text
Keywords: Spirulina cultivation; iron content; light intensity; medium rationalizationSpirulina cultivation; iron content; light intensity; medium rationalization►▼ Figures
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 . The Spirulina production has been estimated to be between 3000 and 20,000 tons/year.
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) . 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 . However, Arthrospira platensis is a cyanobacterium growing at elevated pH (9.5 to 11.0 with an optimum at 10.5 ) and is, therefore, less subject to contaminations .
Cultivation medium has a great impact on the productivity of biomass and other compounds of interest. For example, nitrogen concentration in the medium  (optimum at 2.5 g/L) and also nitrogen source (urea better than ammonium or nitrate)  has a great effect on Spirulina productivity. Additionally, a phosphate concentration of 250 mg/L in the form of K2HPO4 was found to optimize biomass production . 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 . 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 .)
Three media from the literature were selected in this study: Zarrouk’s , Hiri’s , and Jourdan’s  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 . 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 . 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 , 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 . Despite low iron concentrations in its cultivation media, Spirulina contains high amount of iron  (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.
2. Materials and Methods
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 . Its composition is: 50 mg/L of EDTA; 11.4 mg/L of H3BO3; 22 mg/L of ZnSO4,7H2O; 5.06 mg/L of MnCl2,4H2O, 1.61 mg/L of CoCl2,6H2O; 1.57 mg/L of CuSO4,5H2O; and 1.1 mg/L of Mo7O24(NH4)6,4H2O. 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,3H2O, Amsterdam, The Netherlands) and Plantin (Ferro 8 with 8% of EDTA-FeNH4, Courtezon, France).
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  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.
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).
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 .
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). As shown in Figure 2, there is no significant difference in the final OD880 for all experiments. All growth curves reached their stationary phase after 20 days with similar OD880. This was possibly due to higher growth rates measured for culture experiment started at lower initial concentration (Table 2) as this could imply more light availability per cell leading to higher growth rates. In a similar study it was found that higher initial biomass concentrations led to lower growth rates due to the shadowing effect .
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 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 the biomass composition.
3.3. Cost Optimization of Modified Zarrouk’s Medium Composition
Modified ZM gave better growth than the other two media tested but it contains higher amounts of various chemicals leading to significant increased costs. The nitrogen content of modified ZM can theoretically lead to a maximal biomass concentration of 4.6 g/L for Spirulina (if nitrogen was the only limiting element). This would be 74, 31, and 395 g/L for phosphorus, sulfur, and potassium, respectively. These calculations were based on the elemental analyses of three Spirulina strains .
Therefore, dilution of modified ZM was investigated in order to reduce the cost of the cultivation. A growth experiment was conducted using 100%-, 50%-, and 20%-modified ZM (diluted using ultrapure water), in triplicate. Pre-cultures were done in each medium for acclimation. It was found that the 20%-modified ZM showed the best growth (OD880 readings), quite similar to the 50%-modified ZM (Figure 3