Introduction

The water-soluble fluorescent phycobiliproteins (PBPs) are important components of the light-harvesting photosynthetic system, present only in cyanobacteria, red algae, glaucophytes and cryptophytes (Gantt 1980; Glazer 1994; Grossman et al. 1993). The main components of phycobiliproteins are c-phycocyanin, phycoerythrin and allophycocyanin. C-phycocyanin (C-PC) and allophycocyanin (A-PC) are used such as fluorescent markers in diagnostic histochemistry, as dyes in cosmetics and foods, and because of their antioxidant properties may have potential as therapeutic agents (Rimbau et al. 1999; Romay et al. 2003; Fernandéz-Rojas et al. 2014; Manirafasha et al. 2016; Li et al. 2019; Pagels et al. 2019) .

Commercially, the production of C-PC is almost exclusively associated with Arthrospira (Spirulina) platensis, a planktonic, filamentous cyanobacterium (Vernes et al. 2015; Xie et al. 2015; Dejsungkranont et al. 2017; Ho et al. 2018; Hsieh-Lo et al. 2019; Nwoba et al. 2019; Yu et al. 2019; Abalde et al. 1998; Eriksen 2007). The C-PC market is projected to reach US$97 million by 2019 (Santos et al. 2019), and C-PC trades for up to 500 US$ kg−1 for food-grade (Kannaujiya and Sinha 2016) and 10–50 US$ mg−1 for analytical grade (Kuddus et al. 2013). Since the C-PC production process in A. platensis depends on externally supplied light, the productivity of biomass, and therefore C-PC, is low in the open pond/raceway suspension cultures. In phototrophic outdoor cultures of A. platensis, C-PC productivities have been estimated to be on the order of 3–24 mg L−1 day−1 (Schmidt et al. 2005) with dry biomass concentrations usually not exceeding 1 g/L. In addition to low productivity due to seasonal daily fluctuations in temperature, light intensity and uneven mixing, open-pond cultivation also suffers from contamination by other microalgae, predators and pathogens (Richmond et al. 1990; Carbone et al. 2018, 2019; Richmond and Qiang 2010; Olivieri et al. 2014; Gargano et al. 2016; Osorio et al. 2019; Zuccaro et al. 2019).

The thermophilic unicellular red alga G. sulphuraria has recently emerged as a prospective alternative for production of C-PC (Carfagna et al. 2016, 2018; Castenholz and McDermott 2010; Cennamo et al. 2012; Iovinella et al. 2018; Ciniglia et al. 2017), especially when grown heterotrophically (Graverholt and Eriksen 2007; Sloth et al. 2017; Eriksen 2013; Eriksen 2018). G. sulphuraria grows heterotrophically with a large number of organic carbon sources at pH 1–2 (Gross and Schnarrenberger 1995; Gross 1999), where most contaminating organisms will not grow. High biomass densities (> 100 g/L) have been reported under these conditions (Schmidt et al. 2005). Although some isolates of G. sulphuraria maintain C-PC synthesis in the dark, the C-PC concentration is strongly reduced (10–25 mg/g dry biomass) compared to autotrophically grown cultures of both G. sulphuraria and A. platensis (8–15% C-PC in the dry biomass), almost cancelling the advantage of the higher biomass productivities of heterotrophic cultivation. A recent analysis used a two-step cultivation of G. sulphuraria combining heterotrophic growth with subsequent dilution and exposure to high light intensities to achieve a C-PC content of 13% in the dry biomass and a C-PC yield of 0.8 g/L (Wan et al. 2016). However, organic carbon had to be completely removed after heterotrophic cultivation and culture parameters and bioreactors needed to be changed, making this approach technically demanding.

Both A. platensis and G. sulphuraria as well as most other commercially used microalgae are grown in suspension cultures. However, G. sulphuraria and other thermophilic red algae that belong to the subdivision cyanidiophytina (Yoon et al. 2004; Pinto et al. 2007) thrive in geothermal volcanic areas at temperatures around 40 °C and at high sulphuric acid concentration associated with soil, rocks and in cryptoendolithic habitats (Gross et al. 1998; Ciniglia et al. 2004; Pinto 2007). Technical cultivation of G. sulphuraria in biofilm systems could open a new avenue of research.

Porous substrate bioreactors (PSBR), especially of the twin-layer type (TL-PSBR; Nowack et al. 2005; Melkonian and Podola 2010), have recently attracted considerable attention as an alternative to low-density autotrophic suspension cultures (Podola et al. 2017; Pierobon et al. 2018; Zhuang et al. 2018). In brief, algae are immobilized by self-adhesion on a sheet-like (micro)porous substrate that is impermeable to the cells but permeable to the culture medium, the algae forming a biofilm. The culture medium is applied to the surface of the substrate layer opposite the biofilm, usually to a sheet-like (macro)porous source. The culture medium moves down the source layer by gravity flow and nutrients reach the biofilm by diffusion and convection through the substrate layer. The low energy demand for water circulation as well as the low water footprint and content of the biomass and associated ease of harvesting and processing of biomass make these systems attractive to many applications in bioremediation (Shi et al. 2007, 2014; Li et al. 2015, 2016; Piltz and Melkonian 2018), biotechnology and biorefinery processes (Podola et al. 2017; Wang et al. 2015). The low shear forces to which cells are exposed further enable the cultivation of a large diversity of microalgal species (Nowack et al. 2005; Naumann et al. 2013; Benstein et al. 2014; Kiperstok et al. 2017; Carbone et al. 2017a; Ekelhof and Melkonian 2017; Langenbach and Melkonian 2019). Here, we show that G. sulphuraria can be grown successfully in a bench-scale TL-PSBR to high biomass standing crops (230 g dry weight/m−2 growth area) and significant phycobilin amounts (14 g m−2). We evaluate the effects of different light intensities, addition of CO2 and frequency of exchange of culture medium on growth and C-PC/A-PC in production of G. sulphuraria in the TL-PSBR.

Materials and methods

Algal strain and culture medium

G. sulphuraria strain 064 ACUF (http://www.acuf.net) was chosen for the experiment. The stock culture grew in Galdieria medium (Gross and Schnarrenberger 1995) acidified by sulphuric acid at pH 1.5. Stock cultures were inoculated into a 250-ml glass column bioreactor and after 4 weeks, the scale-up was performed in 1 L Erlenmeyer flasks. Cultures were exposed to a photon fluence rate of 30 μmol photons m−2 s−1 with a light/dark cycle of 14/10 h. The temperature was adjusted with a transparent water bath to 34 °C.

Photobioreactor design and set up

The experiments were carried out in a bench-scale twin-layer system (TL-PSBR) as described by Shi et al. (2007) and Schultze et al. (2015).

A sodium discharge lamp (SON-T AGRO 400 W, Philips, Hamburg, Germany) was used to obtain the three light intensities (50 μmol m−2 s−1, 100 μmol m−2 s−1 and 200 μmol m−2 s−1) used in the experiments.

A quantum sensor (LI-190SA, LI-COR Biosciences GmbH, Bad Homburg, Germany) measured the photosynthetic active radiation (PAR); the light/dark cycle was again set to 14/10 h. Atmospheric CO2 (0.04%) or additional CO2 (1% v/v) were used for the experiments. The temperature was kept constant at 34 °C using an aquarium heater.

Inoculation on the twin-layer

When the microalgae achieved a sufficient cell density in suspension, the algae were harvested by centrifugation at 2000 rpm (Sorvall, RC5C) for 30 min and then immobilized on the twin-layer system using polycarbonate membranes (PC40, 0.4 μm pore size, 25 mm diameter, Whatman, Dassel, Germany) as the substrate layer. Microalgae were inoculated to a density of 10 g dry weight m−2 growth area by filtration.

Determination of biomass

During the first experiment, the biomass was harvested from the polycarbonate discs every 2 days in triplicate. In the next experiments, the biomass was harvested every 3 days in triplicate. Only biomass in the inoculated area was harvested; surplus biomass was scraped off (18 mm diameter). Then, the samples were lyophilized in a freeze dryer for 2 h and weighed in an analytical balance (Sartorius Bovenden, Germany).

Extraction and characterization of phycobilins

C-PC and A-PC were extracted after lyophilization from the dried biomass in the following way: the microalgae were removed from the disk and homogenized with quartz sand (Moraes et al. 2011); 4 ml of sodium acetate solution, pH 6.5 and 20 mM were added to the samples and the samples subjected to one freeze thaw cycle (Moon et al. 2014) at − 80 °C and 4 °C. After this step, the algae were centrifuged at 4 °C for 30 min at 10,000 rpm to remove cells debris. The blue-coloured supernatant contained the phycobiliproteins.

C-phycocyanin (C-PC) and allophycocyanin (A-PC) were measured spectrophotometrically using the following equations for quantification (Moon et al. 2014; Pan-Utai et al. 2018; Estrada et al. 2001):

$$ \mathrm{C}-\mathrm{PC}\ \left(\mathrm{mg}\ {\mathrm{ml}}^{-1}\right)=\left({A}_{620}-0.474{A}_{652}\right)/5.34\kern0.5em $$
(1)
$$ \mathrm{APC}\ \left(\mathrm{mg}\ {\mathrm{ml}}^{-1}\right)=\left({A}_{652}-0.208{A}_{620}\right)/5.34 $$
(2)
$$ \mathrm{C}-\mathrm{EP}=\left({A}_{620}/{A}_{280}\right) $$
(3)
$$ \mathrm{APC}-\mathrm{EP}=\left({A}_{652}/{A}_{280}\right) $$
(4)

where C-PC is c-phycocyanin, A-PC is allophycocyanin, C-EP and APC-EP are the purity of c-phycocyanin and allophycocyanin respectively, and A is the absorbance measured at the specified wavelength in quartz cuvettes.

Results

Here, we report, for the first time, growth of an extremophile red microalga in a TL-PSBR. Since no previous data on immobilized growth of G. sulphuraria existed, some basic parameters of growth were first tested such as varying light intensities and carbon dioxide addition (for technical reasons, the growth temperature was kept constant at 35 °C, which is considerably lower than the optimal growth temperature (42 °C) reported for this strain (Sloth et al. 2006).

Three sets of experiments were performed. In the first experiment, growth and phycobilin production were monitored over 14 days at two different carbon dioxide concentrations (atmospheric and 1% (v/v) and a fixed photon fluence rate of 100 μmol m−2 s−1. In the second set of experiments, over an extended experimental period of 30 days, different photon fluence rates (50, 100 and 200 μmol m−2 s−1) were compared at a fixed carbon dioxide concentration of 1% (v/v). Finally, the cultivation time was varied (14, 30 and 43 days) at a fixed photon fluence rate (100 μmol m−2 s−1) and carbon dioxide concentration (1%). In the latter experiment, nutrients were replenished more frequently (every 2 days instead of every 3 days) after 15 days of cultivation to avoid nutrient depletion.

Effect of CO2 addition on growth and phycobiliprotein production

G. sulphuraria strain 064 was tested in a TL-PSBR for 14 days in the presence of atmospheric CO2 (0.04%) or 1% CO2 (v/v, in the gas phase) at light intensities of 100 μmol m−2 s−1 (Fig. 1). Samples were taken every 2 days and the culture medium was exchanged every 3 days. During the experimental period, biomass growth was linear (Fig. 1). Addition of 1% CO2 had a significant positive effect on the biomass growth rate (~ 6 g m−2 day−1 with 1% CO2 compared to only 1.7 g m−2 day−1 at atmospheric CO2 levels; Fig. 1). After 14 days of growth, the biomass standing crop reached ~ 100 g dry weight m−2 with the addition of 1% CO2 (it should be noted that, on average, the water content in one m2 of a TL-PSBR is about 0.5–1 L during operation (Podola et al. 2017).

Fig. 1
figure 1

Biomass density (a), C-PC density and dry weight content (b, c), A-PC density and dry weight content (d, e) over the time during batch growth performed at 100 μmol photons m−2 s−1with air or 1% CO2 (v/v) in the gas phase

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The light yield on biomass was larger in case of 1% CO2 (YX/PH = 0.68 g/mol) than in case of atmospheric CO2 (0.19 g/mol). The production of C-PC and A-PC also benefitted from the addition of CO2, with 3–4 times higher productivity (Fig. 1b–d) and on average 6–7 times higher pigment content in the biomass (Fig. 1c–e). Interestingly, C-PC and A-PC were present in similar amounts in the biomass (Fig. 1c–e).

Effect of different photon flux densities on biomass growth and phycobiliprotein production

To test how long linear growth of G. sulphuraria can be maintained in a TL-PSBR, the experimental period was extended to 30 days. Since the effect of elevated CO2 on growth depends on the photon fluence rate in TL-PSBRs (Schultze et al. 2015), also three different photon fluence rates at 1% CO2 concentration were tested.

The samples were taken every 3 days and the culture medium was exchanged every 3 days (Fig. 2). Depending on the photon fluence rate, the duration of linear growth varied: 15 days at 50 μmol m−2 s−1 (final biomass standing crop 107 g m−2), 22 days at 200 μmol m−2 s−1 (final biomass standing crop 225 g m−2) and 24 days at 100 μmol m−2 day−1(final biomass standing crop 190 g m−2). Biomass growth rates also varied with photon fluence rates being lowest (5.2 g m−2 day−1) at 50 μmol m−2 s−1 and highest (10 g m−2 day−1) at 200 μmol m−2 s−1 with the biomass growth rate at 100 μmol m−2 day−1 intermediate (7.5 g m−2 d−1). The biomass yield was almost constant at 50 and 100 μmol m−2 s−1 light intensity (0.92 and 0.91 g mol−1) and strongly decreased at 200 μmol m−2 s−1 (0.54 g mol−1), indicating the presence of a saturating light effect on photosynthesis and growth rate.

Fig. 2
figure 2

Biomass density (a), C-PC density and dry weight content (b, c), A-PC density and dry weight content (d, e) over the time during batch growth performed at 1% CO2 as gas phase and 50, 100 and 200 μmol m−2 s−1 irradiance

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After 22–24 days, growth also ceased at the two higher photon fluence rates and the biomass density slightly decreased (Fig. 2).

Phycobiliprotein accumulation differed from biomass accumulation in that the highest phycobilin growth rate and phycobilin standing crop were observed at the intermediate photon fluence rate (100 μmol m−2 s−1), the lowest at the lowest photon fluence rate (50 μmol m−2-s−1) with the phycobilin accumulation intermediate at the highest photon fluence rate used (200 μmol m−2 s−1). The highest phycobiliproteins productivity was at 100 μmol photons m−2 s−1.

The highest C-PC value was achieved after 24 days (6.5 g m−2) while the highest A-PC value was achieved after 21 days (4.1 g m−2).

The highest percentage of phycobiliproteins in the biomass dry weight was determined at 100 μmol photons m−2 s−1: C-PC achieved 3.8% of dry weight and A-PC 2.3%. In the presence of 50 μmol photons m−2 s−1, the maximum C-PC percentage achieved 2.4% and A-PC 1.8% while at 200 μmol photons m−2 s−1, 1.8% and 1%.

After days 22–24, the phycobilin standing crop and concentration of phycobilin in the biomass dropped significantly (Fig. 2b–e).

Extending linear growth by increasing the frequency of nutrient replenishment

The previous experiment had shown that linear growth of G. sulphuraria in a TL-PSBR ceased around days 22–24 when the biomass density reached 200–220 g m−2. To investigate whether nutrient limitation could have been responsible for growth arrest and the subsequent drop in biomass and phycobilin standing crops, we extended the cultivation period to 42 days and exchanged the culture medium after 15 days of cultivation every 2 days (instead of every 3 days as in the previous experiments). The growth parameters were 100 μmol m−2 s−1 and addition of 1% CO2. More frequent exchange of the culture medium extended linear growth to 30 days over the 24 days in the previous experiment but the growth rate did not differ (Fig. 3a) compared to the previous experiment. The biomass standing crop reached 225 g m−2 at day 30 (Fig. 3a) compared to 190 g m−2 in the previous experiment. Nevertheless, growth ceased after day 33 (maximal biomass standing crop 232 g m−2) and the standing crop dropped to 210 g m−2 at the end of the experiment (Fig. 3a).

Fig. 3
figure 3

Biomass density (a), C-PC density and dry weight content (b, c), A-PC density and dry weight content (d, e) over the time during batch growth performed at 1% CO2 as gas phase and 100 μmol m−2 s−1 irradiance for the last experiment of 30 days

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Similarly, phycobilin standing crop was higher than in the previous experiment and reached 7.39 g m−2 for C-PC at day 30 and 6.8 g m−2 for A-PC at day 33 (Fig. 3b–d). At day 30, the total phycobilin content in the dry biomass was 63 mg g−1. In this experiment, we also observed the highest productivities of C-PC (0.28 g m−2 day−1) and A-PC (0.23 g m−2 day−1). The drop in phycobilin standing crop after biomass growth ceased (after day 33) was less strong in this experiment than in the previous one. After the frequency of exchange of the culture medium was increased, the growth rates of the phycobilin also increased (after 6 days) despite the continuing linear biomass growth during this time (Fig. 3a, b, d). In consequence, phycobilin content in the biomass also increased between days 21 and 30 (Fig. 3c–e).

Purity level of C-PC and A-PC

In all experiments, the purity ratios of C-PC and A-PC were evaluated (see “Materials and methods” for details). The purity ratios were constant during the experiment but results were different for the two phycobiliproteins. Indeed, the purity ratio was higher for C-PC than A-PC (Fig. 4).

Fig. 4
figure 4

Pigment purity level of the C-PC and A-PC at different light intensities

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Discussion

G. sulphuraria showed a different behaviour in the TL-PSBR compared to other microalgae tested such as green algae or dinoflagellates (Benstein et al. 2014; Schultze et al. 2015). Indeed, G. sulphuraria in the natural environment grows at low light conditions under an atmosphere enriched in CO2 (Albertano et al. 2000; Cozzolino et al. 2000; Toplin et al. 2008; Del Mondo et al. 2019; Eren et al. 2018). So, the addition of CO2 (1%) in a TL-PSBR already enhanced growth at the low photon fluence rate of 50 μmol photons m−2 s−1 (compare Figs. 1a and 2a), whereas addition of CO2 in air usually does not have a measurable effect on biomass growth of microalgae at such low photon fluence rates (Langenbach and Melkonian 2019; Li et al. 2015).

However, G. sulphuraria achieved a good maximum biomass productivity of about 10 g m−2 day−1 at 200 μmol photons m−2 s−1 with addition of 1% CO2, comparable to that obtained in other microalgae such as Haematococcus pluvialis, Scenedesmus vacuolatus and Symbiodinum voratum using the same parameters and cultivation system (Carbone et al. 2017a; Kiperstok et al. 2017; Do et al. 2019; Langenbach and Melkonian 2019). Biomass growth at photon fluence rates exceeding 200 μmol m−2 s−1 was not tested in this study because it was found that phycobilin productivity was higher at the lower photon fluence rate of 100 μmol m−2 s−1. This result corroborates similar work on the LHC antenna carotenoid peridinin in the dinoflagellate Symbiodinium voratum, whose productivity was found to be highest at the same photon fluence rate in a TL-PSBR (Langenbach and Melkonian 2019) and has also been reported for phycobilin-containing algae (Graverholt and Eriksen 2007; Takano et al. 1995; Chen and Zhang 1997; Chen et al. 2017). It is well known that the amount of LHC antenna pigments (chlorophylls, carotenoids, phycobilin) strongly depends on the incident photon fluence rate (Müller et al. 2001). For phycobilin, the ratio of C-PC to A-PC also depends on the nutrient status of cells; under nitrogen limitation for example, C-PC is preferentially degraded (Yamanaka and Glazer 1980). In suspension cultures of G. sulphuraria, either of the two, A-PC (Graziani et al. 2013) or C-PC (Sørensen et al. 2013), was found to be dominant.

The advantages of growing microalgae immobilized in porous substrate bioreactors compared to suspension cultures have been recently summarized (Podola et al. 2017; Pierobon et al. 2018; Zhuang et al. 2018; Carbone et al. 2017b; Li et al. 2017). Since shear forces are minimized through cell immobilization and separation of the bulk culture medium from the immobilized algae by the microporous substrate layer, a large variety of microalgae, including shear-sensitive taxa, have been successfully grown in TL-PSBRs (e.g. Nowack et al. 2005; Ekelhof and Melkonian 2017; Langenbach and Melkonian 2019). Changing cultivation parameters such as culture medium, temperature and pH is easily achieved and does not involve interference with the cultivation process. Although the TL-PSBR is an open cultivation system, contaminations are usually not encountered during the cultivation period needed to obtain the maximal biomass standing crop. For G. sulphuraria, we have not seen contaminating organisms by light microscopy over the 42-day cultivation period under autotrophic culture conditions (results not shown); this was, of course, not unexpected as the pH had been adjusted to 1.5, providing a highly selective environment that minimized the contamination risk. However, when G. sulphuraria was grown mixotrophically in media enriched in organic carbon, fungi quickly developed in this open cultivation system (results not shown). Polycarbonate filters with a pore size of 0.2 μm were used to avoid permeation of the small cells of G. sulphuraria (2–3 μm diameter) through the substrate layer. For scale-up, other inexpensive types of substrate layers need to be chosen and tested for their suitability to maintain the G. sulphuraria biofilm coherent during growth.

In our experiment, the microalgae achieved the maximum growth rate at 200 μmol photons m−2 s−1, while in suspension culture, this organism grows better at lower level of light intensities (Seckbach 2007; Seckbach and Chapman 2010). This result corroborated data obtained from other algae grown in TL-PSBRs that showed high growth rates at very high photon fluence rates exceeding 1000 μmol m−2 s−1 (e.g. Schultze et al. 2015). This is linked to rapid attenuation of light in the subsurface layers of the biofilm, caused by efficient light absorption at the surface of the biofilm as measured with microsensors (Li et al. 2016). At the beginning of the experiment, the biofilm on the twin-layer system was relatively thin and most cells were exposed to higher photon fluence rates. In consequence, during the first 3 days of growth, the biomass growth rate was higher at 100 μmol photons m−2 s−1 than at 200 μmol m−2 s−1 (Fig. 2a). When the biofilm became thicker, the upper biofilm layers shaded the lower ones, thus reducing photoinhibition processes (Gross et al. 1998; Schultze et al. 2015) and allowing the biofilm to achieve higher grow rates at the higher light intensity.

These results suggest that the growth performances of G. sulphuraria should also be tested at photon fluence rates exceeding 200 μmol m−2 s−1 using a two-phase approach (Langenbach and Melkonian 2019) or even a three-phase approach: cultures could initially be grown for a few days at 100 μmol photons m−2 s−1 to minimize photoinhibition and then progressively shifted to higher photon fluence rates to enhance biomass growth. To optimize phycobilin production, it may be necessary to include a final third phase at the lower photon fluence rate and surplus nutrients (in particular nitrogen) to boost synthesis of phycobilin. Addition of CO2 was found to be necessary not only to enhance biomass growth but also phycobilin production. This result was in contrast to observations made in Arthrospira platentis, in which elevated CO2 concentration reduced biomass growth and C-PC concentration (Gordillo et al. 1998) and could be related to the contrasting habitats of both organisms.

Phycobilin concentrations under autotrophy are generally higher than those in heterotrophic condition (Ciniglia et al. 2014; Graziani et al. 2013). C-PC values obtained in this study thus exceeded the highest values obtained in heterotrophic cultivation (C-PC values of ~25 mg g−1 in Eriksen 2018; Sloth et al. 2006, 2017). However, higher phycobilin contents in the biomass than obtained here have been described in G. sulphuraria grown in Japanese hot springs supplemented with NH4. In this case, a C-PC value of ~ 100 mg g−1 was achieved but biomass standing crop was low (2.7 g L−1) in this open-pond system (Hirooka and Miyagishima 2016).

In conclusion, TL-PSBRs appear to be suitable to grow the thermo- and acidophilic red alga G. sulphuraria autotrophically to high biomass densities with acceptable phycobilin productivities and levels. Optimization of the biomass and phycobilin production process should be possible using a multi-phase approach with different photon fluence rates, optimal growth temperatures and manipulation of nutrient concentration, in particular nitrogen.

The possibility to produce phycobiliproteins from Galdieria sulphuraria in a twin-layer system has several advantages. (1) In a TL-PSBR, very high biomass densities exceeding those achievable by fermentation can be obtained, and harvesting the biomass as fresh weight thus does not require a pre-concentration step (Naumann et al. 2013; Wang et al. 2015; Gargano et al. 2016; Carbone et al. 2017a). (2) Despite the fact that the amount of phycocyanin produced by G. sulphuraria is no more or even less than that of other microalgae such as Porphyridium purpureum or A. platensis (Sosa-Hernández et al. 2019a; Coward et al. 2016; da Fontoura et al. 2018; Xie et al. 2015), the pigments show a very high level of purity, an indispensable factor for industrial production and cost effectiveness (Imbimbo et al. 2019; Graziani et al. 2013; Sørensen et al. 2013). Moreover, these results foster continuation of experiments with this microalga not only to optimize phycobiliprotein and biomass production but also to couple biomass production with other industrial applications such as wastewater treatment. (Sosa-Hernández et al. 2019b; da Silva et al. 2016; Rizwan et al. 2018; Centella et al. 2017; Minoda et al. 2015; Ju et al. 2016). Indeed, G. sulphuraria is considered one of the best algal candidates for phytoremediation of metals such as caesium, vanadium and uranium (Jalali et al. 2019; Fukuda et al. 2018) but also for removal of dissolved organic carbon and nutrients (Henkanatte-Gedera et al. 2017).