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Article

Pilot-Scale Phycocyanin Extraction by the Green Two-Step Ultrasound-Based UltraBlu Process

by
Rosaria Lauceri
1,*,
Melissa Pignataro
2,
Antonio Giorgi
2,
Antonio Idà
2 and
Lyudmila Kamburska
1
1
Istituto di Ricerca Sulle Acque, CNR, Largo Tonolli 50, 28922 Verbania, Italy
2
Algaria Srl, Via Ruggiero Settimo 4, 20146 Milano, Italy
*
Author to whom correspondence should be addressed.
Separations 2025, 12(8), 194; https://doi.org/10.3390/separations12080194
Submission received: 17 June 2025 / Revised: 8 July 2025 / Accepted: 10 July 2025 / Published: 25 July 2025
(This article belongs to the Special Issue Application of Sustainable Separation Techniques in Food Processing)
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Abstract

Phycocyanin is a natural, non-toxic, blue pigment-protein with many commercial applications. Its exploitation in various biotechnological sectors strongly depends on its purity grade (P). Phycocyanin is largely used in food industry where a low purity grade is required, while its widespread use in sectors requiring a higher purity is hampered by the cost of large-scale industrial production. Industry, in fact, needs simple, easily scalable and cost-effective procedures to ensure sustainable production of high-quality pigment. In this work we applied the innovative two-step ultrasound-based process UltraBlu to the pilot-scale production of phycocyanin. A total of 50 L of biomass suspension of commercial Spirulina were processed in batch mode. The pigment extract was obtained in one day, including the biomass harvesting. Food/cosmetic grade (P = 1.41–1.76) and a good yield (Y = 59.2–76.1%) were achieved. The initial results obtained suggest that UltraBlu can be an effective scalable process suitable to produce phycocyanin also on an industrial scale.

Graphical Abstract

1. Introduction

Phycocyanin (PC), is a water soluble, brilliant blue and highly fluorescent phycobiliprotein (PBP) of the photosynthetic light-harvesting antenna complexes of cyanobacteria and of some other algae. PC has many biotechnological applications in various sectors, such as medicine, pharmaceutic, diagnostic, cosmetic and, particularly, nutraceutical and food. In fact, PC is one of the few safe natural blue colourants available for food industries. It is valuable because blue is a primary colour, and natural blue pigments are rare. The increasing use of natural pigments is boosted by the consumers’ preference for natural and safe products, since synthetic ones (especially food colorants) are recognized as dangerous for human health [1].
PC is also widely used as a nutraceutical, as it is an excellent protein supplement containing almost all essential amino acids. Furthermore, PC is a bioactive molecule. Its therapeutic properties as antioxidant, anti-inflammatory, anti-cancer, neuroprotective and, in general, as a health-promoting agent have been extensively studied and acknowledged [1,2,3,4,5,6,7].
The principal source of commercial phycocyanin is Spirulina (cyanobacteria), term including various species of the genus Arthrospira and of the new proposed genus Limnospira [8,9] cultivated for commercial purposes (food, feed, production of bio-compounds, such as phycocyanin). This edible, protein-reach (up to 60–70% of biomass dried weight) microalgae group is considered safe worldwide, as it has been consumed by humans for millennia, and it becomes more and more popular thanks to its remarkable nutritional and health-promoting properties [9]. Moreover, Spirulina growth involves CO2 bio-fixation, contributing to the reduction in this greenhouse gas and to climate change mitigation [1,10]. Spirulina cultivation therefore responds to the growing demand and crucial need to exploit eco-sustainable renewable resources to obtain safe products through environmentally friendly green industrial processes.
PC extracted from Spirulina is one of the few foods natural blue colorants approved worldwide. According to European legislation PC is a colourant food, while in Australia it is listed as a food additive, as well as in the USA, where the Food and Drug Administration (FDA) has confirmed the pigment GRAS (Generally Recognized As Safe) status as a colour additive in foods and cosmetics [9,10,11,12]. The commercial value of PC is strongly dependent on its purity grade (P). PC retail price as a colorant in food sector (P > 0.7) is of a few euro (€) per g and can reach about €200 per mg for therapeutic and diagnostic applications (P > 3–4, for example, Merck product 52468), as the price increases significantly due to the high costs of extraction and purification.
The widespread use of PC is actually limited by the high production costs and by some intrinsic characteristics of this compound, such as its sensitivity to light, temperature, high and low pH and deterioration in organic solvents.
Critical issues for PC sustainable large-scale production are (i) biomass production to be optimized to increases PC content, and (ii) efficient extraction and purification procedure to enhance yield and purity grade, limiting pigment deterioration. Indeed, to obtain stable, bioactive, blue PC it is necessary to operate in mild conditions, applying rapid procedures both in the extraction and in the purification process. Finally, the product must be preserved by quickly applying appropriate storage conditions.
Despite these drawbacks, the global market of phycocyanin was assessed around 200 M US$ only for 2024 [13] and it is expected to rise to about 275–280 M US$ by 2030 [7,13,14].
Several studies [4,5,15,16,17] point out that efficient large-scale PC extraction is one of the major critical issues facing industrial PC production. In fact, although literature reports many examples of procedures exploited for laboratory-scale PC extraction, large-scale examples are not common and are usually patent-protected processes. Many methods are to be scalable, but their actual effectiveness on large-scale need still to be tested [18].
Various methodologies considered as environmentally friendly have been applied to PC extraction [4,10,18], such as pulsed electric field-assisted extraction, microwave-assisted extraction or enzymatic-assisted extraction. However, these methods present some disadvantages. In pulsed electric field-assisted and microwave-assisted extraction the increase in temperature of the samples can cause PC degradation. In addition, the possible electrode corrosion and metal leaking in pulsed electric field-assisted extraction can affect PC stability and pose serious limitations to industrial PC applications [19,20]. Enzymatic-assisted extraction is reported to ensure high yield, but it is not particularly suitable to large-scale production of PC because of the high cost of the commercially available enzymes [20]. One of the most widely used methods for extracting PC is repeated freeze-thawing [10]. It is particularly popular thanks to its simplicity, reproducibility, and moderate cost. However, it is quite time consuming, especially on large-scale production. Therefore, it is commonly exploited on laboratory-scale, to extract PC from limited amount of biomass [19,21]. Controversial evaluations are reported on the effectiveness of supercritical fluid extraction method (for example, with respect to equipment cost or limited extraction efficiency for polar compound (such as PC)) [18,22]. However, some studies, such as Deniz et al. [22], report excellent PC extraction yield (90.74%) exploiting supercritical fluid extraction, using supercritical CO2 and 10% ethanol as co-solvent. Ultrasound-assisted extraction of PC is another process considered green. But its effectiveness on large-scale is controversial too. In fact, some studies report that this process is not suitable for industrial production due to high energy consumption and low processing capacity [5,23], while others consider it an efficient and cost-effective method since ultrasonication-assisted extraction requires short processing times and low solvent consumption, allowing to achieve high yields [10,18,24]. Nevertheless, ultrasound-assisted extraction turns out to be one of the most widely used methods to obtain PBPs on laboratory scale [10]. This method has been applied to PC extraction although the temperature increase caused by ultrasonic cavitation must be controlled and avoided to preserve the pigment functionalities [19,25] and a low purity degree was generally achieved [23,26,27,28].
We have recently developed an innovative and rapid two-step extraction process, hereupon UltraBlu, to obtain blue PC crude extracts from fresh biomass of Arthrospira platensis [12] (see also Figure 1). In UltraBlu process, entirely carried out in aqueous medium, extraction phase is decoupled from biomass cell lysis. Cell lysis, executed by ultrasonication in ammonium sulphate (AS) solution, is merged with purification in a single step, before the pigment extraction/recovering phase.
On laboratory scale, concentrated (up to about 5 mg mL−1) brilliant blue PC extracts with high P (within 2.5 and 3.5) were obtained processing small volumes (5 mL) of biomass suspension [12], thus creating products suitable for the most common phycocyanin applications (i.e., foods, nutraceuticals and cosmetics) without applying additional downstream purification steps. Production time, hours instead of days, was reduced to the advantage of the product quality.
In this work, we present a progressive scale-up (up to the pilot-scale) of UltraBlu environmentally friendly process applied to the extraction of PBPs from A. platensis cultivated in laboratory-scale photo-bioreactor and in large-scale facilities for commercial purpose. To the best of our knowledge, this work is the first example of pilot-scale PC production using an ultrasound-based process.

2. Materials and Methods

2.1. Organism and Culture Conditions

2.1.1. Laboratory Scale

Arthrospira platensis, strain M2M, from the culture collection of the Institute of Bioeconomy of CNR (CNR-IBE, Sesto Fiorentino, Firenze, Italy), and strain 001, from Algaria Srl were used. The cells were grown in Zarrouk medium [29] in glass flasks (2000 mL working volume) or in a home-made photobioreactor constituted of four tubes of about 8 L capacity each (Figure 2a). Cultures were exposed to continuous illumination supplied from one side by two 60 × 60 cm LED panel (40 W, 4000 lm, Neutral White, 4000 K, V-TAC EUROPE Ltd., Sofia, Bulgaria). Air was continuously blown in the flasks and in the photobioreactor (from the bottom of the tubes). The temperature of the laboratory was controlled daily. During the biomass cultivation period, temperature was comprised between 22 and 27 °C.

2.1.2. Pilot Scale

Algaria strain 001 was semi-continuously cultivated in a 300 m2 open raceway pond (Figure 2b) exposed to natural sunlight inside a heated greenhouse (30 ± 5 °C). The growth medium used was based on Zarrouk medium, with a few modifications performed by the company.

2.2. Phycocyanin Extraction Procedures

A. platensis contains both PC and allophycocyanin (APC, a bluish PBP produced in smaller quantities). Both PC and APC were extracted applying the procedures used in this study, although referred only to PC extraction. Ultrasonication processes were always conducted in batch mode. CaCl2 0.1 M was used as an extracting solution. The dry weight (d.w.) of each biomass stock suspension was determined (by drying the fresh biomass at 105 °C for 5–6 h) to assess the yield of the recovered PBPs versus the biomass processed.

2.2.1. One-Step Conventional Ultrasound-Assisted Extraction Procedures for PC Control-Extraction and Optimization of Large-Scale Cell Lysis

Control Extraction Procedure
Reference ultrasound-assisted extraction procedure (control) on 5–6 mL of biomass suspension [12] was always performed on aliquots of the same biomass processed (1) by applying the two-step UltraBlu method or (2) by applying one-step direct ultrasound-assisted extraction on larger scale, executed to optimize cell lysis conditions.
Control extraction, based on conventional one-step ultrasound-assisted extraction, was carried out by direct ultrasonication in CaCl2 0.1 M (extracting solution), using a Hielscher Ultrasonic Processor UP200S as described by Lauceri et al. [12]. Briefly, the conditions used were: four ultrasonication cycles of 2 min (total sonication time equal to 8 min), at least 1 min pause between two consecutive ultrasonication cycles, maintaining the sample in an ice-water bath; sonication power 100%; pulse 1 s (i.e., continuous ultrasonication), extraction time at least 2 h (at room temperature), R > 100. R is the volume (mL)/biomass (g) ratio, calculated with respect to the d. w. of the processed biomass.
Finally, the supernatant (PBPs extract) was recovered by centrifuging and PBP content (PC + APC) and PC purity determined by spectrophotometric analysis (Section 2.3). For each biomass batch, mean value of two independent extraction trials was considered.
Optimization of Cell Lysis on Lab-Scale Processing 3–4 L of Biomass Suspension
Spirulina biomass was separated from the culture medium through a stainless-steel test sieve of mesh size 38 mm, washed with deionized water on the same sieve, recovered with a minimum volume of deionized water in a glass beaker (biomass stock suspension) and used quickly after its preparation to avoid osmotic cell lysis. Cell lysis conditions were optimized by applying longer sonication times and processing progressively increasing volumes (from 700 mL to 4 L on laboratory scale) and densities (i.e., decreasing R from 313 to 96.2 on laboratory scale, and from 575 to 61.3 on pilot scale) of biomass suspension (see also Table 1 in the Results section). Conventional direct one-step ultrasound-assisted extraction procedure was followed to establish optimal ultrasonication time to reach efficient PBPs extraction. A Hielscher Ultrasonic Processor UP400ST was used. Continuous ultrasonication at the maximum amplitude (A = 100%) was adopted, since reducing the amplitude caused a reduction in yield. During the ultrasonication process the biomass suspension was stirred and kept in an ice-water bath. The temperature of the suspension was continually checked by a temperature probe. The ultrasonication process was started when the biomass temperature was lowered up to 12 °C. The maximum temperature registered at the end of the ultrasonication process was 31 °C. Two 3 mL aliquots of suspension were taken at increasing ultrasonication times, centrifuged to eliminate cell debris, and PBP content determined by spectrophotometric analysis. Hence, data as the average of two replicates per biomass batch were considered.
Optimization of Cell Lysis on Pilot-Scale
Spirulina biomass was separated from the culture medium through a vibrating sieve of mesh size 25 μm, washed with tap water on the same sieve (biomass stock) and used immediately after. Cell lysis conditions were optimized processing 50 L of biomass suspension at low (R = 575) and high (R = 61) biomass density by applying direct one-step ultrasound-assisted extraction procedure. A Hielscher Ultrasonic Processor UIP2000hd was used. Continuous ultrasonication at the maximum amplitude (A = 100%) was adopted. During the ultrasonication process the biomass suspension was stirred and kept in an ice-water bath. The temperature of the suspension was continually checked by a temperature probe (Tmax = 31 °C) (Figure 3). Two 3 mL aliquots of suspension were taken at increasing ultrasonication times, centrifuged to eliminate cell debris and PBP content determined by spectrophotometric analysis after filtering the solution with a 0.45 μm pore-size membrane (Millex-HV syringe filter, diam. 33 mm, sterile, hydrophilic PVDF, Durapore®, Darmstadt, Germany) or 0.22 μm pore-size membrane (Millex-GV syringe filter, diam. 33 mm, sterile, hydrophilic PVDF, Durapore®, Darmstadt, Germany) to eliminate chlorophyll contamination. The average values of two replicates per biomass batch were considered.

2.2.2. UltraBlu Extraction Process

The UltraBlu process was executed using only Algaria strain 001.
Laboratory-Scale UltraBlu Extraction Process
Once optimal ultrasonication time was established, it was adopted in Step 1 (Cell lysis/purification phase, see Figure 1) of UltraBlu process.
In UltraBlu process the following conditions were used:
  • Step 1—Cell lysis/purification phase. Biomass was suspended in AS 1.2 M (final volume 3 L) and ultrasonicated at A 100% under stirring for 2 h (ultrasonication time), while being kept in an ice-water bath (temperature continually checked by a temperature probe). The lysed biomass was separated from the AS solution by centrifuging.
  • Step 2—Extraction phase. The lysed biomass was resuspended in CaCl2 0.1 M extracting solution (final volume 1.7 L) and stirred for 2 h (extraction time). Finally, the supernatant (crude extract) was recovered by centrifuging and PBP content and PC purity determined by spectrophotometric analysis.
Pilot-Scale UltraBlu Extraction Process
Pilot scale PC extraction was carried out at Spirulina production plant of Algaria in Casalbuttano (Lombardy, Northern Italy) at following conditions:
  • Step 1—Cell lysis/purification phase. Biomass was suspended in AS 1.2 M (final volume 50 L) and ultrasonicated at A 100% under stirring for 1:30 h (ultrasonication time), while being kept in an ice-water bath (temperature continually checked by a temperature probe). The lysed biomass was separated from the AS solution by centrifuging.
  • Step 2—Extraction phase. The lysed biomass was resuspended in CaCl2 0.1 M extracting solution (final volume 50 L) and stirred for 2 h (extraction time). Finally, the supernatant (crude extracts) was recovered by centrifuging and PBP content and PC purity determined by spectrophotometric analysis after having filtered the solution with a 0.45 μm or 0.22 μm pore-size membrane to eliminate chlorophyll contamination.

2.3. Spectrophotometric Analyses

Absorbance analyses were carried out with an UVmc2 (SAFAS Monaco, Monaco) or a XD 7500 (Lovibond, Amesbury, Salisbury, UK) spectrophotometer. Suprasil quartz cuvettes of 1 cm light-path were used.
PC and APC were determined using Equations (1) and (2) [30]. Absorbance values were corrected by subtracting the absorbance at 750 nm [12].
[PC] (mg mL−1) = (APC − 0.474 AAPC)/5.34,
[APC] (mg mL−1) = (AAPC − 0.208 APC)/5.09,
where APC and AAPC are the absorbance maximum of PC (at 615–618 nm) and APC (at 652 nm), respectively. Being PC the most abundant PBP in A. platensis, only the purity grade (P) of PC was considered (Equation (3)); however, the yield of the total extracted (or recovered) blue PBPs (PC + APC) was reported (Y%, Equations (4)–(6)).
P = APC/A280
Y (%) = ((CPBPs × VE)/(Bd.w. × VB)) × 100,
where A280 is the absorbance at 280 nm, related to the total protein content [31], CPBPs is the concentration (mg mL−1) of all the PBPs present in the extract, VE and VB are the recovered extract volume and the biomass stock suspension volume (mL), respectively, and Bd.w. is the biomass stock suspension dry weight (mg mL−1).
On pilot-scale wet biomass, stock was used. Equation (4) was adapted accordingly (Equation (5)):
Y (%) = ((CPBPs × VE)/(Bd.w. × Bw.w.)) × 100,
where Bd.w. is the biomass stock dry weight (mg mg−1), while Bw.w. is the weight (mg) of the wet biomass processed.
Finally, as the PC content of a Spirulina culture is variable over time, the yield Y of the various extraction processes were expressed (Yrel) with respect to the yield obtained extracting the same biomass batch in the reference conditions (control), Ycontr, (Equation (6)):
Yrel (%) = (Y/Ycontr) × 100,

3. Results

Efficient cell lysis is a key challenging step, since ultrasound-assisted extraction of large volumes of dense biomass suspensions can be problematic. Uniform propagation of ultrasounds in the biomass suspension is essential to ensure efficient cell lysis. Cell lysis conditions were optimized by processing increasing volumes (only on laboratory-scale. On pilot-scale we directly processed the largest volume, 50 L) and densities of biomass suspension and testing various ultrasonication times, since volume, suspension density and ultrasonication time are crucial parameters to set.

3.1. Laboratory-Scale Optimization of PC Extraction by UltraBlu Process

Efficient cell lysis conditions were determined by direct one-step ultrasound-assisted extraction in CaCl2 0.1 M. When 700 mL of biomass suspension with R = 461 were processed, 100% pigments (PC + APC) were extracted after 12 min sonication. Increasing the biomass suspension density (R = 223), pigments extraction was incomplete both after 12 min (Yrel = 83.6%) and 15 min (Yrel = 89.7%) of ultrasonication. Reducing the amplitude to 70% caused a reduction in yield in respect to the one reached at A = 100%.
Increasing the volume up to 3 L (R = 313) 90.7% of the PBPs were recovered only after ultrasonication for 1:45 h. When the largest biomass suspension volume planned was processed (4 L), the maximum recovery decreased to about 82.0% both at R = 258.4 and R = 145.6, by applying an ultrasonication time of 2:30 h. A longer ultrasonication period resulted in a slight reduction in the yield (Figure 4). This effect (i.e., reduced yield by applying sonication time longer than 2:30 h) was confirmed by a second experiment using a different biomass batch.
Having determined optimal cell lysis set-up, the two-step UltraBlu process was carried out under conditions designed to ensure a high PBPs concentration in the extract and a high P. Cell lysis/purification phase (Figure 1) was executed on 3 L of biomass suspension in AS 1.2 M, while the lysed biomass (Extraction phase, Figure 1) was suspended in 1.7 L of extracting solution (CaCl2 0.1 M). After an extraction time of 2 h, a concentration of 3.768 mg mL−1 of PBPs was achieved, 86.1% of PBPs were recovered with P = 2.58, while control extract had a much lower purity grade (P = 0.84).

3.2. Pilot-Scale PC Extraction by UltraBlu Process

Efficient cell lysis was the main issue to be addressed on a pilot-scale. Optimal cell lysis conditions were determined by direct one-step ultrasound-assisted extraction in CaCl2 0.1 M on the largest volume (50 L) of biomass suspension planned to be processed. Yrel > 90% was achieved both for low density (R = 575, Yrel = 95.8%) and high density (R = 61.3, Yrel = 92.4%) biomass suspensions following an ultrasonication of 1:30 h. This result was considered a good achievement, hence longer ultrasonication times were not tested.
UltraBlu process was carried out adopting this ultrasonication time: 1:30 h. Yrel = 59.2% and P = 1.72 were achieved processing a relatively low-density biomass suspension (R = 266). The unusual low yield obtained was due to (1) loss of part of the lysed biomass during biomass recovering from the centrifuge bowl before the extraction phase, and (2), probably, the characteristics of the centrifuge used to perform the two-step UltraBlu process on pilot-scale. In particular, the liquid and the solid phase of biomass suspension were separated by a disk stack centrifuge (Figure 5, Mora Marco Separation Technology Srl) operating in continuous at 1400 rpm (centrifugal force in g (gravitational force) unit unknown). Its rotating bowl, having a dead volume of 2.5 L, contained both the solid pellet and part of biomass suspension (Figure 5b). To recover the biomass pellet it was necessary to eliminate the suspension contained in the bowl, wasting part of the biomass.
In order to reduce biomass loss and increase the process yield, the UltraBlu process was repeated trying to transfer the biomass carefully to reduce losses during this operation and slightly modifying the centrifugation procedure. In particular, following cell lysis/purification phase centrifugation (Figure 1), additional 5 L of AS 1.2 M were centrifuged to boost the lysed biomass pellet formation and reduce the amount of biomass in the liquid phase present in the bowl. Processing a biomass suspension with a higher density (R = 70.5) than the previous pilot-scale attempt (R = 266), a higher yield was obtained (Yrel = 76.1%) but a lower purity grade (P = 1.41) was achieved, even if higher of the control extract purity grade (P = 0.65). The lower P was probably in part because of the low purity grade AS (agricultural grade, forming turbid solutions) used in this test, as the analytical grade reagent used on lab-scale and in the first trial of UltraBlu process on pilot-scale was no more available.
Table 1 summarizes all principal parameters/results of the tests executed on laboratory and pilot-scale and of the related controls.

4. Discussion

PC Extraction by UltraBlu Process

Foods is the largest market sector of phycocyanin worldwide, as 80% of globally produced PC is used in the food industry [10], which requires a low purity grade (P > 0.7).
The widespread use of PC in sectors requiring a higher purity grade (from cosmetic to bio-medic field) is in fact limited by the extreme costs, as PC price increases significantly due to the high costs of large-scale purification. Further, extraction and purification must face PC thermal, light and pH sensitivity. Indeed, the preservation of PC chemical physical properties, such as bioactivity, colour, fluorescence, as well as organoleptic characteristics, impose the choice of rapid processes and mild operative conditions.
Ultrasound-assisted extraction is one of the most common methods exploited to obtain phycobiliproteins [4,10,14,18,32,33]. It is considered an efficient and green method suitable on industrial scale [4,10,23]. However, to the best of our knowledge, the literature lacks examples of large-scale ultrasound-assisted extraction procedure applied to phycobiliprotein production [18,34]. Recently Vernès et al. [24] presented a study on green ultrasound-based extraction of proteins from Spirulina, operating in continuous flow rate using an ultrasonic flow-through reactor chamber. The process was performed on laboratory scale (about 800 mL of biomass suspension processed by a Hielscher Ultrasonic Processor UIP1000hd) and on pilot-scale (30 L of biomass suspension processed by a Hielscher Ultrasonic Processor UIP4000hd). The aim of Vernès et al. [24] was the extraction of all proteins produced by Spirulina biomass. It was not focused on PBPs, and conventional one-step direct ultrasound-assisted extraction was applied. However, although different, it represents the most similar study to our two-step UltraBlu process presented here, also addressing the issue of large-scale application and economic evaluation of the ultrasound-based method.
Our results confirmed that efficient cell lysis is a key challenging step, since ultrasound-assisted extraction of large volumes of dense biomass suspensions can be problematic. Uniform propagation of ultrasounds in the biomass suspension is essential to ensure efficient cell lysis. The absorption of an insufficient quantity of energy in all or in part of the processed biomass would lead to reduced cell lyses and ineffective extraction of PBPs. On the other hand, excessive energy absorption would lead to excessive cell lyses, with a larger release of contaminant compounds, as well as excessive temperature increase, damaging the pigments and causing a reduction in the yield and of the quality of the product.
Laboratory scale conventional one-step direct ultrasound-assisted extraction of PBPs in CaCl2 0.1 M evidenced that the efficiency of the process depends heavily on the volume and biomass suspension density. Yrel decreased (Table 1) processing higher volumes and lowering R (i.e., increasing biomass suspension density). Further, sonication time cannot be extended indefinitely, as PBPs can denaturise. PBPs are temperature sensitive and are quite unstable over 40 °C. To ensure a uniform ultrasonication and temperature of the biomass, the suspension was kept under stirring and chilled by immersion in a water-ice bath. In these conditions the maximum sonication time advantageous for 4 L suspension was of 2:30 h (Yrel  ≅ 82.4%). Longer sonication caused a yield reduction (Figure 4), most probably due to partial pigment thermal denaturation. A similar finding was also reported for the extractions of proteins from Spirulina by Vernès et al. [24] in respect to temperature, ultrasonic intensity and pressure increase.
Unlike the yield, P seemed not to be greatly affected by the volume or biomass density. Instead, P was correlated to the type of strain (Table 1). Extracts obtained by one-step direct ultrasound-assisted extraction in CaCl2 0.1 M from M2M strain had higher P (1.54 < P < 1.82) than those obtained from Algaria 001 strain (0.61 < P < 0.84), cultivated both on lab- and large-scale. However, the extract obtained from biomass cultivated on large-scale were always blue-green or greenish due to chlorophyll contamination. Microfiltration through a 0.45 μm or, preferably, 0.22 μm pore size membrane was necessary to eliminate chlorophyll aggregates and obtain a blue extract. The use of a 0.22 μm pore size membrane is particularly advantageous because it guarantees the necessary sterilization of the extract. In fact, high temperature sterilization cannot be applied to PC due to its thermal instability.
UltraBlu two-step process was performed using only Algaria 001 strain, since we were interested to verify UltraBlu efficacy on pilot-scale. In fact, only sufficient biomass of this strain was available to carry out pilot-scale tests, as it was cultivated by Algaria Srl for commercial porpoise. Despite lab- and pilot-scale extracts obtained by one-step direct ultrasound-assisted extraction had similar P, lab- and pilot-scale extracts obtained by UltraBlu process had quite different purity grade. The extract produced by the lab-scale process reached P = 2.58 with a Yrel = 86.1%, while on pilot-scale both the purity grade and yield were much lower, being the maximum purity grade and yield achieved equal to 1.72 and 76.1%, respectively. The P reached on pilot-scale, lower than the minimum value we expected (P > 2), could be result of various causes. For example, a lower pigment content of the biomass cultivated on large-scale (8–9% of PBPs) in respect of the lab-cultivated biomass (23–24% of PBPs), or the low efficiency of centrifugation, and the low purity grade of AS. However, extracts obtained by UltraBlu process had a higher purity grade and were less affected by chlorophyll contamination than extracts produced by direct one-step ultrasound-assisted extraction (Figure 6).

5. Conclusions

While laboratory-scale tests performed on 3 L of biomass suspension confirmed the efficacy of UltraBlu process, still Pilot-scale process need further validation. Our initial results hint UltraBlu as an effective process suitable to produce phycocyanin also on an industrial scale. However, industrial scale processes use very large volumes requiring ultrasonic devices more powerful of that used in this study and, most probably, an operational mode different from the batch mode we have used. Most likely devices such as flow cells would be more effective. Although these issues still need to be studied, we suggest that under optimized operating conditions, the UltraBlu process can be economically viable at the industrial level. Future focus should remain on scaling biomass input, controlling losses, and exploring routes to upgrade product value, ensuring long-term alignment between sustainability and profitability.

6. Patents

The authors declare that the UltraBlu process presented in this manuscript is the object of a patent application (WO2022144704A1, inventors: Rosaria Lauceri, Graziella Chini Zittelli, and Giuseppe Torzillo) submitted by Consiglio Nazionale delle Ricerche (CNR), Italy.

Author Contributions

Conceptualization, R.L.; methodology, R.L.; validation, R.L.; formal analysis, A.I. and R.L.; investigation, R.L., M.P. and A.G.; resources (biomass production), A.G.; data curation, M.P.; writing—original draft preparation, R.L. and A.I.; writing—review and editing, L.K.; visualization, L.K.; supervision, R.L.; project administration, R.L.; funding acquisition, R.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the European Union-NextGenerationEU under the Ultra-Blu project (C28H23000360002) within the framework of AMICO 2 Program of CNR–UVR supported by the PoC-PNRR measure of the Ministry of Enterprise and Made in Italy.

Data Availability Statement

All data generated or analyzed during this study are included in this published article. Additional information could be available upon request.

Acknowledgments

The authors gratefully acknowledge Graziella Chini Zittelli and Giuseppe Torzillo of the Istituto per la BioEconomia (CNR) for kindly providing Arthrospira platensis strain M2M, the Istituto Sperimentale Italiano Lazzaro Spallanzani (Rivolta d’Adda, Italy) for kindly providing the disk stack centrifuge used for the pilot-scale centrifugation in this work, and Luciano Foglio and Lorenzo Proietti for technical support.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Separations 12 00194 g001
Figure 1. Scheme of two-step UltraBlu process for phycocyanin extraction/purification. AS: ammonium sulphate.
Figure 1. Scheme of two-step UltraBlu process for phycocyanin extraction/purification. AS: ammonium sulphate.
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Separations 12 00194 g002
Figure 2. Tubular photobioreactor (a) and open raceway pond (b) for the laboratory and large-scale cultivation of A. platensis.
Figure 2. Tubular photobioreactor (a) and open raceway pond (b) for the laboratory and large-scale cultivation of A. platensis.
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Separations 12 00194 g003
Figure 3. Pilot-scale one-step conventional ultrasound-assisted extraction of PBPs in CaCl2 0.1 M.
Figure 3. Pilot-scale one-step conventional ultrasound-assisted extraction of PBPs in CaCl2 0.1 M.
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Figure 4. Yield of the phycobiliprotein (PBP) extraction process as a function of the ultrasonication time. A volume of 4 L of biomass suspension was processed. The two replicates are shown in different colours (red and blue).
Figure 4. Yield of the phycobiliprotein (PBP) extraction process as a function of the ultrasonication time. A volume of 4 L of biomass suspension was processed. The two replicates are shown in different colours (red and blue).
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Separations 12 00194 g005
Figure 5. Disk stack centrifuge used on pilot-scale (a). The centrifuge rotating bowl containing the biomass pellet and green suspension is also shown (b).
Figure 5. Disk stack centrifuge used on pilot-scale (a). The centrifuge rotating bowl containing the biomass pellet and green suspension is also shown (b).
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Figure 6. Absorbance spectrum of PC extract obtained by UltraBlu process (red curve) on pilot-scale (P = 1.72) and control extract obtained by one-step direct ultrasonication (blue curve) in CaCl2 0.1 M (P = 0.61).
Figure 6. Absorbance spectrum of PC extract obtained by UltraBlu process (red curve) on pilot-scale (P = 1.72) and control extract obtained by one-step direct ultrasonication (blue curve) in CaCl2 0.1 M (P = 0.61).
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Table 1. Principal parameters/results of the extraction tests of phycobiliproteins executed on laboratory and pilot-scale and of the related controls.
Table 1. Principal parameters/results of the extraction tests of phycobiliproteins executed on laboratory and pilot-scale and of the related controls.
Extraction Tests of PBPs: TEstr 1 = 2 hControl: Lab-Scale One-Step Direct Ultrasound-Assisted Extraction
V = 5–6 mL; TUS 2 = 8 min; TEstr = 2 h
A. platensis
Strain		ProcessScaleVolume ProcessedR 3TUS (h:mm)PBPs 4 Yield % (d.w.) ± SD
(no. = 2)		P 5 ± SD
(no. = 2)		RPBPs Yield % (d.w.) ± SD
(no. = 2)		P ± SD
(no. = 2)
M2MOne-step 6Lab-scale700 mL4610:12Yrel 7 = 98.7 ± 0.71.82 ± 0.00711Y 8 = 23.9 ± 1.31.77 ± 0.01
M2MOne-stepLab-scale700 mL2230:15Yrel = 89.7± 011.67 ± 0.01401Y = 23.5 ± 0.11.72 ± 0.02
M2MOne-stepLab-scale3 L3131:45Yrel = 90.7± 0.71.54 ± 0.011560Y = 22.1 ± 0.31.63 ± 0.00
M2MOne-stepLab-scale4 L258.42:30Yrel = 82.5 ± 2.41.73 ± 0.051292Y = 30.1 ± 1.01.75 ± 0,01
M2MOne-stepLab-scale4 L145.62:30Yrel = 82.4 ± 3.31.80 ± 0.01728Y = 24.3 ± 1.01.82 ± 0.01
Algaria-001UltraBlu 9Lab-scale3 L (Step 1)
1.7 L (Step 2) 		96.2 (Step 1) 54.5 (Step 2) 2:00Yrel = 86.1 (no. = 1)2.58 (no. = 1)280Y = 23.9 ± 0.50.84 ± 0.01
Algaria-001One-stepPilot-scale50 L5751:30Yrel = 95.8 ± 3.30.79 ± 0.00575Y = 10.6 ± 0.50.81 ± 0.01
Algaria-001One-stepPilot-scale50 L61.31:30Yrel = 92.4 ± 1.10.67 ± 0.00307Y = 10.6 ± 0.10.66 ± 0.01
Algaria-001UltraBluPilot-scale50 L2661:30Yrel = 59.2 (no. = 1)1.72 (no. = 1)194Y = 8.7 ± 0.20.61 ± 0.00
Algaria-001UltraBluPilot-scale50 L70.51:30Yrel = 76.1 (no. = 1)1.41 (no. = 1)572Y = 8.1 ± 1.40.65 ± 0.01
1. TEstr = extraction time; 2. TUS = ultrasonication time; 3. R = volume (mL)/biomass (g) ratio; 4. PBPs = phycobiliproteins; 5. P = purity grade; 6. One-step = One-step direct ultrasound-assisted extraction process; 7. Yrel = process yield expressed with respect to the control yield (Y); 8. Y = control process extraction yield; 9. UltraBlu = Two-step UltraBlu ultrasound-based extraction process.
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MDPI and ACS Style

Lauceri, R.; Pignataro, M.; Giorgi, A.; Idà, A.; Kamburska, L. Pilot-Scale Phycocyanin Extraction by the Green Two-Step Ultrasound-Based UltraBlu Process. Separations 2025, 12, 194. https://doi.org/10.3390/separations12080194

AMA Style

Lauceri R, Pignataro M, Giorgi A, Idà A, Kamburska L. Pilot-Scale Phycocyanin Extraction by the Green Two-Step Ultrasound-Based UltraBlu Process. Separations. 2025; 12(8):194. https://doi.org/10.3390/separations12080194

Chicago/Turabian Style

Lauceri, Rosaria, Melissa Pignataro, Antonio Giorgi, Antonio Idà, and Lyudmila Kamburska. 2025. "Pilot-Scale Phycocyanin Extraction by the Green Two-Step Ultrasound-Based UltraBlu Process" Separations 12, no. 8: 194. https://doi.org/10.3390/separations12080194

APA Style

Lauceri, R., Pignataro, M., Giorgi, A., Idà, A., & Kamburska, L. (2025). Pilot-Scale Phycocyanin Extraction by the Green Two-Step Ultrasound-Based UltraBlu Process. Separations, 12(8), 194. https://doi.org/10.3390/separations12080194

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