An Easy and Non-Hazardous Extraction Method for Phycobiliproteins and Pigments from Anabaena cylindrica

Abstract

Phycobiliproteins and pigments derived from cyanobacteria hold significant potential for diverse applications in the food, pharmaceutical, and chemical industries. The filamentous cyanobacterium Anabaena cylindrica serves as a valuable resource for extracting these compounds. This study develops a simplified, safe, and cost-effective extraction method that eliminates toxic solvents and minimizes processing steps. This makes the method applicable for all users and allows the easy integration of the extraction into biorefinery concepts in which the biomass is to be used as a fertilizer, for example. Utilizing salts such as ammonium sulfate and calcium chloride (15 gL⁻¹ each) enables the effective extraction of phycocyanin (PC) and allophycocyanin, achieving a PC concentration of 192.34 $\mathrm{mg}{\mathrm{g}}_{\mathrm{CDW}}^{-1}$ and 209.44 $\mathrm{mg}{\mathrm{g}}_{\mathrm{CDW}}^{-1}$, respectively. Ethanol was introduced as a less toxic alternative to methanol for pigment extraction, increasing chlorophyll a and carotenoid recovery by 21% and 37%, respectively.

1. Introduction

The market size for phycocyanin is expected to increase by 33.8% between 2023 and 2030 [1], which means that production is likely to increase in the coming years. This results in increased interest in research on new processing methods. Phycocyanin is produced by cyanobacteria. These are prokaryotes that can grow photoautotrophically, which enables sustainable cultivation, as apart from CO₂ from the air, no other carbon sources are required for cultivation. Just like plants, cyanobacteria require light-harvesting complexes for photosynthesis, which is why they possess chlorophyll a and carotenoids. In addition to these pigments, cyanobacteria also possess other light-harvesting complexes in the form of phycobiliproteins (PBPs). These are made up of a protein component and Phycobilins [2]. In addition to the aforementioned phycocyanin (PC), PBPs include allophycocyanin (APC) and phycoerythrin (PE). These additional light-harvesting complexes enable cyanobacteria to utilize a broader light spectrum than plants [3]. PBPs are used in fluorescence-based diagnostics, cancer therapies, and stress-related treatments, for example [2]. Similarly, carotenoids and chlorophyll are valued for their antioxidant properties and applications in the food and pharmaceutical industries [4,5]. PBPs as well as chlorophyll and carotenoids can also be used as dyes, making cyanobacteria the perfect organisms for the production of natural dyes. PC and APC have a blue color, PE red, chlorophyll green and carotenoids orange. PC is primarily used as a food colorant and is already being produced commercially in large scales for this purpose [6]. Industrially, cyanobacteria hold immense potential due to their ability to produce a wide array of valuable compounds, including over 1100 secondary metabolites [7]. Applications range from biofuels and natural dyes to pharmaceuticals and food additives [8]. The extracellular polymeric substances (EPS) produced by cyanobacteria can improve soil water retention and cyanobacteria can serve as natural fertilizers, enhancing agricultural yields [9]. In this context, the ability of some cyanobacteria to fix nitrogen can be useful. Despite their advantages, challenges such as low growth rates compared to heterotrophic organisms and low product stability limit their industrial scalability.

Due to the wide range of applications, however, biorefinery concepts are conceivable for optimizing costs, which enable the multiple use of the biomass or the production of several valuable products. Such biorefinery concepts have already been described many times in the literature [10,11,12]. One conceivable scenario is the production of PC as a food colorant, chlorophyll as a natural dye and the subsequent use of the biomass as biofertilizer. However, this also entails requirements for the production of the dyes, which are obtained by extraction from the biomass. The extraction of pigments and phycobiliproteins involves mechanical and non-mechanical methods, with cell disruption being a common starting point. Techniques such as ultrasonic and microwave-assisted extraction, alongside traditional solvent-based methods, are widely studied but often require optimization for lab-scale applications [13]. Challenges include the thermal sensitivity of products and the complexity of multi-step procedures.

In this work, a method for the combined extraction of phycobiliproteins and pigments from A. cylindrica was developed to overcome these challenges. Since PBPs are soluble in aqueous solvents and pigments in organic solvents, the separate extraction of PBPs and pigments could not be avoided. As biorefinery concepts can increase cost efficiency, it is assumed that the biomass will be used as biofertilizer after extraction. On this basis, the following requirements for the method were defined: (i) low-cost solvents, (ii) a one-step extraction process without mechanical pre-treatment, (iii) the use of less toxic solvents and (iv) no need to remove residues of the solvents. By fulfilling these requirements, it can be ensured that the method is as simple and cost-effective as possible. The use of less toxic solvents leads to greater safety for operators and improved food safety (for example, when chlorophyll is used as a food colorant), makes the method more environmentally friendly and in turn means that residues of the solvent in the biomass are less problematic. In combination with the simplification of the extraction, the process could theoretically be carried out by non-professionals, which could lead to a further reduction in costs.

2. Materials and Methods

2.1. Cultivation of Cyanobacteria

All experiments were conducted with the cyanobacterium Anabaena cylindrica (strain number 1403-2). The strain originates from the Experimental Phycology and Culture Collection of Algae at the University of Goettingen (EPSAG, Göttingen, Germany). The pre-culture was carried out in 300 mL Erlenmeyer flasks in 100 mL BG11 medium according to Rippka et al. [14]. The flasks were incubated in a shaking incubator at 30 °C and 120 rpm for 14 days. Illumination was provided continuously with a photosynthetically active radiation of 140 µmol m⁻² s⁻¹. The biomass including medium was then transferred to 1 L vertical tubular photobioreactors (PBRs) (V = 1 L, d = 6 cm, Algoliner, Messel, Germany) and cultivated again for 14 days. The tubular PBR was illuminated using red and blue LEDs (red/blue = 4:1) with a photosynthetically active radiation of 100 µmol m⁻² s⁻¹. The PBR was continuously gassed with air at a flow rate of 1 L min⁻¹. Cultivation took place at room temperature. The harvest was carried out by stopping the gassing and allowing the biomass to settle in the lower part of the PBR (approx. 2 to 3 h settling time). The supernatant was removed, and the biomass was transferred to centrifuge tubes. The remaining medium was separated from the biomass by centrifugation at 3005× g (Mega Star 1.6R, VWR, Leuven, Belgium) for 15 min and discarded. The biomass was then lyophilized for 48 h at −20 °C and 1 mbar and the cell dry weight (CDW) was determined gravimetrically.

2.2. Extraction of Phycobiliproteins and Pigments

All extractions in this work were performed with 20 to 30 mg CDW. The original method used for the extraction of PBPs and pigments was the method according to Kollmen et al. [15] (see the red path in Figure 1). The extraction was carried out from the previously dried biomass. The CDW was transferred to 2 mL reaction vessels (maximum 80 mg) and 1 mL of potassium phosphate buffer (pH7, 0.1 M) was added. Subsequently, 50% (w/w) glass beads (diameter: 0.7–0.9 cm) were added to the biomass suspension and the biomass was disrupted in a mixer mill (MM 301, Retsch Technology GmbH, Haan, Germany) for 10 min at a frequency of 30 s⁻¹. The biomass was then transferred to 15 mL reaction vessels. Additional potassium phosphate buffer (PPB) was added until the ratio of PPB to CDW was 0.25 mL mg⁻¹. The extraction was then carried out for 24 h in the dark at 4 °C. Subsequently, the suspension was centrifuged at 3005× g for 10 min and the supernatant was transferred to a new reaction tube and measured spectrophotometrically (Cary 60 UV-Vis, Agilent Technologies, Santa Clara, CA, USA) at the wavelengths 652, 615 and 562 nm. The concentration of the PBPs was calculated using the equations according to Bennett and Bogorad [16].

After the extraction of the PBPs, chlorophyll a and carotenoids were extracted. For this purpose, the resulting pellet was resuspended in methanol saturated with CaCO₃. For each milligram of CDW, 0.5 mL of methanol was added. The suspension was then stored for 24 h at 4 °C in the dark. Afterwards, it was centrifuged at 3005× g for 15 min. The supernatant was transferred to a new reaction tube and measured spectrophotometrically at 665, 652 and 461 nm. The chlorophyll a and carotenoid content were determined using the equations according to Porra et al. [17] and Chamovitz et al. [18], respectively.

The new method (the green path in Figure 1) is based on the combined cell disruption and extraction of PBP. For this purpose, different salt solutions (ammonium sulfate and calcium chloride) with a concentration of 15 g L⁻¹ were used. As a result, the cells are damaged by the osmotic shock and the pigments can be extracted [19]. For this, 5 mL of the salt solution was pipetted onto the CDW and then the PBPs were extracted for 24 h in the dark. This was followed by centrifugation at 3005× g for 10 min to separate the cell pellet from the solved PBPs and then the concentration of PBPs was determined spectrophotometrically as before. These experiments were carried out with ammonium sulfate and calcium chloride solutions. For better comparability, the experiments were also carried out with PPB (pH 7, 0.1 M) and water as the extraction agent.

The extraction of chlorophyll a and carotenoids was carried out in the same way as the previous method, whereby the toxic solvent methanol was replaced by the less toxic solvent ethanol.

2.3. Statistical Analysis

All experiments were performed with seven biological replicates and mean values with standard deviations were calculated. The data were checked for normal distribution using the Shapiro–Wilk test. In addition, the data were tested for equal variance. The t-test was then used to test for significance at a fixed significance level of p < 0.05.

3. Results and Discussion

3.1. Extraction of Phycobiliproteins

The extraction of phycobiliproteins (PBPs) should be simplified and shortened. For this purpose, cell disruption is combined with extraction, so that separate cell disruption in the ball mill can be eliminated. In total, 15 g L⁻¹ of ammonium sulfate and calcium chloride solutions were selected as extraction solvents. Both salts are inexpensive and easily available. At the same time, they facilitate the subsequent use of the biomass as natural fertilizer after extraction as residues do not have to be removed. Both salts, ammonium sulfate and calcium chloride, are already used as fertilizers in the agricultural industry [20,21]. This makes it possible to extend the value chain and thus increase economic efficiency.

A comparison of direct extraction with potassium phosphate buffer (PPB) and extraction with mechanical cell disruption as pretreatment shows that cell disruption is not necessary (see Figure 2). No significant change in the extractable amount of phycocyanin (PC) or allophycocyanin (APC) was observed. Phycoerythrin is not present in all extracts because A. cylindrica produces only small amounts of PE under the cultivation conditions used in this study. Extraction with water in place of PPB does not lead to any significant change in the extractable amount of PBP. By using 15 g L⁻¹ (NH₄)₂SO₄ solution, the extractable amount of APC can be significantly increased and by using 15 g L⁻¹ CaCl₂ solution, the amount of PC can be significantly increased. Extraction with 15 g L⁻¹ (NH₄)₂SO₄ or CaCl₂ is therefore preferable, as larger quantities of PBPs are extracted. The maximum PC concentrations achieved were 192.34 and 209.44 $\mathrm{mg}{\mathrm{g}}_{\mathrm{CDW}}^{-1}$ with (NH₄)₂SO₄ and CaCl₂, respectively. For APC, the maximum concentrations were 112.05 and 91.11 $\mathrm{mg}{\mathrm{g}}_{\mathrm{CDW}}^{-1}$ with (NH₄)₂SO and PPB, respectively. This results in calcium chloride as the optimum solvent for the extraction of PC and ammonium sulfate as the optimum solvent for the extraction of APC from A. cylindrica.

The results obtained in this work show that the extraction of PBP from A. cylindrica is possible without prior mechanical cell disruption. This could indicate that the biomass is already in a digested or damaged state. Damage to the cells may occur during the lyophilization step. Kim et al. [22] investigated lyophilization as a method for cell lysis of Microcystis aeruginosa and obtained a disruption efficiency of 92.2%. Corbett and Parker [23] investigated the influence of lyophilization on 13 different cyanobacterial strains, including strains of the genus Anabaena, and obtained different viabilities depending on the suspension solution during lyophilization. Thus, damage to the cells cannot be ruled out here either. However, this effect is presumably strain-specific and should therefore be checked when transferring the method to other production strains. While the direct extraction of PBP with ammonium sulfate has not yet been investigated, there are already some studies on extraction with calcium chloride, although not from A. cylindrica. Dincoglu et al. [24] investigated the extraction of PBP from a microalgae cyanobacteria mixture consisting of Haematococcus pluvialis, Chlorella vulgaris and Arthrospira platensis. The biomass was lyophilized before extraction, similar to this work. They observed optimal extraction using 20 g L⁻¹ CaCl₂ for 1 h at 100 rpm and a solvent–biomass ratio of 1:100, but no control experiment was performed to test the extraction for completeness. Due to the similar pretreatment of the biomass, it is therefore likely that the method developed in this study can be transferred to other strains. Julianti et al. [25] obtained lower PC concentrations with 10 g L⁻¹ CaCl₂ solution as the extractant compared to 0.01 M phosphate buffer (pH 7). The extraction was performed from dried Spirulina powder and in combination with freeze–thawing cycles. These results contradict the results observed here of better extraction with CaCl₂ compared to phosphate buffer. The different extraction methods and different organisms must be considered as reasons for this. Furthermore, it might be possible to extract PBPs directly from living A. cylindrica using CaCl₂ solution as an extractant. Pott [26] investigated CaCl₂ as an extraction agent for the direct extraction of PC from living Spirulina and achieved approximately 90% PC yield with 55.5 g L⁻¹ CaCl₂ solution in relation to an extraction after cell disruption in the ball mill. Therefore, direct extraction from fresh biomass should also be tested in future experiments. Another criterion that should be considered in the course of optimizing the extraction is the storage stability of the PBPs. The stability of PBP can be increased by the addition of preservatives, whereby it has already been shown that inorganic salts can be used for this purpose [27,28]. Kannaujiya and Sinha [29] showed an increase in the stability of PC and PE for CaCl₂ as a preservative. The extraction with salt solutions could therefore also improve the stability of the PBP, which was not checked within this work. Ammonium sulfate is often used in the literature for precipitation and consequently for the purification of PBPs [30]. However, the concentration is significantly higher than the 15 g L⁻¹ selected here. It must be ensured when using salt solutions that the concentrations are not too high, as otherwise, proteins and thus PBPs may precipitate.

3.2. Extraction of Chlorophyll a and Carotenoids

In addition to PBPs, the pigments chlorophyll a and carotenoids were extracted from the cyanobacteria. It was first investigated whether the method selected for the preceding extraction of PBPs had an influence on the extraction of chlorophyll a and carotenoids (Figure 3A). After the extraction of the PBPs with potassium phosphate buffer, the highest concentrations of chlorophyll a and carotenoids are extracted. The cell disruption has no influence on the extraction of the pigments either. If the PBPs are extracted with water, 15 gL⁻¹ ammonium sulfate or calcium chloride solution, the subsequently extractable amount of chlorophyll a is significantly lower by approx. 25%. The amount of carotenoids is also significantly lower.

One reason for the lower extractable quantities of chlorophyll a and carotenoids could be co-extraction in the previous step. Although chlorophyll a and carotenoids are insoluble in aqueous solvents, contamination by chlorophyll during extraction with water or aqueous solvents has been observed several times in the literature [31,32,33,34]. Thus, there is probably also a slight extraction of chlorophyll a and carotenoids during the extraction of PBPs. This effect can be reduced by using potassium phosphate buffer to extract the PBPs [15]. However, the influence on the measurement of the PBP content is small, as A. cylindrica contains 10 times as much PC as chlorophyll a. Considering the amount of all PBPs, the ratio is approx. 300 $\mathrm{mg}{\mathrm{g}}_{\mathrm{CDW}}^{-1}$ PBPs (extraction with (NH₄)₂SO₄ or CaCl₂) to approx. 24 $\mathrm{mg}{\mathrm{g}}_{\mathrm{CDW}}^{-1}$ chlorophyll a and carotenoids (the previous extraction of PBPs with PPB). Since the problem of chlorophyll contamination in the extraction of PBPs is well known, mathematical methods for eliminating the influence in the calculation of PBP contents are described in the literature [35].

The aim of this work is to develop a simple and safe method for the extraction of PBPs and pigments. The extraction of chlorophyll a and carotenoids is usually carried out with an organic solvent, many of which are highly toxic. This also applies to methanol, which is used in the original method [36]. Therefore, methanol was replaced by a less hazardous solvent, and ethanol was chosen. In addition, methanol was saturated with CaCO₃ in the initial method, although this was eliminated as an additional step. The addition of CaCO₃ to methanol had no significant effect on the extraction of chlorophyll a and the carotenoids (Figure 3B). Therefore, the addition of CaCO₃ can be omitted. However, CaCO₃ can have an influence on the stability of the pigments [37]. By using ethanol as an extraction agent, the chlorophyll a concentration could be significantly increased from 15.97 $\mathrm{mg}{\mathrm{g}}_{\mathrm{CDW}}^{-1}$ (extraction with methanol) to 19.36 $\mathrm{mg}{\mathrm{g}}_{\mathrm{CDW}}^{-1}$. A significant increase in the carotenoid concentration of 37% could also be observed. Thus, ethanol is excellently suited for extracting the pigments from A. cylindrica. An influence on the subsequent use of the biomass as biofertilizer can be prevented by drying the biomass, as this completely evaporates the solvents.

Different solvents are described in the literature as optimal for extracting the respective pigments. Papista et al. [38] described an increase in the extraction of chlorophyll a by extraction with methanol compared to ethanol of 23.1%. Thao et al. [39], on the other hand, used ethanol as an extraction agent for chlorophyll a from A. platensis. For carotenoids, too, the literature is not consistent as to what is the best extraction agent. For example, Tavanandi et al. [40] found ethanol to be the best extraction agent compared to methanol, DMSO, diethyl ether and acetone. Assunção et al. [41] optimized the extraction of carotenoids for Chroococcidiopsis sp. Different extraction agents such as methanol, ethanol and acetone were investigated, with the highest content of carotenoids being achieved with methanol. In this work, ethanol was found to be the optimal solvent, whereby it also had advantages in application due to its lower toxicity compared to methanol.

4. Conclusions

This study presents a straightforward and non-hazardous approach for extracting phycobiliproteins, chlorophyll a and carotenoids from Anabaena cylindrica. By integrating cell disruption and extraction into a single step using salt solutions, the method eliminates the need for mechanical processing and reduces costs. Calcium chloride (15 g L⁻¹) has proven to be the best extraction agent for PC, followed by ammonium sulfate (15 g L⁻¹), achieving PC concentrations of 209.44 $\mathrm{mg}{\mathrm{g}}_{\mathrm{CDW}}^{-1}$ and 192.34 $\mathrm{mg}{\mathrm{g}}_{\mathrm{CDW}}^{-1}$, respectively. For the extraction of APC, ammonium sulfate with 112.05 $\mathrm{mg}{\mathrm{g}}_{\mathrm{CDW}}^{-1}$ is the best solvent. Therefore, the solvent must be adjusted according to the target product. As both salts are used as fertilizers, the biomass can be used as a fertilizer after extraction despite the residues of the salts. During pigment extraction, it was shown that replacing the toxic methanol with ethanol leads to a higher concentration of chlorophyll a and carotenoids by 21% and 37%, respectively. The method demonstrated excellent yields for both phycobiliproteins and pigments, offering an efficient route for producing valuable compounds. Future research could focus on refining product stability and exploring additional applications to maximize industrial scalability.