Optimizing Biomass and Phycobilin Production in Arthrospira platensis Through Carbon Source Variation and Developing a Cost-Effective Purification Method

Optimizing Biomass and Phycobilin Production in Arthrospira platensis Through Carbon Source Variation and Developing a Cost-Effective Purification Method | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Optimizing Biomass and Phycobilin Production in Arthrospira platensis Through Carbon Source Variation and Developing a Cost-Effective Purification Method Zahra Soltani Far, Mohammad Ali Nematollahi, Seyed Vali Hosseini, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7284160/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Arthrospira platensis, commonly known as Spirulina , is an industrially cultivated cyanobacterium due to its high content of phycobiliproteins, mainly phycoerythrin, phycocyanin, and allophycocyanin. In this work, a low-cost strategy for the enhancement of biomass and pigment production, along with the improvement of phycocyanin extraction, is investigated. In this regard, two carbon sources at different dosages were tested for their effect on biomass and phycobilin production. Biomass production was estimated in terms of specific growth rate (SGR), cell count per liter, and increase in dry weight. Phycobilin protein quantification was done through spectrophotometry and gel electrophoresis. The results indicated that the addition of extra carbon sources increased cell proliferation and biomass production. Sodium acetate at 50 mg/L/day significantly increased the biomass, SGR, and phycocyanin production. Glycerol at 1 cc per day enhanced cell proliferation and biomass but did not enhance phycobilin accumulation. The spectrophotometric analysis on 5 mg dry phycocyanin with 10 cc distilled water showed no significant difference (p ≥ 0.05) at 680 nm, while in the absorbance at 620 nm, there is a significant difference between spray and oven drying of the samples (p ≤ 0.05). Spirulina Mixotrophic Culture Carbon Supplementation Phycocyanin Biomass Production Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Cyanobacteria, also known as blue-green algae, are a group of photosynthetic and nitrogen-fixing bacteria living in almost all sorts of moist soils and waters, either freely or in symbiotic relationships (Hamouda et al., 2022 ). These bacteria have been found capable of synthesizing a variety of secondary metabolites, including biologically active compounds such as antibacterial, antiviral, antifungal, and anticancer agents (Richmond, 2013 ; Hu, 2019 ; Nowruzi et al., 2021 ; Zanolla et al., 2022 ). These properties are strain- and culture-condition-dependent (Zanolla et al., 2022 ). Microalgae are generally photoautotrophic, but some strains have great potential to grow under mixotrophic or heterotrophic conditions (Abiusi et al., 2020 ; Jin et al., 2020 ; Smith et al., 2020 ). Spirulina spp. (Arthrospira platensis) is a cyanobacterium widely grown in alkaline brine lakes (Nege et al., 2020 ). The high concentration of protein pigments called phycobiliproteins in spirulina sets it apart from other microalgae (Glazer, 1985 ). Phycobiliproteins consist of three main components: phycoerythrin, phycocyanin Glazer, and allophycocyanin Sonani et al., ( 2017 ). Among them, phycocyanin is a blue-colored natural pigment with different physiological activities, such as antioxidants, antibacterial, and anticancer properties. It could also be applied in the food industry as a bioactive substance and dye (Kumar et al., 2014 ; Liu et al., 2016 ; Lee et al., 2022 ). Although it is true that microalgae, to which Spirulina spp. belong, can use inorganic carbon sources, their biomass productivity is generally low and limited (Zhu et al., 2019 ; Zhu et al., 2022 ), their biomass productivity is often low and limited (Verma et al., 2020 ). It was proposed to provide microalgae cultures with an organic carbon source for maximal production of high-quality biomass (Roostaei et al., 2018 ). Several photoautotrophic microalgae have mechanisms to enhance the utilization of externally added sugars under mixotrophic and heterotrophic conditions (Verma et al., 2020 ). With an organic carbon source, it increases productivity in biomass with higher efficiency and cell density (Roostaei et al., 2018 ; Nowruzi et al., 2021 ). Besides, it also increases levels of desirable compounds like lipids and pigments (Ip et al., 2004 ; Liang et al., 2009 ; Yun et al., 2021 ). The environmental impact of the cultivation stage is significant due to the extensive use of chemicals and nutrients (Lim et al., 2021 ). Therefore, various types of culture media—chemical-based, modified, and alternative—have been reviewed for Spirulina cultivation (Wang et al., 2019 ). Further study is needed to modify or explore alternative culture media utilizing waste, wastewater, or industrial by-products to ensure the long-term sustainability of the environment and nutrient sources for cultivation. In this research, we aim to investigate the effects of adding three different carbon sources in daily doses to Spirulina mixotrophic cultures. These sugars are relatively cheap and easy to use. The carbon sources will be applied in fixed doses but at short intervals, allowing comparison of growth results with conditions where the carbon source is added only once at the beginning and cultures grow solely via photosynthesis. We will measure the consequent effects on cell proliferation, biomass production, and phycocyanin production. In a similar study, de Morais et al., ( 2020 ) investigated the different concentrations of crude glycerol that influence productivity, biomass concentration, and biomolecule content in cultures of Spirulina sp. It states that microalgae grown under different glycerol concentration regimes in Erlenmeyer bioreactors for the 15-day test obtained the best results when this compound was at a value equal to 2.5 g L⁻¹, giving a maximum (0.43 g L⁻¹ day⁻¹) of dry weight production. The scientists also show that proteins compose the main constituent of these biomasses at a yield of 75.7% w/w when considering 2.5 g L⁻¹ as the best glycerol concentration, and their synthesizing and accumulation into these biomass's cells is strictly dependent upon glycerol concentration. Moreover, (da Silva Braga et al., 2018 ) focused on verifying if the production of Spirulina biomass with high carbohydrate content is stimulated by a reduced supply of nitrogen combined with the addition of NaHCO₃ or CO₂ at different flow rates and injection times. The addition of 0.25 g L⁻¹ of NaNO₃ allowed Spirulina to accumulate up to 49.3% (w/w) of carbohydrates with the highest amount of CO₂ (0.3 vvm injected for 5 min). This value reached 59.1% (w/w) when NaHCO₃ was the carbon source. Meanwhile, biomass concentrations achieved were 0.81 and 0.97 g L⁻¹, respectively. In another study, Yun et al., ( 2021 ) reported increased bioresource productivity under both mixotrophic and heterotrophic conditions for Chlorella sp. cultivation. Different conditions were applied to three well-known strains of Chlorella (KNUA104, KNUA114, and KNUA122) to assess biomass productivity. Compositional changes (lipids, protein, and pigments) were evaluated in BG11 media under photoautotrophic, mixotrophic, and heterotrophic conditions, utilizing different initial concentrations of glucose (5, 10, 15, 20, and 25 g L⁻¹). All strains under mixotrophic and heterotrophic conditions were optimally cultured with 15–20 g L⁻¹ initial glucose. Despite their beneficial properties, the incorporation of microalgae metabolites faces several obstacles (Hosseinkhani et al., 2022 ). These obstacles include the hard cell wall of most species, the high sensitivity and reactivity of ingredients, as well as issues like fishy taste, odor, and color, which reduce consumer acceptance. Furthermore, the high costs and complexity of downstream processing, along with legislative issues related to the incorporation of microalgae into food and pharmaceuticals, form serious barriers to industrial-scale production (Zhang et al., 2019 ; Howlader & French, 2020 ; Zahra et al., 2020 ; Ahmad et al., 2022 ). Moreover, the economic and environmental costs of traditional isolation and purification processes pose obstacles to the production of high-value algal metabolites (HVAMs). These processes involve harmful residues, high temperatures, and high time and energy demands, leading to metabolite degradation (Heffernan et al., 2016 ). In the United States, for instance, management of production-related waste has resulted in the release of 25.45 billion pounds of toxic chemicals (Persico & Venator, 2021 ). Risks such as solvent toxicity and the presence of solvent contaminants in extracts, coupled with low yields, have spurred the development of alternative extraction methodologies, such as green and clean methods, which aim to reduce or eliminate the use of organic solvents (Chemat et al., 2012 ; Chemat et al., 2019 ; Picot-Allain et al., 2021 ; Rodriguez Garcia & Raghavan, 2022 ). Sustainable methods for designing innovative and efficient processes are crucial to improving efficiency, time, and costs while ensuring the sustainable use of resources and materials (Jacotet-Navarro et al., 2016 ; Verma et al., 2020 ). Recent reports have highlighted various techniques aimed at maximizing phycocyanin extraction yield and minimizing environmental and financial costs from Arthrospira platensis biomass in different physical forms (dry, wet, and frozen) (Wyman, 1992 ; Sekar & Chandramohan, 2008 ; Chittapun et al., 2020 ; Rahman, 2021 ; Gladfelter et al., 2022 ). Several methods have been proposed to disrupt the cell wall, including homogenization, sonication, microwave treatment, supercritical fluid extraction, and lysozyme disintegration (Duangsee et al., 2009 ; Deniz et al., 2016 ; Martínez et al., 2017 ; Chittapun et al., 2020 ; Ferreira-Santos et al., 2021 ). However, many of these extraction methods are not feasible for large-scale applications due to high costs or challenges with industrial-scale implementation. For example, techniques like ultrasound or freezing thawing may not be applicable on an industrial scale (Howlader & French, 2020 ). Researchers often combine methods to reduce costs and increase the yield or stability of products (Zahra et al., 2020 ). However, when it comes to phycocyanins, one of the main challenges in producing high-quality dry powder is the high solubility and sensitivity of phycocyanin protein pigments to heat (above 30°C) and pH shifts (optimal pH around 7) (Ferreira-Santos et al., 2021 ; Lee et al., 2022 ). This study aims to develop an extraction process from a cost-effective bench-scale perspective with the potential for industrialization. Therefore, methods that minimize chemical and thermal treatments are more effective and financially reasonable. Physical separation of proteins using filters has shown promising results in extracting contents from microalgae (Zhang et al., 2019 ; Howlader & French, 2020 ; Ahmad et al., 2022 ) Over the past few decades, membrane processes have emerged as important methods in the food business. They are frequently regarded as the best available technology (BAT) because of their capacity to gently treat foodstuffs at low to moderate temperatures (Chen et al., 2018 ; Tabani et al., 2018 ; Castro-Muñoz et al., 2021 ; Monesi et al., 2022 ). These processes offer several advantages over conventional separation systems, including high separation precision, better selectivity, operation at room temperature, no chemical damage, high automation, easy operation, energy savings, reduced costs, comprehensive utilization of resources, and reduced pollution. Researchers often combine methods to reduce costs and increase the yield or stability of products (Zahra et al., 2020 ). Specifically, for phycocyanins, a significant challenge in producing high-quality dry powder is the high solubility and sensitivity of phycocyanin protein pigments to heat (above 30°C) and pH shifts (optimal pH around 7) (Ferreira-Santos et al., 2021 ; Lee et al., 2022 ). This study aims to develop a cost-effective bench-scale extraction process with industrialization potential, focusing on minimizing chemical and thermal methods. Physical separation of proteins using filters has shown promising results for extracting contents from microalgae (Zhang et al., 2019 ; Howlader & French, 2020 ; Ahmad et al., 2022 ) Membrane processes have become pivotal in the food industry, recognized as the best available technology (BAT) due to their ability to treat products gently at low-to-moderate temperatures (Chen et al., 2018 ; Tabani et al., 2018 ; Castro-Muñoz et al., 2021 ; Monesi et al., 2022 ). They offer high separation precision, better selectivity, operation at room temperature, no chemical damage, high automation, easy operation, energy savings, reduced costs, comprehensive resource utilization, and reduced pollution (Lipnizki & Ruby-Figueroa, 2013 ; Chen et al., 2018 ; Quezada et al., 2021 ). In this paper, we demonstrate a cost-effective method to enhance biomass and pigment production during cultivation, optimize extraction methods to increase phycocyanin dry powder quantity and quality, and compare different powder drying methods. 2. Materials and Methods 2.1. Chemical and Consumables Arthrospira platensis was provided as fresh stock from the Green Gold Research Institute of Qom Province subordinate to Jihad Agricultural Ministry of Iran where the bacterial and nutrient content tests were conducted. Potassium phosphate buffer saline (PBS) was purchased from Dulbecco Co., Germany. Ultrafiltration centrifugal tubes and cassettes were purchased from Sigma-Aldrich Co., Germany. Zarrouk culture media components were bought from Merck, Germany, imported by Iran Algae Co. Glycerol, Sodium acetate and NaOH purchased from Iran Chemicals Co. Protein detector ladder model PM 1700, a gift from SMOBIO Co., Taiwan. 2.2. Microalgae Cultivation According to the literature review, Zarrouk, ( 1966 ) culture medium was selected for the cultivation of algae, with all parameters of this culture medium kept constant except for the carbon source, which varied between treatments. The composition of the culture medium is reported in Table 1 . Table 1 Chemical composition of Zarrouk culture medium with some modifications (Zarrouk, 1966 ). Solution A5: (gL-1): H3BO3, 2.86- MnCl2.4H2O, 1.81- ZnSO4.7H2O, 0.222 – Na2MoO4.2H2O, 0.390 – CuSO2.5H2O, 0.79 Solution B6(mg L-1): NH4VO3, 22.86 – KCr (SO4)2.12H2O, 192 – NiSO4.6H2O, 44.8 – Na2WO4.2H2O, 17.94 – TiOSO4.8H2O, 61.1 – CO(NO3)2.6H2O, 43.98 Reagents Amount(gL-1) NaHCO3 16.8 K2HPO4 0.50 NaNO3 2.5 SK2SO4 1.00 NaCl 1.00 MgSO4.7H2O 0.20 CaCl2 0.04 FeSO4.7H2O 0.01 EDTA 0.08 Solution A5 1 ml Solution B6 1ml Cultivation was carried out in 18 one-liter containers made of compressed plastic with rotating lids, allowing the placement of an air/stirrer tube in a batch culture regime for 20 days. Cultivation parameters were adjusted continuously during cultivation (measurements every 5 days). The culture temperature was maintained at 26 ± 2°C, and the pH was adjusted to approximately 9. The culture medium was sterilized by microwave at 180°F for 10 minutes, except for solution B6, which contained minerals and vitamin B12, and was passed through 0.2 µm membrane filters for decontamination. Inoculation was performed at a ratio of 1:10, achieving a concentration of about 20,000 cells per milliliter (20,000 ± 200). Illumination was provided by LED lights at about 10,000 lux (10,000 ± 50) of mixed yellow and white colors, equalized for all containers, and measured every five days by a lux meter (Model Testo Inc., Sparta, NJ 542, USA). Light exposure was set to 16 hours of light and 8 hours of darkness, controlled manually using an alarm clock. The physical distance of all culture containers from the light source was kept uniform. To prevent dehydration, 1–3 cc of distilled water was added daily, usually dissolving the extra added carbon source. pH was measured every five days using a digital pH meter (model HANNA HI98100, made in the USA). Aeration was performed using two aquarium air pumps at a rate of 0.3 vvm. Six treatments and three replications were defined, making a total of 18 culture containers: Containers 1 to 3: Control / Photoautotrophic culture (Control) Containers 4 to 6: Mixotrophic culture with 25 mg/L sodium acetate daily dose as an extra carbon source (T1) Containers 7 to 9: Mixotrophic culture with 50 mg/L sodium acetate daily dose as an extra carbon source (T2) Containers 10 to 12: Mixotrophic culture with 75 mg/L sodium acetate daily dose as an extra carbon source (T3) Containers 13 to 15: Mixotrophic culture with 0.5 cc of glycerol per liter daily dose (T4) Containers 16 to 18: Mixotrophic culture with 1 cc of glycerol per liter daily dose (T5) Algal biomass was measured during the logarithmic phase (fifth and tenth days), the stationary phase (fifteenth day), and just before the decline phase (twentieth day). To measure algal biomass, a hemocytometer slide was used. However, due to the high concentration of samples, the samples were diluted with distilled water at a ratio of 1:20 on the tenth day and at a ratio of 1:100 on the fifteenth and twentieth days. The following Eq. 1 was used to calculate the number of cells per milliliter (Lund et al., 1958 ): $$\:\:Total\:Cells/mL=\frac{Total\:Cells\:count\times\:Dilution\:Factor}{\left(Number\:of\:squares\right)\times\:1000cells/ml}$$ 1 Moreover, the biomass concentration was calculated by measuring the optical density using spectrophotometry at 680 nm (Unico Model S-2150UV) and plotting the optical density calibration curve against the biomass concentration (g dry weight per liter). The following Eq. 2 , as described by Soltani et al., ( 2006 ), was used: $$\:{\mu\:}=\frac{\text{ln}\left(\text{O}\text{D}\text{t}2\right)-\text{ln}\left(\text{O}\text{D}\text{t}1\right)}{\text{t}2-\text{t}1}$$ 2 Where µ is the special growth rate, and ODt1 and ODt2 are the optical densities measured at times t1 and t2 respectively. Calibration curves demonstrating changes in spectrophotometric concentration relative to biomass production levels were generated following the method outlined by Wood et al., ( 2012 ). The dry weight of biomass was measured as follows: The culture medium was initially centrifuged (SIGMA model 3-16L) for 20 minutes at 4°C, after which the supernatant was discarded. The resulting pellets were washed twice with 200 mL of distilled water each time, shaken for 10 minutes to achieve a uniform suspension, and centrifuged again under the same conditions. After discarding the supernatant, the algal pellet was dried at room temperature (25°C) for 24 hours. 2.2. Calculating Grow Rate The Eq. 3 was used to calculate the Specific Growth Rate (SGR) (Trenkenshu & Novikova, 2019 ): $$\:SGR=\frac{Ln\left(\frac{m2}{m1}\right)}{t2-t1}$$ 3 Where m2 is the cell density on the last day (cell/mL), m1 is the cell density on the first day (cell/mL), t1 is the first day, t2 is the last day, and t2 > = t1. 2.3. Harvest and Preparation of Samples Algae were harvested at the end of the 20th day, when the biomass reached the end of the stationary phase, just before the beginning of the death phase. To harvest the algae, filter nets with a mesh size of 22 microns were used. The culture medium from each container was passed through the net, collecting the algae on top. The resulting algae paste was dried at room temperature, wrapped in aluminum foil, and kept frozen at -18°C for further studies. 2.4. Optimization The Taguchi-based optimization studies were conducted using Minitab Software (Version 16). To find the optimum condition, a Taguchi orthogonal (L9) array was used. Based on preliminary experimental trials and literature support, the parameters chosen for optimization included extraction methods, solid-to-solvent ratio, and time of exposure (Naghdi et al., 2021 ; Safarzadeh Markhali, 2021 ). A three-parameter, three-level design was used and suitably input into the software to determine the 9 sets of experimental conditions suggested by Taguchi. The solid-to-solvent ratio was experimentally considered using 0.5 g in 10 ml, 1 g in 10 ml, and 1.5 g in 10 ml of algae dry matter in solvent for the ratios of 1:20, 1:10, and 1:6.7, respectively. The treatment with the maximum phycocyanin content in the extract was selected as the optimum treatment. 2.5. Extraction Potassium Phosphate Buffer was used to extract intracellular components according to the method of Liao et al., ( 2011 ). The researchers identified this buffer as the most effective for stabilizing the pH in the neutral range, which is ideal for the extraction of phycocyanin from spirulina. Potassium Phosphate Buffer 1 M (PBS buffer 10x, Dulbecco A0965, 9010) was used as the main solvent. The frozen algae were weighed and transferred to 50 ml Falcon tubes. For each gram of dried algae, 10 ml of 1 M buffer was added to the samples, which were then homogenized in a medium-speed mixer for 10 minutes. The samples were then transferred to a freezer at -18°C and kept frozen for 24 hours. After 24 h, the samples were removed and thawed at refrigerator temperature (5°C). They were stirred again for 10 min and refrozen under the same conditions. The freezing-thawing was repeated three times. After complete thawing, the samples were centrifuged at 4°C for 15 minutes (Tb7000 refrigerator model). The supernatant was collected and filtered with a laboratory 0.45 µ syringe filter to eliminate impurities. A sample was taken from the crude extract and was kept at 5°C (refrigerator) for further experiments. 2.6. Identification of Proteins in the Crude Extract The maximum light absorption peaks for phycoerythrin, phycocyanin, and allophycocyanin are at 565 nm, 620 nm, and 650 nm, respectively (Gantt & Lipschultz, 1973 ; Canaani et al., 1980 ; Beattie et al., 2018 ; Bharmoria et al., 2020 ). These pigments can be identified and quantified based on their specific spectral absorption characteristics. To determine the purity of each pigment in the extract, the absorption ratios are calculated relative to the total absorption at 280 nm, which represents the crude extract's protein content. The purity percentages are calculated as follows: Phycoerythrin : Purity percentage = \(\:\left(\frac{A565}{A280}\right)\times\:100\) Phycocyanin : Purity percentage = \(\:\left(\frac{A620}{A280}\right)\times\:100\) Allophycocyanin : Purity percentage = \(\:\left(\frac{A650}{A280}\right)\times\:100\) 2.7. Isolation and Purification of Phycobiliprotein After initial impurity absorption, each phycobiliprotein pigment needs to be separated from other biological compounds in the extract. Ultrafilters in the form of disposable centrifugal Falcon tubes made of Polyether sulfone (PES) are highly suitable for this purpose. PES filters are hydrophobic, offering high resistance to mechanical pressure and temperatures (up to 230°C). They allow for both the storage of the filtered solution (Filtrate Permeate) and the remaining solution above the filter (Retentate) for further analysis. These filters are also efficient with acidic solutions, alkalis, and buffers. The molecular weight of phycocyanins ranges between 18 and 20 kDa (types α and β) (Eriksson-Quensel, 1938 ; Julianti et al., 2019 ; Ashaolu et al., 2021 ). To separate these proteins, 20 mL Vivaspin 6 centrifuge Falcon tubes with a 100 kDa molecular weight cut-off (MWCO) were employed. Phycocyanin molecules passed through the filter membrane and were collected in the lower part of the tube (permeate), while phycoerythrin and allophycocyanin, which have higher molecular weights, remained in the upper part (retentate). The samples were centrifuged at 6000 × g and 4°C for 10 minutes to achieve this separation. To separate phycoerythrins, which have a molecular weight of 240 kDa, and allophycocyanin, which ranges between 104–110 kDa, an Advantec USY-20 Disposable Ultrafiltration Unit with a 200 kDa molecular weight cut-off (MWCO) was employed. This unit allows retention of both upper (retentate) and lower (permeate) solutions. After obtaining phycoerythrin in the retentate, it was diluted tenfold with distilled water to increase sample volume. Allophycocyanin, collected as a colorless solution at the bottom of the tube, was separated and all samples were transferred to a round plastic dish for drying in a freeze dryer. 2.8. Polyacrylamide Gel Electrophoresis The protein profile of the crude extract was determined according to the method described by Laemmli (1970), to detect the specific pigments targeted in this study, comparing qualitatively the phycobilin proteins and their molecular weights between treatments. Protein Ladder Model PM 1700 from SMOBIO was used as a marker; it is suitable for identification between 10 and 240 kDa. Electrophoresis was performed at 90 volts using a 1 × 110 × 140 mm gel size for approximately 4 hours; that is until bromophenol blue dye reached the bottom edge of the gel. The electrophoresis system used was SLABGEL xi Π PROTEIN (RAD Co., China) set at 30 milliamperes. After electrophoresis, gels were stained in a solution containing 0.3 g Coomassie Brilliant Blue R-250, 4% glacial acetic acid, and 4% methanol for 2 hours and destaining in 5% glycolic acid and 2% methanol solution for 2 hours. Gels were then washed with distilled water and results were captured using a scanner and camera. 2.9. Drying Three drying methods were employed for the samples: freeze-drying using a glass tank freeze-dryer (model FD-5003-BT, Sanat Pardaz Dena Co.), spray drying with a spray dryer (model DSD-06, Dersa Behsaz Co.), and oven drying in a 55-liter digital oven (model G602). 2.10. Statistical Analysis All experiments were in triplicate. One-way ANOVA was used to test the differences among data for significance using IBM SPSS Statistics version 16 software. The differences in mean values between treatments were taken as significant at P ≤ 0.05 and were assessed using LSD. Pearson's parametric correlation test was done to establish the relationship between the applied carbon source and biomass and phycobilin yield. The graphs were charged with the help of Origin Pro software version 2021 and Microsoft Excel version 365. 3. Results 3.1. pH Changes in pH under different treatments during the experiment were summarized in Table 2 . At the initial cultivation, the pH was 9.2 in all treatments. As the experiment progressed, the observed pH varied between approximately 9.7 to 10.2 in different treatments. By statistical analysis employing One-Way ANOVA, the obtained results testify to a significant difference concerning the levels of pH compared with Control within all treatments except the 75 mg/L sodium acetate one at P ≤ 0.05. Table 2 Arthrospira platensis Cultivation specifications by treatment for one-liter batch culture during 20 days of cultivation. (*Significance level at α = 0.05 *Significant changes are shown in alphabetical order) Treatments pH first−day pH 20th day Biomass First-day Cell/L Biomass last day Cell/L Algae weight gr DM SGR (µ/d) Control (photoautotroph) 9.2 ± 0.001 9.63 ± 0.047 2×10 7 ± 0.047 32 × 10 7 ± 4714045 3.41 ± 0.004 0.14 ± 0.001 Sodium acetate 25 mg/L (T1) 9.2 ± 0.001 9.90 ± 0.16 a 2×10 7 ± 0.047 38 × 10 7 ± 12472191 c 4.04 ± 0.22 c 0.15 ± 0.001 ab Sodium acetate 50 mg/L (T2) 9.2 ± 0.001 10.7 ± 0.12 b 2×10 7 ± 0.047 47 × 10 7 ± 12472191 a 4.96 ± 0.07 a 0.16 ± 0.001 a Sodium acetate 75 mg/L (T3) 9.2 ± 0.001 9.80 ± 0.08 ab 2×10 7 ± 0.047 39 × 10 7 ± 16996732 c 4.03 ± 0.028 c 0.15 ± 0.002 b Glycerol 0.5 cc (T4) 9.2 ± 0.001 10.7 ± 0.12 c 2×10 7 ± 0.047 44 × 10 7 ± 9428090 b 4.07 ± 0.085 a 0.16 ± 0.000 a Glycerol 1 cc (T5) 9.2 ± 0.001 10 ± 0.08 ac 2×10 7 ± 0.047 44 × 10 7 ± 8164965 b 4.54 ± 0.08 b 0.15 ± 0.001 b 3.2. Biomass Production Table 2 shows that the maximum biomass production was achieved in T2 (Sodium acetate 50 mg/L), as evidenced by both cell count and dry weight measurements. T5 (Glycerol 1 cc/L) ranked second, exhibiting values significantly different from the Control. 3.3. SGR The specific growth rate (SGR) reaches its peak in T4 (Glycerol 0.5 cc/L), which is not significantly different from T2 (Sodium acetate 50 mg/L), indicating that SGR is highly influenced by the type of carbon source rather than the dosage. Higher doses do not necessarily increase the growth rates in the culture. This is evidenced by total biomass production values, which were measured by cell count and weight methods. The specific growth diagram (Fig. 1 ) shows that algae in treatments with added sugars adjusted faster to the culture medium compared with the Control, as expressed by the steepness of the slope from days 0 to 5. Treatment 3 (75 mg/L sodium acetate daily) exhibited the highest slope value during the growth phase (Table 3 ), suggesting the most rapid adaptation to the culture medium with algae reaching maximum exponential growth rate (0.24226 ± 0.0086). Conversely, this treatment also showed the fastest decline phase with a slope of 0.22354 ± 0.0042. Immediately following treatment with 0.5 cc of glycerol, the control group remains relatively stable for 5 days before entering a decline phase, which is delayed by 5 days compared to other treatments. Figure 2 depicts the production of biomass of Arthrospira platensis based on cell count over a culture period of 20 days. From the graph, it can be observed that the highest cell production occurred in T2, which produced approximately 47 × 10^7 cells per liter. From Table 3 , this treatment also produced the highest dry-weight production at 4.96 ± 0.07 g/liter. The SGR was also highest for T2 at 0.16 ± 0.001. After T2, 50 mg/L sodium acetate treatment, the next highest biomass was produced by the 0.5 cc glycerol treatment with a dry weight of 4.8 ± 0.08 g/liter at specific growth rate ranked second to the ± 0.0012. Table 3 weight gain statistics obtained by Origin Pro 2021 during 20 days of cultivation for Arthrospira platensis. Carbon Source Growth equation (SLOPE) Control Y = a + b*x 0.1765 ± 0.019 25 mg Dm/l Sodium acetate (T1) Y = a + b*x 0.1981 ± 0.0127 50 mg Dm/l Sodium acetate (T2) Y = a + b*x 0.2278 ± 0.0057 75 mg Dm/l Sodium acetate (T3) Y = a + b*x 0.24226 ± 0.0086 0.5 cc Glycerol (T4) Y = a + b*x 0.23204 ± 0.011 1 cc Glycerol (T5) Y = a + b*x 0.23217 ± 0.015 The correlation test results (Table 4 ) show that there is a significant positive correlation between sodium acetate and biomass dry weight (r = 0.576). This infers that the addition of sodium acetate has contributed to increased biomass dry weight. The strength of this correlation falls within the average range of 0.4–0.6, indicating a moderate relationship (Miller & Haden, 2006 ). There is also a strong positive correlation between sodium acetate and the number of cells per liter, at r = 0.612, which proves that sodium acetate can affect biomass increase with a huge positive effect. Table 4 The results of the one-way ANOVA to check the significance of the differences between the optical absorption of samples dried by spray drying, freeze-drying, and oven. (٭Significant differences at α = 0.05) Sum of squares df Mean square F Sig. 680 nm Between groups 0.283 2 0.141 1.397 0.273 Within groups 1.820 18 0.101 Total 2.103 20 620 nm Between groups 23.611 2 11.806 4.211 0.032 * Within groups 50.466 18 2.804 Total 74.078 20 280 nm Between groups 67.029 2 33.514 2.347 0.124 Within groups 257.017 18 14.279 Total 324.046 20 This is in addition to a high positive relationship of sodium acetate with the percentage of extracted phycocyanin compared to the total crude extract, as presented by the correlation analysis, at α = 0.01. The close-to-1 correlation coefficient shows a very significant and strong correlation. The aforementioned statistical observation underlines that sodium acetate significantly enhanced the yield of Spirulina biomass and phycocyanin production by the cells. In addition, a more intensive relationship between the given conditions with the production of phycocyanin implies a highly marked stimulation in its biosynthesis by the sodium salt. Sodium acetate acts positively on other parameters like specific growth coefficient. Pearson's correlation test indicated that glycerol was strongly positively correlated with the production of dry biomass weight. However, no significant correlations were found with other growth-related parameters such as specific growth rate, cell density, or biomass concentration measured by spectrophotometric absorption. This indicates that while glycerol was able to improve cell proliferation and enhance biomass weight, it hardly affected these other growth parameters. Therefore, it could be put that supplementation with glycerol mainly enhanced cell proliferation and biomass weight production, with hardly any contribution to the production of pigments inside the cells. 3.4. Pigment Production Figure 3 presents the overall production of pigments for all treatments during the 20-day cultivation period. It can be seen that the highest phycocyanin production was from T3 (75 mg Sodium acetate), followed by T2 (50 mg Sodium acetate) in the second position, while T5 (1 cc Glycerol) secured the third position. On the other hand, the graph for allophycocyanin shows that there is no much difference between the treatments T1 to T5, which all have performed far better than the control. Higher carbon source concentration in treatments like T3 and T5 resulted in a comparably low production of the allophycocyanin with others, showing possible negative impacts of high carbon concentration on allophycocyanin yield. Regarding phycoerythrins, the highest production was obtained for T2 (50 mg Sodium acetate) and T4 (0.5 cc Glycerol), which were significantly different from T3 (75 mg Sodium acetate) and T5 (1 cc Glycerol). More importantly, all the treatments showed significantly higher phycoerythrin production compared to the control, which would indicate that while additional carbon sources enhance biomass and may increase phycoerythrin production, lower sugar concentrations may favor higher yields of phycoerythrin. These trends are consistent with the findings for allophycocyanin production. 3.5. Protein Profile and Detection of Protein Pigments in the Crude Extract Figure 4 : SDS-PAGE gel profile of protein from Arthrospira platensis separated on a polyacrylamide gel. In gel, within the range of 20 to 25 kDa, sharp and prominent bands of phycocyanin are present, and band intensity is indicative of its high protein concentration in the samples. Also, allophycocyanin reveals particular bands at about 100 kDa that are more conspicuously thick in the treatments of 1 cc glycerol and 50 mg sodium acetate. Phycoerythrin bands are also present in the gel, which was observed in the range of 240 kDa. The results of polyacrylamide gel electrophoresis support the data of spectrophotometry for the confirmation of the presence, concentration, and qualitative assessment of every phycobilin protein in the samples under consideration (Fig. 4 ). 3.6. Drying Various drying methods were used in this experiment to compare visual and spectral differences in the phycocyanin powder of different samples. The freeze-drying method should produce higher quality powder than the other methods because there is less sample heating during this method (the differing rates of protein denaturation will not be explored within this experiment). Distinct visual disparities were observed among the samples. Oven-dried samples displayed a blue-green color (dark blue with a greenish hue), while spray-dried samples appeared solid blue without showing any green tendency (as is typical of many market samples). Freeze-dried samples displayed a very light sky-blue color, with a stark visual contrast compared to the dry powder appearance. For spectral analysis, 5 mg of each method of dry phycocyanin powder was dissolved in 10 mL distilled water, and their optical absorption at 680 nm, 620 nm (phycocyanin wavelength), and 280 nm was analyzed. In optical absorption at 680 nm, no significant difference appeared among the drying methods at p ≥ 0.05. However, significant differences were realized at 620 nm p ≤ 0.05 between spray-dried and oven-dried samples, which might indicate phycocyanin denaturation by the more prolonged heating in the oven. Visually, it was not possible to notice any difference in appearance with the naked eye. 4. Discussion The experiment focused on the cultivation and growth performance of Arthrospira platensis , a significant algal species, using varying concentrations of sodium acetate and glycerol as additional carbon sources alongside the Zarrouk, ( 1966 ) basic medium in all treatments and controls. Initial cell density was consistent at 2×10^7 ± 0.047 cells/L across treatments, peaking at 47 × 10^7 cells/L with Sodium acetate 50 mg/L. Different cell growth and biomass accumulation due to varying carbon levels and sources were observed within the study. The optimum level of carbon was required for normal cellular growth and hyperproduction of phycobilin proteins intra-cellularly. More carbon led to faster cell growth but, however, resulted in slow production of phycobilin protein and entry into early death stage. These findings underscore the importance of using an optimal, consistent level of carbon that is appropriate for the used carbon source for the normal weight of cells and phycobilin production during its growth. The carbon source is one of the most important factors in the cultivation of microalgal species, such as Arthrospira platensis, commonly known as Spirulina. Different media and carbon sources have been tested concerning their influence on the growth and performance of these microalgae. Rahman, ( 2021 ) studied the evaluation of Spirulina platensis for their culture and growth performance using different concentrations of supernatant of digested rotten potato (DRP) with 20%, 40%, and 60% of DRP with Kosaric medium as a control in 26 days of digestion and Spirulina was also inoculated into DRP supplemented with 9.0 g/L NaHCO3 and micronutrients including KM for a period of 14 days in this study. Spirulina reached its maximum cell density in KM on the 10th day of culture at 12.42 ± 0.21 mg/L, followed by 9.505 ± 0.43 mg/L in the 60% DRP supernatant, 8.352 ± 0.21 mg/L in the 40% DRP supernatant, and 6.256 ± 2.34 mg/L in the 20% DRP supernatant. The same trend was observed in the optical density, chlorophyll A content, total biomass, and specific growth rate. These results reflect significant differences in cell density between KM and the DRP supernatants at p < 0.01, which were positively related to chlorophyll content and total biomass. Indeed, this reflects that growth performance in Spirulina is influenced by the added concentration of the DRP supernatant; usually, higher concentrations support lower cell densities compared to KM. The growth performance of Spirulina platensis was notably enhanced when cultivated in the supernatant of 60% digested rotten potato (DRP) compared to lower concentrations (20% and 40% DRP). This suggests that mass cultivation of Spirulina can be effectively carried out using a 60% DRP supernatant due to its superior growth-promoting properties observed in the study. Mia et al., ( 2019 ) conducted an experiment to study the culture and growth performance of Spirulina platensis on various rotten apple medium (RAM) concentrations and Kosaric Medium (KM). In the experiment, S. platensis was cultured in 1.0L glass flasks containing three different RAM concentrations, namely 2.5%, 5.0%, and 10% along with KM, and each medium was replicated thrice. These cultures were maintained for 14 days under a fluorescent light regime composed of 12 hr of light and 12 hr of dark. There is a significant difference in the growth performance of S. platensis on different media formulations. Initially, S. platensis was recorded as 0.0023 mg/l cell weight. The 10th-day maximum record of cell weight in KM and RAM at 10% was 12.44mg/l and 10.468 mg/l respectively. Similarly, the initial Chlorophyll content of S. The highest amount of platensis was 0.0015 mg/L, reaching a maximum value of 10.54 mg/L in KM and 12.35 mg/L in RAM (at 10% concentration) on the 10th day. The weight of the cell started to decline after the 10th day of cultivation. S. platensis growth in 5.0% DRAM was significantly higher (p < 0.05) than the growths in the other two RAM concentrations of 2.5% and 10% DRAM. In a related study, Habib and Kohinoor, ( 2018 ) recorded that Spirulina platensis showed better growth when cultured in the supernatant of 60% DRGM as the carbon source, rather than at lower concentrations of 20% and 40% DRGM. Their observation agrees with previous studies that the supernatant of 45% digested poultry waste also supported healthy growth of Spirulina, indicating that the concentration of the carbon source is an important component in the improvement of conditions for growth. Although many studies have been carried out on the phycocyanin content of Spirulina during its growth, few have been conducted that simultaneously studied the effects of carbon sources on cell proliferation and phycobilin content. These two aspects are important in relation to how carbon sources influence biomass production and pigment synthesis in Spirulina platensis. Chaiklahan et al., ( 2022 ) recently published their findings on the cultivation of Arthrospira ( Spirulina ) platensis BP in a photobioreactor under varying light intensities (635, 980, 1300, and 2300 µmol m − 2 s − 1) using a semi-continuous mode to maintain cell concentrations at optical densities (OD) of 0.4, 0.6, and 0.8. They also noted that the highest biomass productivity of 0.62 g L–1 d–1 and phycocyanin yield of 123 mg L–1 d–1 occurred at a light intensity of 2300 µmol m − 2 s − 1 at OD 0.6. The energy consumption efficiency in this case for algal biomass was around 2.26–2.31 g·(kW h)-1 d-1, while the photosynthetic efficiency at a light intensity of 635 µmol·m-2·s-1 at OD 0.8 was around 8.02%. This indeed signifies the importance of light intensity, cellular concentration, and light/dark period for biomass improvement and the production of phycocyanin in Arthrospira platensis while pointing toward the most ideal conditions required for maximum yield. Gladfelter et al., ( 2022 ) conducted a 2-week field experiment using 1,100-L plastic limnocorrals to investigate the response of the cyanobacterial community to different nitrogen forms: nitrate, ammonium, and urea (added at 600 µg N/L). They monitored cell pigments and counts to calculate cell-specific pigment concentrations and measured cell-associated microcystin concentrations to assess the toxin response to nitrogen source variations. The results indicated that upon nitrogen addition, extracellular nitrogen levels rapidly decreased, corresponding with an increase in cellular phycocyanin levels 72 hours after fertilization. Ammonium and urea treatments exhibited higher phycocyanin/cell ratios compared to nitrate or control treatments at the 72-hour mark. Extraction methods significantly influence the quality, quantity, and stability of phycobilin pigments extracted from algae. Extensive research has focused on maximizing phycocyanin production in dry powder form. For instance, Chittapun et al., ( 2020 ) investigated the extraction of C-phycocyanin from Nostoc commune TUBT05 and Oscillatoria okeni TISTR8549 using freezing and thawing, as well as pulsed electric field treatments. Their findings demonstrated that freezing and thawing are effective methods for extracting C-phycocyanin from both cyanobacterial strains. In contrast, pulsed electric field treatment was successful only with N. commune due to its cell structure being compatible with this technology. The number of freeze/thaw cycles, the composition of the extraction solution, and the number of electric pulses exerted significant statistical influence on C-phycocyanin concentration, purity, yield, and total protein content in the crude extracts (p ≤ 0.005). Patel et al., ( 2005 ) in the year 2005, developed an effective single-step chromatographic method for the purification of C-Phycocyanin from three cyanobacterial species, namely Spirulina sp., Phormidium sp., and Lyngbya sp. The procedure for purification consisted of successive steps involving fractional precipitation with ammonium sulfate, followed by chromatography on a DEAE–Sepharose CL-6B column. From Spirulina, Phormidium, and Lyngbya spp., C-Phycocyanin was thus obtained in purity ratios (A620/A280) of 4.42, 4.43, and 4.59, respectively. Native and SDS–PAGE were also used to further verify purity and homogeneity. The objective of the present study has been to establish in detail the major parameters affecting natural pigment production, above all phycocyanins, right from the levels of algae cultivation to the final steps of extraction and product preparation. Results presented in this work are derived from laboratory-scale studies and hence not directly generalized to industrial settings. Nevertheless, they provide valuable insights that could facilitate the commercialization of phycocyanin production. 5. Conclusion Generally, this review highlights the importance of the addition of extra carbon sources to enhance cell proliferation and biomass accumulation in cultures of Arthrospira platensis . These additional supplements of carbon sources were truly crucial regarding dosage and timing for biomass, SGR, and even the pigment production of the organism under investigation. Importantly, the type of carbon source proved to be one of the decisive factors for optimization in biomass yield and phycobilin production. Furthermore, our investigation has pointed out the great influence of extraction and drying methods on the quality of phycocyanin and phycoerythrin dry powders. Since these protein pigments are temperature-sensitive, we suggest non-thermal and non-chemical extraction techniques to keep their integrity and maximize product quality. Although our findings are based on the laboratory scale and need further validation at an industrial scale, they provide a basis on which to improve the commercial production of phycocyanin. Further research is thus needed for scale-up at the industrial level, taking into account the complexities and economic viability of large-scale production. The present study therefore, helped to fill basic knowledge gaps lacking in the area of producing natural pigments in A. platensis and will be useful for pointing out insights on how conditions of cultivation and extraction procedures should be optimized with regard to elevated levels of phycocyanin and phycoerythrin production. Abbreviations Spirulina ( Arthrospira platensis ), Chlorella ( Chlorella vulgaris ), PC ( Phycocyanin ), PE ( Phycoerythrin ), ALP ( Allophycocyanin ). Declarations Funding Declaration Paragraph : This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. 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Rapid quantification of reducing sugars in biomass hydrolysates: Improving the speed and precision of the dinitrosalicylic acid assay. Biomass and bioenergy , 44 , 117–121. Wyman, M. (1992). An in vivo method for the estimation of phycoerythrin concentrations in marine cyanobacteria (Synechoccus spp.). Limnology and Oceanography , 37 (6), 1300–1306. Yun, H.-S., Kim, Y.-S., & Yoon, H.-S. (2021). Effect of different cultivation modes (photoautotrophic, mixotrophic, and heterotrophic) on the growth of Chlorella sp. and biocompositions. Frontiers in Bioengineering and Biotechnology , 9 , 774143. Zahra, Z., Choo, D. H., Lee, H., & Parveen, A. (2020). Cyanobacteria: Review of current potentials and applications. Environments , 7 (2), 13. Zanolla, V., Biondi, N., Niccolai, A., Abiusi, F., Adessi, A., Rodolfi, L., & Tredici, M. R. (2022). Protein, phycocyanin, and polysaccharide production by Arthrospira platensis grown with LED light in annular photobioreactors. Journal of Applied Phycology , 34 (3), 1189–1199. Zarrouk, C. (1966). Contribution a l'etude d'une Cyanophycee. Influence de Divers Facteurs Physiques et Chimiques sur la croissance et la photosynthese de Spirulina mixima. Thesis. University of Paris, France. Zhang, R., Parniakov, O., Grimi, N., Lebovka, N., Marchal, L., & Vorobiev, E. (2019). Emerging techniques for cell disruption and extraction of valuable bio-molecules of microalgae Nannochloropsis sp. Bioprocess and biosystems engineering , 42 , 173–186. Zhu, C., Chen, S., Ji, Y., Schwaneberg, U., & Chi, Z. (2022). Progress toward a bicarbonate-based microalgae production system. Trends in Biotechnology , 40 (2), 180–193. Zhu, C., Han, D., Li, Y., Zhai, X., Chi, Z., Zhao, Y., & Cai, H. (2019). Cultivation of aquaculture feed Isochrysis zhangjiangensis in low-cost wave driven floating photobioreactor without aeration device. Bioresource technology , 293 , 122018. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7284160","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":506829903,"identity":"3e65a180-e3d2-4134-b78f-86cde6630bf3","order_by":0,"name":"Zahra Soltani Far","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Zahra","middleName":"Soltani","lastName":"Far","suffix":""},{"id":506829904,"identity":"2760def7-30ef-4950-97c8-27060e9c4227","order_by":1,"name":"Mohammad Ali Nematollahi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArUlEQVRIiWNgGAWjYFAC5gZmBgMbKMeAKC2MIC1pDDwkamE4DNVCDNBtP9j4uaDgfOJ+BuaHHxgK7hHWYnYmsVl6hsHtxB4GNmMJBoNiIrQcSGyQ5gFrYTAD+iWBCC3nHzb/5jE4B9TC/o1ILTcS24C2HABq4SHWlhsP26x5DJKNew7zFEskEOew5MO3ef7Yyba3t2/88OEPEVoQABg7DCRpGAWjYBSMglGAGwAANT0zQGLz7csAAAAASUVORK5CYII=","orcid":"","institution":"University of Tehran","correspondingAuthor":true,"prefix":"","firstName":"Mohammad","middleName":"Ali","lastName":"Nematollahi","suffix":""},{"id":506829906,"identity":"77a6a50f-e958-4085-87d6-4e24f3a107a5","order_by":2,"name":"Seyed Vali Hosseini","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Seyed","middleName":"Vali","lastName":"Hosseini","suffix":""},{"id":506829909,"identity":"4f9a049a-d661-4150-bfdf-025248678c9d","order_by":3,"name":"Pouya Farshbaf Aghajani","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Pouya","middleName":"Farshbaf","lastName":"Aghajani","suffix":""}],"badges":[],"createdAt":"2025-08-03 15:08:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7284160/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7284160/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90320238,"identity":"5f304fb7-2c35-457c-be14-b9faaebb7b71","added_by":"auto","created_at":"2025-09-01 10:43:33","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":32258,"visible":true,"origin":"","legend":"\u003cp\u003eSGR comparison between treatments during 20 days of cultivation of \u003cem\u003eArthrospira platensis.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7284160/v1/a986e5fab9cceb29a08f7e42.jpg"},{"id":90320236,"identity":"2d7c04c2-c230-4791-a8e3-391210f20b6e","added_by":"auto","created_at":"2025-09-01 10:43:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":42143,"visible":true,"origin":"","legend":"\u003cp\u003eBiomass production for \u003cem\u003eArthrospira platensis\u003c/em\u003e based on the number of cells produced per 20 days of culture.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7284160/v1/f9acc1e8e67d81ea03b78664.png"},{"id":90320237,"identity":"0ec08bf1-0f53-4dff-996a-105ed82e3e80","added_by":"auto","created_at":"2025-09-01 10:43:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":57480,"visible":true,"origin":"","legend":"\u003cp\u003ePhycobilin pigment production in cultures of \u003cem\u003eArthrospira platensis\u003c/em\u003e after 20 days of cultivation (Data shown as % of pigment/crude extract).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7284160/v1/ebea18a9ea65d2aa7c799c6e.png"},{"id":90320239,"identity":"94baa4ba-2862-4662-9232-74cf7d9523ba","added_by":"auto","created_at":"2025-09-01 10:43:33","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33953,"visible":true,"origin":"","legend":"\u003cp\u003eSDS-PAGE results for crude extracts of \u003cem\u003eArthrospira platensis\u003c/em\u003e samples cultivated using different carbon sources. Bands for Phycocyanin (20-25 kDa), Phycoerythrin (240 kDa), and Allophycocyanin (100-110 kDa) are visible.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7284160/v1/7df21799d18a39444e948086.jpg"},{"id":101753750,"identity":"bcf532c8-04b0-49af-9a7d-5ba93da7213b","added_by":"auto","created_at":"2026-02-03 10:40:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1283963,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7284160/v1/8387a195-db5b-48ae-8286-31a662ae9c0b.pdf"},{"id":90320881,"identity":"6c9afc55-36c2-4dc2-bbea-a0fc623147f0","added_by":"auto","created_at":"2025-09-01 10:51:33","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":377634,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7284160/v1/0f56af3267eca449c87b7984.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Optimizing Biomass and Phycobilin Production in Arthrospira platensis Through Carbon Source Variation and Developing a Cost-Effective Purification Method","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCyanobacteria, also known as blue-green algae, are a group of photosynthetic and nitrogen-fixing bacteria living in almost all sorts of moist soils and waters, either freely or in symbiotic relationships (Hamouda et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These bacteria have been found capable of synthesizing a variety of secondary metabolites, including biologically active compounds such as antibacterial, antiviral, antifungal, and anticancer agents (Richmond, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hu, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Nowruzi et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zanolla et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These properties are strain- and culture-condition-dependent (Zanolla et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Microalgae are generally photoautotrophic, but some strains have great potential to grow under mixotrophic or heterotrophic conditions (Abiusi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Jin et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Smith et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSpirulina spp. (Arthrospira platensis) is a cyanobacterium widely grown in alkaline brine lakes (Nege et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The high concentration of protein pigments called phycobiliproteins in spirulina sets it apart from other microalgae (Glazer, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). Phycobiliproteins consist of three main components: phycoerythrin, phycocyanin Glazer, and allophycocyanin Sonani et al., (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Among them, phycocyanin is a blue-colored natural pigment with different physiological activities, such as antioxidants, antibacterial, and anticancer properties. It could also be applied in the food industry as a bioactive substance and dye (Kumar et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough it is true that microalgae, to which Spirulina spp. belong, can use inorganic carbon sources, their biomass productivity is generally low and limited (Zhu et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), their biomass productivity is often low and limited (Verma et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It was proposed to provide microalgae cultures with an organic carbon source for maximal production of high-quality biomass (Roostaei et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Several photoautotrophic microalgae have mechanisms to enhance the utilization of externally added sugars under mixotrophic and heterotrophic conditions (Verma et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). With an organic carbon source, it increases productivity in biomass with higher efficiency and cell density (Roostaei et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nowruzi et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Besides, it also increases levels of desirable compounds like lipids and pigments (Ip et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Liang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Yun et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe environmental impact of the cultivation stage is significant due to the extensive use of chemicals and nutrients (Lim et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, various types of culture media\u0026mdash;chemical-based, modified, and alternative\u0026mdash;have been reviewed for Spirulina cultivation (Wang et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Further study is needed to modify or explore alternative culture media utilizing waste, wastewater, or industrial by-products to ensure the long-term sustainability of the environment and nutrient sources for cultivation.\u003c/p\u003e\u003cp\u003eIn this research, we aim to investigate the effects of adding three different carbon sources in daily doses to Spirulina mixotrophic cultures. These sugars are relatively cheap and easy to use. The carbon sources will be applied in fixed doses but at short intervals, allowing comparison of growth results with conditions where the carbon source is added only once at the beginning and cultures grow solely via photosynthesis. We will measure the consequent effects on cell proliferation, biomass production, and phycocyanin production.\u003c/p\u003e\u003cp\u003eIn a similar study, de Morais et al., (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) investigated the different concentrations of crude glycerol that influence productivity, biomass concentration, and biomolecule content in cultures of Spirulina sp. It states that microalgae grown under different glycerol concentration regimes in Erlenmeyer bioreactors for the 15-day test obtained the best results when this compound was at a value equal to 2.5 g L⁻\u0026sup1;, giving a maximum (0.43 g L⁻\u0026sup1; day⁻\u0026sup1;) of dry weight production. The scientists also show that proteins compose the main constituent of these biomasses at a yield of 75.7% w/w when considering 2.5 g L⁻\u0026sup1; as the best glycerol concentration, and their synthesizing and accumulation into these biomass's cells is strictly dependent upon glycerol concentration.\u003c/p\u003e\u003cp\u003eMoreover, (da Silva Braga et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) focused on verifying if the production of Spirulina biomass with high carbohydrate content is stimulated by a reduced supply of nitrogen combined with the addition of NaHCO₃ or CO₂ at different flow rates and injection times. The addition of 0.25 g L⁻\u0026sup1; of NaNO₃ allowed Spirulina to accumulate up to 49.3% (w/w) of carbohydrates with the highest amount of CO₂ (0.3 vvm injected for 5 min). This value reached 59.1% (w/w) when NaHCO₃ was the carbon source. Meanwhile, biomass concentrations achieved were 0.81 and 0.97 g L⁻\u0026sup1;, respectively.\u003c/p\u003e\u003cp\u003eIn another study, Yun et al., (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported increased bioresource productivity under both mixotrophic and heterotrophic conditions for Chlorella sp. cultivation. Different conditions were applied to three well-known strains of Chlorella (KNUA104, KNUA114, and KNUA122) to assess biomass productivity. Compositional changes (lipids, protein, and pigments) were evaluated in BG11 media under photoautotrophic, mixotrophic, and heterotrophic conditions, utilizing different initial concentrations of glucose (5, 10, 15, 20, and 25 g L⁻\u0026sup1;). All strains under mixotrophic and heterotrophic conditions were optimally cultured with 15\u0026ndash;20 g L⁻\u0026sup1; initial glucose.\u003c/p\u003e\u003cp\u003eDespite their beneficial properties, the incorporation of microalgae metabolites faces several obstacles (Hosseinkhani et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These obstacles include the hard cell wall of most species, the high sensitivity and reactivity of ingredients, as well as issues like fishy taste, odor, and color, which reduce consumer acceptance. Furthermore, the high costs and complexity of downstream processing, along with legislative issues related to the incorporation of microalgae into food and pharmaceuticals, form serious barriers to industrial-scale production (Zhang et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Howlader \u0026amp; French, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zahra et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ahmad et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMoreover, the economic and environmental costs of traditional isolation and purification processes pose obstacles to the production of high-value algal metabolites (HVAMs). These processes involve harmful residues, high temperatures, and high time and energy demands, leading to metabolite degradation (Heffernan et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In the United States, for instance, management of production-related waste has resulted in the release of 25.45\u0026nbsp;billion pounds of toxic chemicals (Persico \u0026amp; Venator, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Risks such as solvent toxicity and the presence of solvent contaminants in extracts, coupled with low yields, have spurred the development of alternative extraction methodologies, such as green and clean methods, which aim to reduce or eliminate the use of organic solvents (Chemat et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Chemat et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Picot-Allain et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Rodriguez Garcia \u0026amp; Raghavan, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSustainable methods for designing innovative and efficient processes are crucial to improving efficiency, time, and costs while ensuring the sustainable use of resources and materials (Jacotet-Navarro et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Verma et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Recent reports have highlighted various techniques aimed at maximizing phycocyanin extraction yield and minimizing environmental and financial costs from \u003cem\u003eArthrospira platensis\u003c/em\u003e biomass in different physical forms (dry, wet, and frozen) (Wyman, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Sekar \u0026amp; Chandramohan, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Chittapun et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Rahman, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gladfelter et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSeveral methods have been proposed to disrupt the cell wall, including homogenization, sonication, microwave treatment, supercritical fluid extraction, and lysozyme disintegration (Duangsee et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Deniz et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Mart\u0026iacute;nez et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Chittapun et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ferreira-Santos et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, many of these extraction methods are not feasible for large-scale applications due to high costs or challenges with industrial-scale implementation. For example, techniques like ultrasound or freezing thawing may not be applicable on an industrial scale (Howlader \u0026amp; French, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eResearchers often combine methods to reduce costs and increase the yield or stability of products (Zahra et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, when it comes to phycocyanins, one of the main challenges in producing high-quality dry powder is the high solubility and sensitivity of phycocyanin protein pigments to heat (above 30\u0026deg;C) and pH shifts (optimal pH around 7) (Ferreira-Santos et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study aims to develop an extraction process from a cost-effective bench-scale perspective with the potential for industrialization. Therefore, methods that minimize chemical and thermal treatments are more effective and financially reasonable. Physical separation of proteins using filters has shown promising results in extracting contents from microalgae (Zhang et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Howlader \u0026amp; French, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ahmad et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eOver the past few decades, membrane processes have emerged as important methods in the food business. They are frequently regarded as the best available technology (BAT) because of their capacity to gently treat foodstuffs at low to moderate temperatures (Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Tabani et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Castro-Mu\u0026ntilde;oz et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Monesi et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These processes offer several advantages over conventional separation systems, including high separation precision, better selectivity, operation at room temperature, no chemical damage, high automation, easy operation, energy savings, reduced costs, comprehensive utilization of resources, and reduced pollution.\u003c/p\u003e\u003cp\u003eResearchers often combine methods to reduce costs and increase the yield or stability of products (Zahra et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Specifically, for phycocyanins, a significant challenge in producing high-quality dry powder is the high solubility and sensitivity of phycocyanin protein pigments to heat (above 30\u0026deg;C) and pH shifts (optimal pH around 7) (Ferreira-Santos et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study aims to develop a cost-effective bench-scale extraction process with industrialization potential, focusing on minimizing chemical and thermal methods. Physical separation of proteins using filters has shown promising results for extracting contents from microalgae (Zhang et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Howlader \u0026amp; French, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ahmad et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eMembrane processes have become pivotal in the food industry, recognized as the best available technology (BAT) due to their ability to treat products gently at low-to-moderate temperatures (Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Tabani et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Castro-Mu\u0026ntilde;oz et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Monesi et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). They offer high separation precision, better selectivity, operation at room temperature, no chemical damage, high automation, easy operation, energy savings, reduced costs, comprehensive resource utilization, and reduced pollution (Lipnizki \u0026amp; Ruby-Figueroa, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Quezada et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this paper, we demonstrate a cost-effective method to enhance biomass and pigment production during cultivation, optimize extraction methods to increase phycocyanin dry powder quantity and quality, and compare different powder drying methods.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Chemical and Consumables\u003c/h2\u003e\u003cp\u003e\u003cem\u003eArthrospira platensis\u003c/em\u003e was provided as fresh stock from the Green Gold Research Institute of Qom Province subordinate to Jihad Agricultural Ministry of Iran where the bacterial and nutrient content tests were conducted. Potassium phosphate buffer saline (PBS) was purchased from Dulbecco Co., Germany. Ultrafiltration centrifugal tubes and cassettes were purchased from Sigma-Aldrich Co., Germany. Zarrouk culture media components were bought from Merck, Germany, imported by Iran Algae Co. Glycerol, Sodium acetate and NaOH purchased from Iran Chemicals Co. Protein detector ladder model PM 1700, a gift from SMOBIO Co., Taiwan.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Microalgae Cultivation\u003c/h2\u003e\u003cp\u003eAccording to the literature review, Zarrouk, (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e1966\u003c/span\u003e) culture medium was selected for the cultivation of algae, with all parameters of this culture medium kept constant except for the carbon source, which varied between treatments. The composition of the culture medium is reported in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eChemical composition of Zarrouk culture medium with some modifications (Zarrouk, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e1966\u003c/span\u003e). Solution A5: (gL-1): H3BO3, 2.86- MnCl2.4H2O, 1.81- ZnSO4.7H2O, 0.222 \u0026ndash; Na2MoO4.2H2O, 0.390 \u0026ndash; CuSO2.5H2O, 0.79 Solution B6(mg L-1): NH4VO3, 22.86 \u0026ndash; KCr (SO4)2.12H2O, 192 \u0026ndash; NiSO4.6H2O, 44.8 \u0026ndash; Na2WO4.2H2O, 17.94 \u0026ndash; TiOSO4.8H2O, 61.1 \u0026ndash; CO(NO3)2.6H2O, 43.98\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eReagents\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAmount(gL-1)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNaHCO3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16.8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eK2HPO4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNaNO3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSK2SO4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNaCl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMgSO4.7H2O\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCaCl2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFeSO4.7H2O\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEDTA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSolution A5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1 ml\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSolution B6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1ml\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eCultivation was carried out in 18 one-liter containers made of compressed plastic with rotating lids, allowing the placement of an air/stirrer tube in a batch culture regime for 20 days. Cultivation parameters were adjusted continuously during cultivation (measurements every 5 days). The culture temperature was maintained at 26\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, and the pH was adjusted to approximately 9. The culture medium was sterilized by microwave at 180\u0026deg;F for 10 minutes, except for solution B6, which contained minerals and vitamin B12, and was passed through 0.2 \u0026micro;m membrane filters for decontamination. Inoculation was performed at a ratio of 1:10, achieving a concentration of about 20,000 cells per milliliter (20,000\u0026thinsp;\u0026plusmn;\u0026thinsp;200). Illumination was provided by LED lights at about 10,000 lux (10,000\u0026thinsp;\u0026plusmn;\u0026thinsp;50) of mixed yellow and white colors, equalized for all containers, and measured every five days by a lux meter (Model Testo Inc., Sparta, NJ 542, USA). Light exposure was set to 16 hours of light and 8 hours of darkness, controlled manually using an alarm clock. The physical distance of all culture containers from the light source was kept uniform. To prevent dehydration, 1\u0026ndash;3 cc of distilled water was added daily, usually dissolving the extra added carbon source. pH was measured every five days using a digital pH meter (model HANNA HI98100, made in the USA). Aeration was performed using two aquarium air pumps at a rate of 0.3 vvm. Six treatments and three replications were defined, making a total of 18 culture containers:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eContainers 1 to 3: Control / Photoautotrophic culture (Control)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eContainers 4 to 6: Mixotrophic culture with 25 mg/L sodium acetate daily dose as an extra carbon source (T1)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eContainers 7 to 9: Mixotrophic culture with 50 mg/L sodium acetate daily dose as an extra carbon source (T2)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eContainers 10 to 12: Mixotrophic culture with 75 mg/L sodium acetate daily dose as an extra carbon source (T3)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eContainers 13 to 15: Mixotrophic culture with 0.5 cc of glycerol per liter daily dose (T4)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eContainers 16 to 18: Mixotrophic culture with 1 cc of glycerol per liter daily dose (T5)\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eAlgal biomass was measured during the logarithmic phase (fifth and tenth days), the stationary phase (fifteenth day), and just before the decline phase (twentieth day). To measure algal biomass, a hemocytometer slide was used. However, due to the high concentration of samples, the samples were diluted with distilled water at a ratio of 1:20 on the tenth day and at a ratio of 1:100 on the fifteenth and twentieth days. The following Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e was used to calculate the number of cells per milliliter (Lund et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1958\u003c/span\u003e):\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\:Total\\:Cells/mL=\\frac{Total\\:Cells\\:count\\times\\:Dilution\\:Factor}{\\left(Number\\:of\\:squares\\right)\\times\\:1000cells/ml}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eMoreover, the biomass concentration was calculated by measuring the optical density using spectrophotometry at 680 nm (Unico Model S-2150UV) and plotting the optical density calibration curve against the biomass concentration (g dry weight per liter). The following Eq.\u0026nbsp;\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, as described by Soltani et al., (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), was used:\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:{\\mu\\:}=\\frac{\\text{ln}\\left(\\text{O}\\text{D}\\text{t}2\\right)-\\text{ln}\\left(\\text{O}\\text{D}\\text{t}1\\right)}{\\text{t}2-\\text{t}1}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWhere \u0026micro; is the special growth rate, and ODt1 and ODt2 are the optical densities measured at times t1 and t2 respectively.\u003c/p\u003e\u003cp\u003eCalibration curves demonstrating changes in spectrophotometric concentration relative to biomass production levels were generated following the method outlined by Wood et al., (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The dry weight of biomass was measured as follows: The culture medium was initially centrifuged (SIGMA model 3-16L) for 20 minutes at 4\u0026deg;C, after which the supernatant was discarded. The resulting pellets were washed twice with 200 mL of distilled water each time, shaken for 10 minutes to achieve a uniform suspension, and centrifuged again under the same conditions. After discarding the supernatant, the algal pellet was dried at room temperature (25\u0026deg;C) for 24 hours.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Calculating Grow Rate\u003c/h2\u003e\u003cp\u003eThe Eq.\u0026nbsp;\u003cspan refid=\"Equ3\" class=\"InternalRef\"\u003e3\u003c/span\u003e was used to calculate the Specific Growth Rate (SGR) (Trenkenshu \u0026amp; Novikova, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2019\u003c/span\u003e):\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:SGR=\\frac{Ln\\left(\\frac{m2}{m1}\\right)}{t2-t1}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWhere m2 is the cell density on the last day (cell/mL), m1 is the cell density on the first day (cell/mL), t1 is the first day, t2 is the last day, and t2\u0026thinsp;\u0026gt;\u0026thinsp;=\u0026thinsp;t1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Harvest and Preparation of Samples\u003c/h2\u003e\u003cp\u003eAlgae were harvested at the end of the 20th day, when the biomass reached the end of the stationary phase, just before the beginning of the death phase. To harvest the algae, filter nets with a mesh size of 22 microns were used. The culture medium from each container was passed through the net, collecting the algae on top. The resulting algae paste was dried at room temperature, wrapped in aluminum foil, and kept frozen at -18\u0026deg;C for further studies.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Optimization\u003c/h2\u003e\u003cp\u003eThe Taguchi-based optimization studies were conducted using Minitab Software (Version 16). To find the optimum condition, a Taguchi orthogonal (L9) array was used. Based on preliminary experimental trials and literature support, the parameters chosen for optimization included extraction methods, solid-to-solvent ratio, and time of exposure (Naghdi et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Safarzadeh Markhali, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). A three-parameter, three-level design was used and suitably input into the software to determine the 9 sets of experimental conditions suggested by Taguchi. The solid-to-solvent ratio was experimentally considered using 0.5 g in 10 ml, 1 g in 10 ml, and 1.5 g in 10 ml of algae dry matter in solvent for the ratios of 1:20, 1:10, and 1:6.7, respectively. The treatment with the maximum phycocyanin content in the extract was selected as the optimum treatment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Extraction\u003c/h2\u003e\u003cp\u003ePotassium Phosphate Buffer was used to extract intracellular components according to the method of Liao et al., (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The researchers identified this buffer as the most effective for stabilizing the pH in the neutral range, which is ideal for the extraction of phycocyanin from spirulina.\u003c/p\u003e\u003cp\u003ePotassium Phosphate Buffer 1 M (PBS buffer 10x, Dulbecco A0965, 9010) was used as the main solvent. The frozen algae were weighed and transferred to 50 ml Falcon tubes. For each gram of dried algae, 10 ml of 1 M buffer was added to the samples, which were then homogenized in a medium-speed mixer for 10 minutes. The samples were then transferred to a freezer at -18\u0026deg;C and kept frozen for 24 hours.\u003c/p\u003e\u003cp\u003eAfter 24 h, the samples were removed and thawed at refrigerator temperature (5\u0026deg;C). They were stirred again for 10 min and refrozen under the same conditions. The freezing-thawing was repeated three times.\u003c/p\u003e\u003cp\u003eAfter complete thawing, the samples were centrifuged at 4\u0026deg;C for 15 minutes (Tb7000 refrigerator model). The supernatant was collected and filtered with a laboratory 0.45 \u0026micro; syringe filter to eliminate impurities. A sample was taken from the crude extract and was kept at 5\u0026deg;C (refrigerator) for further experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Identification of Proteins in the Crude Extract\u003c/h2\u003e\u003cp\u003eThe maximum light absorption peaks for phycoerythrin, phycocyanin, and allophycocyanin are at 565 nm, 620 nm, and 650 nm, respectively (Gantt \u0026amp; Lipschultz, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1973\u003c/span\u003e; Canaani et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Beattie et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Bharmoria et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These pigments can be identified and quantified based on their specific spectral absorption characteristics.\u003c/p\u003e\u003cp\u003eTo determine the purity of each pigment in the extract, the absorption ratios are calculated relative to the total absorption at 280 nm, which represents the crude extract's protein content. The purity percentages are calculated as follows:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003ePhycoerythrin\u003c/b\u003e: Purity percentage = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\left(\\frac{A565}{A280}\\right)\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003ePhycocyanin\u003c/b\u003e: Purity percentage = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\left(\\frac{A620}{A280}\\right)\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eAllophycocyanin\u003c/b\u003e: Purity percentage = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\left(\\frac{A650}{A280}\\right)\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Isolation and Purification of Phycobiliprotein\u003c/h2\u003e\u003cp\u003eAfter initial impurity absorption, each phycobiliprotein pigment needs to be separated from other biological compounds in the extract. Ultrafilters in the form of disposable centrifugal Falcon tubes made of Polyether sulfone (PES) are highly suitable for this purpose. PES filters are hydrophobic, offering high resistance to mechanical pressure and temperatures (up to 230\u0026deg;C). They allow for both the storage of the filtered solution (Filtrate Permeate) and the remaining solution above the filter (Retentate) for further analysis. These filters are also efficient with acidic solutions, alkalis, and buffers.\u003c/p\u003e\u003cp\u003eThe molecular weight of phycocyanins ranges between 18 and 20 kDa (types α and β) (Eriksson-Quensel, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1938\u003c/span\u003e; Julianti et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ashaolu et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To separate these proteins, 20 mL Vivaspin 6 centrifuge Falcon tubes with a 100 kDa molecular weight cut-off (MWCO) were employed. Phycocyanin molecules passed through the filter membrane and were collected in the lower part of the tube (permeate), while phycoerythrin and allophycocyanin, which have higher molecular weights, remained in the upper part (retentate). The samples were centrifuged at 6000 \u0026times; g and 4\u0026deg;C for 10 minutes to achieve this separation.\u003c/p\u003e\u003cp\u003eTo separate phycoerythrins, which have a molecular weight of 240 kDa, and allophycocyanin, which ranges between 104\u0026ndash;110 kDa, an Advantec USY-20 Disposable Ultrafiltration Unit with a 200 kDa molecular weight cut-off (MWCO) was employed. This unit allows retention of both upper (retentate) and lower (permeate) solutions. After obtaining phycoerythrin in the retentate, it was diluted tenfold with distilled water to increase sample volume. Allophycocyanin, collected as a colorless solution at the bottom of the tube, was separated and all samples were transferred to a round plastic dish for drying in a freeze dryer.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Polyacrylamide Gel Electrophoresis\u003c/h2\u003e\u003cp\u003eThe protein profile of the crude extract was determined according to the method described by Laemmli (1970), to detect the specific pigments targeted in this study, comparing qualitatively the phycobilin proteins and their molecular weights between treatments. Protein Ladder Model PM 1700 from SMOBIO was used as a marker; it is suitable for identification between 10 and 240 kDa. Electrophoresis was performed at 90 volts using a 1 \u0026times; 110 \u0026times; 140 mm gel size for approximately 4 hours; that is until bromophenol blue dye reached the bottom edge of the gel. The electrophoresis system used was SLABGEL xi Π PROTEIN (RAD Co., China) set at 30 milliamperes.\u003c/p\u003e\u003cp\u003eAfter electrophoresis, gels were stained in a solution containing 0.3 g Coomassie Brilliant Blue R-250, 4% glacial acetic acid, and 4% methanol for 2 hours and destaining in 5% glycolic acid and 2% methanol solution for 2 hours. Gels were then washed with distilled water and results were captured using a scanner and camera.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Drying\u003c/h2\u003e\u003cp\u003eThree drying methods were employed for the samples: freeze-drying using a glass tank freeze-dryer (model FD-5003-BT, Sanat Pardaz Dena Co.), spray drying with a spray dryer (model DSD-06, Dersa Behsaz Co.), and oven drying in a 55-liter digital oven (model G602).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll experiments were in triplicate. One-way ANOVA was used to test the differences among data for significance using IBM SPSS Statistics version 16 software. The differences in mean values between treatments were taken as significant at P\u0026thinsp;\u0026le;\u0026thinsp;0.05 and were assessed using LSD. Pearson's parametric correlation test was done to establish the relationship between the applied carbon source and biomass and phycobilin yield. The graphs were charged with the help of Origin Pro software version 2021 and Microsoft Excel version 365.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1. pH\u003c/h2\u003e\u003cp\u003eChanges in pH under different treatments during the experiment were summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. At the initial cultivation, the pH was 9.2 in all treatments. As the experiment progressed, the observed pH varied between approximately 9.7 to 10.2 in different treatments. By statistical analysis employing One-Way ANOVA, the obtained results testify to a significant difference concerning the levels of pH compared with Control within all treatments except the 75 mg/L sodium acetate one at P\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cem\u003eArthrospira platensis\u003c/em\u003e Cultivation specifications by treatment for one-liter batch culture during 20 days of cultivation. (*Significance level at α\u0026thinsp;=\u0026thinsp;0.05 *Significant changes are shown in alphabetical order)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026times;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatments\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003epH \u003csub\u003efirst\u0026minus;day\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003epH\u003csub\u003e20th day\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBiomass First-day Cell/L\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eBiomass last day\u003c/p\u003e\u003cp\u003eCell/L\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAlgae weight\u003c/p\u003e\u003cp\u003egr DM\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eSGR\u003c/p\u003e\u003cp\u003e(\u0026micro;/d)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl (photoautotroph)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e9.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e\u003cp\u003e2\u0026times;10\u003csup\u003e7\u003c/sup\u003e \u0026plusmn; 0.047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e32 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e \u0026plusmn; 4714045\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSodium acetate 25 mg/L (T1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e9.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e\u003cp\u003e2\u0026times;10\u003csup\u003e7\u003c/sup\u003e \u0026plusmn; 0.047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e38 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e \u0026plusmn; 12472191\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSodium acetate 50 mg/L (T2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e9.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e\u003cp\u003e2\u0026times;10\u003csup\u003e7\u003c/sup\u003e \u0026plusmn; 0.047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e47 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e \u0026plusmn; 12472191\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSodium acetate 75 mg/L (T3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e9.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e\u003cp\u003e2\u0026times;10\u003csup\u003e7\u003c/sup\u003e \u0026plusmn; 0.047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e39 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e \u0026plusmn; 16996732\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlycerol 0.5 cc (T4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e9.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e\u003cp\u003e2\u0026times;10\u003csup\u003e7\u003c/sup\u003e \u0026plusmn; 0.047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e44 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e \u0026plusmn; 9428090\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.085 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.000\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlycerol 1 cc (T5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e9.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 \u003csup\u003eac\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026times;\" colname=\"c4\"\u003e\u003cp\u003e2\u0026times;10\u003csup\u003e7\u003c/sup\u003e \u0026plusmn; 0.047\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e44 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e \u0026plusmn; 8164965\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Biomass Production\u003c/h2\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows that the maximum biomass production was achieved in T2 (Sodium acetate 50 mg/L), as evidenced by both cell count and dry weight measurements. T5 (Glycerol 1 cc/L) ranked second, exhibiting values significantly different from the Control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3. SGR\u003c/h2\u003e\u003cp\u003eThe specific growth rate (SGR) reaches its peak in T4 (Glycerol 0.5 cc/L), which is not significantly different from T2 (Sodium acetate 50 mg/L), indicating that SGR is highly influenced by the type of carbon source rather than the dosage. Higher doses do not necessarily increase the growth rates in the culture. This is evidenced by total biomass production values, which were measured by cell count and weight methods.\u003c/p\u003e\u003cp\u003eThe specific growth diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) shows that algae in treatments with added sugars adjusted faster to the culture medium compared with the Control, as expressed by the steepness of the slope from days 0 to 5. Treatment 3 (75 mg/L sodium acetate daily) exhibited the highest slope value during the growth phase (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), suggesting the most rapid adaptation to the culture medium with algae reaching maximum exponential growth rate (0.24226\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0086). Conversely, this treatment also showed the fastest decline phase with a slope of 0.22354\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0042.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eImmediately following treatment with 0.5 cc of glycerol, the control group remains relatively stable for 5 days before entering a decline phase, which is delayed by 5 days compared to other treatments. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e depicts the production of biomass of Arthrospira platensis based on cell count over a culture period of 20 days. From the graph, it can be observed that the highest cell production occurred in T2, which produced approximately 47 \u0026times; 10^7 cells per liter. From Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, this treatment also produced the highest dry-weight production at 4.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 g/liter. The SGR was also highest for T2 at 0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001.\u003c/p\u003e\u003cp\u003eAfter T2, 50 mg/L sodium acetate treatment, the next highest biomass was produced by the 0.5 cc glycerol treatment with a dry weight of 4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 g/liter at specific growth rate ranked second to the \u0026plusmn;\u0026thinsp;0.0012.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eweight gain statistics obtained by Origin Pro 2021 during 20 days of cultivation for \u003cem\u003eArthrospira platensis.\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCarbon Source\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGrowth equation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e(SLOPE)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eY\u0026thinsp;=\u0026thinsp;a\u0026thinsp;+\u0026thinsp;b*x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.1765\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e25 mg Dm/l Sodium acetate (T1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eY\u0026thinsp;=\u0026thinsp;a\u0026thinsp;+\u0026thinsp;b*x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.1981\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0127\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e50 mg Dm/l Sodium acetate (T2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eY\u0026thinsp;=\u0026thinsp;a\u0026thinsp;+\u0026thinsp;b*x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.2278\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0057\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e75 mg Dm/l Sodium acetate (T3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eY\u0026thinsp;=\u0026thinsp;a\u0026thinsp;+\u0026thinsp;b*x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.24226\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0086\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.5 cc Glycerol (T4)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eY\u0026thinsp;=\u0026thinsp;a\u0026thinsp;+\u0026thinsp;b*x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.23204\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1 cc Glycerol (T5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eY\u0026thinsp;=\u0026thinsp;a\u0026thinsp;+\u0026thinsp;b*x\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e0.23217\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe correlation test results (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) show that there is a significant positive correlation between sodium acetate and biomass dry weight (r\u0026thinsp;=\u0026thinsp;0.576). This infers that the addition of sodium acetate has contributed to increased biomass dry weight. The strength of this correlation falls within the average range of 0.4\u0026ndash;0.6, indicating a moderate relationship (Miller \u0026amp; Haden, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThere is also a strong positive correlation between sodium acetate and the number of cells per liter, at r\u0026thinsp;=\u0026thinsp;0.612, which proves that sodium acetate can affect biomass increase with a huge positive effect.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe results of the one-way ANOVA to check the significance of the differences between the optical absorption of samples dried by spray drying, freeze-drying, and oven. (٭Significant differences at α\u0026thinsp;=\u0026thinsp;0.05)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSum of squares\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003edf\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMean square\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eSig.\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e680 nm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBetween groups\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.283\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.141\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.397\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.273\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWithin groups\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.820\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.101\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.103\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e620 nm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBetween groups\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e23.611\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e11.806\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e4.211\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.032 *\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWithin groups\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e50.466\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2.804\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e74.078\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e280 nm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBetween groups\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e67.029\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e33.514\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2.347\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.124\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWithin groups\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e257.017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e14.279\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e324.046\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThis is in addition to a high positive relationship of sodium acetate with the percentage of extracted phycocyanin compared to the total crude extract, as presented by the correlation analysis, at α\u0026thinsp;=\u0026thinsp;0.01. The close-to-1 correlation coefficient shows a very significant and strong correlation. The aforementioned statistical observation underlines that sodium acetate significantly enhanced the yield of Spirulina biomass and phycocyanin production by the cells.\u003c/p\u003e\u003cp\u003eIn addition, a more intensive relationship between the given conditions with the production of phycocyanin implies a highly marked stimulation in its biosynthesis by the sodium salt. Sodium acetate acts positively on other parameters like specific growth coefficient.\u003c/p\u003e\u003cp\u003ePearson's correlation test indicated that glycerol was strongly positively correlated with the production of dry biomass weight. However, no significant correlations were found with other growth-related parameters such as specific growth rate, cell density, or biomass concentration measured by spectrophotometric absorption. This indicates that while glycerol was able to improve cell proliferation and enhance biomass weight, it hardly affected these other growth parameters.\u003c/p\u003e\u003cp\u003eTherefore, it could be put that supplementation with glycerol mainly enhanced cell proliferation and biomass weight production, with hardly any contribution to the production of pigments inside the cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Pigment Production\u003c/h2\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents the overall production of pigments for all treatments during the 20-day cultivation period. It can be seen that the highest phycocyanin production was from T3 (75 mg Sodium acetate), followed by T2 (50 mg Sodium acetate) in the second position, while T5 (1 cc Glycerol) secured the third position. On the other hand, the graph for allophycocyanin shows that there is no much difference between the treatments T1 to T5, which all have performed far better than the control. Higher carbon source concentration in treatments like T3 and T5 resulted in a comparably low production of the allophycocyanin with others, showing possible negative impacts of high carbon concentration on allophycocyanin yield.\u003c/p\u003e\u003cp\u003eRegarding phycoerythrins, the highest production was obtained for T2 (50 mg Sodium acetate) and T4 (0.5 cc Glycerol), which were significantly different from T3 (75 mg Sodium acetate) and T5 (1 cc Glycerol). More importantly, all the treatments showed significantly higher phycoerythrin production compared to the control, which would indicate that while additional carbon sources enhance biomass and may increase phycoerythrin production, lower sugar concentrations may favor higher yields of phycoerythrin. These trends are consistent with the findings for allophycocyanin production.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Protein Profile and Detection of Protein Pigments in the Crude Extract\u003c/h2\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e: SDS-PAGE gel profile of protein from Arthrospira platensis separated on a polyacrylamide gel. In gel, within the range of 20 to 25 kDa, sharp and prominent bands of phycocyanin are present, and band intensity is indicative of its high protein concentration in the samples. Also, allophycocyanin reveals particular bands at about 100 kDa that are more conspicuously thick in the treatments of 1 cc glycerol and 50 mg sodium acetate. Phycoerythrin bands are also present in the gel, which was observed in the range of 240 kDa.\u003c/p\u003e\u003cp\u003eThe results of polyacrylamide gel electrophoresis support the data of spectrophotometry for the confirmation of the presence, concentration, and qualitative assessment of every phycobilin protein in the samples under consideration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Drying\u003c/h2\u003e\u003cp\u003eVarious drying methods were used in this experiment to compare visual and spectral differences in the phycocyanin powder of different samples. The freeze-drying method should produce higher quality powder than the other methods because there is less sample heating during this method (the differing rates of protein denaturation will not be explored within this experiment).\u003c/p\u003e\u003cp\u003eDistinct visual disparities were observed among the samples. Oven-dried samples displayed a blue-green color (dark blue with a greenish hue), while spray-dried samples appeared solid blue without showing any green tendency (as is typical of many market samples). Freeze-dried samples displayed a very light sky-blue color, with a stark visual contrast compared to the dry powder appearance.\u003c/p\u003e\u003cp\u003eFor spectral analysis, 5 mg of each method of dry phycocyanin powder was dissolved in 10 mL distilled water, and their optical absorption at 680 nm, 620 nm (phycocyanin wavelength), and 280 nm was analyzed. In optical absorption at 680 nm, no significant difference appeared among the drying methods at p\u0026thinsp;\u0026ge;\u0026thinsp;0.05. However, significant differences were realized at 620 nm p\u0026thinsp;\u0026le;\u0026thinsp;0.05 between spray-dried and oven-dried samples, which might indicate phycocyanin denaturation by the more prolonged heating in the oven. Visually, it was not possible to notice any difference in appearance with the naked eye.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe experiment focused on the cultivation and growth performance of \u003cem\u003eArthrospira platensis\u003c/em\u003e, a significant algal species, using varying concentrations of sodium acetate and glycerol as additional carbon sources alongside the Zarrouk, (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e1966\u003c/span\u003e) basic medium in all treatments and controls. Initial cell density was consistent at 2\u0026times;10^7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.047 cells/L across treatments, peaking at 47 \u0026times; 10^7 cells/L with Sodium acetate 50 mg/L.\u003c/p\u003e\u003cp\u003eDifferent cell growth and biomass accumulation due to varying carbon levels and sources were observed within the study. The optimum level of carbon was required for normal cellular growth and hyperproduction of phycobilin proteins intra-cellularly. More carbon led to faster cell growth but, however, resulted in slow production of phycobilin protein and entry into early death stage.\u003c/p\u003e\u003cp\u003eThese findings underscore the importance of using an optimal, consistent level of carbon that is appropriate for the used carbon source for the normal weight of cells and phycobilin production during its growth.\u003c/p\u003e\u003cp\u003eThe carbon source is one of the most important factors in the cultivation of microalgal species, such as Arthrospira platensis, commonly known as Spirulina. Different media and carbon sources have been tested concerning their influence on the growth and performance of these microalgae.\u003c/p\u003e\u003cp\u003eRahman, (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) studied the evaluation of Spirulina platensis for their culture and growth performance using different concentrations of supernatant of digested rotten potato (DRP) with 20%, 40%, and 60% of DRP with Kosaric medium as a control in 26 days of digestion and Spirulina was also inoculated into DRP supplemented with 9.0 g/L NaHCO3 and micronutrients including KM for a period of 14 days in this study.\u003c/p\u003e\u003cp\u003eSpirulina reached its maximum cell density in KM on the 10th day of culture at 12.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 mg/L, followed by 9.505\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43 mg/L in the 60% DRP supernatant, 8.352\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 mg/L in the 40% DRP supernatant, and 6.256\u0026thinsp;\u0026plusmn;\u0026thinsp;2.34 mg/L in the 20% DRP supernatant. The same trend was observed in the optical density, chlorophyll A content, total biomass, and specific growth rate.\u003c/p\u003e\u003cp\u003eThese results reflect significant differences in cell density between KM and the DRP supernatants at p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, which were positively related to chlorophyll content and total biomass. Indeed, this reflects that growth performance in Spirulina is influenced by the added concentration of the DRP supernatant; usually, higher concentrations support lower cell densities compared to KM.\u003c/p\u003e\u003cp\u003eThe growth performance of Spirulina platensis was notably enhanced when cultivated in the supernatant of 60% digested rotten potato (DRP) compared to lower concentrations (20% and 40% DRP). This suggests that mass cultivation of Spirulina can be effectively carried out using a 60% DRP supernatant due to its superior growth-promoting properties observed in the study.\u003c/p\u003e\u003cp\u003eMia et al., (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) conducted an experiment to study the culture and growth performance of Spirulina platensis on various rotten apple medium (RAM) concentrations and Kosaric Medium (KM). In the experiment, S. platensis was cultured in 1.0L glass flasks containing three different RAM concentrations, namely 2.5%, 5.0%, and 10% along with KM, and each medium was replicated thrice. These cultures were maintained for 14 days under a fluorescent light regime composed of 12 hr of light and 12 hr of dark.\u003c/p\u003e\u003cp\u003eThere is a significant difference in the growth performance of S. platensis on different media formulations. Initially, S. platensis was recorded as 0.0023 mg/l cell weight. The 10th-day maximum record of cell weight in KM and RAM at 10% was 12.44mg/l and 10.468 mg/l respectively. Similarly, the initial Chlorophyll content of S. The highest amount of platensis was 0.0015 mg/L, reaching a maximum value of 10.54 mg/L in KM and 12.35 mg/L in RAM (at 10% concentration) on the 10th day.\u003c/p\u003e\u003cp\u003eThe weight of the cell started to decline after the 10th day of cultivation. S. platensis growth in 5.0% DRAM was significantly higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than the growths in the other two RAM concentrations of 2.5% and 10% DRAM.\u003c/p\u003e\u003cp\u003eIn a related study, Habib and Kohinoor, (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) recorded that Spirulina platensis showed better growth when cultured in the supernatant of 60% DRGM as the carbon source, rather than at lower concentrations of 20% and 40% DRGM. Their observation agrees with previous studies that the supernatant of 45% digested poultry waste also supported healthy growth of Spirulina, indicating that the concentration of the carbon source is an important component in the improvement of conditions for growth.\u003c/p\u003e\u003cp\u003eAlthough many studies have been carried out on the phycocyanin content of Spirulina during its growth, few have been conducted that simultaneously studied the effects of carbon sources on cell proliferation and phycobilin content. These two aspects are important in relation to how carbon sources influence biomass production and pigment synthesis in Spirulina platensis.\u003c/p\u003e\u003cp\u003eChaiklahan et al., (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) recently published their findings on the cultivation of \u003cem\u003eArthrospira\u003c/em\u003e (\u003cem\u003eSpirulina\u003c/em\u003e) platensis BP in a photobioreactor under varying light intensities (635, 980, 1300, and 2300 \u0026micro;mol m\u0026thinsp;\u0026minus;\u0026thinsp;2 s\u0026thinsp;\u0026minus;\u0026thinsp;1) using a semi-continuous mode to maintain cell concentrations at optical densities (OD) of 0.4, 0.6, and 0.8. They also noted that the highest biomass productivity of 0.62 g L\u0026ndash;1 d\u0026ndash;1 and phycocyanin yield of 123 mg L\u0026ndash;1 d\u0026ndash;1 occurred at a light intensity of 2300 \u0026micro;mol m\u0026thinsp;\u0026minus;\u0026thinsp;2 s\u0026thinsp;\u0026minus;\u0026thinsp;1 at OD 0.6.\u003c/p\u003e\u003cp\u003eThe energy consumption efficiency in this case for algal biomass was around 2.26\u0026ndash;2.31 g\u0026middot;(kW h)-1 d-1, while the photosynthetic efficiency at a light intensity of 635 \u0026micro;mol\u0026middot;m-2\u0026middot;s-1 at OD 0.8 was around 8.02%. This indeed signifies the importance of light intensity, cellular concentration, and light/dark period for biomass improvement and the production of phycocyanin in Arthrospira platensis while pointing toward the most ideal conditions required for maximum yield.\u003c/p\u003e\u003cp\u003eGladfelter et al., (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) conducted a 2-week field experiment using 1,100-L plastic limnocorrals to investigate the response of the cyanobacterial community to different nitrogen forms: nitrate, ammonium, and urea (added at 600 \u0026micro;g N/L). They monitored cell pigments and counts to calculate cell-specific pigment concentrations and measured cell-associated microcystin concentrations to assess the toxin response to nitrogen source variations.\u003c/p\u003e\u003cp\u003eThe results indicated that upon nitrogen addition, extracellular nitrogen levels rapidly decreased, corresponding with an increase in cellular phycocyanin levels 72 hours after fertilization. Ammonium and urea treatments exhibited higher phycocyanin/cell ratios compared to nitrate or control treatments at the 72-hour mark.\u003c/p\u003e\u003cp\u003eExtraction methods significantly influence the quality, quantity, and stability of phycobilin pigments extracted from algae. Extensive research has focused on maximizing phycocyanin production in dry powder form. For instance, Chittapun et al., (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) investigated the extraction of C-phycocyanin from Nostoc commune TUBT05 and Oscillatoria okeni TISTR8549 using freezing and thawing, as well as pulsed electric field treatments. Their findings demonstrated that freezing and thawing are effective methods for extracting C-phycocyanin from both cyanobacterial strains. In contrast, pulsed electric field treatment was successful only with N. commune due to its cell structure being compatible with this technology. The number of freeze/thaw cycles, the composition of the extraction solution, and the number of electric pulses exerted significant statistical influence on C-phycocyanin concentration, purity, yield, and total protein content in the crude extracts (p\u0026thinsp;\u0026le;\u0026thinsp;0.005).\u003c/p\u003e\u003cp\u003ePatel et al., (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) in the year 2005, developed an effective single-step chromatographic method for the purification of C-Phycocyanin from three cyanobacterial species, namely Spirulina sp., Phormidium sp., and Lyngbya sp. The procedure for purification consisted of successive steps involving fractional precipitation with ammonium sulfate, followed by chromatography on a DEAE\u0026ndash;Sepharose CL-6B column. From Spirulina, Phormidium, and Lyngbya spp., C-Phycocyanin was thus obtained in purity ratios (A620/A280) of 4.42, 4.43, and 4.59, respectively. Native and SDS\u0026ndash;PAGE were also used to further verify purity and homogeneity.\u003c/p\u003e\u003cp\u003eThe objective of the present study has been to establish in detail the major parameters affecting natural pigment production, above all phycocyanins, right from the levels of algae cultivation to the final steps of extraction and product preparation. Results presented in this work are derived from laboratory-scale studies and hence not directly generalized to industrial settings. Nevertheless, they provide valuable insights that could facilitate the commercialization of phycocyanin production.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eGenerally, this review highlights the importance of the addition of extra carbon sources to enhance cell proliferation and biomass accumulation in cultures of \u003cem\u003eArthrospira platensis\u003c/em\u003e. These additional supplements of carbon sources were truly crucial regarding dosage and timing for biomass, SGR, and even the pigment production of the organism under investigation. Importantly, the type of carbon source proved to be one of the decisive factors for optimization in biomass yield and phycobilin production. Furthermore, our investigation has pointed out the great influence of extraction and drying methods on the quality of phycocyanin and phycoerythrin dry powders. Since these protein pigments are temperature-sensitive, we suggest non-thermal and non-chemical extraction techniques to keep their integrity and maximize product quality. Although our findings are based on the laboratory scale and need further validation at an industrial scale, they provide a basis on which to improve the commercial production of phycocyanin. Further research is thus needed for scale-up at the industrial level, taking into account the complexities and economic viability of large-scale production. The present study therefore, helped to fill basic knowledge gaps lacking in the area of producing natural pigments in A. platensis and will be useful for pointing out insights on how conditions of cultivation and extraction procedures should be optimized with regard to elevated levels of phycocyanin and phycoerythrin production.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eSpirulina\u003c/em\u003e (\u003cem\u003eArthrospira platensis\u003c/em\u003e), \u003cem\u003eChlorella\u003c/em\u003e (\u003cem\u003eChlorella vulgaris\u003c/em\u003e), PC (\u003cem\u003ePhycocyanin\u003c/em\u003e), PE (\u003cem\u003ePhycoerythrin\u003c/em\u003e), ALP (\u003cem\u003eAllophycocyanin\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cb\u003eFunding Declaration Paragraph\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e1- Zahra Soltani Far:Conceptualization, Data curation, Formal analysis, Funding acquisition.2- Mohammad Ali Nematollahi:Investigation, Methodology, Project administration, Resources.3- Mahmoud Soltani Firouz:Software, Supervision, Validation, Visualization.Seyed Vali Hosseini: Methodology, Resources, Visualization.4- Pouya Farshbaf Aghajani:Data curation, Formal analysis, Resources, Software, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbiusi, F., Wijffels, R. 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Cultivation of aquaculture feed Isochrysis zhangjiangensis in low-cost wave driven floating photobioreactor without aeration device. \u003cem\u003eBioresource technology\u003c/em\u003e, \u003cem\u003e293\u003c/em\u003e, 122018.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Spirulina, Mixotrophic Culture, Carbon Supplementation, Phycocyanin, Biomass Production","lastPublishedDoi":"10.21203/rs.3.rs-7284160/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7284160/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eArthrospira platensis, commonly known as \u003cem\u003eSpirulina\u003c/em\u003e, is an industrially cultivated cyanobacterium due to its high content of phycobiliproteins, mainly phycoerythrin, phycocyanin, and allophycocyanin. In this work, a low-cost strategy for the enhancement of biomass and pigment production, along with the improvement of phycocyanin extraction, is investigated. In this regard, two carbon sources at different dosages were tested for their effect on biomass and phycobilin production. Biomass production was estimated in terms of specific growth rate (SGR), cell count per liter, and increase in dry weight. Phycobilin protein quantification was done through spectrophotometry and gel electrophoresis. The results indicated that the addition of extra carbon sources increased cell proliferation and biomass production. Sodium acetate at 50 mg/L/day significantly increased the biomass, SGR, and phycocyanin production. Glycerol at 1 cc per day enhanced cell proliferation and biomass but did not enhance phycobilin accumulation. The spectrophotometric analysis on 5 mg dry phycocyanin with 10 cc distilled water showed no significant difference (p\u0026thinsp;\u0026ge;\u0026thinsp;0.05) at 680 nm, while in the absorbance at 620 nm, there is a significant difference between spray and oven drying of the samples (p\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e","manuscriptTitle":"Optimizing Biomass and Phycobilin Production in Arthrospira platensis Through Carbon Source Variation and Developing a Cost-Effective Purification Method","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-01 10:43:29","doi":"10.21203/rs.3.rs-7284160/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"49663168-c492-4b91-aacc-6e0c729265d0","owner":[],"postedDate":"September 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-28T09:55:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-01 10:43:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7284160","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7284160","identity":"rs-7284160","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}