Influence of Sulphate Nutrition on Growth Performance and Antioxidant Enzymes Activities of Spirulina platensis

The growth of Spirulina platensis is dependent on culture conditions. This study has established adequate conditions for the quality and quantity production of S. platensis. The effect of sulphate salts nutrition on growth performance and biochemical status of S. platensis was assessed in vitro. Prior to culture, the Paracas strain of S. platensis from SAGRIC pond was analysed in different magnesium sulphate (MgSO4; 0.08, 0.16, 0.32, 0.64 and 1.28 g/L), potassium sulphate (K2SO4; 0.08, 0.16, 0.32, 0.64 and 1.28 g/L) and MgSO4/K2SO4 (0.16/0.00, 0.08/0.08, 0.04/0.12, 0.02/0.14 and 0.01/0.15 g/L) concentrations. Culture media pH, total dissolved solids (TDS) and conductivity rate were monitored. Microscopic analysis revealed sulphate salt concentrations influenced the number of whorls and filaments of S. platensis. K2SO4 (1.28 g/L) produced the highest number of whorls and filaments. Moreover, pH level fluctuated by sulphate treatments. K2SO4 (1.28 g/L) had a pH level of 8.77±0.01 (day 5 of culture incubation). TDS and conductivity rate, protein and cysteine contents increased with culture age and K2SO4 concentration in a culture medium. Conversely, negative correlations between protein and cysteine contents were observed, and sugar content decreased. Sulphate salt type and concentrations affected polyphenol oxidase (PPO) and peroxidase (POD) activities. MgSO4/K2SO4 (0.02/0.14 g/L) displayed the best PPO and POD activities. Both enzymes appeared to be negatively correlated to the decreasing sugar content. These results indicate growth performances and biochemical status of S. platensis are significantly improved with the adequate supplementation of sulphate salts (MgSO4 and K2SO4) in culture media.


Introduction
Spirulina platensis is a multicellular, filamentous and microscopic photosynthetic cyanobacterium commonly found in the brackish lakes of Central Africa and Mexico. S. platensis has been consumed for centuries by the Aztecs and bordering populations on Lake Chad (Shigekatsu et al., 2019). This microalgae is characterized by a high content of protein (including enzymes such as polyphenol oxidase and peroxidase) and high amounts of essential fatty acids, essential amino acids, minerals, vitamins (especially B12), polysaccharides and antioxidant pigments (chlorophyll, carotenoids, phycobiliprotein, phycocyanin and carotenoids) (Budiyono et al., 2014;Ben Amor et al., 2017;Jung et al., 2019;Fatemeh & Choopani, 2020).
This microalgae is being studied, not only for its nutritional properties but also for its reported therapeutic properties related to its hypolipidemic effect (Al-Saman et al., 2020), protective effect against diabetes and obesity (Azabji-Kenfack et al., 2011;Gómez-Téllez et al., 2020), inhibitory effect on anemia and cancer (Abdel-Daim et al., 2013;Barakat et al., 2015), stimulatory effect on the immunological system Ama Moor et al., 2020), nephrotoxicity effect on pharmaceuticals and toxic metals and protective effect against harmful radiation (Mohan et al., 2006;Priyanka Yadav et al., 2019). Because of its multiple properties, the production of S. platensis has gained worldwide attention for use in human food supplements, animal feed and pharmaceuticals industries.
In Cameroon, culture of spirulina remains rudimentary, not controlled by producers and its biochemical status uncertain. Biomass, specific growth and biochemical composition of spirulina depend on many factors which include farming practices, environmental parameters and culture medium composition (Madkour et al., 2012). However, composition of culture medium is a major factor which influences growth rate, biomass production and biochemical status of this cyanobacterium. Hence, it is possible to improve the growth performance, biochemical status and antioxidant activity of S. platensis while acting on composition of culture medium in order to fulfil pharmaceutical and nutritional requirements. Therefore, the present study was undertaken to study the incidence of exogenous sulphate salts (K 2 SO 4 and MgSO 4 ) supplementation on growth performance and biochemical profile (including antioxidant enzymes activities) of S. platensis cultured in vitro. Green algae growth is negatively influenced by harmful reactive oxygen species (ROS) from diver's physiological metabolic processes. Polyphenol oxidase and peroxidase are important antioxidant enzymes in stress control of the cell (Yakelín et al., 2001;Mostafa Mahmoud et al., 2016).

Microorganism Strain
The cyanobacterium S. platensis strain «Paracas» used in the present study was obtained from the freshwater culture pond of SAGRIC Common Initiative Group (CIG) farm, Douala-Cameroon. The strain was grown and maintained in 500 mL sterilized Erlenmeyer flasks containing 100 mL Jourdan's medium (Table 1) (Jourdan, 2013) at pH 9 in an illuminated growth room at 28±0.5 °C under 12/12 hours photoperiod and daily manually shake (thrice).

Culture Media and Experimental Design
Jourdan's medium (Jourdan, 2013) was used as the reference medium. Sulphate salts (MgSO 4 and K 2 SO 4 ) were brought in variable concentrations in Jourdan's medium. MgSO 4 was varied in absence of K 2 SO 4 . Conversely, K 2 SO 4 was varied in absence of MgSO 4 . Also, the ratios MgSO 4 /K 2 SO 4 varied with fixed content of SO 4 2- (Table  1). The algae S. platensis cells were inoculated at a concentration of 15% (V inoculation/V media) in 1000 mL erlenmeyer flasks. The pH of all culture media was adjusted to 9 before sterilization, cool and addition of S. platensis cells (15% v/v). Cultures were incubated at 12/12 hours (light-darkness) photoperiod under temperature 28±0.5 °C for 5 days. Cultures were manually shook (for 3 min) thrice daily. Samples were collected every day for assessment of the cyanobacteria growth as well as estimation of biochemical status and antioxidant enzymes activities. All experiments were carried out in triplicate.

Monitoring of Physico-Chemical Parameters of Culture Media
Physico-chemical parameters (temperature, pH, conductivity, and total dissolved solids (TDS) of media were recorded daily using of multi-parameters (HI 98130, HANNA Instruments, Rhodes Island, USA).

Assessment of S. platensis Growth Parameters
S. platensis cell populations (number of filaments and whorls) were evaluated using light and fluorescence microscope (Cyscope® HP, Sysmex-Partec, Japan) by direct microscopic counting method described by Usharani et al. (2012). Biomass concentration (g/L) was determined every day by measuring the optical density at 560 nm. A standard concentration was used to determine the biomass of individual samples (culture media and daily monitoring) based on optical density and use the coefficient of correlation (C = 0.782 X, where X is the biomass concentration (g /L) according to Tsarahevitra et al. (2003). The calculated biomass was used to obtain maximum specific growth rate (μ m ) and productivity (P) from the following equation of (Madkour et al., 2012): where, µ m = specific growth rate (div/day); X 1 = biomass concentration at time t 1 ; X 2 = biomass concentration at the time t 2 .
Productivity (P) was estimated as follow: where, P = productivity (mg /L/day); X i = initial biomass density (g/L); X m = biomass density at time m (g/L); t m = time interval (day) between X i and X m .

Chemical and Biochemical Analysis
2.5.1 Chemical Composition Analysis of S. platensis Strain Used The S. platensis sample from SAGRIC pond was aseptically filtered and dried during 48 hours at 50 °C in a sterilizer (Binder, Germany). Subsequently, the sample was analyzed to find out its chemical composition. Total protein was determined by the conventional Micro-Kjeldahl method (AOAC, 1995). Lipids were extracted using a Soxhlet apparatus and analyzed according to the AOAC (1990) method. Total ash and fibers were determined by the standards method of AOAC (1990) and Wolff (1968). The determination of minerals (Ca, Mg, K, Na, Fe and P) was carried out using atomic absorption spectrophotometer after extraction in a mixture of nitrichydrochloric acid (75v/ 25v).

Biochemical Analysis of S. platensis in Experimental Design
(1) Reducing Sugars and Cysteine Extraction and Analysis Reducing sugars and cysteine were extracted in 80% ethanol. One mL of homogenized algal suspension was added in 5 mL of 80% ethanol in the mortar and then centrifuged (3000 g, 10 min). The supernatant was collected and used for reducing sugars and cysteine contents quantification. Reducing sugars were assayed by mixing 0.1 mL of reducing sugars crude extract with 1.5 mL of water and 0.5 mL of Müller reagent [1% (w/v) DNS (3,5-dinitro salicylic acid), 1.6% NaOH (w/v) and 30% (w/v) sodium-potassium tartrate]. The mixture was homogenized and incubated at 100 °C for 10 min in the water bath to allow colour development. Optical density was measured at 575 nm using glucose as standard.
Cysteine content was estimated by the method describe by Gaitonde (1967). Cysteine crude extract (0.15 mL) was added to 0.35 mL of acidic ninhydrin reagent [1, 3% (w/v) Ninhydrine in 1:4 HCl:CH 3 COOH conc]. The mixture was homogenized and heated at 100 °C for 10 min followed cooling in ice. A volume of 1 mL ethanol 95° was added and the optical density read 560 nm against a blank where cysteine crude extract was replaced equal volumeof ethanol 80°.
(2) Antioxidant Enzymes and Proteins Extraction Polyphenol oxidase and peroxidase were extracted by homogenizing 1g of fresh S. platensis sample in a mortar containing 10 mL potassium phosphate buffer (50 mM, pH 6.0). The homogenate was subsequently centrifuged (6000g, 30 min at 4°C) and the supernatant was collected. The pellet was re-suspended in the same buffer centrifuged under the same conditions as previously. The second supernatant was added to the first to obtain polyphenol oxidase and peroxidase preparation extract which was used for the analysis of proteins contents, polyphenol oxidase and peroxidase activities.
(3) Proteins Quantification The protein content was determined by the method of Bradford (1976) using bovine serum albumin (BSA) as a blank.
(4) Polyphenol Oxidase and Peroxidase Activities (a) Polyphenol Oxidase Activity Polyphenol oxidase (PPO) activity was determined by measuring the increase in absorbance at 330 nm using the method of Van Kammen and Broumer (1964). The reaction mixture incubated at 25 °C was made of: 2.7 mL of phosphate buffer (1/15 M, pH 6.1) and 0.3 mL catechol (10 mM). The reaction was initiated by adding 40 µL of enzymatic extract. The enzyme activity was monitored through change of optical density at 330 nm after 30 s. PPO activity was expressed in unit per µg of proteins content.

Data A
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Discussion
This study highlights that sulphate nutrition has an important influence on the growth performance and biochemical status including antioxidant enzymes activity of the cyanobacterium Spirulina platensis.
In the mass culture of microalgae, the medium quality is one of the key factors controlling growth, productivity and biochemical status (Madkour et al., 2012). The algae S. platensis has been studied with the basic aim of screening for magnesium sulphate (MgSO 4 ) and potassium sulphate (K 2 SO 4 ) concentrations.
The cyanobacterium S. platensis was cultured at pH 9 and temperature of 28 °C. However, we noticed progressive increase of conductivity and total dissolved solids (TDS) in media with different concentration of MgSO 4 , K 2 SO 4 and the MgSO 4 /K 2 SO 4 combination. This increase of conductivity and total dissolved solids (TDS) could be explained by the presence of electrically charged atoms which increase with the evaporation of water in media and to the change of the other variables of the culture media due to uptake of nutrients brought by the different concentration of sulphate salt (Anna, 2018).
The morphological feature of S. platensis identified like a blue-green filamentous cyanobacterium was reported by Luo and Jiang (2015). Microscopic analysis revealed that the number of whorls and filaments are influenced by sulphate salts concentration. Growth of S. platensis as number of filaments, biomass, cell productivity and maximum specific growth rate in 0.16 g/L MgSO 4 , 1.28 g/L K 2 SO 4 and 0.02/0.14 g/L MgSO 4 /K 2 SO 4 combination was significantly higher than those obtained in media supplemented with others concentrations of MgSO 4 , K 2 SO 4 and combination of MgSO 4 /K 2 SO 4 ( Table 2). These results could be explained by the fact that the higher concentration of MgSO 4 (0.32, 0.64 and 1.28 g/L) have negative effect in media involving the reduction of photosynthetic activity of S. platensis (Nyabuto et al., 2015). According to Ndjouondo et al. (2017), 0.1 and 0.2 g/L of magnesium sulphate are used as optimum concentration for growth of S. platensis and growth delay was observed at concentration higher than 0.2 g/L and the low biomass yield at the highest concentration could be attributed to substrate toxicity (Wakte et al., 2011). The lower number of filaments, biomass, cell productivity and maximum specific growth rate at lower concentration of K 2 SO 4 (0.16 g/L) are in agreement with those reported by Wagih El-Shouny et al. (2015) which showed that the reduction of sulphur in the culture medium involved non significant reduction in the growth of the biomass and productivity of S. platensis. Moreover low yield of growth recorded in media in with low potassium sulphate concentrations could be because sulphur deficiency could cause a reduction in the cell multiplication while influencing on metabolism of carbon in photosynthetic activity as reported by Carfagna et al. (2015) to Chlorella sorokiniana. Therefore, in media supplemented with different concentrations of sulphate salts, the best number of filaments, biomass, cell productivity and maximum specific growth rate were recorded on medium supplemented with MgSO 4 /K 2 SO 4 (0.02/0.14 g/L). This could be due to the presence of mineral nutrients (K and Mg) brought by the MgSO 4 /K 2 SO 4 combination that might play a critical role in the metabolic activities, as essential components of enzymes and other cellular components (Kaushik et al,. 2006) and the presence in the medium of ions Mg 2+ and K + which could play a significant role in the mechanism of photosynthesis.
Culture medium composition has been reported as one of the most important factors with determining role in biochemical status of microalgae (Çelekli et al., 2016). The biochemical profile of S. platensis strain in pond of SAGRIC Common Initiative Group (CIG) farm, Douala (Cameroon) has shown highest content of protein (63.9%) compared to the 37.5% of protein harvested by Ama Moor et al. (2016) in Nomayos-Cameroon, 58.6% and 50.2% of protein by Ngakou et al. (2012) but lower than 69.2% of protein obtained by Mbaïguinam et al. (2006). This could be attributed to the availability of essential elements (N and P) in Jourdan's medium as well as the tendency of algae for bioaccumulation and incorporation of these elements into their macromolecules. Furthermore this analysis revealed that total ash and some minerals (Ca, Mg, K, Fe and P) were much higher than those reported by Ngakou et al. (2012) and Ama Moor et al. (2016). These differences could be explained by either the influence of culture media, the difference in climate, or caused by the differentiated cellular metabolism in as much as these elements are actively involved in the metabolism of S. platensis. Reversely, lipids, fibers and sodium contents appeared to be lower. This could be due particularly for lipids content to a variation of the extraction method or the type of solvent used (Ama Moor et al., 2016). Thus the strain of S. platensis used in this experiment contains macronutrients and essential micronutrients absorbed from its growth medium become chelated with amino acids and are therefore more easily assimilated by the body and is considered as an excellent food supplement, nephrotoxicity effect of pharmaceuticals and toxic metals, immunological properties and acts as a potent antioxidant.
Considering, magnesium sulphate (MgSO 4 ), the concentration 0.16 g/L gave higher content of protein. Protein contents were increased with K 2 SO 4 content in culture media (the highest protein contents were obtained with 1.28 g/L). The combination of both salt generated the highest protein content with 0.02/0.14 g/L MgSO 4 /K 2 SO 4 . These results highlight the benefit effect of sulphate salts nutrition on proteins accumulation in S. platensis biomass. However, this benefit effect depends on sulphate salt type and Mg 2+ /K + ratio. S. platensis seems to not tolerate high concentration of Mg 2+ . This might indicate the toxicity of magnesium sulphate at highest concentration in S. platensis biomass (Wakte et al., 2011). Reversely, high K + promotes protein accumulation in S. platensis biomass.
Cysteine content analysis in S. platensis biomass (relation sulphate nutrition) revealed the influence of sulphate salt type and Mg 2+ /K + ratio on content of this sulphurous amino acid (as with protein content). The accumulation of cysteine with increasing content of K 2 SO 4 might indicate that, the availability of sulphate promote the assimilation of sulphur for the synthesis of cysteine (a sulphur-containing amino acid). However, this promoting effect appeared to be stimulate by high content of K + in culture media; but altered by high content of Mg 2+ (above 0.16 g/L) in culture media. The increase in SO 4 2concentration caused an increase in cysteine due to essential role of sulphur in synthesis of amino acids like cysteine which make up proteins and enters in the composition of chlorophyll and has a direct implication in the enzymatic catalysis (Schwenk, 2012).
Reducing sugars contents in S. platensis biomass displayed a reverse pattern compared to protein and cysteine contents. This might reveal that, the sulphate supply in culture media leads the use of reducing sugars for synthesis of cysteine which is utilized for protein building. Negative and significant correlation was found between cysteine contents, protein contents and reducing sugars.
These changes in biochemical composition could be correlated with the essential role played by the ions K + and Mg 2+ in the assimilation of sulphur and the growth of S. platensis (Dea Prianka et al., 2019).
Algae are negatively affected by harmful reactive oxygen species (ROS) produced by photosynthetic electron transport, photorespiration, respiration and other metabolic processes which may cause the deterioration of cell metabolism and damage cellular components (Foyer et al., 2011;Mostafa Mahmoud et al., 2016). To alleviate the harmful effects of ROS, S. platensis have developed several mechanisms such as antioxidants enzymes in which the polyphenol oxidase (PPO) and peroxidase (POD) play a significant role. The enhanced activity of PPO and POD in S. platensis biomass in lower MgSO 4 and higher K 2 SO 4 concentrations observed in the present study may suggest a cooperative role of these antioxidants enzymes in protection of S. platensis cells against ROS.
Highest PPO and POD activities matched with highest protein, cysteine contents and biomass production. These set of results might reveal that an adequate sulphate supply leads to optimal biomass production, protein and cysteine accumulation under appropriate PPO and POD activities which preserved spirulina cells against ROS (Mostafa Mahmoud et al., 2016;Panahi et al., 2019).

Conclusion
Medium composition is one of the key factors that control S. platensis growth, biochemical status and antioxidant activity. From the present study, it could be concluded that high yield of biomass (number of filaments, biomass concentration, cell productivity and specific growth rate), biochemical status (protein, cysteine and reducing sugars) and antioxidant enzymes activities (PPO and POD) were sulphate salt type and concentration dependent. S. platensis cultured in medium supplemented with both MgSO 4 and K 2 SO 4 was characterized by highest growth performance, biochemical status (protein, cysteine and reducing sugars) and antioxidant activities (PPO and POD). These sets of results draw attention to the importance of selecting the source and concentration of sulphate salts for S. platensis culture. Sulphate nutrition appeared to be useful to improve growth performance and biochemical status (nutritional value and antioxidant activity) of this cyanobacterium which is important in further explored for their use for medicinal products and additives in pharmaceutical, food, cosmetic or other industrial applications.