# Profiling the Spirulina Dietary Supplements Available on the Romanian Market
## 3 Results and Discussion
### Analysis of Spirulina Profile  
The employment of PCA on the isotope and elemental profile (i.e., 24 characteristics) of the analyzed data set, which comprised 66 commercial samples and 1 in-house spirulina sample, has revealed the presence of several outliers, which can be seen in Figure 1. The farthest point from the main cluster observed in the biplot corresponding to the first two principal components proved to be the in-house cultivated spirulina sample, characterized by higher concentrations of several macro-minerals (Ca, Na, and K), micro-elements (Fe and Zn), and trace elements (Ba, Cr, Pb, and Mo). This could be easily explained since the salts utilized in the CCAP Spirulina Medium for cultivating the in-house sample were of reagent quality. Higher purity and proper storing conditions maintained in scientific laboratories prevent most of the different timely alterations in different salts. Calcium chloride especially is a good example of such timely alterations, since it can easily absorb atmospheric humidity, altering the weight of the salt. Another explanation could be the one-batch approach, meaning that for the in-house sample, the culture medium was used once for 3 weeks. At an industrial level, especially in open raceway ponds, nutrients and inoculum are supplied periodically on the basis of each company's internal standards. An example provided by [PERSON] et al. [33], from an Italian producer, describes daily harvesting, with weekly basis addition of nutrients, based on biomass production and chemical composition, and yearly addition of spirulina inoculum. The same paper does not mention the use of any form of calcium addition other than mentioning that the spirulina is grown in Zarrouk medium, which has a 0.04 g/L concentration. Deciding to not add calcium chloride periodically could be a cost-effective decision if the tap water used for open race ponds can provide an adequate calcium level (according to [PERSON] et al. [34], Italy is in the top 10 European countries with the highest calcium concentration in tap water). Thus, higher concentrations of elements in the in-house sample are to be expected. Additionally, two commercial samples, labeled S3 and S12 in Figure 1, exhibited atypical elemental profiles due to their high Ni, Cd, Rb, Al, and Li contents. These spirulina samples could not be correlated with any of the investigated differentiation criteria (i.e., packaging type, packaging material, type, origin, or growing regime), with the only notable factor linking these samples being the shared producing company. Other anomalies detected through this unsupervised statistical method were represented by samples S65 and S66, which illustrated elevated levels of B (22.29 mg/kg and 22.90 mg/kg, respectively) and Na (41704 mg/kg and 38,849 mg/kg, respectively). From a biological perspective, B is an elemental requirement with a delicate equilibrium with functions in the proper development of cell walls. This delicate equilibrium can be easily thrown off by inadequate B levels in the water supply. Studies on other photosynthetic microorganisms showed that a mechanism of B tolerance can be mediated by increased alkalinity provided by NaHCO3 [35], which is abundant in Spirulina Medium. Nonetheless, sample S18 exhibited higher concentrations of Mn (60.05 mg/kg) and Al (679.26 mg/kg) as opposed to the other commercial spirulina samples. Higher levels of trace metal and metalloid elements can have a twofold reason. Firstly, the particular ability of this group of bacteria to photosynthetic gives them unique requirements such as cobalt, copper, manganese, nickel, and zinc [36]. Mn, for example, is a must in any oxygenic photosynthetic organism, since it is an integral part of the water-splitting site of PSII [37]. Secondly, these elements are abundant in seawater, which can be used to grow spirulina. Not only that, but spirulina grown like this has a higher content of carbohydrates [33].