Comparative Analysis of three Growth Medium for Arthrospira platensis Cultivation based on Lab-Scale Results

Arthrospira platensis is a rich source of essential amino acids, vitamins and it is used as a feedstock for energy sources. The high cost of the growth medium used in its cultivation is a problem for increasing the production viability. The present study aimed to compare the technical viability and the cost elementsin different growth medium for A. platensis cultivation. For that purpose, it was proposed the use of three different growth medium, named as M1, M2 and M3 in a lab scale. The growth of the treatments presented a microbial process with characteristic phases. M1, M2 and M3 maximum concentration (Xmax), productivity (Px) and the maximum specific growth rate (μmax) showed no significant difference among treatments. However, M3 presented the lowest cost element, about 45.75% less than the M1 and 38.92% lower than M2. Therefore, the comparison enabled the result that M3 presented the best performance to be used, thus increasing profitability of this production in a lab-scale analysis.


I. INTRODUCTION
The provision of food security for the growing population, higher oil prices, environmental concerns and increased interest in energy security have encouraged a private and public investment in microalgae biofuels and microalgae food research. It was a challenge to be aware of this potential thus far has been finding a process that can be scaled in a cost competitive manner [1]. Microalgae are simple organisms that are mostly in farm water [2,3]. Throughout its evolution, microalgae have become a very diverse group of photosynthetic organisms, and a particular example is the Arthrospira Platensis (A. platensis) [4].
For food, microalgae A. platensis represents one of the most promising sources of compounds with biological activities possibly used as functional ingredients [5]. Its use is stimulated in order to increase the nutritional value of the food because it is a source of pigments, unsaturated fat, vitamins, sterols,among others [6]. This microorganism has received considerable interest as a potential feedstock for biofuel production, thereby leading to a marketing appeal by decreasing greenhouse gas emissions, as well as using renewable energy sources. Its big advantage is that it easily adapts to the environmental conditions of cultivation, high photosynthetic efficiency and is not a competitor in food production areas [7]. In addition, it can produce large amounts of polysaccharides (sugar) and triglyceride (oil), these are materials for the production of bioethanol and biodiesel [8].
Regarding the national production of microalgae, companies which started producing bio compounds claim about the lack of investment in production technology and competitive pricing, which ended up preventing the advance of large-scale production projects. Consequently, proposals must concern about lower production costs and high efficiency. Furthermore, the new growth medium sources should be encouraged to achieve this purpose. Raoof et al. [9] emphasized that the growth medium is a significant part of production costs. Several lab-scale recent studies demonstrated the potential of alternative growth methods in increasing the production of A. platensis biomass [1,3,5,6,7]. However, regarding its importance, a few number of scientific articles concern about comparing a new growth medium in order to verify their gains. In this context, the objective of this paper is to perform a comparison analysis related to the technical feasibility and the cost element of three different growth medium for

A. platensis
The specie A. platensis, from Oscillatoriaceae family, includes multicellular filamentous cyanobacterium group (blue-green microalgae),formed by cylindrical cells arranged in helical trichomes [15]. Humans learned early how to use the A. platensis as a power source, by noting the migration of birds being safely fed and also encouraging them to use it [16]. Kebede [17] reported that, in Ethiopia, farmers and shepherds living close to the alkaline lakes made the cattle feed mixed with A. platensis water monthly, and they believed it had therapeutic effect and was a supplement in the daily diet. They are able to adapt in certain environments where other microorganisms would not survive, such as those in media with high salt concentration. Through photosynthesis, it converts nutrients into cellular material and produces oxygen. Necessary nutrients are water and a source of carbon, nitrogen, phosphorus, potassium, iron and other minerals [15]. Microalgae are also vectors for the production of bioactive agents that can be applied to medical field, compounds with specific biochemical characteristics, and compounds which are energy sources such as bio-diesel, bio-methane, and hydrogen biobutanol, biomethanol [18,19]. A. platensis is legally permitted as a food supplement in Europe, Japan and in the United States by the Food and Drug Administration (FDA), with no toxic effects on the human body [20]. In Brazil, ANVISA (National Health Surveillance Agency) allowed its sale provided that the final product, which microalgae have been added, is properly registered [21].

Growth Medium
The current interest in expansion of industrial use of microorganisms is leading to a greater attention to the growth medium used. As a matter of profitability and environmental protection, it is essential to ensure the productivity optimization with the lowest cost [22]. The growth of A. platensis and the composition of the biomass produced depend on many factors. The most importants are temperature, light intensity and specially the amount of available nutrients. Therefore, the costs elements are considered the second major influence that impacts on its production [23]. A. platensis growth requires carbon because their cells contain approximately 50% (v/v) of this element. Carbon costs represent a higher impact, for the reason that this element being the most important chemical constitution. For an autotrophic growth (recommended to open largescale cultivation), carbon can be replaced by CO2, carbonate or bicarbonate. If bicarbonate is used, it will represent about 60% of the costs of nutrients [24]. Scientific literature lacks information while proposing alternative growth medium components and their impacts in the synthesis of compounds [25].

III. MATERIALS AND METHODS Microorganism:
It was used the cyanobacterium A. platensis, classified as Arthrospira platensis (Nordstead) by Gomont [26], obtained from Federal University of Santa Catarina, Brazil.

Culture medium and cultivations:
The inoculum was prepared according to the criteria established in previous studies: 8-day process, temperature ± 30 ° C using shaker Marq Labor CFW 08, fitted with artificial light. Initial concentration of inoculum was 50 mg.L -1 [27].

International Journal of Advanced Engineering Research and Science (IJAERS)
[ The experimental design was a completely randomized design (C.R.D.), using a factorial 3x4, with three proposed medium and 4 replicates. Three culture media were proposed and analyzed: a standard medium [29] -Medium 1 (M1); a standard modified medium [30] with no trace elements -Medium 2 (M2); and finally, a medium proposed with alternative commercial substances replacing natural elements -Medium 3 (M3). All three growth mediums elements are presented in Table 1.

Determinations and analyzes:
The parameters monitored were growth with biomass maximum concentration (Xmax), productivity (Px), maximum specific growth rate (μmax) and the cost element. The comparison between these three mediums, with the same day sampling, was conducted by using Tukey test, also considering a significance level of 5% (p <0.05) [28].

Growth and biomass maximum concentration (Xmax):
Biomass concentration was determined daily by turbidimetry at 560 nm [30] in 8-day process.

Productivity (Px):
[  Thirumala [33] also studied A. platensis biomass growth by using different growth medium from distinct combinations. As a result obtained from growth medium, slightly different from this paper findings, it established that it had a maximum concentration on the 9 th and 12 th day of incubation. The experimental results and statistical analysis maximum concentration (Xmax), productivity (Px) and specific maximum growth rates (μmax) are shown in table 2. Also cultivating A. platensis in a low-cost medium, Madkour et al. [23] affirmed maximum concentration values (Xmax) starting from 591 ± 0.018 mg. L -1 for different concentrations of nitrate ammonia. Finally, Pandey et al. [35] in his research cultivated A. platensis in Zarrouk medium confirmed maximum concentration values (Xmax) from 390 ± 0.020 mg. L -1 .

Productivity (Px)
In Table 2 The results indicated that there is no effect of treatments on productivity because, although numerically the result of M2 was higher, statistical Tukey (p <0.05) test analysis showed that the three treatments were equal. Jitendra et al. [37] presented similar results in A. platensis cultivation. For productivity results, it was obtained values between 60% and 90% mg.L -1 .d -1 . Madkour et al. [24] also found results between 16 ± 0.0005 mg.L -1 .d -1 and 52 ± 0.0005 mg.L -1 .d -1 Specific growth rate (μmax) According to  [24], showing that this specific maximum growth rate found in this study indicates good results. which are similar to those found in this work.
The cost element for 1 kg of A. platensis biomass, at labscale, in M1 was US$ 1212.69 and the biggest costs identified, based on the percentage over the total, were NaHCO3 (45.07%) followed by Na2CO3 (27.5%) and Na2SO4 (10.91%). For M2 the cost element was US$ 1037.36 and the biggest costs were NaHCO3 (45.09%) followed by Na2CO3 (27.51%) and Na2SO4 (10.92%). For M3 the cost was US$ 633.55 and the biggest costs were Na2CO3 (47.02%), followed by Na2SO4 (18.66%) and Fe-EDTA solution (12.11%). M1 showed the highest cost (US$ 1212.69), it is probably because it is needed a higher growth medium amount to reach 1Kg of A. platensis biomass. The biggest costs were almost the same as M2. M2 cost (US$ 1037.36) represents a 14.5% lower cost than M1 and it is still approximately 38.92% higher than in M3. The element that most charges the growth medium cost, NaHCO3 (45.09%), was replaced by commercial sodium bicarbonate in M3. M3 cost (US$ 633.55) was the lowest among the three growth medium analyzed. Some of the higher costs in the other growth medium were replaced in M3. It represents about 47.75% lower cost than M1 and 38.92% lower than M2. Several authors have also proposed the use of alternative growth medium for this cyanobacterium growth process. For the purposes of biofuels production, commercially viable biomass production today is only 5 Kt/ year with a production cost of $ 25.00 / ton [14]. However, to occupy only 5% of biofuel demand for a country as the United States, it would be required a production of more than 66.000 Kt/year of biomass rich in oil costing less than US$ 400/t [40]. Analyzing the European market to get more acquainted with the whole transport network of microalgae biodiesel, a 9.25 million ha crop would be required resulting in an output of 40.000 liters/ ha.day [41]. Thus, this number shows that according to the results in this paper, productivity should be improved in order to become viable regarding profitability. Norsker et al. [42] estimated the cost of producing algal biomass on a commercial scale. As a conclusion, the important factors resulted in the irradiation conditions, mixing, efficient photosynthetic system, the medium and the cost of carbon dioxide, in agreement with this study also demonstrating that carbon source has one of the higher costs. Acien et al.
[43] conducted a cost analysis for the actual production of the microalgae Scenedesmus almeriensis and Madkour et al. [24] studied a low cost medium of cultivation for the large-scale production of A. platensis, both resulted in the economic analysis showing the production itself and depreciation as the main offenders of the cost. Also concluded that the simplification of the technology used and the scaling tests could increase productivity and reduce production costs.
In lab-scale process, the costs will probably be higher than the commercial value of the product, mainly due to the productivity scale and the small amount of purchased resources [24,40].
V. CONCLUSIONS As stated in experimental results, this research conducted efficient conversion. It had specifically grown from 0,50 mg.L -1 until for M1 373.29 ± 100.62 mg. L -1 , for M2 436.17 ± 107.48 mg. L -1 and for M3 417.88 ± 113.72. M1 biomass productivity had results statistically identical to the other culture medium (M1 and M2) which did not contain macroelements confirming such a no need of these elements in order to increase A. platensis biomass. Productivity (Px) and specific rate (μmax) also showed good results regarding the ones presenting their similarity among other carried out researches. Even though M3 cost element for 1 kg of A. platensis production was the lowest between them, it maximum concentration, productivity and specific growth rate had no statistical difference from the others. Therefore, the use of M3 is strongly recommended in this comparative study.