1.1.1 Carbon

Since S. platensis is composed of approximately 50% carbon [30], the use of carbon dioxide in the cyanobacterium cultivation may contribute significantly to reducing the cost of production and, at the same time, to reducing the emission of this green­house gas. Photosynthetic microorganisms can efficiently assimilate carbon dioxide from different sources, including the atmosphere, industrial exhaust gases, and soluble carbonate salts [112] . In fact, carbon dioxide taken up by photosynthetic

microorganisms is among the most productive biological methods of treating industrial waste emissions, and the yield of biomass per acre is three — to fivefold greater than in typical crops [2, 55]. The most important trace gases, which intensify the greenhouse effect, are carbon dioxide (CO2) , methane (CH4) , nitrous oxide (N2O), and ozone (O3). Among all these emissions, carbon dioxide makes the great­est contribution to global warming. For this reason, most of the measures for miti­gating climate change target the reduction of carbon dioxide emissions to the atmosphere [77]. In this sense, there are several studies that point out the use of carbon dioxide in the cultivation of Arthrospira (Spirulina) platensis [8,15,41,63, 79, 96, 114].

Traditionally, Arthrospira spp. grow autotrophically in open tanks (Fig. 2) in the presence of high sodium carbonate and bicarbonate levels as carbon sources because they are relatively inexpensive and provide a high pH in the culture medium. Bicarbonate, the main carbon source, is actively transported into the cell, where the enzyme carbonic anhydrase, present intracellularly and/or in the periplasmic mem­brane, promotes the release of carbon dioxide. This is incorporated in the Calvin cycle to produce organic molecules such as carbohydrates, proteins, and lipids [52] .

At low extracellular concentration of bicarbonate, cyanobacteria have the ability to accumulate bicarbonate intracellularly [30] and use carbon dioxide as a carbon source for its metabolism. In medium containing only carbonate, there is no increase in cell concentration and the pH remains almost unchanged, emphasizing the impor­tance of bicarbonate in cyanobacterial metabolism [8].

Taking into account that cultivations in tubular photobioreactors (Fig. 3) lead to a high cell concentration, it is essential that CO2 is added during cell growth to sus­tain it [63], thus justifying the fed-batch process for this carbon source. Soletto et al. [96] evaluated the performance of a bench-scale helical photobioreactor in fed-batch

cultures of S. platensis under different conditions of light intensity and CO2 feeding rate. The optimum feeding rate of carbon dioxide for the microalgal growth was cor­related with the light intensity to which it was exposed. In general, the behavior of S. platensis was more influenced by the CO2 feeding pattern at low PPFD. Irrespective of the light intensity studied, at a CO2 feeding rate of 1.03 g L-1 d-1, the excessive amount of CO2 caused an inhibition of biomass growth due to excess carbon levels and likely due to pH reduction.

Matsudo et al. [63] studied the use of CO2 released from alcoholic fermentation, without any prior treatment, for carbon source replacement and pH control, in the continuous cultivation of A. platensis, using urea as nitrogen source, in a bench scale tubular photobioreactor. Irrespective of the carbon source used in the cultiva­tion of this cyanobacterium (pure CO2 or from alcoholic fermentation), it was obtained similar behavior in cell growth. In both cases, the maximum cell concen­tration in steady state condition (X) occurred at dilution rate (D) of 0.2 d-1, being
obtained Xs = 2,446 ± 75 and 2,261 ± 71 mg L-1 in cultivations carried out using pure CO2 and CO2 from alcoholic fermentation, respectively. It was not observed any difference in the protein content of the dry biomass when these two kinds of carbon sources were used. The higher the D, the higher the protein contents of the dry bio­mass, which were as high as about 50% when the cultivations were carried with D=0.8 d-1. These results indicated that the possibility of using such cost-free carbon source and a cheap nitrogen source like urea may contribute to reduce the cost of culture medium. Besides, this work suggests that the biofixation of CO2 released from alcoholic fermentation of sugar raw materials, which represents about 33% of the whole CO2 involved in the use of ethanol as fuel, may help to mitigate the green­house effect.

Carvalho et al. [15] have proposed methods for the recovery and purification of CO2 from alcoholic fermentation and/or burning of lignocellulosic materials and feed it into the cultivation of photosynthetic microorganisms. Application of the fed-batch process would be particularly important for the fixation of CO2 from industrial plants. Such statement can be evidenced by the fact that the ethanol pro­duction in Brazil was as high as 27.5 billion liters in 2008/2009 [107], and it can be estimated that the release of CO2 associated only with this fermentation process was about 20.8 billion kg. Moreover, considering that the all correspondent sugar cane bagasse was burned, an additional CO2 production of about 83 billion kg would occur [108].

Regardless of the importance of the fed-batch process when using CO2 as a car­bon source, organic carbon sources are the most cited cases in which the fed-batch process is employed in the cultivation of photosynthetic microorganisms. Despite the increase in the risk of contamination, which requires running the process in closed reactors under aseptic conditions, the use of an organic carbon source can provide readily usable energy and make it possible to reach a high final biomass concentration. It can be done under dark (heterotrophic) or light (mixotrophic) con­ditions. Taking into account that the organic carbon source can lead to inhibition of the growth beyond a limit concentration [103] or even repress the formation of a desired metabolite, several fed-batch processes have been used to improve cell or metabolite production by different microalgae and cyanobacteria [20, 103] or even to remove organic pollutants from wastewater [58].

Marquez et al. [61] showed that S. platensis can grow heterotrophically in a medium containing glucose in aerobic and dark conditions but also mixotrophically under illuminated conditions. Chojnacka and Noworyta [20] also observed Spirulina sp. growth under heterotrophic conditions using glucose as carbon and energy sources. Chen et al. [19] showed that acetate may be used as a carbon source, in mixotrophic S. platensis cultivations, for the production of several photosynthetic pigments.

S. platensis can also use glycerol as the sole carbon source. Nevertheless, when this microorganism takes up glycerol for the first time, it forms aggregates of cells and a lag phase takes place. After the lag phase, however, it was possible to achieve a cell concentration higher than with cultivation using bicarbonate as the sole car­bon source [68] .

Three organic carbon sources differing in molecular complexity (glucose, acetate, and propionate) were tested by Lodi et al. [58] in a fed-batch mixotrophic process with minimum volume variation. To avoid carbon source accumulation in the medium, acetate and proprionate were added by pulse feeding equimolar amounts about 12 h after their complete depletion, namely acetate every 3.4 days and propi­onate every 4.0 days, whereas glucose was added once a day. The results of cultiva­tions performed under continuous illumination show that glucose was metabolized for algal growth faster than acetate and proprionate. Besides, the values of nitrate and phosphate removals are near those observed with traditional biological treat­ment plants [29]. This suggests that mixotrophic metabolism of S. platensis could be exploited in a tertiary treatment system for simultaneous removals of mixtures of organic pollutants, nitrate, and phosphate [58] .

The chlorophyll content in the biomass did not appreciably vary during the course of cultivations (2-5%), thereby indicating that most of the dry weight increase was the result of microbial growth. Besides, it was shown to be almost independent on the type of organic carbon source and about 17% lower than that determined by Danesi et al. [33] under autotrophic conditions, thus confirming the underutilization of the photosynthetic apparatus when cyanobacteria are grown with an organic car­bon source [58] .

Chen and Zhang [18] reported that S. platensis cell concentration in a fed-batch culture with intermittent glucose feeding with a peristaltic pump was 4.25-fold higher than in the mixotrophic batch culture and 5.1-fold higher than in the photoau­totrophic batch culture. The biomass output rate of the fed-batch culture was 3.1-fold higher than in the mixotrophic batch culture and 3.8-fold that of the photoautotrophic culture. Additionally, these authors demonstrated that a mixotrophic fed-batch cul­ture of S. platensis with glucose is suitable for the production of high-value products, particularly the light-induced pigments such as phycocyanin.