Nutrient feeding during a fed-batch process can be done utilizing either constant or variable mass flow rate. Additionally, the addition regime can be either intermittent or continuous [14]. Pulse feeding is easier and less expensive because of the absence of pumping costs. On the other hand, it could lead to lower cell growth, productivity and nitrogen-to-cell conversion factor. During the continuous addition, a feed pump is always used for the continuous addition of substrate during cell growth. With intermittent addition, the substrate is added by pulses and the time between two pulses is another variable to be studied.

Danesi et al. [33] have evaluated the S. platensis growth using urea as a nitrogen source in a fed-batch process, and they tested different protocols for urea addition, namely:

1. Intermittent—exponentially increasing the amount added, every 24 h

2. Intermittent—exponentially increasing the amount added, every 48 h

3. Continuous—exponentially increasing the mass flow rate

4. Continuous—constant mass fl ow rate, using a controlled fl ow peristaltic pump

The addition of urea at intervals of 48 h led to the lowest maximum cell concentra­tion, as a consequence of the lack of availability of the nitrogen to the microorganism between feeding times due to the lost of the nitrogen source in the ammonia form.

The best results, in terms of cell growth, show that the continuous form (protocol (iii)) is appropriate, bringing about better use of the nitrogen source and increasing biomass growth. It was possible to achieve a gain of 37% in biomass and consequently larger total amounts of chlorophyll at a lower cost when compared with cultures grown with KNO3. The fact that the water is being continually replaced (and not just once a day) in such experiment is also favorable, because it avoids the build up of high salin­ity conditions in the culture medium, that can hinder cellular growth. Nevertheless, comparing the results obtained at three temperatures studied (27, 30, and 33°C), the average maximum cell concentration was only 5.7% higher when urea fed continu­ously instead of intermittent addition every 24 h.

On the other hand, Sanchez-Luna et al. [86] compared the influence of the proto­col of urea addition (pulse or continuous), under variable conditions of temperature and total feeding time, in order to select the best feeding regime for S. platensis fed — batch cultivation with constant flow rate. Urea was added to the culture by a fed-batch process at constant mass flow rate, following two different protocols: (a) intermittent addition every 24 h and (b) continuous feeding. Fed-batch cultiva­tion of this cyanobacterium with constant urea feeding rate by pulse and continuous additions exhibited statistically coincident results. Because of the large solubility of this nitrogen source in water, it could be intermittently added avoiding the use of pumping equipments. Therefore, the addition of urea by daily pulse feeding at con­stant flow rates could be a useful protocol to be used in large-scale aquaculture facilities, implying lower costs for A. platensis biomass production.

An exponentially increasing feeding rate to supply urea is suitable for those cul­tures in which microbial growth can be inhibited by this nutrient or its derivatives. In fact, the highest nutrient supply takes place just at the end of the run, when bio­mass concentration achieves its maximum value. In this way, despite the inhibitory effect of ammonia coming from urea hydrolysis under alkaline conditions, the use of such a nitrogen source allowed S. platensis to reach a concentration comparable with that obtained with potassium nitrate in a batch run [33]. On the other hand, Sanchez-Luna et al. [87] observed that fed-batch autotrophic A. platensis cultiva­tions, carried out at 6.0 klux (72 pmol photons m-2 s-1) in 5.0-L open tanks at vari­able pH, temperature, and urea molar flow rate (K) resulted in a cell growth that followed a linear trend likely due to light limitation [13], thus justifying the use of constant mass flow rates. The use of urea as a nitrogen source prevented the inhibi­tory effect observed with ammonium chloride, and the growth curves did not exhibit any lag phase. The yield of biomass based on nitrogen progressively decreased with increasing urea molar flow rate. The statistical model pointed out pH = 9.5, T= 29°C, and K = 0.551 mM d-1 as the best conditions optimizing cell concentration and pro­ductivity, which do not differ much from the experimental observations.

Ammonium sulfate and urea have been tested as nitrogen sources for S. platensis cultivations, according to different batch and fed-batch protocols. The results showed that the use of urea in fed-batch culture led to better growth kinetics. Adoption of an appropriate slowly increasing urea feeding rate prevented the accumulation of ammonia in the medium as well as its well-known inhibition of biomass growth [97]. Preliminary batch cultivations were carried out to determine the actual nitrogen requirements of biomass as well as to establish the inhibition threshold of ammonia. Subsequent fed-batch runs, performed according to different feeding protocols, allowed the selection of the best conditions for urea supply and demonstrated that growth kinetics may be comparable and even better than that obtained with the tra­ditional nitrate-based culture media. Ammonia accumulation that usually inhibits biomass growth was in fact prevented following an appropriate pulse-feeding pat­tern. Although the highest productivity during the start-up was obtained with linearly increasing feeding rate, the use of a more slowly increasing pattern, aimed at mini­mizing ammonia accumulation, was shown to be the most suited for long-term culti­vation. Therefore, the use of urea in a fed-batch process should be recognized as a possible way to decrease the costs of a large-scale plant for the production of this cyanobacterium.

In a recent study, Ferreira et al. [41] cultivating A. platensis in a tubular photo­bioreactor, which is useful in preventing ammonia loss by evaporation, observed that parabolic protocol for ammonium sulfate addition appeared to be the best one for biomass production comparable to those obtained using sodium nitrate as the conventional nitrogen source. Additionally, due to high cell growth observed in the cultivations, the demand for nitrogen was extremely high, reaching values around 12 mM per day. Taking into account that the inhibitory levels of ammonia is around 6 mM [13], in this case, considering the whole period of cultivation, the addition of 12 mM of ammonium sulfate per day in a single daily addition would probably lead to cell death. Thus, the daily addition was divided into eight times, which allowed a maximum cell concentration of approximately 14 g L-1 . In this work, a parabolic protocol for ammonium sulfate addition, in which the cells with 7% nitrogen was considered, led to biomass protein contents (35.6 ± 1.7%), comparable to that obtained in standard runs (35.5 ± 0.9%), thus demonstrating that the use of ammo­nium salts does not modify the cell composition.

Feeding time is a variable of great importance in the fed-batch process, where it is responsible for nutrient availability for the microorganisms. Concerning urea and ammonium salts for A. platensis cultivation, it is also very important to avoid any toxic effect of ammonia. A strict control of the feeding time in fed-batch culture in open ponds, at the same time, prevented the toxic effect exerted by excess ammonia or its loss by off-gassing [13]. As ammonia is the predominant nitrogenous species in the medium when ammonium chloride is used as a nitrogen source, an appropri­ate feeding time should be selected to maintain optimum ammonia levels through­out the whole cultivation. In fact, longer times limited growth because of the shortage of the nitrogen source, while shorter times affected the growth due to the occurrence of a displacement between nitrogen source supply and utilization [7] .

Bezerra et al. [7] reported that a short feeding time results in the partial loss of ammonia when using NH4Cl as nitrogen source and, consequently, growth limita­tion can occur. As evidenced by the increase in maximum cell concentration (Xm) from 1,111 to 1,633 mg L-1, a longer feeding time favored A. platensis growth. In addition, the run with the longest time to achieve the stationary phase exhibited a lower value of Xm (1,561 mg L-1), likely due to growth limitation by nitrogen begin­ning with the feeding step. This is in agreement with Carvalho et al. [13], who reported the existence of an optimum feeding time for cell production, below and above which biomass growth was affected by nitrogen source accumulation and consequent loss in the form of ammonia (feeding time of 12 days) or limitation (feeding time of 20 days) in the medium. Both situations led to decreases in Xm and suggested some discrepancy between biological demand and availability of the nitrogen source.

The fed-batch cultivation can be carried out as a repeated fed-batch process, in which once the cultivation reaches a certain stage after which it is not effective any­more, a portion of the culture medium is removed from the bioreactor and replaced by fresh nutrient medium. It allows keeping part of the medium in the reactor at the end of cultivation, reusing the exponentially growing cells for subsequent runs, cheaply ensuring high starting cell levels, and avoiding long stopping of the process. Moreover, it is expected to increase cell productivity, ensuring high cell growth rate. These features could be usefully exploited to evaluate the possibility of using this process with urea as a nitrogen source in large-scale cultivations. This process is characterized by removing a constant fraction of volume of culture at fixed time intervals and can be maintained indefinitely, where the volume is replenished to its maximum value by adding medium culture with appropriate flow rate [76, 115]. This type of process was employed by Matsudo et al. [62] to evaluate if urea could be successfully employed when using repeated fed-batch cultivation of A. platensis in open ponds. This study showed that the repeated fed-batch process using urea as a nitrogen source was suited for long-term A. platensis cultivation, during which the maximum cell concentration, the nitrogen-to-cell conversion factor and the kinetic parameters remained stable when using an appropriate ratio of renewed to total volume and experimental protocols of urea addition. Moreover, it should be noted that the biomass protein content was not influenced by the experimental conditions. The maintenance of maximum cell concentration during three consecutive cycles and the absence of contamination probably take place due to the well-known ability of this microorganism to grow well in saline and alkaline inorganic environments.

In conclusion, the fed-batch process is a useful tool for supplying carbon and nitrogen in cultivations of A. platensis under different photobioreactor configurations. For each specific carbon and nitrogen source added, it is necessary to evaluate the experimental conditions that lead to both desired cell growth and composition. Besides classical factors that affect photosynthetic cell growth, such as carbon source, nitrogen source, other nutrients, pH, temperature, light intensity and salin­ity, the typical parameters of the fed-batch process, such as feeding time, addition protocol and flow rate, should be evaluated. The comments presented in this chapter demonstrate that the use of inexpensive nitrogen sources, such as urea, ammonium salts and nitrogen-rich wastewaters can be used for A. platensis cultivation, with results that can be comparable to those with classical nitrate sources. The results also show that closed photobioreactor is useful for preventing ammonia loss during A. platensis cultivation. The best form of supplying organic carbon needs to be evaluated, where different strategies are necessary for each organic carbon source employed. In cases of using organic carbon, the process needs to be carried out under aseptic conditions. Considering inorganic carbon sources, CO2 has an impor­tant role in the growth of such cyanobacteria, where it needs to be added to the medium to maintain the carbon source level as well as pH. Finally, the fed-batch process was demonstrated to be useful for the production of A. platensis using CO2 from industrial plants, particularly from industrial alcoholic fermentations.