Fed-batch fermentation is one of the most employed cultivation regimes when process microorganisms present catabolic repression, i. e., when high substrate concentrations inhibit specific metabolic processes like those related to cell growth rate. For this reason, the microorganisms grow faster at low substrate concentrations. In fact, the cultivation of S. cerevisiae to produce baker’s yeasts is accomplished by this process. Nevertheless, the application of fed-batch culti­vation to ethanolic fermentation has also offered important results by maintain­ing low substrate levels as ethanol is accumulated in the medium. This type of cultivation regime along with the cell recycling is the most utilized technology in Brazil for bioethanol production due to the possibility of achieving higher volu­metric productivities (Sanchez and Cardona, 2008). To implement such a process, conventional batch fermentation is performed though using a less-concentrated medium. Once the sugars have been consumed, the bioreactor is fed with por­tions of fresh medium or by adding a small amount of medium permanently until the end of fermentation. This continuous feeding of the medium can be done in a linear way (with a constant feeding rate) or according to a more complex func­tion defining the rate with which the fresh medium is added to the fermenter, e. g., by an exponential feeding rate. Control of flow rate of medium feeding is quite advantageous because the inhibitory effect caused by high concentrations of substrate or product in fermentation broth is neutralized. It was observed that the addition of sucrose in a linear or exponentially decreasing way leads to 10 to 14% increase in ethanol productivity (Echegaray et al., 2000). It has been reported that immobilized yeast cells have better performance in fed-batch cultures regard­ing ethanol production (Roukas 1996). Aeration is an important factor during this fermentation regime as well. For fed-batch cultures, Alfenore et al. (2004)

Some Fermentation Processes for Ethanol Production from Sugarcane Molasses Using Saccharomyces cerevisiae

Culture broth

Stillage recycle

FIGURE 7.2 Reutilization of yeast cells and stillage during batch fermentation: (1) fer­menter, (2) separation of cells by centrifugation, (3) distillation column.

have shown that higher ethanol concentrations (147 g/L) could be obtained during cultivation without oxygen limitation (0.2 volumes of air per volume of broth in 1 min or vvm) during only 45 h in comparison to microaerobic conditions.

In the case of multiple or repeated batch fermentation, the use of flocculating strains of yeasts is of great importance. In this type of culture, after starting a conventional batch, the yeasts are decanted in the same vessel where they were cultivated and then the clarified culture broth located in the upper zone of the fermenter is removed. Then, an equal amount of fresh culture medium is added for the following batch. In this way, high cell concentrations are reached and inhi­bition effect by ethanol is reduced without the need of adding flocculation aids or using separation or recirculation devices. These repeated batches can be accom­plished until the moment when the activity and viability of culture are lost as a consequence of a high exposition to fermentation environment. When this occurs, the system should be reinoculated (Sanchez and Cardona, 2008). Some factors, such as agitation, allow flock size to be optimum for reaching higher ethanol concentrations. Even small levels of dissolved oxygen in the medium can facili­tate the neutralization of inhibition effect by ethanol, as suggested by Hawgood et al. (1985). Maia and Nelson (1993) point out that the addition of unsaturated fatty acids can reduce or eliminate the need for microaeration because the oxygen requirement is related to the synthesis of these acids. These authors evaluated the supplementation of sucrose-based medium with fatty acids sources (soy and corn flours) in repeated-batch cultures obtaining best results with corn flour and justi­fying the traditional and empiric use of this component during the fermentation step by the small Brazilian distillers. Some examples of fed-batch and repeated batch fermentations for bioethanol production from sugarcane molasses can be observed in Table 7.1.

Q

S

CD-413

V, X, S, P

(a)

7.1.2.1 Continuous Fermentation

Continuous fermentation consists of the cultivation of cells in a bioreactor to which the fresh medium is permanently added and from which an effluent stream of culture broth is permanently removed, as shown in Figure 7.3. The microor­ganisms are reproduced within the bioreactor at a grow rate that offsets the cells withdrawal with the effluent achieving the corresponding steady state. To ensure the system homogeneity and reduce concentration gradients in culture broth, continuous stirred-tank reactors (CSTR) are employed. In this way, a constant production of fermented wort can be obtained without the need of stopping the bioreactor operation in order to perform the periodic procedures typical of batch processes, such as filling-up and unloading. This allows a remarkable increase in volumetric productivity compared to discontinuous or semicontinuous processes (see Table 7.1).

The design and development of continuous fermentation systems have allowed the implementation of more effective processes from the viewpoint of the produc­tion costs. Continuous processes have a series of advantages in comparison to conventional batch processes mainly due to reduced construction costs of the bio­reactors, lower requirements of maintenance and operation, better control of the process, and higher productivities (Sanchez and Cardona, 2008). For those very reasons, 30% of ethanol production facilities in Brazil utilize continuous fermen­tation processes (Monte Alegre et al., 2003). Most of these advantages are due to the high cell concentration found in this type of bioreactor. Such high densities can be reached by immobilization techniques, recovery and recycling of biomass, or control of cell growth. The major drawback is that cultivation of yeasts during a long time under anaerobic conditions diminishes their ability to produce etha­nol. In addition, at high dilution rates (a magnitude proportional to the feed or effluent flow rate) ensuring elevated productivities, the substrate is not completely
consumed and yields are reduced. In general, in commercial processes for etha­nol production, although the productivity is important, it is more relevant for the substrate conversion considering that the main part of the production costs cor­respond to feedstocks (Gil et al., 1991). On the other hand, aeration also plays an important role during continuous ethanolic cultivations. Cell concentration, cell yield from glucose, and yeast viability may be enhanced by increasing air supply, whereas ethanol concentration decreases under both microaerobic and aerobic conditions. Cell growth inhibition by ethanol is reduced at microaerobic condi­tions compared to fully anaerobic cultivation, and specific ethanol productivity is stimulated with the increase of oxygen percentage in the feed stream (Alfenore et al., 2004; Sanchez and Cardona, 2008).

An important feature of continuous processes is related to the diminution of the product inhibition effect. Through cascade of continuous reactors, ethanol obtained in the first reactors is easily transported to the next ones reducing in this way its inhibitory effect (see Figure 7.3b). On the other hand, other configurations employing one fermenter can contribute to the reduction of product inhibition. In particular, the Swedish company Alfa Laval implemented a continuous process for producing 150,000 L EtOH/d in Brazil by Biostill technology (Kosaric and Velikonja, 1995). This process is based on yeast cultivation carried out in a fer­mentation vessel from which a liquid stream is continuously withdrawn to be sent to a centrifuge. From the centrifuge, one concentrated yeast stream is continu­ously removed and recycled back to the fermenter. The other yeast-free stream is directed to a distillation tower. From this tower, concentrated solution of ethanol and stillage are removed. A portion of the stillage is also recycled back to the fermenter in order to maintain the mass balance necessary for conserving steady — state conditions according to the original configuration patented by Alfa Laval (Ehnstroem, 1984), which is shown in Figure 7.4. In this process, there is signifi­cant savings in process water, which reduces stillage volumes and low residence times (3 to 6 h) in the fermenter can be achieved. A modification to this process using no recirculation stream from a distillation column and reaching yields of 96% of theoretical has been patented as well (Da Silva and Vaz, 1989).

Other alternatives of continuous fermentation have been proposed, but many of them still have not reached the commercial level. Some of them require the use of highly flocculating yeast strains similar to the tower and fluidized-bed reactors. These types of reactors allow much higher cell concentrations (70 to 100 g/L) and ethanol productivities, and have a long-term stability due to the self-replenishing of fresh yeasts. Moreover, these fermenters do not require stir­ring devices or centrifugation (Gong et al., 1999). S. uvarum is one of the most promising yeasts to be employed in these configurations thanks to its flocculating properties. All of these efforts have been directed to the increase of productivity and yield, as can be seen in Table 7.1. Another approach for increasing process productivity is the continuous ethanol removal from culture broth during fer­mentation by means of a vacuum or membranes. These configurations enhance efficiency of the process remarkably well, but imply an increase in capital costs. The use of vacuum flash coupled with continuous fermenters could eliminate the

need of heat exchangers and increase the productivity (Costa et al., 2001). These types of configurations involving the application of reaction-separation integra­tion are discussed in Chapter 9.