Through recombinant DNA technology, amylolytic yeast strains have been “con­structed.” This allows for the design of ethanol production processes, excluding the liquefaction and saccharification steps using exogenous enzymes, and the uti­lization of only one bioagent during the transformation, the yeast (consolidated bioprocessing, CBP). The savings obtained during the commercial implementa­tion of such a process could offset by far the lower growth rates and the longer fermentation times. In this way, a single microorganism can directly convert the starch into ethanol (Cardona and Sanchez, 2007). Some examples of these efforts are shown in Table 6.3. Lynd et al. (2002) mention, among the saccharolytic genes that have been introduced into microorganisms as S. cerevisiae and Klebsiella oxytoca, those encoding a-amylase, glucoamylase, amylopullulanase, pectate lyase, and polygalacturonase obtained from bacterial and fungal sources.

Many of the investigated recombinant strains have demonstrated the pro­duction of ethanol from starch, but in some cases, the results are not definitive. Surprisingly, reduced starch hydrolysis and fermentation rates have been observed for yeast strains expressing a set of genes previously considered as appropriate, as is the case of the work of Knox et al. (2004). This example shows the difficul­ties that arise during the research using recombinant microorganisms. Although the methods of genetic transformation are relatively developed, the results can be unexpected. This is a key factor when these microorganisms are evaluated from an industrial point of view. For this reason, deep studies on the effects of genetic modifications on engineered strains are required.

The processes with microorganisms modified by genetic engineering involve the optimization not only of microbial physiology parameters, but also of cell culture parameters (retention and stability of plasmids, nutritional factors, cell growth, and protein synthesis). Therefore, the modeling of these processes and the application of the principles of biochemical engineering can be helpful con­sidering the uncertainties and complexities inherent to these biological systems (Cardona and Sanchez, 2007). An example of this type of modeling is the work of Kobayashi and Nakamura (2004), who corroborated experimentally at laboratory scale the higher productivity of the continuous fermentation process from starch using recombinant yeast cells immobilized in calcium alginate beads in compari­son with the free cell system.

Another approach employed for modeling this process is the so-called flux balance analysis. Qakir et al. (2004) have employed and experimentally validated this methodology in the case of yeasts. They have determined that if the split ratio in the branch point of the glucose-6-phosphate corresponding to the glucolytic

Some Examples of Recombinant Microorganisms with Potential Use for Fuel Ethanol Production

Glucose and xylose containing medium; acid pretreated starch industry effluents	Saccharomyces sp. strain is the product of fusion between S. diastaticus and S. uvarum and is capable of fermenting xylose to ethanol and utilizing it for aerobic growth; batch cultures; 48 h (sugars medium), 8 d (starch effluents) cultivation; EtOH cone. 60 g/L; yield 84% Introduced genes codify xylose assimilation and metabolism; batch culture; 48 h cultivation; EtOH cone. 62 g/L Hexose and pentose — assimilating E. coli acquires the ability for producing ethanol by introducing ethanol pathway genes; productivity 1.4 g/(L. h) from glucose. 0.64 g/(L. h) from xylose; EtOH cone. 41 g/L from xylose, 57 g/L from glucose; patented strain	Ho et al. (1998); Zaldivar et al. (2005)
Glucose and xylose containing medium
Leksawasdi et al. (2001)
Glucose or xylose containing medium
Ingram et al. (1991)
Continued

Some Examples of Recombinant Microorganisms with Potential Use for Fuel Ethanol Production

S. cerevisiae L26126GC Endo/exoglucanase p-glucosidase Bacillus sp. strain DO4 B. circulans Crystalline cellulose Batch SSF; 12 h cultivation; saccharolytic yeast capable of enzyme production; EtOH conc. 20.4 g/L; a considerable amount of commercial enzymes was reduced; potential strain for DMC of cellulose Cho and Yoo (1999) Clostridium cellulolyticum Pyruvate decarboxylase Alcohol dehydrogenase Z. mobilis Cellulose Native cellulolytic strategy Guedon et al. (2002) S. cerevisiae Cellobiohydrolase Thermoascus aurantiacus Crystalline cellulose Recombinant cellulytic strategy Hong et al. (2003)

metabolic pathway is changed by genetic manipulations, ethanol yield from starch can be considerably affected. This shows that the improvement in ethanol produc­tion goes with a rational design of metabolic pathways. In the future, this infor­mation could be crucial when different bioprocesses are designed, although it is necessary to analyze the costs and the complexity for acquiring this information in comparison with other “more traditional” procedures of design and control.

Similarly, important parameters, such as the stability of the plasmids used for introducing the desired traits to yeast cells, depend on the definition of the best environmental conditions during the cultivation of recombinant microorganisms. For example, Mete Altinta§ et al. (2002) showed that culture media containing specific salts and yeast extract drastically enhance plasmid stability during fed — batch cultures of yeasts using starch as feedstock.