Identification of pathways and environmental conditions affecting the metabolism of glycerol under anaerobic conditions by wild-type E. coli provide opportunities to manipulate the microorganism for enhancement of ethanol yield and productivity. Fermentation of glycerol to either ethanol and H2 or ethanol and formate is one of the most effective ways of exploiting the reduced property of glycerol for bioproducts production.

Ethanol is predominantly produced following glycerol fermentation by E. coli. The product mixture, however, contains succinate, acetate, and formate. Succinate and acetate are com­peting byproducts because some substrates that would have been used for the production of ethanol are diverted toward the production of succinate and acetate (Murarka et al. 2008- , resulting in decreased ethanol yield. Blocking pathways for succinate and acetate synthesis are possible ways to enhance ethanol yield (Figure 6.7

For the ethanol-H2 production pathway, we carried out two mutations in E. coli to disrupt genes that encode fumarate reductase (FRD) and phosphotransacetylase (PTA; Figure 6.7). FRD and PTA are two key enzymes involved in the production of succinate and acetate, respectively. The resultant strain, SY03, produced an almost equimolar amount of ethanol and hydrogen and yield comparable to theoretical maximum of 1 mol of each product per mole of glycerol (Yazdani and Gonzalez 2008). To facilitate coproduction of formate and ethanol, another mutation was introduced in the gene (fdhF) encoding component of FHL in the strain SY03 (Figure 6.7). FHL is responsible for the oxidation of formate into H2 and CO2 . This triple mutant strain, called SY04, produced exclusively ethanol and formate at yields of 92%-96 % of the theoretical maximum.

The strategies used in the generation of these mutants led to a decrease in the growth rates of E. coli mutants (Yazdani and Gonzalez 2008). The overexpression of GldA and dihydroxy — acetone kinase (DHAK), which are responsible for converting glycerol into glycolytic intermediate DHAP, was assessed for improving the growth rate of E. coli mutants. We overexpressed GldA and DHAK separately and in combination with plasmids pZSgldA, expressing GldA, pZSKLM expressing DHAK, and pZSKLMgldA expressing both GldA and DHAK to improve the rate of glycerol fermentation (Yazdani and Gonzalez 2008) . Transformation of these plasmids in E. coli MG1655 led to a 10-20-fold increase in GldA and a 5-6-fold increase in DHAK activities. When the effect of overexpression of GldA and DHAK genes in E. coli MG1655 was tested on glycerol fermentation, individual overexpres­sion of either GldA or DHAK did not have a significant effect on glycerol fermentation. Simultaneous overexpression of GldA and DHAK from plasmid pZSKLMgldA led to a 3.4- fold increase in the amount of glycerol fermented in E. coli MG1655. Overexpression of GldA and DHAK in the host SY04 with mutations in three genes, f rdA, pta, and f dhF, for the coproduction of ethanol-formate led to the production of ethanol and formate at maximum volumetric rates of 3.58 and 3.18 mmol/L/h, respectively.

Further experiments involving various mutants confirmed role of both respiratory and fermentative pathways of glycerol utilization under microaerobic condition (Durnin et al. 2009). Enzymes involved in the respiratory pathway of glycerol metabolism, glycerol kinase (GlpK), and glycerol-3 .phosphate dehydrogenase (GlpD) exhibited higher activities in the initial aerobic phase of glycerol utilization. The transition to microaerobic conditions, char­acterized by undetectable amounts of dissolved oxygen, resulted in a 2.5- fold increase in glycerol-3-phosphate dehydrogenase activity and a 10-fold increase in both GldA and DHAK activities. The GlpK-GlpD pathway predominated during the early phase of fermentation, while the GldA-DHAK pathway predominated during the later stage of cultivation. Under microaerobic conditions, the engineered strains were able to utilize glycerol to produce ethanol and hydrogen, or ethanol and formate in basal media.

Conclusions and Future Outlook

Considering the worldwide surplus of crude glycerol and the need to find new uses for this cheap abundant carbon source, the use of anaerobic fermentation to convert low-value crude glycerol streams generated during the production of biodiesel to value-added products represents a promising step toward achieving an economically viable biodiesel industry. A number of organisms are able to ferment glycerol to different bioproducts with a wide range of applications. The success of these strategies will depend on the use of robust microorganisms that are amenable to industrial applications.