While hydrocarbon-based biofuel production relies on the biosynthetic pathways discussed in the previous section, the source of feedstock plays an important role in the overall produc­tion process. As discussed in the Introduction to this chapter, there are two main feedstocks for biofuel production: lignocellulosic biomass and gaseous CO2 supporting the production of second and third generation biofuels, respectively (Figure 1). Both processes ultimately rely on CO2 and sunlight as the carbon and energy source, but the microbial conversion processes are distinctly different between the two feedstocks. Lignocellulosic biomass deconstruction produces organic carbon, mostly in the form of hexoses and pentoses (C5 and C6 sugars); this feedstock requires heterotrophic microorganisms to convert the organic carbon into biofuel. Alternatively, the fixation of inorganic carbon feedstock (CO2/HCO3-) into biofuel is reliant upon autotrophic microbes. The heterotroph vs. autotroph requirement of the respective feedstocks is an important distinction from both the metabolic engineering and biofuel production perspectives. Only a few model microorganisms are capable of both heterotrophy and autotrophy, resulting in different host candidates for second and third generation biofuel production. The feedstock will also influence the metabolic engineering targets, as hetero- trophs utilize glycolysis and oxidative phosphorylation pathways for carbon consumption and energy production while oxygen-generating autotrophs utilize the Calvin-Benson-Bassham cycle and photosynthesis under light conditions (Figure 4). This section will discuss the host