The selection of organic or inorganic carbon feedstock for biofuel production has downstream ramifications on host selection, product yields, and process requirements. Clearly, the feedstock choice will determine whether a heterotrophic or autotrophic host is required, and in turn, this will influence the metabolic engineering strategy. In general, heterotrophic hosts have generated higher fuel titers than autotrophic hosts, with more than 10-fold higher concentrations of FFAs, FAEEs, fatty alcohols, and alkanes/alkenes (Table 1). This does not imply that heterotrophic production is more advantageous than autotrophic production, for the entire production process must be considered (Figure 1). The sugars from lignocellulosic biomass deconstruction (heterotrophic feedstock) have a higher energy content compared to inorganic carbon (autotrophic feedstock). The overall balances for obtaining one molecule of GAP from heterotrophic and autotrophic metabolisms provide evidence for this:

Heterotrophic: V Glc + ATP ® GAP + ADP

Autotrophic: 3 CO2 + 9 ATP + 6 NADPH + 5 H2O ® GAP + 9 ADP + 6 NADP+ + 8 Pi

While autotrophic GAP generation requires a significant investment of energy (9 ATP) and reducing equivalents (6 NADPH), heterotrophic GAP production only requires one energy equivalent. However, if a life cycle perspective is considered, the carbon from lignocellulosic feedstocks is ultimately derived from photosynthesis, requiring the same energy and reducing equivalent input as autotrophic microorganisms. Overlooking this fact will bias a direct comparison between heterotrophic and autotrophic fuel production.

One major difference between heterotrophic and autotrophic fuel production is the design considerations for the bioreactor. Heterotrophic microbes, such as E. coli and S. cerevisiae, are traditional industrial microorganisms with well-established, large-scale cultivation practices and bioreactors. On the other hand, autotrophic hosts like algae and cyanobacteria require light as the energy source to drive photosynthesis and inorganic carbon fixation. This can have a dramatic effect on bioreactor design. Transparent materials can be used with traditional bioreactor designs to allow for light penetration. Light availability, however, will ultimately limit the cell densities of photosynthetic microalgae, and the surface area of light exposure with traditional bioreactor designs is not optimal. Some have proposed to use fiber-optics within the liquid culture to improve light availability [114], but a costly solution such as this is not feasible for a low-value, commodity product like fuel. A wide-range of photobioreactor (PBR) designs have been proposed [115], yet generally, PBRs are characterized by the use of transparent materials, high surface area to volume ratios, and a relatively short pathlength for light. Other PBR design factors include a mechanism for air/CO2 delivery, dissipation of radiative heat, and removal of inhibitory O2 [115]. Due to the low value of fuel products, PBRs for fuel synthesis favor low-tech designs and inexpensive materials to reduce both capital and operating costs. In fact, NASA has proposed to float plastic bags of algal cultures in wastewater to allow for nutrient exchange [116]. Alternatively, open pond systems, traditionally a raceway configuration with a paddle-wheel for mixing, have proven successful for cultivating micro­algae at scale [117]. Unlike PBRs, ponds are open to the environment, allowing for evaporative water loss and pond crash due to contamination by predators and competitors. However, the low capital cost of an open pond system makes this design a contender for fuel production. Clearly, the large-scale cultivation techniques for autotrophic fuel production still require additional development and optimization compared to heterotrophic cultivation.