(721d) Progress and Economic Considerations for the Biological Production of Triterpene Biofuels From Gases

We present our progress in producing triterpene fuel precursors  from CO₂, H₂ and O₂as part of the ARPA-E electrofuels program.  While increasing product titer provides an obvious improvement to process economic feasibility, we also present that the maintenance energy of bacteria, or the energy needed to keep it alive, is a significant economic consideration for fuel production.

The Advanced Research Projects Agency (ARPA) program seeks to produce liquid transportation fuels from renewable electricity and CO₂. Botryococcenes (C₃₀ hydrocarbons) from Botryococcus braunii is a very good potential fuel molecule for this purpose due to its high energy density, hydrophobicity and similarity to petroleum crude.  A primary research milestone is the genetic engineering of the isoprene metabolic pathway through isopentenyl diphosphate (IPP) to triterpenes. This is being accomplished using the genes from Mevalonate and MEP pathways as well as triterpene synthase genes from an algae, Botryococcus braunii, known to produce hydrocarbons. We were able to use the vector systems that have been used earlier to transform other Rhodobacterspecies with constitutive promoters (pLac) and native promoters to successfully produce isoprenes. Current efforts are undertaking a "breadth screen" for different vectors, promoters, knock-in and hosts to examine the "landscape" for triterpene production. A "depth" screen is underway to better understand the rate limiting enzymes and metabolic fluxes. The current status of these screens will be presented.

We are using economic considerations of the envisioned process to help drive research priorities in addition to the goal of genetically engineering the botryococcenes pathway from B. braunii into a chemolithoautotrophic bacteria – Rhodobacter capsulatus. Since the process involves the production of a moderate value fuel molecule from useful gaseous substrates (H₂ and CO₂), the growth of the bacteria has to be minimized to maximize the yield to to final product. Therefore the maintenance coefficient of the bacterial host becomes important as this is the absolute minimum energy wasted in the process without generating any product. To this end, we have undertaken the measurement of the growth yield and maintenance coefficient of Rhodobacter capsulatus and Ralstonia eutropha (another well-studied autotrophic production host) and determine their relative impact on the overall economics of the process. While it is relatively straight-forward to close a mass balance in the liquid phase, it is quite difficult to do it accurately in the gas phase because of effects of temperature, pressure and composition on the measurements. As such, literature references for growth yield and maintenance coefficient of autrophic bacteria are scarce. We have carried out chemostat studies under various conditions using an elaborate setup to continuously measure the inlet and outlet gas flow rate and composition from the reactor to ensure accurate determination of the parameter values. Our results indicate that under oxygen limited conditions the maintenance coefficient of R. capsulatus is significantly lower than that of R. eutropha. On the other hand, the growth yield of R. eutropha is much better than R. capsulatus. This represents critical trade-offs in terms of process design. The other important parameter affecting the process is the gas-liquid mass transfer of the bioreactor employed as this would affect the volumetric productivity of the process and thus the capital cost. We have carried out a detailed process design and economic analysis (incorporating operating, fixed and capital costs at large industrial scale) to determine the overall feasibility of the process. There exist multiple routes of going from gaseous substrates to liquid fuels. We will present the results of the different economic scenarios incorporating both literature and experimentally obtained values for the organism and reactor performance. This analysis will be used to draw conclusions about the most probable path forward for this technology.

The results of this project thus far challenge the typical paradigm of working with organisms that are inherently fast growing and easy to culture.  We suggest that as genetic engineering capabilities extend to more difficult organisms, some of the more fringe ‘tougher, more efficient’ organisms may be better suited for fuels production than their fast-growing ‘easy to manipulate’ counterparts.