Maximizing fatty acid production by Rhodobacter sphaeroides grown on corn stover hydrolysate

The use of cellulosic biomass residues, such as corn stover, for production of advanced biofuels at industrial scale has gained renewed interest since the passage of the Energy Independence and Security Act in 2007. Biomass-based biodiesel, composed of fatty acid alkyl esters produced from biologically-derived fatty acids (FA), is an alternative fuel of commercial importance. In our laboratory, we are focusing on developing Rhodobacter sphaeroides strains capable of producing FA at high concentrations as a microbially-based technology for production of biodiesel precursors.R. sphaeroides is a versatile microorganism that can grow heterotrophically, autotrophically, and photoheterotrophically. This bacterium has a unique ability to increase phospholipid membrane synthesis when grown anaerobically in order to harbor the membrane-bound photosynthetic apparatus. However, to grow efficiently under anaerobic conditions, R. sphaeroides requires light energy for photosynthesis, and since light may be difficult and costly to provide at an industrial scale, we have constructed R. sphaeroides mutants capable of high FA production in the absence of light and the presence of O2. Under laboratory conditions, one of the mutants can accumulate 12.5% of dry cell weight as FA under aerobic conditions, which is about 2.5 fold higher than the aerobic FA accumulation in wild type cells. FA yields from other mutants are currently being studied.We are able to grow R. sphaeroides aerobically on corn stover hydrolysates. Under these conditions wild type R. sphaeroides accumulates FA as 3 to 5% of the dry cell weight, similar to the yield on defined minimal media. This yield is expected to increase when using the high FA mutants described above. However, since one of the bottlenecks in converting corn stover hydrolysates to biofuels is the presence of plant-derived inhibitory compounds, we are also developing strategies to eliminate inhibition in R. sphaeroides cultures and maximize the volumetric density of the culture. The first strategy is the use of fed-batch technology to maintain concentrations of inhibitory compounds at non-inhibitory concentrations. A second strategy is to apply a biological detoxification approach to remove specific inhibitory components.When using a defined mineral medium with succinate as the organic substrate, we have been able to reach biomass concentrations as high as 14.6 g dry cell weight/L by fed-batch technology, which is equivalent to a fatty acids concentration of 0.7 g/L. When using glucose at concentrations found in corn stover hydrolysates, biomass yields in defined media have been as high as 22.4 g dry cell weight/L, which is equivalent to a fatty acid concentration of 1.0 g/L. In these initial experiments with wild type cultures, the FA yield from succinate or glucose is 2% (g FA/g organic substrate), indicating that there is plenty of room for improvement, considering that the theoretical yield is 35% (g FA/g glucose) and 29% (g FA/g succinate). In conclusion, the combination of fed-batch technology and genetic improvement of FA production in R. sphaeroides hold promise for increasing the yields and concentrations of FA from corn stover hydrolysates. Experiments to characterize FA production with the high FA mutants grown on corn stover hydrolysate are underway.