Engineering of a Stable, Syntrophic Microbial Coculture for Enhanced H2 Production

Hydrogen gas is an important commodity chemical and a promising fuel that can be produced biologically from inexpensive renewable resources. Anaerobic fermentative bacteria, such as Escherichia coli, produce H₂ from carbohydrates but at a low yield due to obligate co-production of organic acids and alcohols. In contrast, anoxygenic photoheterotrophic bacteria, such as Rhodopseudomonas palustris, cannot consume carbohydrates but use electrons in fermentation products to produce H₂ via nitrogenase. Several studies have combined the complementary metabolic traits of fermentative and photoheterotrophic bacteria to circumvent the limitations of single species. The fermentative bacterium ferments the carbohydrates to H₂ and fermentation products while the photoheterotroph oxidizes fermentation products to H₂. These studies have confirmed that coculturing can increase the H₂ yield. Unfortunately, the coculturing techniques used did not promote culture stability, as conditions allowed for unrestrained growth of the fermentative bacterium. Overall, these cocultures have given highly variable trends and have been inadequate as an experimental system. We hypothesized that engineering metabolic interdependency between microbes would resolve these issues by stabilizing species population dynamics.

Using defined genetic mutations and environmental conditions, we developed a stable coculture between E. coli and R. palustris that functions via obligate, bi-directional exchange of nutrients. R. palustris acquires carbon from fermentation products excreted by E. coli while it simultaneously fixes N₂ gas and provides essential nitrogen to E. coli; growth of each species is dependent on the metabolic activity of the other. Under these conditions, the coculture achieves higher H₂ yields than E. coli monocultures while accessing electron sources otherwise unavailable to R. palustris. Importantly, unlike traditional cocultures, this coculture is stable and gives reproducible results over multiple serial transfers, essentially serving as a coculture that can be treated like a monoculture. We have found that we can alter environmental conditions to limit N₂ availability to a subpopulation of bacteria in the coculture, resulting in even higher H₂ yields while still maintaining stability over serial transfers. We have also determined that the R. palustris Calvin cycle competes for electrons against nitrogenase under coculture conditions. Eliminating Calvin cycle activity results in higher H₂ yields and improved coculture growth rates, likely through a redirection of electrons to nitrogenase.

The reproducible metabolic traits of this coculture make it an attractive platform to adapt ¹³C-labeling approaches to determine metabolic fluxes in single species within a simple defined community. Furthermore, the modularity of this approach will allow for the incorporation of other microbes to make use of alternative feedstocks and to produce other chemicals of value to society.