(592a) Invited Talk: The Challenge of Growing and Sustaining Acetogens on CO2 & H2 to High Cell Densities: Bioenergetics Combined with Deficient H2 uptake Limit CO2 Utilization

Developing a carbon neutral (CNeu) or negative (CNeg) platform for production of chemicals is a compelling approach for sustainable biomanufacturing. Sugar catabolism leads to loss of a third of the carbon as CO₂. One approach to resolve this issue is acetogenic mixotrophy with one organism, whereby sugar substrates are used together with an electron-rich sources (eg, H₂) and additional CO₂ (Nature Commun. 7: 12800 (2016). To attain modularity and increase process intensification, synthetic syntrophic cocultures offer potent opportunities thus extending the potential of acetogenic mixotrophy. Syntrophy is obligately mutualistic metabolism to enable culture stability. We have demonstrated the usefulness of a syntrophic system that can be engineered at the molecular and process levels is made up of the solventogen, sugar utilizing Clostridium acetobutylicum (Cac) and the acetogen Clostridium ljungdahlii (Clj). Cac utilizes sugars and produces CO₂, H₂ and metabolites, while Clj feeds on CO₂ and H₂ to enable CNeu and CNeg processes to produce native or non-native metabolites. While acetogens grow well on CO and syngas, all systems engaging acetogens alone or in coculture are limited by the apparently inherent inability of acetogens to grow to high cell densities using CO₂ and H₂ as the only substrates. Why do they stop growing after reaching rather low cell densities? Why not continue to high cell densities like when growing on CO or syngas or some sugar? What is preventing that?

We will address these questions using Clj, a well-known model acetogen. One limitation is the inferior bioenergetics of the system: the low ATP yield of using CO₂/H₂ significantly constrains the growth, activity, and product profile of acetogens relative to their growth on higher energy substrates such as CO, syngas, or carbohydrates. While this could be resolved by acetogenic mixotrophy, catabolite repression of CO₂/H₂ utilization by the sugar constrains this approach. Analysis and data will be used to demonstrate that a second major limitation is H₂ transport and H₂ uptake by the cells, the latter never previously entertained in the literature. We will finally propose and demonstrate technological approaches to resolve these crucial limitations, and thus enable the development of technologies for CNeu and CNeg bioprocessing.

Supported by the U.S. Department of Energy ARPA-E project under contract AR0001505.

N.B.W. and J.H. were supported in part by a U.S. Department of Education GAANN Fellowship under grant P200A210065.