Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Model-Guided Design of Synthetic Microbial Consortia for Next-Generation Biofuel Production

Authors:

Deepan Thiruppathy1* (dthirupp@eng.ucsd.edu), Lizzette Moreno García2, Gustavo Lastiri-Pancardo1, Neal N. Hengge2, Loryn Chin1, Violeta Sànchez i Nogué2, Jeffrey G. Linger2, Michael T. Guarnieri2, Karsten Zengler1

Institutions:

1University of California–San Diego; 2National Renewable Energy Laboratory

Goals

This project aims to establish the foundation for bioproduction using multifaceted microbial communities. Researchers will build metabolic community models of increasing complexity by integrating multiomics datasets. These models will guide engineering designs for optimized production of biofuels from lignocellulosic biomass. Furthermore, the team will use innovative approaches to augment existing communities for improved bioproduction and complete conversion of different biomass feedstocks. Overall, these strategies will provide knowledge of the functional metabolic exchanges driving interspecies interactions in microbial communities, thus providing insights into fundamental biological processes. Lessons learned here will be crucial for researchers’ ability to design stable microbial communities for various biotechnology applications in the future.

Abstract

The multiplicity of intertwined, interspecies interactions within microbial communities regulates their functional organization and assembly. This allows these communities to perform complex functional tasks unreachable by axenic cultures, such as the breakdown of recalcitrant lignocellulosic materials into high-energy volatile fatty acids (VFAs). Bioproduction of one such fatty acid, butyric acid (BA), from sustainable lignocellulosic sources has gained attention owing to BA’s versatile applications as a precursor for a range of products, including sustainable aviation fuel, polymers, fibers, and cosmetics. However, the current necessity for costly enzymatic pretreatment of the lignocellulosic substrate that is currently required undermines the economic advantages of a biosustainable process.

A mutualistic co-culture of the thermophilic strains Clostridium thermocellum and Clostridium thermobutyricum was recently shown to be effective in converting lignocellulose to BA without expensive enzymatic pretreatment (Chi et al. 2018). However, the process is still suboptimal, leaving ample room for improvement in substrate utilization and product formation. While the co-culture of C. thermocellum and C. thermobutyricum resulted in a >100% improvement in substrate utilization compared to a monoculture of C. thermocellum, notable amounts of carbohydrates−primarily constituted by xylans−and the fermentation end products ethanol and acetate, were left unutilized.

Here, researchers characterized the metabolic interactions and exchanges of this thermophilic co-culture using high-quality, manually curated genome-scale metabolic models (GEMs) for both species. Compartmentalized as a community metabolic model (CM-model) comprising 1,777 reactions, 1,679 metabolites and 1,569 genes, the constructed CM-model enabled researchers to identify predicted metabolic bottlenecks that account for the co-culture’s constrained BA production and incomplete substrate utilization. The group aimed to release these bottlenecks via targeted and untargeted augmentation of the community to generate a reproducible synthetic community (SynCom), thereby improving substrate utilization and BA production. Targeted augmentation involves conducting bibliographic research and using CM-model guidance to select characterized microbial strains compatible with the system. Conversely, untargeted expansion entails isolating and selecting microbes with desirable metabolic traits.

For the targeted augmentation, the researchers considered specific roles fulfilled by individual members of the co-culture (with C. thermocellum specializing in recalcitrant carbohydrate biomass hydrolysis and C. thermobutyricum in BA production from soluble sugars) and structured the expanded SynCom around functional modules. This organizational framework integrates Thermoanaerobacterium xylanolyticum along with C. thermocellum into a “lytic” module, specializing in complex carbohydrate oligomer breakdown. Moorella thermoacetica was chosen for the “scavenging” module, aimed at recapturing and redirecting residual byproducts. Together, these augmented modules combined with the “yield” module, focused on C. thermobutyricum-mediated BA production, completed the setup.

Simultaneously, researchers explored augmenting the community in an untargeted approach. Soil samples were collected and grown on lignocellulosic substrates of varying recalcitrance under thermophilic, anoxic conditions to enrich thermophilic bacteria capable of hydrolyzing the complex oligomers in lignocellulose. Additional selection pressures were applied by growing these enrichments on spent supernatants from the co-culture’s growth on deacetylated, and mechanically refined corn-stover (DMR). From these, the group isolated and characterized thermophilic strains capable of growing solely on DMR and other non-pretreated lignocellulosic substrates, indicating possible new metabolic capabilities. Next, the group constructed three-member communities from these strains by pairing them with C. thermocellum and C. thermobutyricum and observed improved BA titers from DMR compared to the co-culture. Hence, this study establishes the foundation for advanced bioproduction using multifaceted microbial communities.

References

Chi, X., et al. 2018. “Hyper-Production of Butyric Acid from Delignified Rice Straw by a Novel Consolidated Bioprocess,” Bioresource Technology 254, 115–20. DOI:10.1016/j.biortech.2018.01.042.

Funding Information

This material is based upon work supported by the U.S. DOE, Office of Science, BER program under Award DE- SC0022137.