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

Developing, Understanding, and Harnessing Modular Carbon/Nitrogen-Fixing Tripartite Microbial Consortia for Versatile Production of Biofuel and Platform Chemicals

Authors:

Xiaoxia Nina Lin1* (ninalin@umich.edu), Yanmeng Liu1, David Carruthers1, Yi Dai1, Josie Mcquillan2, Maciek Antoniewicz1, Sujit Datta3, Jagroop Pandhal2, Andrew Allman1

Institutions:

1University of Michigan–Ann Arbor; 2University of Sheffield–United Kingdom; 3Princeton University–Princeton

Goals

The overall goal of this project is to design, construct, analyze and optimize a synthetic microbial consortium system consisting of three closely interacting members—a carbon dioxide (CO2)-fixing photosynthetic specialist, a nitrogen (N2)-fixing specialist, and a third specialist that can convert organic carbon (C) and N generated by the first two specialists to synthesize a desired product. By integrating complimentary expertise from multiple research laboratories at three institutions, researchers are pursuing three specific objectives: (1) develop tripartite microbial consortia for C or N fixation and production of biomolecules with various N or C ratios; (2) investigate molecular and cellular mechanisms governing the tripartite consortia via omics study and predictive modeling; and (3) explore alternative spatial configurations and develop scalable design principles.

Abstract

Microbial communities are ubiquitous in nature, exhibiting incredibly versatile metabolic capabilities and remarkable robustness. Inspired by these synergistic microbial ecosystems, rationally designed synthetic microbial consortia is emerging as a new paradigm for bioprocessing and offers tremendous potential for solving some of the biggest challenges the society faces. In this project, researchers focus on a tripartite consortium consisting of a CO2-fixing photosynthetic specialist, a N2-fixing specialist, and a third specialist that can convert organic C and N generated by the first two specialists to synthesize a desired product. In addition to CO2 fixation, a noteworthy feature of this design is the elimination of the requirement for N fertilizer, which has been produced through ammonia synthesis using the Haber-Bosch process and accounts for an estimated 2% of global energy expenditure. Researchers aim to develop a modular and flexible model system capable of producing diverse biomolecules (varying C:N ratio) as advanced biofuel or platform chemicals, to dissect this complex ecosystem using a spectrum of cutting-edge systems approaches, and to ultimately derive scalable and broadly applicable design principles for maximizing the system performance.

The team’s first prototype tripartite consortium employs genetically modified strains of photosynthetic cyanobacterium Synechococcus elongatus that secretes sucrose and N-fixing bacterium Azotobacter vinelandii that secretes ammonia respectively, to form a symbiotic foundation for supporting a third producer member (Abramson et al. 2016; Barney et al. 2015). Utilizing a customized bioreactor system consisting of multiple chambers separated with permeable membranes and allowing control of growth rates of individual consortium members, researchers demonstrate this platform technology with selected representative production specialists, including a sucrose-metabolizing Escherichia coli K-12 derivative strain, Corynebacterium glutamicum, and Bacillus subtilis (Carruthers et al. 2020; Carruthers et al. 2024).

The team’s ongoing work aims to develop new methods for creating novel spatial configurations that provide individualized environmental niches for each consortium member and thereby maximize their performance on intended functionalities. One initial focus is to dissect spatially separated and spatially consolidated cultivations of Azotobacter vinelandii and Synechococcus elongatus. Omics studies are conducted to unravel regulatory mechanisms. This allows the ability to gain fundamental insights on species interactions and their contributions to the robustness of the biculture system, which will guide future efforts in optimization of the whole system.

Other ongoing work includes: (1) development of predictive mathematical models to systematically explore the parameter space to understand how different biological parameters and operating strategies impact the system performance such as yield and productivity; and (2) investigation of spatially organized cocultures using 3D-printed communities in hydrogel matrices, which render high-resolution control and analysis capabilities.

References

Abramson, B. W., et al. 2016. “Increased Photochemical Efficiency In Cyanobacteria Via An Engineered Sucrose Sink,” Plant and Cell Physiology, 57(12):2451-2460, 2016.

Barney, B., et al. 2015. “Gene Deletions Resulting in Increased Nitrogen Release by Azotobacter Vinelandii: Application of a Novel Nitrogen Biosensor,” Applied and Environmental Microbiology 81(13), 4316–28.

Carruthers, D., et al. 2020. “Random Chromosomal Integration and Screening Yields E. Coli K-12 Derivatives Capable of Efficient Sucrose Utilization,” ACS Synthetic Biology 9(12), 3311–21.

Carruthers, D., et al. To be submitted 2024. “Engineering Modular Carbon- and Nitrogen-Fixing Microbial Consortia for Sustainable Biochemical Production.”

Funding Information

This research was supported by the DOE Office of Science, BER program, grant no. DE-SC0022136.