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

The Enigma of N2 Fixation in Energy-Limited Anaerobic Methane-Oxidizing Microbial Consortia


Ranjani Murali2* (, Kyle Craig1, Filipe Liu3, Jose P. Faria3, Nidhi Gupta3, Chris Henry3, Sergio Parra2, Shahram Asgari1, Keunyoung Kim4, Mark Ellisman4, Christof Meile1, Victoria Orphan2


1University of Georgia–Athens; 2California Institute of Technology–Pasadena; 3Argonne National Laboratory; 4National Center for Microscopy and Imaging Research, University of California–San Diego


The goals of this project are to (1) develop a mechanistic understanding of the anaerobic oxidation of methane (AOM) syntrophic interactions; (2) define and functionally characterize the microbial community, including viruses, associated with methanotrophic consortia under changing environmental conditions; and (3) create an integrative modeling framework to explore the ecophysiology of AOM consortia and their community interactions in an environmental context.


Most elemental cycles in Earth’s surface environment are mediated by microbial reactions. To quantify these transformations and predict the distribution of nutrients and other chemicals of interest, biogeochemical models need to capture the transport and reaction rates of these processes, which depend on environmental conditions, such as the availability of substrates, metabolic pathway expression, and physiological constraints.

In this project, researchers explore environment-microbe interactions through reactive transport modeling of the microbially mediated AOM. First, researchers show simulations of spatially resolved microbial consortia composed of methanotrophic archaea (ANME) coupled metabolically to sulfate-reducing bacteria (SRB) via direct interspecies electron transfer (He et al. 2021). Growth efficiencies derived from estimates of catabolic energy yields, anabolic energy requirements and energy dissipation (Heijnen and Van Dijken 1992) resulted in growth-yields consistent with observations when using ammonium as the nitrogen (N) source.

The team’s model simulations showed that N demands can likely be fulfilled without causing significant ammonium drawdown within or surrounding the microbial aggregates. Nevertheless, some archaea and bacteria involved in AOM, a process with limited energy yield, have been shown to fix N2 (Dekas et al. 2018; Metcalfe et al. 2021), which requires a significant amount of ATP and reducing equivalents. When extending this model to allow for N2 as the N source, the predicted growth yields decreased but remained substantially higher than yields derived from measurements when N2 fixation was active, suggesting that physiological controls are important.

To further investigate possible triggers for this energy-consuming process, researcherse studied growth and its dependence on N processing using a flux balance model of ANME (Faria et al. 2023). The simulations showed that even significant leakage of N-rich compounds is unlikely to induce N2 fixation. Researchers therefore explored the potential of the use of N2 fixation to maintain intracellular redox homeostasis as has recently been proposed for Geobacter sulfurreducens (Ortiz-Medina et al. 2023). However, researchers were unsuccessful at inducing N2 fixation in the model under environmentally relevant conditions, which points to as yet poorly understood features of this energy-limited syntrophic partnership and the need for additional studies of the metabolic controls in methanotrophic ANME archaea.

Finally, environmental conditions within sediments, soils, and rock matrices may also vary on small spatial scales, depending on the pore connectivity, which could lead to conditions that trigger different metabolic activities. To explore the potential for the formation of distinct microenvironments within carbonate rocks that are formed through the process of sulfate-coupled anaerobic methane oxidation, the team developed a Lattice-Boltzmann porescale reactive transport model (CompLab3D). In these simulations, researchers established the model domain from CT scans of carbonate rocks, and then quantified the connectivity of their pore spaces by computing the distribution of water ages. This distinguishes well-connected regions from isolated pores, which may support different microbiological processes and levels of activity within the carbonate structure.


Dekas, A. E., et al. 2018. “Widespread Nitrogen Fixation in Sediments from Diverse Deep‐Sea Sites of Elevated Carbon Loading,” Environmental Microbiology 20(12), 4281–96.

Faria, J. P., et al. 2023. “ModelSEED v2: High-Throughput Genome-Scale Metabolic Model Reconstruction with Enhanced Energy Biosynthesis Pathway Prediction,” bioRxiv.

He, X., et al. 2021. “Controls on Interspecies Electron Transport and Size Limitation of Anaerobically Methane Oxidizing Microbial Consortia,” mBIO, 12(3), e03620-20.

Heijnen, J. J., and J. P. Van Dijken. 1992. “In Search of a Thermodynamic Description of Biomass Yields for the Chemotrophic Growth of Microorganisms,” Biotechnology and Bioengineering 39(8), 833–58.

Metcalfe, K. S., et al. 2021. “Experimentally Validated Correlation Analysis Reveals New Anaerobic Methane Oxidation Partnerships with Consortium-Level Heterogeneity in Diazotrophy,” The ISME Journal 15(2), 377–96.

Ortiz-Medina, J. F., et al. 2023. “Nitrogen Fixation and Ammonium Assimilation Pathway Expression of Geobacter sulfurreducens Changes in Response to the Anode Potential in Microbial Electrochemical Cells,”Applied and Environmental Microbiology 89(4), e02073-22.

Funding Information

This work was supported by the DOE, BER program, GSP under award number DE-SC0022991.