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

The Use of Synthetic Communities Reveals Disturbance of Process Partitioning Among Denitrifying Microbes Leads to Increased Nitrous Oxide Emissions


Jacob J. Valenzuela1* (, J. Wilson1, A. Carr1, S. Turkarslan1, S. Altenburg2, H. Smith2, A. Otwell3, K. Hunt3, J. Goff4, F. Poole4, X. Ge4, M. Thorgersen4, P. Walian5, V. Mutalik5, A. M. Deutschbauer5, T. R. Northen5, M. W. W. Adams4, R. Chakraborty5, D. A. Elias5, D. A. Stahl3, M. W. Fields2, N. S. Baliga1,2, A. P. Arkin5,6, and Paul D. Adams5,6


1Institute for Systems Biology; 2Montana State University; 3University of Washington; 4University of Georgia; 5Lawrence Berkeley National Laboratory; and 6University of California–Berkeley



The Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA) Science Focus Area (SFA) uses a systems biology approach to understand the interaction between microbial communities and the ecosystems they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. Thus, ENIGMA has been organized into several campaigns involving multiple institutes with varying expertise. An overarching goal of the Environmental Simulations and Modelling Campaign is to simulate, model, and predict the mechanistic underpinnings of field-observed phenomena. This includes characterizing the process partitioning of N2O emissions in varying ecological contexts (pH, metal availability, oxygen, and phage attack) using field isolates assembled into synthetic communities (SynComs).


The Field Research Center (FRC) at Oak Ridge, Tenn., has some of the highest subsurface nitrate concentrations (>10g/L) ever recorded. This pool of subsurface nitrate, a remnant of legacy activities, can end up as the greenhouse gas nitrous oxide (N2O) via incomplete denitrification or as nitrogen gas (N2) when completely denitrified. Based on spatiotemporal field surveys of biogeochemistry, hydrology, metagenomics, and activity measurements the ENIGMA team is formulating mechanistic hypotheses to explain ecologically important phenomena, such as the emissions of increased N2O emissions in wells that transiently transition from a neutral to an acidic pH. Here, using the N2O emission phenomenon, researchers explicate how such phenomena are being dissected through laboratory studies using synthetic communities assembled from field isolates of relevant organisms. The ultimate goal is to characterize the mechanism(s) responsible for each field phenomenon and test generalizability of findings back at the field site.

Analysis of FRC field isolates with denitrifying capabilities showed that more than half of the isolates were missing at least one step in the denitrification pathway. Researchers, therefore, hypothesized that multiple microbes with complementary enzymatic steps likely work together in communities to complete denitrification through the exchange of nitrogen intermediates. Further, the team hypothesized that different abiotic and biotic factors that inhibit specific enzymatic reactions would likely decouple complete denitrification, contributing to significant levels of N2O emissions at the FRC. To test this hypothesis, the team established a synthetic community (SynCom) of two field isolates—Rhodanobacter sp. R12 and Acidovorax sp. 3H11—which together can perform complete denitrification but cannot independently. Therefore, a cross-campaign initiative was generated to elucidate different mechanisms of abiotic control, including pH shifts, microaerobic environments, and metal availability. Using time course experiments, researchers determined that a shift in pH from neutral pH 7 to pH 6 or an increase in nickel (Ni) concentrations was enough to decouple the complete denitrification process of the SynCom by different mechanisms. Strikingly, both perturbations resulted in significant increases in N2O emissions. Transcriptome analysis of the SynCom at differing pH or Ni conditions suggests dynamic changes in community composition and physiological states. Current experiments are focused on shifts in pH at varying C/N ratios, oxygen, and even phage attack that may result in the decoupling of denitrification partitioning. Transmission electron microscope imaging suggests very different morphologies between the two field isolates that may play an essential role in carbon, nitrogen, and phosphorous fluxes between the organisms. Together, all these efforts are leading to the construction of a context-specific gene regulatory network that can be used to predict how environmental fluctuations at the field site will impact emissions of N2O.

Funding Information

This material by ENIGMA SFA Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Biological and Environmental Research (BER) Program under contract number DE-AC02-05CH11231.