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

2024 Abstracts

Enhancing Biological Nitrogen Fixation in Sorghum (Sorghum bicolor) Aerial Roots Through Engineering Diazotrophic Communities


Rafael E. Venado2*, Claire Palmer1 ([email protected]), Paulo Ivan Fernandes-Júnior2,3, Biswajit Samal2, April MacIntyre2,5, Ophelia Venturelli1, Jean-Michel Ané2,4


1Department of Biochemistry, University of Wisconsin–Madison; 2Department of Bacteriology, University of Wisconsin–Madison; 3Brazilian Agricultural Research Corporation (Embrapa) Petrolina; 4Department of Plant and Agroecosystem Sciences, University of Wisconsin–Madison; 5Valent Biosciences Corporation



We aim to reduce the dependency of bioenergy production on synthetic nitrogen fertilizers by taking better advantage of biological nitrogen fixation. We specifically focus on nitrogen fixation in the mucilage produced by aerial sorghum roots (Sorghum bicolor). Alongside our collaborators exploring this plant trait, we investigate the sorghum-associated bacterial communities that contribute to biological nitrogen fixation. Our plan includes (1) isolating and characterizing bacterial strains, including diazotrophs, from aerial root mucilage; (2) assessing bacterial interspecies interactions that influence biological nitrogen fixation; and (3) developing and testing synthetic communities with robust biological nitrogen fixation capabilities.


Sorghum (S. bicolor) is a promising bioenergy crop due to its high biomass yield, resilience to harsh environmental conditions, and ability to grow in diverse geographical regions. However, sorghum production relies on synthetic nitrogen fertilizers, which have negative economic and ecological consequences such as leaching and greenhouse gas production. We identified sorghum accessions harboring high rates of biological nitrogen fixation in a carbohydrate-rich gel/mucilage produced by their aerial roots. We demonstrated that high rates of nitrogenase activity occur in the mucilage using acetylene reduction assays and the transfer of this fixed nitrogen to the plant using nitrogen (15N) gas enrichments. We also determined that these sorghum accessions obtain up to 43% of their nitrogen from the atmosphere using 15N isotope dilution experiments (Venado et al. 2023). In this part of this DOE-funded project, we aimed to (1) isolate bacteria, including diazotrophs, from the sorghum mucilage; (2) explore bacteria interspecies interactions using synthetic communities (SynComs); and (3) explore the potential of engineering diazotrophs from the mucilage to further enhance nitrogen fixation (Chakraborty et al. 2023;Venkataraman et al. 2023).

  1. We isolated over 200 unique bacteria from sorghum mucilage. We assessed carbon utilization, plant growth-promoting activities, and nitrogenase activity of 34 promising diazotrophs along with Azospirillum brasilense FP2 and Klebsiella variicola A3 as reference strains. Among the 34 new strains tested, at least 23 showed robust nitrogenase activity, with 11 strains showing higher nitrogenase activity than our two reference Pairwise coculture assay indicated that several strains, including non-diazotrophs, enhanced the nitrogenase activity of A. brasilense and K. variicola, indicating that sorghum mucilage harbors “helper strains” in addition to diazotrophs.
  2. We used a SynCom approach to investigate the bacterial interspecies interactions impacting biological nitrogen fixation. We assessed community nitrogenase activity and time-resolved composition from 93 different subcommunities of PComm1, leading to a dataset containing a range of diazotroph growth and fixation profiles. We found that nitrogenase activity is positively correlated with diazotroph abundance. However, there were several notable exceptions, suggesting the presence of interspecies competition impacting community fixation that is not growth-mediated. We observed pairwise cocultures containing one diazotrophic and one non-diazotrophic species can lead to improved nitrogenase activity compared to the monoculture, indicating again the presence of strains that can function as helpers for diazotrophs in a species-specific We are further exploring this dataset using a paired modeling approach.
  3. We genetically engineered Klebsiella strains isolated from the mucilage to fix more nitrogen and release this fixed nitrogen as ammonium. Using biosensors, some of these “ammonium-excreting” diazotrophs were further engineered to release nitrogen only in the presence of arabinose, an abundant sugar in the sorghum mucilage, to improve the fitness of the engineered strains. Additional biosensors are currently being investigated.

Altogether, our project allowed us to identify efficient diazotrophs from the sorghum mucilage, better understand interactions between bacteria within this unique environment, and engineer these bacteria to increase further nitrogen fixation rates and the delivery of fixed nitrogen to sorghum and enhance the efficiency and sustainability of bioenergy production.


Chakraborty, S., et al. 2023. “Scripting a New Dialogue between Diazotrophs and Crops,” Trends in Microbiology. DOI:10.1016/j.tim.2023.08.007.

Venado, R. E., et al. 2023. “Mucilage Produced by Sorghum (Sorghum Bicolor) Aerial Roots Supports a Nitrogen-Fixing Community,” bioRxiv. DOI:10.1101/2023.08.05.552127.

Venkataraman, M., et al. 2023. “Synthetic Biology Toolbox for Nitrogen-Fixing Soil Microbes,” ACS Synthetic Biology 12(12): 3623–34. DOI:10.1021/acssynbio.3c00414.

Funding Information

The authors gratefully acknowledge funding from the U.S. DOE BER program grant no. DE-SC0021052.