Nitrogen is essential for all life on Earth, forming amino acids, proteins, and nucleic acids and powering organisms’ ability to metabolize and grow. Most nitrogen comes from Earth’s atmosphere, where inert dinitrogen (N2) makes up 78 percent by weight. However, this vital element is unusable by most organisms until it is transformed, or fixed, into a reactive molecular form that can be metabolized.
Plants naturally absorb ammonium and other nitrogenous molecules through their roots. When organisms higher on the food chain eat the plants, the nitrogen they ingest transfers throughout the food web. Today, the majority of crop nitrogen fertilizing is done using industrially produced ammonia (NH3) and other nitrogen-containing compounds. These chemicals are often produced via the Haber–Bosch process, a high- temperature-and-pressure industrial process that consumes approximately 2 percent of the world’s energy output.
Beyond its energy demands and environmental impacts, fertilizer production is currently dominated by Russia and China, and despite being among the world’s largest ammonia producers, the United States still imports a significant amount of all fertilizers. One way to relieve foreign reliance on fertilizers is to ensure their efficient domestic production. (See S&TR, December 2024, Lowering Barriers to Cleaner Ammonia Production.) However, most crops uptake less than half of the nitrogen chemical fertilizers applied to their soil, while the remaining volume risks aerosolizing or leaching away and threatening downstream ecosystems. Livermore staff scientist Cody Madsen is pursuing another approach: augmenting the nitrogen-uptake efficiency of crops by enhancing the nitrogen-fixing abilities and biological compatibility of the microbes living among their roots.
Growth from the Ground Up
Among major crops, legumes such as soybeans, peas, and other beans stand out for their high nitrogen-uptake efficiency (NUE). Their roots create nodules supporting fruitful, symbiotic relationships with specialized soil- dwelling bacteria (diazotrophs) that naturally convert N2 into ammonium, allowing for convenient nutrient intake. Madsen aims to increase plants’ NUE via the microbial life they host, reducing the volume of synthetic fertilizers farmers need to apply to their fields and improving efficiency of what is already applied. Doing so would also reduce the fertilizer leaching, and fixed nitrogen remaining in the soil could benefit next season’s growth. However, other row crops such as corn have different root physiology that require more fertilizer and nitrogen-fixing activity from free-living microbes in the soil. Madsen is investigating how microbes known to help retain nitrogen fertilizers in the soil can be engineered to fix nitrogen themselves, as well as how they can partner with free-living diazotrophs to support crop growth as combinatorial fertilizers—those that blend multiple nutrient sources or additives.
Agricultural scientists have long wrestled with increasing NUE by introducing solutions of microorganisms (biofertilizers) to fields and applying biostimulants to improve plant hardiness, but results have been inconsistent. Just because one microbial species is beneficial to one crop does not guarantee similar performance for other crops. Biofertilizers do not always persist in the soil, and they may not interact well with the local soil microbiome, driving down their effectiveness. (See S&TR, March 2025, Digging into the Soil Microbiome.) Madsen is familiar with the agricultural industry’s desire for new biological solutions to spur crop growth. “I am a first-generation scientist who grew up on a farm in the Midwest where ‘magic microbe’ biologics for improving soil health have been sold for decades,” he says. “For example, California-based researchers could identify a strong microbial candidate to boost NUE, but soil microbiome variation across the continent significantly impacts an organism’s ability to thrive in different locations. We need to be able to demonstrate that a biofertilizer will work on a farmer’s own plot of land.”
While from an economic standpoint, biofertilizer manufacturers would want microbes to rapidly fix as much nitrogen as possible, Madsen says this approach risks overworking the organisms, leaving them vulnerable to environmental stressors, competition from other species in the soil, and less stable crop interactions. “Nitrogen fixation is energetically demanding for microbes—akin to a human doing a long, intense cardiovascular workout with minimal sustenance,” says Madsen. “Microbes are highly sensitive to environmental cues including soil nitrogen availability, heat, drought, and salinity, so they must be strategic about when they perform these processes.” Biofertilizers need to survive to deliver nitrogen throughout the growing season, especially when drastic plant growth and maturity occur midway and toward the end.
Introducing Synthetic Communities
In a Laboratory Directed Research and Development (LDRD) project, Madsen designed a high-throughput laboratory assay to determine how biofertilizers could fare in soil microbiomes across the country and how to boost their effectiveness. Tailoring biofertilizers to local soil microbiomes and identifying beneficial microbial partnerships for a compounding effect can increase the efficacy of nitrogen-fixing activity and improve crop growth, but determining the best microbial match for a plot of land and for a particular microbiome requires satisfying numerous biological and abiotic variables.
The organism must endure myriad soil conditions and interaction with other species, many of which are still unknown to science. Microbes forge a spectrum of allies and enemies in pursuit of finite nutrients in their vicinity, so scientists need to ensure biofertilizers are not outcompeted. “Life at the microbial level is a dog-eat-dog world,” says Madsen. “One microbe may produce a metabolite that feeds another species. Meanwhile, other microbe species may fight over the same resources. We want to discover what relationships a biofertilizer candidate forms with other species and how well it persists in the soil.” Madsen identified a bioengineering framework involving mechanizable laboratory equipment, statistical methods, and computational tools to create a microbial interaction pipeline that analyzes the compatibility of biofertilizers with microbial communities found in different soils. “With this pipeline, we can capture with statistical rigor key portions of native microbiome complexity that mirrors soil ecology principles and results in a reproducible laboratory setting,” he says.
Tracking Nitrogen with NanoSIMS
Throughout his fertilizer research, Livermore staff scientist Cody Madsen focused on the bacterium Bacillus subtilis because the organism can withstand traditionally demanding environments and has been shown to have multiple modes of action to support plants by retaining nitrogen in soil, protecting against stressors, and supplying nutrients. However, B. subtilis does not fix nitrogen in the wild. Madsen needed to equip the organism with this ability through genetic engineering. He transplanted genes from a closely related bacterium (Paenibacillus polymyxa) to make B. subtilis fix nitrogen in full capacity under the elevated heat and salinity conditions that cause stress in crops, especially later in the growing season. Madsen assessed B. subtilis’s new nitrogen- fixing ability using Lawrence Livermore’s Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS) capability to measure nitrogen fixation on a cell-by-cell basis. NanoSIMS detects radioisotope concentration, so Madsen grew B. subtilis in an environment where the microbe would fix the nitrogen isotope 15N2 to act as a tracer. With NanoSIMS’s impressive spatial resolution, Madsen verified that the bioengineered strain could convert dinitrogen into ammonium, and by varying the temperature and salinity of the medium, he quantified how the rate of nitrogen fixation varied with different environmental conditions. Results indicated the engineered strain is a resilient nitrogen-fixing organism that can withstand demanding environments.
Madsen is pioneering the concept of synthetic communities (SynComs) for soil applications as the foundation for this large-scale, data-heavy approach to studying microbial interactions. To create SynComs, researchers first place a soil sample into a solution and allow the organisms present to dissociate from the soil particles. After two days, the solution is divided into well plates and provided different growth media. Throughout repeated dilutions and regrowth, the free- floating microbes are left to interact head-to-head, either cooperating, clashing, or merely coexisting in the well plate. At the end of the growth phase, species identified via DNA sequencing are those that have achieved a biological steady state through partnership with other species. The numerous samples this methodology returns enables researchers to test the effects of different energy sources and environmental conditions while capturing stochastic variation in microbial interactions. “Using this approach, we have observed complex interactions in the laboratory even cultivating microbes that have not been characterized in any database, likely because SynComs maintains more natural interactions from the soil than is possible with conventional growing techniques,” says Madsen.
With thousands of SynComs established, the methods shift to biofertilizer assessment. Researchers introduce the biofertilizer organism of interest into SynComs to measure its interspecies interactions and determine its suitability for each microbial community. During the LDRD study, Madsen evaluated the species Bacillus subtilis, a resilient bacterium shown to benefit plants experiencing environmental stress. “In this stage, we aim to identify the species that most frequently partner with the biofertilizer candidate regardless of media type or soil. The outcome is the list of microbes that consistently support the organism of interest—that is, those it partners with best,” says Madsen.
As microbial interactions play out, SynComs are screened for surviving biofertilizer, which might not persist in each case. Since sequencing DNA from every SynComs would be cost-prohibitive, Madsen used a visible marker as an economical way to identify the SynComs where B. subtilis survived. He bioengineered a strain of B. subtilis to produce the enzyme ß-galactosidase, which acts like a molecular beacon. When the enzyme was exposed to a substrate, it discolored the growth medium of the SynComs, highlighting the wells where B. subtilis persisted.
Next, Madsen sequenced the DNA from the remaining wells to determine their overall species composition and discover species that tend to cooperate with B. subtilis. After introducing B. subtilis to SynComs grown from three distinct soil sources, Madsen found the bacterium persisted in multiple SynComs structures, and sometimes it improved the overall health of microbiome communities present. Within these SynComs structures, B. subtilis interacted with other microbes known to partner with the species in the wild, indicating the SynComs were capturing and reproducing real-world results. “We identified partners to B. subtilis and mirrored microbial soil ecology principles with statistical strength to give us confidence that we are truly capturing real-world behaviors,” he says.
Bridging Computation and Agriculture
Madsen’s LDRD study validated the microbial interaction pipeline’s proof-of-principle and established a high-throughput process for future trials. Looking to the future, he aims to scale up the process and help accelerate production of locally tailored and combinatorial biofertilizers. “These methods are intended to be universally applicable for predicting real- world soil compatibility. The guiding question for this work was whether we could mirror in the laboratory what naturally occurs in soil, and furthermore, whether we could automate the process,” says Madsen. Fortunately, robotic technologies already exist for much of the pipetting, growing, and image analysis methods inherent to the microbial interaction pipeline. Thus, future experimentation could be performed by an autonomous laboratory, drastically reducing the time to findings.
Madsen envisions autonomous systems building a comprehensive database of microbes known to pair well together in soils across the country (especially in regions where crop growth has stunted) and machine-learning methods aiding in prescribing soils with the biofertilizer most likely to increase NUE. He explains, “Suppose an agricultural technology company is searching for a microbe to help provide nitrogen, phosphorus, or potassium to cropland in Missouri. They could consult a database to find which microbes are native to the local soil and identify the organism most likely to provide the desired effects and that thrives in that microbiome. These results would de-risk biofertilizer production and likely encourage adoption by local farmers.”
So far, Madsen’s methods have drawn interest from the private agricultural sector, and they feature in a Department of Energy Earthshot Initiative aimed at improving soil health by identifying microbial partnerships that increase mineral weathering. The approach stands to decrease reliance on foreign, artificial fertilizers in favor of naturally occurring metabolic pathways of microbial matches waiting to be discovered just below the surface.
—Elliot Jaffe
For further information contact Cody Madsen (925) 423-1216 (madsen16 [at] llnl.gov (madsen16[at]llnl[dot]gov)).
