Plant growth is supported by millions of tiny soil microbes competing and cooperating with each other as they perform important roles at the plant root, including improving access to nutrients and protecting against pathogens. As a byproduct of their metabolism, soil microbes can also produce nitrous oxide, or N2O, a potent greenhouse gas that has mostly been studied for its impact on the climate. While some N2O occurs naturally, its production can spike due to fertilizer application and other factors.
While it has long been believed that nitrous oxide doesn’t meaningfully interact with living organisms, a new paper by two MIT researchers shows that it may in fact shape microbial communities, making some bacterial strains more likely to grow than others.
Based on the prevalence of the biological processes disrupted by nitrous oxide, the researchers estimate about 30 percent of all bacteria with sequenced genomes are susceptible to nitrous oxide toxicity, suggesting the substance could play an important and underappreciated role in the intricate microbial ecosystems that influence plant growth.
The researchers have published their findings today in mBio, a journal of the American Society for Microbiology. If their lab findings carry over to agricultural settings, it could influence the way farmers go about everyday tasks that expose crops to spikes in nitrous oxide, such as watering and fertilization.
“This work suggests N2O production in agricultural settings is worth paying attention to for plant health,” says senior author Darcy McRose, MIT’s Thomas D. and Virginia W. Cabot Career Development Professor, who wrote the paper with lead author and PhD student Philip Wasson. “It hasn’t been on people’s radar, but it is particularly harmful for certain microbes. This could be another knock against N2O in addition to its climate impact. With more research, you might be able to understand how the timing of N2O production influences these microbial relationships, and that timing could be managed to improve crop health.”
A toxic gas
Nitrous oxide was shown to be toxic decades ago when researchers realized it can deactivate vitamin B12 in the human body. Since then, it has mostly drawn attention as a long-lived greenhouse gas that can eat away at the ozone. But when it comes to agricultural settings, most people have assumed it doesn’t interact with organisms growing in the soil around the plant root, a region called the rhizosphere.
“In general, there’s an assumption that N2O is not harmful at all despite this history of published studies showing that it can be toxic in specific contexts,” says McRose, who joined the faculty of the Department of Civil and Environmental Engineering in 2022. “People have not extended that understanding to microbial communities in the rhizosphere.”
While some studies have shown nitrous oxide sensitivity in a handful of microorganisms, less is known about how it impacts the distribution of microbial communities at the plant root. McRose and Wasson sought to fill that research gap.
They started by looking at a ubiquitous process that cells use to grow called methionine biosynthesis. Methionine biosynthesis can be carried out by enzymes that are dependent on B12 — and by other enzymes that are not. Many bacteria have both types.
Using a well-studied microbe named Pseudomonas aeruginosa, the researchers genetically removed the enzyme that isn’t dependent on B12 and found the microbe became sensitive to nitrous oxide, with its growth harmed even by nitrous oxide it produced itself.
Next the researchers looked at a synthetic microbial community from the plant Arabidopsis thaliana, finding many root-based microbes were also sensitive to nitrous oxide. Combining sensitive microbes with nitrous oxide-producing bacteria hampered their growth.
“This suggests that N2O-producing bacteria can affect the survival of their immediate neighbors,” Wasson explains. Together, the experiments confirmed the researchers’ suspicion that the production of nitrous oxide can hamper the growth of soil bacteria dependent on vitamin B12 to make methionine.
“These results suggest nitrous oxide producers shape microbial communities,” McRose says. “In the lab the result is very clear, and the work goes beyond just looking at a single organism. The co-culture experiments aren’t the same as a study in the field, but it’s a strong demonstration.”
From the lab to the farm
In farms, soil commonly experiences spikes of nitrous oxide for days or weeks from the addition of nitrogen fertilizer, rainfall, thawing, and other events. The researchers caution that their lab experiments are only the first step toward understanding how nitrous oxide affects microbial populations in agricultural settings.
Wasson calls the paper a proof of concept and plans to study agricultural soil next.
“In agricultural environments, N2O has been historically high,” Wasson says. “We want to see if we can detect a signature for this N2O exposure through genome sequencing studies, where the only microbes sticking around are not sensitive to N2O. This is the obvious next step.”
McRose says the findings could lead to a new way for researchers and farmers to think about nitrous oxide.
“What’s important and exciting about this case is it predicts that microbes with one version of an enzyme are going to be sensitive to N2O and those with a different version of the enzyme are not going to be sensitive,” McRose says. “This suggests that in the environment, exposure to N2O is going to select for certain types of organisms based on their genomic content, which is a highly testable hypothesis.”
The work was supported, in part, by the MIT Research Support Committee and a MIT Health and Life Sciences Collaborative Graduate Fellowship (HEALS).
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Written by Zach Winn, MIT News
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