Sound Researchers Directly Measure Nitrogen Fixation with SOURCE® in Exciting New Experiment

Expertise provided by Alex Greenlon and Iris Moore; written by Rachel Sim.

Understanding how nutrients move through the soil and into the plant can be challenging, and not just for growers. Researchers want to give growers the best information they can about the products and practices that might boost yield, increase ROI and improve sustainability. Soil tests might tell growers what’s in their soil, but that doesn’t mean the nutrients are getting to the plant, and while tissue tests can show what’s in the crop, it can be difficult to know for sure which practice is responsible.

Researchers from Sound Agriculture recently conducted an experiment tracking nitrogen gas from the air all the way into the plant, linking nitrogen in the plant tissues directly to nitrogen fixation by soil microbes. In this comprehensive experiment, the researchers were able to see clear links between SOURCE®, Sound’s microbiome activator, and increased nitrogen fixation.

“​The advantage of doing this study is that there is absolutely no ambiguity,” says Alex Greenlon, Research Scientist at Sound.

Iris Moore is a Research Associate at Sound. ​“This is the one experiment where you can actually link the increased supply of nitrogen found in plant tissue to the increased nitrogen fixation that’s happening from SOURCE activating those nitrogen-fixing microbes,” she explains.

For most of human history, a lot of the nitrogen that crops needed was acquired through nitrogen fixation, whether directly or indirectly. Direct nitrogen fixation comes from soil microbes, which convert nitrogen gas in the air into forms that plants can use. Legumes famously form such close symbiotic relationships with some of these microbes that nodules are formed on the roots, where the microbes can turn atmospheric nitrogen into ammonia for the plants. 

Plants also access nitrogen from inorganic sources (fertilizer) and organic sources (like manure). Still, the origin of the nitrogen in the manure ultimately comes from soil microbes. Animals acquire nitrogen through the food they eat, and it was soil microbes that fixed the nitrogen for those plants that the cows ate in the first place. 

This is still a hot area of research; scientists are still trying to better understand how much nitrogen plants get in a natural system and how much, if any, comes from these indirect, inorganic processes like mineral weathering versus nitrogen-fixing microbes. Regardless, the nitrogen plants get from these natural processes is generally small compared to the amount of nitrogen growers apply to their fields each season.

Sound scientists wanted to directly measure the amount of atmospheric nitrogen that is incorporated into plant biomass when SOURCE is applied. While it’s relatively straightforward to measure the nitrogen in the plant or applied to the soil, understanding how much nitrogen comes from N‑fixing bacteria alone is a challenge. Tissue testing can show how much nitrogen is in a plant’s tissue, but it doesn’t tell growers how much nitrogen was available to the plant or whether that nitrogen came from nitrogen-fixing microbes or fertilizer. 

Common methods of trying to measure nitrogen fixation are very indirect, measuring features associated with nitrogen fixation rather than nitrogen fixation itself. The first method is an acetylene reduction assay, which shows that the enzymatic activity associated with nitrogen fixation increased but doesn’t reveal how much nitrogen the plant got through that process. Another method is to look for nitrogen-fixing microbes in the soil and then test the gene expression of those fixers, but this test reveals very little about the impact on the plants themselves; nitrogen fixation might be active in soil microbes, but how much actually makes it to the plant?

Enter 15N, a stable nitrogen isotope. An isotope is a form of element where the atoms have the same number of protons but a different number of neutrons. Elements are defined by the number of protons in an atom; all hydrogen atoms have one proton, all oxygen atoms have eight. However, the number of neutrons an atom has can vary. The most common form of naturally occurring nitrogen is nitrogen-14 or 14N; it has seven protons and seven neutrons. The only meaningful difference between it and 15N is that 15N has an extra neutron. 

“15N is the exact same atom, it’s just slightly heavier but in a way that doesn’t affect any of its chemical properties,” says Alex. ​“It behaves exactly like normal nitrogen.” 15N isn’t harmful or radioactive, just a bit bigger, a lot rarer, and most importantly, it’s detectable. 

Over 99% of the nitrogen on earth is 14N. The rarity and stability of 15N makes it ideal for nitrogen tracing — if researchers introduce 15N to a system, they know that any 15N that they find came from the introduced nitrogen. For nitrogen tracing, 15N is referred to as a ​“labeled isotope.”

“There is absolutely no ambiguity when doing 15N testing,” says Alex.

“What separates this 15N experiment from other tests is that it’s a direct linkage between nitrogen fixation and nitrogen being stored in the plant itself,” adds Iris. 

That’s why the Sound team’s 15N test is the gold standard of nitrogen fixation tracing. The study allowed them to track the labeled nitrogen from the air all the way into the plant tissue and know that nitrogen fixation is how it ended up there.

To control exactly how much 15N gas each plant received and to limit any other confounding variables, the plants were grown in sealed, air-tight containers. Iris says one of the things she wanted to find out was if she could successfully grow a plant in a completely closed system. 

“You couldn’t give them any supplemental water, and it can get pretty humid in there,” she says. ​“I had to figure out how to regulate the moisture and irrigation without opening the jar back up.” 

The team inoculated the sterile substrate with nitrogen-fixing bacteria and planted the corn into pre-autoclaved quart-sized mason jars. They also had a soybean plant, grown in the same sterile system as a positive control. 

“Because there’s a symbiotic relationship between legumes like soy and nitrogen-fixing bacteria, with this system, we know that there was nitrogen fixation occurring,” she explains. 

After the plants had grown their first true leaves, a foliar application of SOURCE was administered to the test plants. The same day, air was vacuumed out of each jar and replaced with the 15N gas. Once the plants had grown some more, tissue was sent off to a third-party lab for analysis.

“It turned out that there was about the same proportion of 15N and 14N in each plant,” Alex says. ​“But the SOURCE treated plants were bigger than the untreated plants, which meant they had more total 15N.”

“We saw approximately 40% more nitrogen in the SOURCE treated plants,” adds Iris.

For growers, size does matter. Nitrogen is a key nutrient for increasing plant and leaf size and an important component of chlorophyll; nitrogen deficiency often results in reduced growth and yellowed leaves. Bigger, greener SOURCE-treated plants suggest that even if the proportion of 15N and 14N is the same, the plants are benefiting from an overall increase in nitrogen availability.

While the results were encouraging, what’s just as exciting for Iris and Alex is the experiment itself. It demonstrated that nitrogen from the air is being used by the plants, and while it’s well known that this happens with legumes, it doesn’t generally happen in cereal crops like corn. Additionally, using nitrogen gas in a closed system to measure nitrogen fixation just isn’t how 15N is commonly used in agricultural science, even when it comes to nitrogen fixation. 

Sometimes, 15N is used as the nitrogen in a fertilizer. The isotope is then tracked to measure things like conversion or denitrification and occasionally even nitrogen fixation. ​“But it’s just a much less robust method,” Alex says.

In these studies, researchers extrapolate how much nitrogen in the plant came from the air versus the fertilizer, rather than measuring it directly. In contrast, the 15N gas experiment is a closed system, with labeled nitrogen and bacteria added in controlled amounts. This allowed Sound’s scientists to track the 15N in the plant with a much higher level of confidence, linking the 15N in the plant directly to the 15N gas. 

“Sound is the only company in the biological or biological-adjacent space to have done this kind of experiment and it is publicly available information,” says Iris. ​“It takes away that ambiguity with a direct line of evidence of nitrogen fixation. In the agricultural research space, that was pretty huge.” 

Iris says that as a research scientist, it can be easy to get sucked into the lab work and end up forgetting about the real-world impact for growers. Doing experiments like this one, she is reminded of how impactful this research can be to agronomists and growers. 

“I was born and raised in Indiana, and I think about all the farms I grew up around and how easy it is to disconnect them from the work I do as a scientist,” she says. But as more information is revealed about the mechanisms at work within plants and the soil, Iris may soon be driving past fields that her work has impacted. 

“For me, the coolest part was unveiling this information, and knowing how excited our team was to share what we learned with growers.”