Inspired by nature, SDSU scientists engineer tiny 'nitrogen factories' that could help future cereal crops

A new study from South Dakota State University demonstrates how hydrogel beads create a microenvironment needed for naturally supplying nitrogen to crops. 

As fertilizer prices remain elevated, researchers at South Dakota State University are developing an innovative solution to corn's nitrogen supply problems by taking inspiration from an ancient Mexican variety and root nodules found on soybeans. In a new study, published in Plant and Soil, the researchers demonstrate how hydrogel beads may act as a promising solution in naturally supplying plants nitrogen.

Nitrogen is a key building block for life and is the most common gas in our atmosphere. While life on Earth is almost entirely dependent on nitrogen, only a tiny percentage of organisms can actually convert the gas into a usable form needed, in the case of plants, for making food from sunlight or growing new cells.

Researchers around the world have been working to leverage the ability of these select few organisms to naturally fix nitrogen into useable forms for crop production. The goal is to reduce the need for synthetic nitrogen fertilizers, which are not only environmentally harmful but are skyrocketing in costs, creating financial instability for farmers. 

Microenvironments

In the highlands of Oaxaca, Mexico, scientists observed, first in the 1980s, how a specific variety of corn was able to grow in nitrogen-deficient soils. This was a considerable discovery and was later described in more detail in a 2018 study.

Previously, scientists had observed how leguminous crops, like soybeans, are able to naturally fix nitrogen through their roots. The key to this biological nitrogen fixation process is the relationship between free-living bacteria — known as diazotrophs, which are one of the tiny percentages of organisms that can convert nitrogen — and oxygen. In order for bacteria to convert nitrogen into a useable form, an enzyme — known as nitrogenase — must be stable enough to split nitrogen atoms to create molecules of ammonia, releasing hydrogen as a coproduct.

Nitrogenase is highly sensitive to oxygen. When exposed, bacteria are limited in how much nitrogen they can fix. What allows soybeans to successfully convert nitrogen from the air is their root nodules. Small nodules grow on the plant's roots, which work in tandem with bacteria to create ammonia in a sort of oxygen-free nitrogen-conversion laboratory in the soil.

In Mexico, scientists observed a similar system. During certain times of the year, the corn's aerial roots secreted a gel-like substance that provides a low-oxygen microenvironment required to attract the type of bacteria that can convert nitrogen into ammonia from the air.

"The observations from maize landraces in Mexico demonstrated that sustained biological nitrogen fixation is possible when beneficial microorganisms are provided with the right physical and chemical environment," said Srinivas Janaswamy, associate professor in SDSU's College of Agriculture, Food and Environmental Sciences.

For Janaswamy and Senthil Subramanian, dean of SDSU's College of Natural Sciences and a co-investigator on the study, this presented an interesting idea. While other research teams would be looking to breed these specific traits into new varieties of corn, they would take inspiration from nature and utilize Janaswamy's expertise in biodegradable packaging to create a sort of microenvironment that would mimic the low-oxygen conditions needed for nitrogenase activity — similar to soybean's root nodules.

“One of the biggest challenges in using free-living nitrogen-fixing bacteria in agriculture is protecting the nitrogenase enzyme from oxygen while still allowing the microbes to remain active," Subramanian said. 

"Rather than using encapsulation simply to deliver beneficial bacteria, we designed biodegradable hydrogel beads to create the oxygen-regulated microenvironments these microorganisms need to convert atmospheric nitrogen into plant-available forms," Janaswamy said. “Our work builds on that concept by engineering biodegradable hydrogel microenvironments that could potentially extend this capability to conventional cropping systems.”

Hydrogel beads

To build their version of a root nodule, Janaswamy and Sen turned to alginate — a natural, biodegradable polymer derived from seaweed that's already widely used in food packaging, wound dressings and slow-release drug delivery. The team's method was straightforward in concept: drop a solution of alginate into a bath of dissolved metal salts and nitrogen-fixing bacteria, and the alginate instantly crosslinks into small, gel-like beads.

The researchers tested six different metals for the gels — calcium, strontium, zinc, nickel, copper, and aluminum — and two nitrogen-fixing bacteria — Azospirillum brasilense and Herbaspirillum seropedicae — and found that beads made with calcium consistently performed best. Calcium beads had the lowest oxygen levels of any formulation tested, encapsulated more than 70% of the bacteria added to them, and kept those bacteria alive longer than beads made with other metals.

Most importantly, the calcium beads didn't just protect the bacteria from oxygen. They let the bacteria keep working. When researchers measured nitrogenase activity inside the beads, the calcium-encapsulated bacteria performed comparably to bacteria grown in ideal laboratory conditions specifically designed to support nitrogen fixation.

“Nature already provides examples of microorganisms fixing nitrogen efficiently when they live in the right microenvironment, "Janaswamy said. "Our research demonstrates that biodegradable hydrogels can recreate these conditions, supporting biological nitrogen fixation by maintaining the environment required for the nitrogenase enzyme to remain active.

The team also tested whether feeding the bacteria extra sugar — a source of energy — would boost the nitrogen-fixing output. The results were mixed: added glucose significantly boosted nitrogenase activity in Herbaspirillum seropedicae, more than doubling it in some cases, but had the opposite effect in Azospirillum brasilense whose activity dropped when sugar was introduced. The mixed results underline the innate challenges and nuances of working with these specialized bacteria.

A critical component of the beads is their ability to biodegrade in the soil. The team found the beads break down gradually over 120 days, demonstrating the encapsulation technology's sustainability feature.

Future work 

The hydrogel beads — essentially tiny nitrogen factories — are a proof of concept for maintaining nitrogenase activity in the soil, which would facilitate nitrogen supply to crops like corn and wheat. Future studies will bring this research into the field to determine if the beads can fix nitrogen in true soil and plant growth conditions.

"Our laboratory findings have laid the foundation for field evaluation of these engineered microenvironments," Janaswamy explained. "If they perform as expected under agricultural conditions, they could become an important tool for enhancing biological nitrogen fixation and reducing dependence on synthetic nitrogen fertilizers."

As the global fertilizer market continues to fluctuate, the hydrogel beads represent a promising step forward in finding natural ways to supply nitrogen to crops. 

“This research exemplifies how interdisciplinary science can address some of agriculture's most pressing challenges," Subramanian said "By bringing together expertise in microbiology, plant science and biomaterials, our team is advancing new approaches that could help make crop production more sustainable while reducing reliance on conventional nitrogen fertilizers."

Funding for this research was provided by the National Science Foundation, the National Institute of Food and Agriculture, and the South Dakota Nutrient Research Education Council. Contributing authors include Prashant Dahal, a former SDSU graduate research assistant and currently a laboratory manager in North Carolina State University's Department of Food, Bioprocessing and Nutritional Sciences.