Nitrous oxide (N2O) is the third-biggest contributor to global climate change, with each molecule having nearly 300 times the atmosphere-warming power of a molecule of CO2. Most of the global N2O emissions come from the agricultural application of synthetic nitrogen fertilizers.
As a part of the planet’s natural nitrogen cycle, N2O can be broken down (reduced) by soil microbes to harmless dinitrogen gas (N2). Understanding the kind of microbes capable of that chemistry, and how they grow, will go a long way toward predicting greenhouse gas emissions from agricultural soil.
Unfortunately, the use of synthetic fertilizers makes soils more acidic, which in turn leads to greater release of N2O—and, it was long believed, deactivation of the catalysts (enzymes) that break down the greenhouse gas.
“One of the unresolved problems in soil science is that the known microorganisms that convert N2O to N2 don’t do it at acidic pH,” said Frank Loeffler, a professor and Goodrich Chair of Excellence in the Department of Civil and Environmental Engineering.
A few years ago, working with Kostas Konstantinidis from the Georgia Institute of Technology, Loeffler investigated soils from El Yunque National Forest in Puerto Rico, a tropical rainforest where the soil pH is 4.5—about as acidic as tomato juice.
Within the samples, they found genes that suggest that the native microbes have the potential to consume N2O. However, the presence of those genes does not guarantee that N2O consumption is occurring in the soil, and the researchers were unable to find the responsible microbes.
When postdoctoral researcher Guang He (’23) began his PhD research under Loeffler’s guidance, this puzzle nagged at him, too.
“That was the gap,” He said. “I wanted to prove that if these microbes exist in acidic soil, they must be functional there.”
“Guang knows a lot about nitrogen cycling, has strong computational skills, allowing him to analyze large genomic and metabolomic (omics) datasets, and has the knowledge and patience to cultivate fastidious bacteria in the laboratory,” Loeffler said. “Knowing that, I challenged him to find organisms that reduce N2O at acidic pH.”
Years of Microbial Cultivation
Initially, He’s goal seemed simple enough: identify a microbe in the acidic soil that could reduce N2O.
The process took him more than two years.
“I don’t even remember how many different growth conditions I tested,” He said, “but I remember that I had been testing different medium recipes over a 16-month period before one finally worked.”
However, as soon as He transferred the microbes to fresh medium with the same nutrients, the N2O consumption stopped.
“I was very discouraged,” He said. “I was about to set up a Plan B for my PhD program.”
With encouragement from Loeffler, He kept at it. After several more months, he found the recipe that resulted in consistent N2O consumption.
“For this kind of cultivation-based study, you need to possess both patience and impatience at the same time,” He said. “Without patience, I would not have altered the medium recipe over years until I achieved growth of the N2O consumers. Without impatience, I would not have checked the cultures six times per day—so I would have completely missed the minor, transient features that told me what was needed to succeed.”
Combining Cultivation and Cutting-Edge Bioinformatics
The N2O-consuming microbe in He’s samples turned out to be a previously unknown species, a bacterium that he and his colleagues have dubbed Desulfosporosinus nitrosoreducens.
Unlike most known N2O consumers, D. nitrosoreducens has specific nutritional requirements that must be filled by other microorganisms. This is why finding the right conditions to sustain N2O consumption proved so difficult: although He did not know it initially, he was establishing a co-culture in which another microbe supplied D. nitrosoreducens with essential nutrients.
To convert N2O to N2, the new bacterium also requires hydrogen gas—as well as a complete lack of oxygen.
“It sounds trivial, but very few laboratories have the understanding, patience, and skill sets required to handle strict anaerobic bacteria,” Loeffler said. “With modern omics approaches, you just extract and sequence all DNA in a sample, and then make sense of the data using bioinformatics. That timetable is more attractive to many student researchers than anaerobic cultivation experiments, which can last for many weeks.”
Loeffler and He believe that emphasis on omics studies over cultivation is holding back the discovery of N2O reducers in many environments. They hope their discovery of D. nitrosoreducens will demonstrate that combining these techniques is an effective way to move soil science forward.
“We integrated more classical techniques with cutting-edge omics and bioinformatics approaches and made a new discovery,” Loeffler said. “Sometimes there are challenges, but if you give up too early, you will not get over the hurdle and you will not make a new discovery.”
Contact
Izzie Gall (865-974-7203, egall4@utk.edu)