Can ancient bacteria help solve one of agriculture's biggest challenges?

In search of a natural replacement for synthetic fertilizers, South Dakota State University is
leading research that aims to harness the power of ancient — even mystical — bacteria.

During the Archaen Eon — roughly 4 billion years ago — the Earth was a lifeless planet. The atmosphere lacked oxygen, and there were few, if any, organisms to be found anywhere on the globe. Then something incredible happened. Microscopic bacteria appeared in freshwater lakes, likely in what is now present-day Australia. These bacteria — known now as cyanobacteria — had the near magical ability to convert sunlight and water into oxygen and other chemical energy. This systemically changed the Earth's atmosphere over the next billion-plus years, creating the conditions needed for most forms of life that now exist on our planet.

Located in a quiet, refrigerator-like lab on the second floor of South Dakota State University's Edgar S. McFadden Biostress Laboratory sits hundreds of petri dishes full of cyanobacteria samples. Despite the importance of these ancient organisms in creating our world, humanity has not taken full advantage of the practical opportunities afforded by these incredible bacteria, says professor Ruanbao Zhou.

"More than 2.8 billion years ago, tiny cyanobacteria did something truly extraordinary. They used sunlight to split water and release oxygen. In doing so, they turned a lifeless planet into one that could breathe," Zhou said. "Today, these same solar-powered, nitrogen-fixing microbes may once again help us reshape our world."

Zhou is the director of the National Science Foundation-backed BioNitrogen Economy Research Center. Through a collaborative effort from SDSU, South Dakota Mines, University of South Dakota, Oglala Lakota College and Houdek, South Dakota's top biologists and scientists are working to harness the power of these ancient organisms.

Ammonia and agriculture

Nitrogen is an essential colorless and tasteless gas that makes up 78% of the Earth's atmosphere. It is also, in the form of ammonium nitrate fertilizer, required for large-scale crop production. To keep up with the demands of the world's agriculture production, more than 130 million metric tons of ammonia is used globally for nitrogen fertilizer. This number is only expected to increase as the world's population — and its demand for food — grows.

Ammonia, a nitrogen-rich compound, is currently produced through a century-old, energy-intensive process known globally as the Haber-Bosch process. Developed by two German chemists who eventually won Nobel Prizes for their work, this process, which converts atmospheric nitrogen into ammonia through a reaction with hydrogen, greatly increased the amount of food farmers could produce. The mass production of ammonium nitrate fertilizer enabled the Earth to feed an additional 2 billion to 3 billion people, researchers estimate.

But the process also created problems. Because natural gas — a fossil fuel — is required to create the reaction, ammonia production is one of the largest generators of gases that trap heat in the atmosphere. Approximately 1.8 tons of carbon dioxide are emitted to produce just one ton of ammonia. Ammonia production accounts for around 1.2% of global emissions, per the World Economic Forum. Ammonium nitrate fertilizers, a type of synthetic fertilizer, also cause serious problems for water resources. As excess fertilizer runs off into lakes, rivers and streams, the nitrogen can create a phenomenon known as eutrophication, which results in toxic algae blooms, pollution and the death of aquatic life.

It’s not just the environmental issues that are problematic either. In recent years, costs for synthetic fertilizers have skyrocketed, and the American Farm Bureau Federation expects prices to continue to climb, thanks in large part to export restrictions, the rising cost of natural gas and geopolitical instability.

Finding an alternative method for producing ammonia has become a global priority. While some researchers are focused on refining the Haber-Bosch process to make it less energy-intensive, Zhou is taking a different approach. He believes his research team can produce sustainable nitrogen-rich fertilizers by genetically engineering cyanobacteria — bypassing the Haber-Bosch process altogether.

Cyanobacteria: Where are they and how do they work?

Three years ago, Zhou and his SDSU co professor Lan Xu and Liping Gu, were deep in Badlands National Park in search of something too small for the naked eye to see. They were hunting for microscopic cyanobacteria that survive in drought-like conditions. On Badlands' colorful rock formations, he found what he was looking for. Zhou collected samples from the rocks and brought them back to his lab. As he explains, cyanobacteria can be found nearly everywhere — from the Artic ice to hot springs to the gut of a bison — and different strains of bacteria have different characteristics. Zhou is interested in the strains that have the "ancient brilliance" to fix nitrogen using sunlight as the sole energy source.

Analyzing the Badlands samples back in Brookings, Zhou can see the "magic" of cyanobacteria at work. Underneath the microscope, cyanobacteria appear as long, worm-like structures segmented by cells (known together as a filament). When a nitrogen shortage is sensed, the bacteria will signal itself to form a specialized cell-type called heterocyst to fix nitrogen. Heterocyst, one of the segmented cells, soon become noticeably larger and more rounded than the others. Heterocyst creates a mirco-oxic condition and activates the enzyme nitrogenase. This creates an oxygenless "nitrogen conversion facility" in the cell. These specific conditions allow for nitrogenase to convert atmospheric nitrogen into a useable form, like ammonia, through biological nitrogen fixation.

"It's pretty amazing that cyanobacteria can count," Zhou said. "Normally, approximately 10% of the cells selected becoming heterocysts that are present singly at semiregular intervals along the filaments, forming a spacing pattern. By sequestering nitrogenase within heterocysts, this cyanobacterium can perform, simultaneously, oxygenic photosynthesis and the oxygen-labile nitrogen fixation, under the room temperature with 1 atmosphere pressure (atm) while the Haber-Bosch process requires between 400 and 550 degrees Celsius with 150-200 atm."

This process, however, does not generate enough useable nutrients for large-scale crop production. To counter this challenge, Zhou and his researchers are identifying the key genes responsible for nitrogen fixation by utilizing artificial intelligence models and genomic sequencing.

"The primary players in nitrogen fixation are the nif genes, which collectively encode the complex of the nitrogenase," Zhou said.

Cutting-edge technologies, like synthetic biology tools combined with artificial intelligence models, allow Zhou and his research team to create more powerful strains of cyanobacteria, allowing for improved efficiency and nitrogen fixation productivity.

"Genetic engineering is being used to introduce new traits such as improved photosynthesis efficiency and resistance to environmental stresses to cyanobacteria biofertilizers as well," Zhou said.

From lab to field

In an on-campus greenhouse, Zhou has conducted various experiments comparing wheat plants grown with different cyanobacteria-fertilizer strains and standard synthetic fertilizers. Without the labels on the pots, it would be difficult to discern which plant used which fertilizer. All reached similar heights and had similar looks — a signal to Zhou and the research team that cyanobacteria are a viable option for biofertilizers.

In another greenhouse, Lin Wei, professor in SDSU's Department of Agricultural and Biosystems Engineering, is building the equipment needed to scale-up the production of biofertilizers. In lab space, cyanobacteria are grown in photobioreactors — a closed box-like system that allows the researchers to control light, gases and nutrients. But these photobioreactors are not nearly big enough to grow the amount of cyanobacteria needed for farmers to use in their fields. Instead, Wei and his team of graduate student researchers built what they call a "raceway pond" photobioreactor in the greenhouse.

"Lab-scale photobioreactors only have the capacity for 15 to 20 liters," Wei said. "We need to mass produce cyanobacteria, so we built our own photobioreactor."

The raceway pond, which has the same oval shape as a racetrack, has the capacity for around 400 to 500 liters. Wei then selected a cyanobacteria strain and grew it in the raceway pond for three months. To harvest the algae-like biomass, Wei and his team designed and built a harvesting machine.

"We built a harvester with a rotating drum," Wei explained. "The machine also has a separator, washing system and a vacuum drum component that will allow for the biomass to be dried."

Coined the "Cyanobacterial Biomass Harvester," it greatly enhances the efficiency in which cyanobacteria can be turned into a useable product for producers. The dried biomass, which will have a greenish hue, will end up like a powder in its final product that can be spread over crop fields. Rather than spreading ammonium nitrate fertilizer in the fields two or three times during the growing season, the powder — full of cyanobacteria — can be applied once to the field and will naturally fix nitrogen from the air, while providing the soil with the nutrients needed for
plant growth.

"Through their activity in the surface of soil or surface of plant leaves, nitrogen-fixing cyanobacteria release bioavailable nitrogen into the soil, creating a more favorable environment for crop growth and improving overall soil health," Zhou said. "These living, tiny fertilizer factories benefit both current and subsequent crops, fostering long-term soil fertility and sustainable agricultural productivity."

This biofertilizer may be especially impactful in regions with nitrogen-deficient soils, Zhou notes.

"Cyanobacteria provides an effective, environmentally-friendly solution for enhancing nutrient availability and optimizing yields where conventional fertilizers may be limited or economically unfeasible," Zhou said.

The economic potential for these biofertilizers is surprising. The global ammonia market size is projected to reach $313 billion annually by 2030. If cyanobacteria-based bioproducts can capture just 1% of the market, they would be worth approximately $3 billion.

"I am incredibly proud of Dr. Zhou and his team, who are leading the BioNitrogen Economy Research Center," said John Blanton Jr., director of the South Dakota Agricultural Experiment Station and the associate dean for research for SDSU's College of Agriculture, Food and Environmental Sciences. "The vision to grow the bionitrogen economy with both sunlight and biological innovation has the potential to lower costs for producers, create new biomanufacturing industries, and open high-quality STEM career pathways for South Dakotans. This is exactly the kind of bold, collaborative research that can strengthen our rural communities and position South Dakota as a national leader in sustainable agriculture and the bioeconomy."

Beyond the field

Fertilizer production has been one of the first focuses of the BioNitrogen Economy Research Center project, but Zhou believes cyanobacteria can make an impact far beyond corn and soybean fields. He likes to point to the makeup of the Earth's atmosphere and the untapped potential of cyanobacteria to underline the future possibilities.

"Sunlight and nitrogen gas are the two most abundant resources," Zhou explained. "Nitrogen gas is 2,000 times more abundant than carbon in the atmosphere."

Despite the abundance of nitrogen, humans have focused on carbon and have centered our world around a carbon-based economy, which relies almost exclusively on the availability of cheap nonrenewable fuels. The reality is that these fuels are a finite resource and eventually, humans will need to find alternative sources for fueling the economy. Zhou believes we can shift to a more abundant element — nitrogen — and create a biosolar nitrogen economy, centered on the near boundless resources of sunlight, nitrogen gas and cyanobacteria.

“When we team up with these ancient, amazing microbes—and mix nature with modern engineering—we can build a solar-powered bionitrogen economy” Zhou said. “That means greener industries, healthier soils and a planet that stays clean, vibrant and full of life for the next generation.”

One of the more interesting future-focused developments is the team's research into biofuels. Using a synthetic biology approach, Zhou's research group has already developed cellular "cyanofactories" (genetically engineered cyanobacteria) that are able to convert air and water into fuel molecules — such as limonene and linalool for jet fuel — using sunlight.

Cyanobacteria also produce hydrogen gas as a byproduct of biological nitrogen fixation. While still in the very early stages of development, the hydrogen gas could eventually be combined with other elements or chemicals to create a relatively clean biofuel — one that could even power airplanes and potentially reverse the world's dependence on fossil fuels.

"Just as cyanobacteria once transformed the Earth, they can inspire us to transform it again," Zhou said. "Toward a brighter, greener, and more sustainable future for all."