Nitrogen is necessary for the existence of life as we know it. It is an important component of essential biomolecules, including DNA and proteins. It is also the most abundant element in the atmosphere. The element cycles between the atmosphere, Earth's surface and living organisms-similar to how water is ferried from the atmosphere onto Earth, and ultimately back again. Atmospheric nitrogen exists mainly in the form of nitrogen gas (N2), which is unavailable to the majority of living organisms, including plants and animals. However, nitrogen gas can be made useful by nitrogen-fixing bacteria, which use special enzymes to transform nitrogen gas into nitrogen oxide (NO) and dioxide (NO2) in soils. These gases can be further transformed into other nitrogen-containing compounds such as nitrate (NO3-) and ammonium (NH4+), the latter of which plants can use for growth. Without sufficient nitrogen, plants cannot build essential photosynthesis pigments like chlorophyll.
In addition to useful nitrogen products, such as NH4+, some more problematic gases can also be formed during nitrogen cycling, including the potent greenhouse gas nitrous oxide (N2O) familiar to many as 'laughing gas.' Nitrous oxide is produced as an intermediate during de-nitrification, a process where microbes turn nitrate in soils into nitrogen gas that is released back into the atmosphere. The majority of nitrous oxide emissions worldwide are caused by agriculture. Most are attributed to nitrogen fertilizers that are essential for sustaining the level of food production required to feed the planet. This tension underlines a need to discover ways to reduce emissions without compromising crop yields and food security.
Mining Organic Waste for N2O-Respiring Bacteria
Researchers are turning back to soil bacteria for answers, and they may have found a prime candidate for the job. Cloacibacterium sp. CB-01 is a non-denitrifying nitrous oxide-respiring bacterium-which means it can produce nitrogen gas from nitrous oxide, but cannot perform any other steps in the denitrification process. This type of metabolism is important because it increases the potential of CB-01 to act as a sink for nitrous oxide: the bacterium can avoid becoming metabolically distracted and funnel more cellular resources into this desirable reaction.
Many bacteria contribute to the nitrogen cycle at different stages, but there is only 1 enzyme known to produce nitrogen gas from nitrous oxide, called NosZ, which CB-01 encodes. However, CB-01 is not the only nitrous oxide-respiring bacterium known to science, nor is it the best. The researchers compared CB-01 to a panel of 18 other strains that also encode enzymes for nitrous oxide reduction and found that CB-01 comes out in the middle of the pack. Still, CB-01 has something that the others do not-incredible survival skills.
Unlike many other nitrous oxide-respiring strains, CB-01 is particularly fit for life in soil. The strain was originally isolated in an experiment that was designed to farm hardy, nitrous oxide-respiring bacteria from biogas reactor waste, where it dominated over other strains. Biogas reactor waste is particularly suited for this purpose as it is often nitrogen-rich, harbouring ideal conditions for discovery of bacteria involved in nitrogen processing. CB-01 grows well on this biogas reactor waste, possibly due to its ability to attach itself and feast on sugar molecules found in the waste, alongside carrying out nitrous oxide respiration.
Applications for N2O-Respiring Bacteria: From Lab Bench to Buckets and Fields
After quantifying CB-01's ability to turn nitrous oxide into nitrogen gas by measuring the concentration of those 2 gases (among others) during growth in airtight vials, the researchers introduced the bacteria to a new challenge. They inoculated CB-01 into buckets that contained varying types of soil, along with ryegrass seeds to simulate crop growth, and the biogas reactor waste that CB-01 likes to eat. Next, the scientists monitored nitrous oxide emission from the buckets filled with different types of soils over a 90-day period. They also re-fertilized the buckets at intervals to produce transient surges in emissions. This work revealed that CB-01 could reduce nitrous oxide emissions from the buckets by as much as 94% compared to control buckets fertilized with dead CB-01. The effect of CB-01 declined over time, showing that even this hardy bacterium cannot last forever, although it remained more abundant in the buckets for longer than in a laboratory incubation under similar conditions.
Following these promising results, the researchers moved on to a field plot experiment, again fertilizing the soil with CB-01 and biogas reactor waste. Here, CB-01 was most effective early on, with its effect on nitrous oxide emissions waning throughout the 280-day experiment. At best, CB-01 could reduce soil emissions by 64% compared to the control without CB-01. The researchers attributed this decline in performance, compared to the soil bucket experiment, to unusually low soil temperatures during the field experiment, highlighting how weather conditions could impact the effectiveness of these kinds of interventions.
Some concerns that need to be addressed before microbes are deployed to help manage farmland emissions include, unwanted effects on other microbes already present in the ecosystem, as well as the potential of these newly introduced nitrous oxide-respiring bacteria to contribute to antibiotic resistance or to cause disease. However, the researchers were unable to identify effects on the native soil microbiota, nor could they find any antibiotic resistance or pathogenicity genes in CB-01's genome-suggesting that this bacterium has potential as a deployable microbe in agricultural settings.
Despite its promising activity, the researchers acknowledge that more strains like CB-01 will need to be discovered, and there is a need to identify species that can thrive on other materials besides biogas reactor waste, which may not always be readily available in different areas. There is also a need to identify species that can tolerate different kinds of stresses that may be encountered in various soil types and environments. Much is still unknown about how these nitrous oxide-reducing bacteria live in the environment-for example, it is still unclear whether close interactions between nitrous oxide producers and consumers are important for determining net nitrous oxide emissions from soils.
It is becoming increasingly clear that many of the potential solutions to current challenges across society, including farmland greenhouse gas emissions, are microbial. Many agents, such as CB-01, have only recently been discovered and described, and many more lie just out of reach, their potential still unknown and untapped. This makes the work of discovering and isolating such microbes from all kinds of habitats incredibly important to solving some of our time's most pressing challenges. Indeed, bacteria are also being investigated as a replacement for widely used synthetic fertilizers, reducing costs to farmers and CO2 emissions from the resource-intensive production of chemical fertilizers.
Learn more about how microbial probiotics in agriculture, such as biofertilizers and biochar, promote crop health and global food security. Read this next article, Microbial Biofertilizers to Bolster Food Security, from our Spring 2025 issue of Microcosm, our flagship member magazine! Not a member? For a limited time, ASM Membership is 50% off for the remainder of the 2025 membership year!
