Raising the pH solves the problem of N2O greenhouse gas emissions

by Pawel Lycus

For the last decades, our population multiplied more rapidly than ever before, what have resulted in the growth in food demand. Modern agronomic practices have greatly improved crop production per unit land entailing excessive application of nitrogen (N) fertilizers to soils over the past century. Providing more nitrogen, than can be utilized by plants results in reactive N leaching to environment, thus causing its pollution.

One feet under – respiration in the absence of oxygen

Soil is a peculiar ecosystem, where the oxygen concentrations change rapidly and nutrient availability is generally low, still, soil is densely colonized by microorganisms, who, as all living creatures have to respire in order to provide energy needed for life processes and microbes prefer aerobic respiration (utilizing oxygen as an electron acceptor). However, when experiencing oxygen limitations, microbes are able to gain energy by exploiting other electron acceptors, and nitrate is the most favorable one. The step-wise reduction of soluble nitrate and nitrite (NO3 and NO2) through gaseous intermediates nitric oxide and nitrous oxide (NO and N2O) to dinitrogen gas (N2), called denitrification (Fig.1) is an alternative respiratory pathway in microorganisms and drives the nitrogen cycle in soil.


Figure 1. Denitrification pathway. NAR, NIR, NOR, N2OR are the enzymes catalyzing following steps of nitrate reduction to dinitrogen gas. NO, N2O and N2 are gases, and therefore can escape the soil and reach atmosphere.

Backstage greenhouse gas nitrous oxide

Nitrous oxide gas, the intermediate product of denitrification has a great global warming potential, 300 times greater than CO2 and together with methane, these three are the most dangerous greenhouse gases. Although its threatening nature and quick rise of emission (Fig.2), the N2O has been paid not much attention until recently. The total N2O emission from agricultural soils is assigned to 50% of the estimated global N2O emission. Soil denitrification, furthermore, is the main source of N2O that affects the stratospheric ozone layer, thus contributing to its depletion. In order to develop mitigation strategies for N2O emission from anthropogenic ecosystems we need to improve our understanding of processes involved in its production and neutralization.


Figure 2. N2O greenhous gas emission through the last decades. (Earth System Research Lab.)

Soil pH drives the N2O emissions

Due to intensive agronomics and overuse of fertilizers, many of soils are undergoing progressive acidification, and those are yet understudied, although available empirical data assert that these soils are the main source of N2O emissions. Soil microbial emissions of N2O depend on several environmental factors, like: soil N, carbon (C), pH, temperature, oxygen supply and water content, as well as the structure of microbial community (who is there), however, the overall mechanism has not yet been comprehended. Soil pH among others is the key driving factor that affects N2O:N2 product ratio of denitrification process, the lower the pH the more N2O is being released to the atmosphere, without being transformed to N2 (Fig.3). Intra-scales examinations of model organisms, indigenous bacterial communities and soil ecosystems indicate that the phenomenon is more general and pH steers the overall emission of N2O.


Figure 3.The N2O/(N2O+N2) product ratio of denitrification and its dependence on the pH of soil. (Collaborative work of NMBU Nitrogen Group)

Problem of EU concern

The arising problem concerning antropogenic N2O emission attracted the attention of EU authorities, and the Marie Curie ITN Project NORA (Nitrous Oxide Research Alliance) has been founded in the year 2013. The joined forces of top class scientists involved in the nitrogen cycle research in Europe are working together trying to find explanations and solutions for N2O greenhouse gas emissions.

N2O reductase enzyme, the only thing we have

The N2O is a chemically inert gas and the only available weapon that can help us fighting the anthropogenic N2O emissions is the N2O reductase enzyme (NosZ) carried by several microorganisms. The enzyme does perform N2O reduction at the physiological level above 6 pH units, however. In the face of progressing soil acidification the enzyme tends to fail, thus leading to increasing emissions of N2O from these soils. The experimental approach and data provided by NMBU Nitrogen Group has paved the road for the basic understanding of the phenomenon. Researchers from NMBU-NG have been speculating that low pH affects the machinery for correct assembly of the NosZ, thus making the enzyme unable to perform N2O reduction. I joined the Nitrogen Group in 2013 as one of the Marie Curie ITN NORA fellows. My research focuses on the ecophysiology of denitrifying organisms and their roles in N2O emission. Unravelling the enigmas of impaired N2O reductase caused by low pH I work with the model organism in denitrification – Paracoccus denitrificans. My “pet” has been well studied and characterized, and this available knowledge helped me and my advisors to develop a novel approach for resolving “the pH question” concerning N2O reduction at the molecular level, which has a global effect, however. The solution is just around the corner!

Just a Pinch of Copper Against Global Warming

by Maria Conthe

fields of gold

Soil dwelling microbes need copper to stop the release of nitrous oxide, a greenhouse gas, from fields into the atmosphere. (Photo: Stock)

There is more than meets the eye among the fields of barley. Underneath the rows of neatly planted grass, sown and fertilized each year, lies a rich microscopic world, teeming with life. Microorganisms living in the soil are key players in nature’s cycling of nutrients as they grow, metabolize and respire. As such, they have a direct effect on the environment and even the climate.

Enter N2O, short for nitrous oxide, a gas produced and consumed by these microbes in the soil. A part of Earth’s nitrogen cycle, N2O gas is naturally present in very small quantities (in parts per million!) in the environment.


However, like CO2, N2O levels in the atmosphere have increased significantly during the past century.  If the main reason for the increase of CO2 is the use of fossil fuels as an energy source, the main reason for the increase of N2O is the use of synthetic fertilizers to grow crops. The extra input of nitrogen from man-made fertilizers, since they were first developed at the beginning of the 20th century, is altering Earth’s nitrogen cycle. Microbial processes in the soil are affected, and with increasing agriculture to feed the world’s increasing population, N2O levels are rising.

Despite its more common name “laughing gas” (in reference to the euphoric state it induces in users who inhale it as a party drug) nitrous oxide increase in the atmosphere is actually no laughing matter.  N2O is a very strong greenhouse gas, with a global warming potential over 300 times greater than CO2. It is also considered the major ozone-depleting substance of the 21st century.

Important copper

But why exactly does the increased nitrogen turnover in the environment caused by excessive fertilizer use lead to more emissions of nitrous oxide? “In a system as complex as soil, the answer is surely not a simple one” warns Manuel Soriano Laguna, a young researcher at the University of East Anglia (UEA) funded by the European initiative NORA (Nitrous Oxide Research Alliance). However Manuel’s team may have part of the answer, and it involves copper.

In soils and other ecosystems, N2O production is kept in check by N2O consumption. “Some microbes produce N2O as a product of their metabolism, but many can also consume N2O for respiration, much like we respire oxygen,” explains Manuel. “Copper is an essential ingredient of the cell machinery needed for respiration of nitrous oxide. Our research in the lab clearly shows that N2O respiration stops when microorganisms don’t have access to enough copper”.

A simple solution?

Extrapolated to the environment these results suggest that in soils lacking copper, microbes will not be able to consume N2O, leading to its accumulation and emission into the atmosphere. A very serious concern considering that up to 40% of agricultural soils worldwide may be currently copper deficient.

The bright side is that the remedy may be straightforward: “judicious use of copper in fertilizer regimes” says David Richardson, the head researcher at the UEA group. Just a pinch of copper may keep our fields of barley, romantic- and greenhouse gas free- fields of gold.


Have Your Cake and Eat It Too: Bacteria Can Hedge Their Bets When Living in Unpredictable Environments

by Daria Kaptsan

New research suggests that bacteria divide their risk when facing the unknown, by allowing a sub-population to develop alternative pathways to survive.

This new study is an outcome of collaborative work by Marie Sklodowska-Curie ITN NORA partners from the Norwegian University of Life Sciences and the University of East Anglia in the United Kingdom.

The study has been performed in the model soil denitrifying bacterium Paracoccus denitrificans and such bet-hedging is a novel discovered fitness trait.

When oxygen is limited

Generally, bacteria use oxygen, as it is a preferable electron acceptor. When oxygen becomes a limiting factor for growth, the transcription of denitrification enzymes under tight regulation will be initiated. The denitrification enzymes are nitrate-, nitrite-, nitric oxide- and nitrous oxide- reductases. However this process requires a lot of energy and it is uncertain if anoxic condition will last long.

Scientists suggested a strategy that would secure survival under oxygen depletion to the fraction of population that expresses nitrite reductase, and minimize the energy costs for the fraction that does not.

Old hypothesis, new tools

Last decade it has been speculated that only a minor fraction of P. denitrificans population switches to denitrification when oxygen starts to deplete, and to support this hypothesis the dynamic modeling approach has been used. This approach postulates that: “initial transcription of the nirS gene is stochastic with a low probability (r=0.5% h-1), consistent with only a fraction of cells switching to denitrification in response to oxygen depletion”.

To support the hypothesis and to provide the experimental proof, two young researches within NORA group, Pawel Lycus and Manuel-Soriano-Laguna and their PI’s, have developed molecular tools to investigate this phenomenon. The young scientists have used genetic engineering to develop a fluorescent construct. The fluorescent red protein mCherry was fused to nitrite reductase (NirS). The maturation of mCherry is rapid and allows to see the results soon after activating transcription. This way they could capture the small fraction of bacterial population that has expressed anaerobic respiration machinery and to deliver a strong proof of a concept.

Microbe Hunters Cultivate Greenhouse Gas Breathing Bacteria

by Christoph Keuschnig


Monica Conthe is cultivating N2O consuming bacteria at the TU Delft. (Photo: NORA)

A turbid liquid is bubbling in a closed glass pot connected to tubes, pumps and sensors monitoring it’s vital signs. What sounds more like a patient at an intensive care unit of a hospital, is actually a bioreactor containing bacteria situated at the Delft University of Technology. “The bacteria we are cultivating and studying use N2O the same way as we do O2, for respiration” explains Monica Conthe, a PhD student and researcher studying these special group of bacteria “they are the only sink for N2O that we know”.

Laughing Gas

As carbon dioxide (CO2), nitrous oxide (N2O) is a greenhouse gas. The compound, commonly known as “laughing gas”, is increasing in the atmosphere since the beginning of the 20th century, when mankind started using chemical fertilizers to increase crop yields. The IPCC (Intergovernmental Panel on Climate Change) estimated the contribution of one molcule N2O to the greenhouse effect to be 300 times higher than one molecule of CO2. This compensates for the relative low concentrations od N2O measured in the atmosphere and explains why this gas comes more and more into focus of scientists. Unlike for CO2, we are just beginning to understand the reasons for the increase of N2O and prediction studies already called it the “greenhouse gas of the 21st century”.

Where does this increase in N2O come from? “This is rather complex,” says Monica, “as there are different microbial processes that can produce N2O and in some cases the mechanism by which it is produced remain obscure”. Traces of N2O are therefore naturally found in the environment.


With the application of chemical nitrogen fertilizers the input of nitrogen in natural environments has been increased. This disturbs a natural balance between N2O producers and consumers resulting in the emission of the greenhouse gas to our atmosphere.  “Some microorganisms produce N2O, but many others consume it” explains Monica. Instead of using oxygen like we humans do, these organisms can generate energy by “breathing” N2O. The end product of that consummation is N2, which is a natural compound of the air we breathe and a climate neutral gas.

Monica describes her research group as “microbe hunters”. In her case she is hunting bacteria specialized in consuming N2O. This is achieved by a natural selection from a starting material containing all kinds of bacteria.

If microbiologists want to isolate organisms which can grow on diesel or other pollutants, they “feed” a diverse community with the pollutant of interest. Organisms able to consume these compounds have an advantage over the others and grow and proliferate over time.

Monica applied the same principle by feeding her reactor with N2O gas and over time organisms able to breathe it were left over.  “We need to learn as much as we can about these bacteria” she explains further “since they are the only sink of N2O we know”.

Helping the “good” bacteria

A potential mitigation strategy to avoid emissions of this greenhouse gas into the atmosphere would be to favor the presence of N2O consuming bacteria in ecosystems where it is being produced. “Rather than trying to avoid it’s production, why not focus on favoring its consumption?“ The findings from the research at TU Delft can be used to develop strategies for N2O emissions reduction in natural as well as engineered ecosystems.

As an example of such an engineered system, Monica Conthe talks about waste water treatment plants. “If research continues on the topic, I foresee N2O-free wastewater treatment plants in the near future.” Waste water treatment plants are known to contribute to increasing N2O concentrations on our planet.

The outgoing gas streams could be conducted through a bio-filter containing Monicas N2O breathing bacteria to clear it from the greenhouse gas before it exits to the atmosphere. The same principle can be used in other industries where N2O in off-streams is present, like in nylon production.

The bacteria grown and studied in the big glass pots of TU Delft are not the only example where nature provides us a solution for an environmental problem we face. “The tools are out there” Monica Conthe finishes our tour through the lab “we just have to figure out how to use and apply them.“

New EU Project to Reduce Greenhouse Emissions

by Manuel Soriano LagunaDPP_1067Demonstration of the field robot. (Photo: NORA)

Last 2015 was the warmest year on record according to the World Meteorological Organization. This situation has been happening for the last one hundred years since the discovery of the Haber–Bosch process. This is the process of artificial nitrogen fixation, a way of producing fertilizers and a landmark for understanding the actual global population. However, the constant addition of nitrogen-based fertilizers to the soil has an unexpected side effect: global warming. Plants are not able to take all the extra nitrogen that is added to the soil. This excess fertilizer is a fantastic food source for soil microbes that eventually transform them into one of the worst greenhouse gases, nitrous oxide.


The Nitrous Oxide Research Alliance (NORA) is a Marie Sklodowska Curie Initial Training Network (ITN) research project under the EU’s seventh framework programme (FP7). This project aims to generate specific recommendations, strategies and solutions to reduce nitrous oxide emissions. NORA will run for 48 months and has a total budget of five million euros, this money will finance the research of eleven industrial and academic partners from Norway, the Netherlands, the United Kingdom, France, Sweden and Germany.

“Understanding nitrous oxide emissions require an international initiative,” argued Monica Conte and Christoph Keuschnig.

The NORA project has ambitious objectives and as such, it requires a multidisciplinary approach, this is why the partners have been divided in three different work packages: ranging from work package one that studies molecules and genes up to work package (WP) three that studies fields and ecosystems.

(article continues under photo)


Members of NORA at their first meeting in Norway. (Photo: NORA)

Potent gas

“Nitrous oxide is 300 times more potent than carbon dioxide and it can last in the atmosphere for more than 200 years,” said Pawel Lycus, a member of WP01 based at the Norwegian University of Life Sciences. “I will specifically try to characterise denitrification phenotypes with particular relevance to N2O emissions”. On the other hand, WP02 will pay special attention to the optimization of molecular diagnostic tools for the analysis and prediction of functional   characteristics   of   soils   and   wastewater   treatment   systems. Finally yet importantly, WP3 stands out for its huge display of technology. Since traditional manual techniques in the field are work intensive and present a very limited spatiotemporal resolution a completely new approach has been taken. NMBU, in collaboration with ADIGO, has developed a cutting-edge electrically driven automated platform. This robot is expected  to  permit  autonomous  and  continuous  nitrous oxide  measurements  with  an  analytical precision.

On top of everything mentioned above, NORA members will regularly communicate their progress in scientific conferences, press releases, academic publications, etc. At the end of the project, the consortium will deliver its recommendations and tools to the European Commission.