A break-through in developing tools for reducing N2O emissions!
The project follows two tracks for finding efficient ways to reduce N2O emissions from agricultural land: 1) using the waste from biogas production (=digestates) to strengthen the soils? capacity to reduce the climate gas N2O to harmless N2 , and 2) reduce N2O emission by using nitrification inhibitors.
The strategy for using digestates is to isolate bacteria which a) grow fast in digestates b) are strong sinks for N2O and c) survive in soil, and then to grow these organisms to high cell densities in the digestate before it is used as a fertilizer.
Bacteria which are strong sinks for N2O are called NRB. NRB-performance can be due to gene regulatory properties that secures that the enzyme for N2O-reduction (Nos) is strongly expressed compared to the expression of the enzymes that produce N2O (Nar/Nir/Nor). Or, at best, the NRB organism has the gene coding for Nos, but not the gene coding for N2O-producing steps.
We have used enrichment culturing to find and isolate suitable NRB, monitoring the enrichment cultures with a plethora of molecular tools in order to understand the metabolic network of the enrichment culture, to identify the NRB which are growing, their denitrification genes, and which of these genes that are expressed during the enrichment.
At an early stage we proved that this is possible, and that the isolated NRB were able to transform the digestate to an instrument to reduce N2O emission from soil. The isolated NRB were far from ideal, however: 1) their metabolism was streamlined for growing in a digestate (very narrow specter of C substrates), thus plausibly unable to survive in soil 2) all of them had the genes for the entire denitrification pathway, thus they are both producing and reducing N2O.
We then developed a novel enrichment strategy targeting more adequate NRB?s. This led to a break-through: we have found NRB?s that 1) lack the genes coding for the N2O-producing steps of denitrification 2) grow fast in digestates 3) exploit a broad specter of C-substrates, thus increasing their chance to survive in soil.
Our Chinese partners have followed our scheme meticulously, but with different digestates. The results are very good, and they have isolated a set of NRB?s, some of which resemble ours both phenotypically and genetically.
We have recently upscaled the use of NRB in digestates to a field plot experiments, where the N2O emission was measured with our field robots. These measurements show that NRB in the digestate reduced the N2O emission by 95% right after fertilization. The NRB was had a surprisingly persistence effect: even 30 days after fertilization, we fund a 50% reduction of N2O emission.
We have tested the effect of the nitrification inhibitor DMPP (3,4-Dimethylpyrazone) in four different soils, in collaboration with Chinese colleagues. The results show a n increasing effect with increasing soil pH, which corroborates the current understanding of DMPP as a selective inhibitor of ammonia oxidizing bacteria (AOB), and the relative importance of AOB versus the archaeal ammonia oxidizers AOA increase with soil pH. An analysis of the results according to this model (selective inhibition of AOB), we find that the DMPP concentration needed to achieve 50% inhibition of AOB is strikingly similar for the four soils (0.3-0.6 mg DMPP pr L). AOB produce approximately one order of magnitude more N2O than AOA, and our measured N2O emission reflect this nicely: The product ratio N2O/NO3 declines dramatic with increasing DMPP concentrations. DMPP therefor qualifies as a perfect nitrification inhibitor for achieving a reduction of N2O emission from nitrification.
The use of such agrochemicals on soil has not yet been implemented on a large scale, but it may be attempted in the near future. If so, we foresee opposition to such ?poisoning? of our soils, and possibly a prohibition.
Anaerobic digestion (AD) will become the standard way of treating sewage sludge, urban organic wastes and animal manure, both in Norway and China, and the digestates are increasingly used to fertilize farmland. One important motivation for these huge investments in AD-technology is to reduce climate forcing by eliminating methane emission from storage and to replace fossil fuel with biogas. Here we propose to add a new dimension to the climate performance of AD: by growing aggressively N2O reducing bacteria (ANB) in the digestates, we can reduce the N2O emission which is otherwise induced by the fertilization with the digestate. We will search for biodigestate-competent ANB that also grow/survive in soil, thus securing a more long lasting reduction of the N2O emission from farmland. Finally, we will explore if retardation of nitrification by modest doses of nitrification inhibitors can reduce the aerobic N2O emission caused by ammonia oxidation.
We have recently identified several types of bacteria that can potentially act as strong N2O sinks, either because their only denitrification gene is the one coding for N2O reductase (nosZ), or because they express nosZ more than the other denitrification genes. A primary challenge will be to enrich such organisms in the digestates. We will use either digestates or soils as initial inoculum and select for ANB by providing a suitable electron acceptor. Metagenomics and proteomics will be used to track and identify the dominating organisms in the enrichment, and to guide attempts to isolate ANBs. Microcosms will be used to test the effects of ANB on N2O kinetics in soils, and to test the effects of nitrification inhibitors. Upscaling to field experiments will be done by cultivation in pilot plants, and possible implementations in existing AD systems will be explored in collaboration with AD industries that are associated with the project.