Work Package 1 - Regulatory Biology
Biochemical analyses of model strains have been performed, which revealed the important role of NosR in Cu regulation of N2O reduction; and led to the detection of novel, putative Cu chaperones. An mCherry mutant for bet-hedging studies has confirmed hypotheses based on mathematical models, which have been constructed for the denitrifier model strain P. denitrificans. Co-cultivation of model strain mutants has been performed and first models constructed for prediction of gas kinetics. The enzyme N2O reductase has been analyzed from P. denitrificans cells grown at pH 6 and 7, confirming the absence of Cu in the active site of the enzyme when assembled at low pH, giving support to the hypothesis that low pH impairs the making of this important enzyme. (ESRs 1, 2, 3).
Determination of detailed denitrification regulatory phenotypes, and genome sequencing, have been done for ca 30 strains in the new collection of denitrifying bacteria. Detailed kinetics studies were also performed for selected nitrifier strains (bacteria and archaea). Kinetic parameters and cell specific rates of NO turnover (VNO) and N2O production (VN2O) as a function of oxygen concentration at the cell surface ([O2]s. Calculations and modelling of the electron flow to denitrification and terminal oxidases in N. europaea demonstrate a minuscule electron flow to denitrification even at sub-micromolar oxygen concentrations, indicating that denitrification by AOB is a very weak source of N2O. (ESRs 1, 5).
- Several regulatory mechanisms were revealed, which determine the efficacy by which different denitrifying bacteria act as sinks or sources for N2O, including bet-hedging, NO and NO2- accumulation.
- The pH dependency for correct assembly of the N2O reductase was confirmed, as well as the importance of sufficiently high Cu concentrations, and novel, putative Cu chaperones for Cu insertion were detected.
- N2O production by ammonia oxidizing bacteria (AOB) was higher than that of ammonia oxidising archaea (AOA), but yet, AOB contributed only marginally to N2O emissions compared to heterotrophic denitrifying bacteria.
Work Package 2 - Systems
A novel isolation protocol that identifies all possible combinations of truncated denitrification chains by end-point analyses was developed during the former reporting period, and a biobank of >180 isolates was built, of which 70 isolates performed one or more steps in the anaerobic reduction of nitrogen oxides (ESR1). Key N2O reducing strains were identified. Moreover, fungal denitrification was investigated for a large number of fungal soil isolates, the results demonstrating N2O production about 1000 times lower than for bacteria, contradicting earlier results by other researcher groups that fungi are important N2O sources (ESR8).
The effects of agricultural management and fertilizer treatments on N2O emissions have been studied in long-term field experiments at several sites over Europe (performed by ESRs 5, 6, 7 and 9). The results from the studies show that complete denitrifiers carrying nosZI were more abundant than nosZII bacteria; that denitrification was more important N2O source than DNRA; and that the richness and diversity of nosZII but not nosZI was influenced by fertilization. Deliverables 27, 30, 32. A toolbox for amplicon-based and metaomics analyses of 16S rRNA and several functional genes involved in N transformations was developed and tested on sludge and granules from anammox reactors, and results were correlated with to process parameters (ER3; ESR 8). Studies of different wastewater systems are ongoing, and the results demonstrate a high capacity for N2O reduction in these systems, with consistently higher rates (2-10 times) for N2O reduction compared to NO3- reduction. Affinity constants (Ks) for O2 and N2O have been determined. N2O reduction took place in presence of O2, although at slower rates than under anoxic conditions.
Moreover, enrichment studies with N2O as sole electron acceptor showed that N2O respiration can fully sustain the bioenergetics needs of the bacterial cells, with growth rates comparable to that of complete denitrification. Metagenomic analyses have been performed to determine the main functional groups responsible for the transformations (ESRs1, 4; ER3).
The spatial localization of biochemical processes in soil aggregates was investigated by size fractionation of soil aggregates and analyses of microbial community composition and N cycling genes. Surprisingly, there was no separation between N reducing and N oxidizing organisms (based on functional genes) between inner and outer compartments (ESR8).
Metagenomics/metatranscriptomics analyses of a reference soil was done, and effects of the addition of different types of organic matter was investigated. The results indicated that fungi is the microbial group mainly responsible for C degradation, while N-cycling transcriptomal responses were mainly seen in the bacteria. Bioinformatics analyses are ongoing (ESR8; ER3). The effects of NH4+ vs NO3- based fertilizers on N2O emissions was investigated in a series of experiments (including quantification of AOA/AOB genes and transcripts, and isotope tracing techniques). The results demonstrated high nitrification activity and N2O emissions in response to NH4+-based fertilization, dominated by AOB (as seen from selective inhibition approach) while AOA and AOB accounted for similar amounts of N2O from non-fertilized soil (ESR5). Comparison of fast-release vs slow release fertilizer further supported the observed niche-separation between AOA and AOB, showing stimulation of AOA under slow-release fertilizer regiment, and thus lower N2O emissions. Analyses are ongoing (ESRs 5, 9). To evaluate the importance of DNRA (dissimilatory reduction of nitrate to ammonia) for N2O emissions, 15N tracer experiments were performed, which demonstrated that N2O production from arable soils is predominantly from denitrification, not DNRA (ESR9).
Production of N2O from increased mineralization of soil organic matter, resulting from N fertilizers, was investigated using 15N tracing techniques and the results support such a "N2O priming effect (ESR 9; ER2).
- Fungal denitrification is an insignificant source of biologically produced N2O emissions, which are dominated by heterotrophic, denitrifying bacteria.
- Slow-release fertilizers lead to lower N2O emissions than fast-release fertilizers; and NO3- based fertilizers lead to lower N2O emissions than urea and NH4+ based fertilizers.
- Non-denitrifier bacteria carrying nosII could act as sinks for N2O emissions from agricultural soils, but there are no clear solutions as to how to accomplish selective stimulation of these organisms.
- Wastewater treatments plants may have great N2O reducing potential which should be further exploited to reduce point emissions into the environment.
Work Package 3 - Emissions / Processes
Detailed data sets for N2O emissions in response to various fertilizer programs, as well as climatic data, have been collected, analyzed, and used in mathematical modelling. The field robot, developed as a collaboration between the NORA partners UMB and Adigo, is fully operational (ER 1) and has been used for N2O emission field campaigns. (ERs 1, 2). It has also been used to collect data from an ongoing field experiment evaluating the effect of different carbonate-free minerals for slow pH increase of soils. Please klick here to see the robot in action. The field robot has been promoted at various meetings and conferences, including
- EGU General Assembly (European Geosciences Union ) symposium in Vienna, Austria April-May 2013 and 2014 (Jan Reent Koster); http://meetingorganizer.copernicus.org/EGU2014/orals/14222
- 1st ICOS Science conference on greenhouse gases and biogeochemical cycles, Brussels, Belgium 23-25 Sept 2014 (Jan Reent Koster). http://www.icos-infrastructure.eu/scienceconference
- the NORA outreach workshop on N2O and trace gas emissions, "Gas flux measurements in terrestrial ecosystems - state of the art and emerging technologies" held at the University of Gothenburg (UGOT) 10-13 May 2015 (see below);
- the"Annual conference for agronomists and policy makers" organized by Bioforsk on Hamar, Norway, 4-5 February 2015; the GHG Robot meeting 02.02.2015, Osnabrlick, Germany Hosted by ICOS-Germany and Thlinen Institut. Participants: 15, representing ICOS Germany (https://www.icos-ri.eu/home ), Thlinen Institut (https://www.thuenen.de/en/), IOTEC (http://iotec-gmbh.de/EN/index.html), Farm Systems (http://www.farmsystem.de/ ), and NORA (0yvind Overskeid & Lars Bakken). Purpose: Workshsop for discussion of field flux robot designs, pimarily comparing archetcture and performance of the ADIGO/NORA Field Flux Robot (http://www.adigo.no/portfolio/field-flux-robot-2/?lang=en ) and BoniRob (https://www.deepfield-robotics.com/en/BoniRob.html). Outcome: agreement to future collaboration for further development of robot for field flux measurements, based on the architecture of the ADIGO/NORA robot.
- A novel and fully functional field robot for measurements of N2O emissions has been developed, allowing collection of large data sets and with substantially reduced manpower hours hitherto needed for manual measurements.
- Implementation of the mathematical models "LandscapeDNDC and DayCENT" showed good correspondence between predicted and measured N2O emissions in a three-year field trial on fertilization regimes of agricultural soils.