Biogas digestate as substrate and vector for the introduction of N2O-respiring bacteria to agricultural soil
Year: 2021
Type: dissertation
Abstract: Anthropogenic nitrous oxide (N2O) emissions are largely driven by the input of N-based fertilizers in agriculture. N2O emissions from agricultural soils in Europe are estimated to 0.51 Tg annually (Fig. I), which sums to 48 % of total European N2O emissions and 35 % of the climate forcing from European agriculture. Yet, N2O emission mitigation from agriculture is still hampered by a lack of implemented abatement options.\n\nWhilst several biogeochemical reactions may release N2O (Fig. I) the enzyme nitrous oxide reductase (Nos) is the only known enzyme to reduce nitrous oxide. Nos is expressed in denitrifying and non-denitrifying prokaryotes and catalyzes the reduction of N2O to N2. The complete denitrification pathway is the stepwise reduction NO3- → NO2- → NO → N2O → N2, catalyzed by the enzymes Nar/Nap, Nir, Nor, and Nos that are encoded by the genes nar/nap, nirK/nirS, nor, and nosZ, respectively (Fig. I). A significant proportion of the denitrifying community in soils have truncated denitrification pathways, i.e. lacking one to three of the genes encoding the enzymes in the stepwise reduction of NO3- to N2. The consequence of such modularity is that organisms lacking nosZ are net N2O emitters, while organisms with nosZ only are net sinks for N2O. However, organisms equipped with a complete denitrification pathway can also be strong sinks or sources of N2O depending on their regulatory biology.\n\nN2O emissions from soils make up a substantial fraction of the climate forcing from food production and mitigation beyond that achieved by “good management practices” are needed if we are to limit global warming by 2 °C, as set in the Paris Agreement. One approach for reducing N2O emissions is to modify the soil microbiome, increasing the proportion of N2O-respiring bacteria (NRB) resulting in reduced N2O emissions. This would, however, be costly and impractical as a standalone operation.\n\nAs an element towards a low-carbon circular economy, the volume of organic wastes channeled through AD is expected to increase in the coming decades. This presents a unique possibility for mitigation of N2O emissions as the residues of biogas production, digestates, destined as bio-fertilizers in agriculture, could be enriched with N2O-respiring bacteria before soil fertilization. Thus, providing a cost-efficient N2O mitigation measure (Fig. I). Here we demonstrate the use of biogas digestates from anaerobic digestion (AD) as a widely available, low-cost vector for NRB to agricultural soils.\n\nA primary task was to search for suitable organisms that 1) could grow to high cell densities in digestate and 2) would act as net N2O sinks in soil. To achieve this, enrichment culturing under anaerobic conditions with N2O as the sole electron acceptor was used. The enrichment cultures were monitored both by measuring the gas kinetics and by inspecting the composition of the microbiota by genomics and proteomics. Based on genomic information and targeted isolation, we obtained axenic cultures of the organisms that became dominant in the enrichment cultures.\n \nAs a first approach, we enriched indigenous N2O-respiring bacteria in anaerobically digested sewage sludge (digestate) by anoxic incubation with N2O. The gas kinetics predicted that N2O-respiring organisms grew to high cell densities, which was confirmed by metagenomic and metaproteomic (omics-) analyses of the enriched digestate. The omics demonstrated dominance of organisms equipped with the nosZ clade II (coding for N2O-reductase), but also with the genes for the preceding steps of the denitrification pathway. Three digestate-derived N2O-reducing bacteria were isolated, of which one (Azonexus sp.) matched the recovered Metagenome-Assembled Genome (MAG) of the dominant N2O reducer with an average nucleotide identity (ANI) of 98.2%. This MAG also demonstrated a high complement of Nos in the enrichment as quantified by metaproteomics. Gas kinetics and meta-omics indicated that the anaerobic consortium of the digestate remained active during anaerobic incubation with N2O and that N2O-respiring bacteria grew by harvesting fermentation intermediates. The latter was supported by screening carbon catabolism profiles of the isolated organisms. The isolated Azonexus sp. demonstrated regulatory traits that would predict the organism to be a strong N2O sink, and it reduced immediate N2O emissions from digestate-amended soils. However, the Azonexus sp. was probably not an ideal N2O-respiring inoculant in soil because it was equipped with a full-fledged denitrification pathway and because its capacity to utilize soil carbon was limited. The importance of an active methanogenic community throughout the enrichments, providing fermentation intermediates as a carbon source for the N2O-respiring organisms, would predict a selective advantage for organisms with a streamlined (narrow) catabolic capacity, which was the case for the Azonexus sp.. It was evident that we needed to refine our search, to find organisms with a broader catabolic repertoire.\n\nA new procedure to obtain more ideal isolates was designed, involving a deliberate enrichment of N2O-respiring organisms with the characteristics of strong growth both in digestate and soil. We thought this could be achieved by “dual enrichment culturing”, i.e. a sequence of enrichment cultures where a fraction of a batch enrichment was passaged to the next batch, alternating between sterile soil and sterile digestate as substrate. Our point of departure was to model this approach, using a simple logistic model for the competition for a common substrate, between three distinctive groups; 1: Organisms with a competitive advantage in digestate (digestate specialists), 2: Organisms with a competitive advantage in soil (soil specialists), and 3: organisms capable of sustaining growth in both environments (generalists). The modelling revealed that generalists could indeed become dominant within a limited number of batch cultures, depending on their competitive edge vis a vis the specialists. Based on this we realized a dual enrichment experiment, using the microbiota of wastewater digestate and soil as initial inocula, sterile digestate and sterile soil as substrate, and monitored the gas kinetics and the community composition (by 16S rDNA amplicon sequencing) throughout seven consecutive enrichment cultures. The gas kinetics corroborated the model’s prediction of a gradual enrichment of organisms that grew both in soil and digestate, and the generalists that became dominant were identified as a limited number of Operational Taxonomic Units (OTUs, based on 16S rDNA sequencing). OTUs that became dominant circumscribed isolates obtained from the enrichment cultures. These OTUs also portrayed the targeted generalist as predicted by the modelling. Most isolates obtained had traits of strong N2O sinks, of which a dominating Cloacibacterium sp., carrying Nos (Clade II) as the sole N-reductase, significantly reduced N2O emissions in digestate amended soils of both neutral and acidic pH. A full-fledged denitrifying Pseudomonas sp. was able to persist in the soil for at least one month whereby significant N2O emissions reduction was obtained upon a fertilization event. Genome analysis of the isolated organisms shed some light as to why these organisms had a competitive advantage in both soil and digestate.\n\nAlthough the ideal isolate is yet to be found, we’ve opened an avenue to a concept that, within the expected expansion of AD, could be scaled to secure a substantial reduction in N2O emissions.
Author: Jonassen, Kjell Rune
Language: English
Subfield: Soil Science
Domain: Life Sciences
SDGs: Climate action, Life on land
Open Access status: green
OpenAlex ID: W6989655443