Unlocking bacterial potential to reduce farmland N2O emissions.

Farmed soils contribute substantially to global warming by emitting N2O (ref. 1), and mitigation has proved difficult2. Several microbial nitrogen transformations produce N2O, but the only biological sink for N2O is the enzyme NosZ, catalysing the reduction of N2O to N2 (ref. 3). Although strengthening the NosZ activity in soils would reduce N2O emissions, such bioengineering of the soil microbiota is considered challenging4,5. However, we have developed a technology to achieve this, using organic waste as a substrate and vector for N2O-respiring bacteria selected for their capacity to thrive in soil6-8. Here we have analysed the biokinetics of N2O reduction by our most promising N2O-respiring bacterium, Cloacibacterium sp. CB-01, its survival in soil and its effect on N2O emissions in field experiments. Fertilization with waste from biogas production, in which CB-01 had grown aerobically to about 6 × 109 cells per millilitre, reduced N2O emissions by 50-95%, depending on soil type. The strong and long-lasting effect of CB-01 is ascribed to its tenacity in soil, rather than its biokinetic parameters, which were inferior to those of other strains of N2O-respiring bacteria. Scaling our data up to the European level, we find that national anthropogenic N2O emissions could be reduced by 5-20%, and more if including other organic wastes. This opens an avenue for cost-effective reduction of N2O emissions for which other mitigation options are lacking at present.

A Dual Enrichment Strategy Provides Soil- and Digestate-Competent Nitrous Oxide-Respiring Bacteria for Mitigating Climate Forcing in Agriculture.

Manipulating soil metabolism through heavy inoculation with microbes is feasible if organic wastes can be utilized as the substrate for growth and vector as a fertilizer. This, however, requires organisms active in both digestate and soil (generalists). Here, we present a dual enrichment strategy to enrich and isolate such generalists among N2O-respiring bacteria (NRB) in soil and digestates, to be used as an inoculum for strengthening the N2O-reduction capacity of soils. The enrichment strategy utilizes sequential batch enrichment cultures alternating between sterilized digestate and soil as substrates, with each batch initiated with limited O2 and unlimited N2O. The cultures were monitored for gas kinetics and community composition. As predicted by a Lotka-Volterra competition model, cluster analysis identified generalist operational taxonomic units (OTUs) which became dominant, digestate/soil-specialists which did not, and a majority that were gradually diluted out. We isolated several NRBs circumscribed by generalist OTUs. Their denitrification genes and phenotypes predicted a variable capacity to act as N2O-sinks, while all genomes predicted broad catabolic capacity. The latter contrasts with previous attempts to enrich NRB by anaerobic incubation of unsterilized digestate only, which selected for organisms with a catabolic capacity limited to fermentation products. The two isolates with the most promising characteristics as N2O sinks were a Pseudomonas sp. with a full-fledged denitrification-pathway and a Cloacibacterium sp. carrying only N2O reductase (clade II), and soil experiments confirmed their capacity to reduce N2O-emissions from soil. The successful enrichment of NRB with broad catabolic spectra suggests that the concept of dual enrichment should also be applicable for enrichment of generalists with traits other than N2O reduction. IMPORTANCE N2O emissions from farmed soils are a major source of climate forcing. Here, denitrifying bacteria act as both source and sink for N2O, determined by regulatory traits or the absence of genes coding for the enzymes producing or reducing N2O. One approach to reducing emissions is to amend large numbers of N2O-reducing bacteria (NRB) to soil. This was shown to be feasible by growing NRB to high densities in organic wastes and then applying them as fertilizers. The effect on N2O emissions, however, was transient because the isolated NRBs were unsuited to soil. Here, we have developed an enrichment strategy selecting for organisms with generalist lifestyles, tolerant of rapid environmental changes. This was used to isolate robust NRBs that grow both in digestate and when amended to soils. This strategy opens an avenue for obtaining not just robust NRBs to reduce N2O emissions, but any organism destined for application to complex environments.

Nitrous oxide respiring bacteria in biogas digestates for reduced agricultural emissions.

Inoculating agricultural soils with nitrous oxide respiring bacteria (NRB) can reduce N2O-emission, but would be impractical as a standalone operation. Here we demonstrate that digestates obtained after biogas production are suitable substrates and vectors for NRB. We show that indigenous NRB in digestates grew to high abundance during anaerobic enrichment under N2O. Gas-kinetics and meta-omic analyses showed that these NRB's, recovered as metagenome-assembled genomes (MAGs), grew by harvesting fermentation intermediates of the methanogenic consortium. Three NRB's were isolated, one of which matched the recovered MAG of a Dechloromonas, deemed by proteomics to be the dominant producer of N2O-reductase in the enrichment. While the isolates harbored genes required for a full denitrification pathway and could thus both produce and sequester N2O, their regulatory traits predicted that they act as N2O sinks in soil, which was confirmed experimentally. The isolates were grown by aerobic respiration in digestates, and fertilization with these NRB-enriched digestates reduced N2O emissions from soil. Our use of digestates for low-cost and large-scale inoculation with NRB in soil can be taken as a blueprint for future applications of this powerful instrument to engineer the soil microbiome, be it for enhancing plant growth, bioremediation, or any other desirable function.

Linking meta-omics to the kinetics of denitrification intermediates reveals pH-dependent causes of N2O emissions and nitrite accumulation in soil.

Soil pH is a key controller of denitrification. We analysed the metagenomics/transcriptomics and phenomics of two soils from a long-term liming experiment, SoilN (pH 6.8) and un-limed SoilA (pH 3.8). SoilA had severely delayed N2O reduction despite early transcription of nosZ (mainly clade I), encoding N2O reductase, by diverse denitrifiers. This shows that post-transcriptionally hampered maturation of the NosZ apo-protein at low pH is a generic phenomenon. Identification of transcript reads of several accessory genes in the nos cluster indicated that enzymes for NosZ maturation were present across a range of organisms, eliminating their absence as an explanation for the failure to produce a functional enzyme. nir transcript abundances (for NO2- reductase) in SoilA suggest that low NO2- concentrations in acidic soils, often ascribed to abiotic degradation, are primarily due to biological activity. The accumulation of NO2- in neutral soil was ascribed to high nar expression (nitrate reductase). The -omics results revealed dominance of nirK over nirS in both soils while qPCR showed the opposite, demonstrating that standard primer pairs only capture a fraction of the nirK pool. qnor encoding NO reductase was strongly expressed in SoilA, implying an important role in controlling NO. Production of HONO, for which some studies claim higher, others lower, emissions from NO2- accumulating soil, was estimated to be ten times higher from SoilA than from SoilN. The study extends our understanding of denitrification-driven gas emissions and the diversity of bacteria involved and demonstrates that gene and transcript quantifications cannot always reliably predict community phenotypes.

Delving into defence: identifying the Pseudomonas protegens Pf-5 gene suite involved in defence against secreted products of fungal, oomycete and bacterial rhizosphere competitors.

Competitive behaviours of plant growth promoting rhizobacteria (PGPR) are integral to their ability to colonize and persist on plant roots and outcompete phytopathogenic fungi, oomycetes and bacteria. PGPR engage in a range of antagonistic behaviours that have been studied in detail, such as the production and secretion of compounds inhibitory to other microbes. In contrast, their defensive activities that enable them to tolerate exposure to inhibitory compounds produced by their neighbours are less well understood. In this study, the genes involved in the Pseudomonas protegens Pf-5 response to metabolites from eight diverse rhizosphere competitor organisms, Fusarium oxysporum, Rhizoctonia solani, Gaeumannomyces graminis var. tritici, Pythium spinosum, Bacillus subtilis QST713, Pseudomonas sp. Q2-87, Streptomyces griseus and Streptomyces bikiniensis subspecies bikiniensi, were examined. Proximity induced excreted metabolite responses were confirmed for Pf-5 with all partner organisms through HPLC before culturing a dense Pf-5 transposon mutant library adjacent to each of these microbes. This was followed by transposon-directed insertion site sequencing (TraDIS), which identified genes that influence Pf-5 fitness during these competitive interactions. A set of 148 genes was identified that were associated with increased fitness during competition, including cell surface modification, electron transport, nucleotide metabolism, as well as regulatory genes. In addition, 51 genes were identified for which loss of function resulted in fitness gains during competition. These included genes involved in flagella biosynthesis and cell division. Considerable overlap was observed in the set of genes observed to provide a fitness benefit during competition with all eight test organisms, indicating commonalities in the competitive response to phylogenetically diverse micro-organisms and providing new insight into competitive processes likely to take place in the rhizosphere.

Draft Genome Sequence of Dietzia sp. Strain SYD-A1, Isolated from Coal Seam Formation Water.

Subsurface coal seams harbor an array of diverse microbial species subsisting as a community on the organic matter present in coal. Here, we present the annotated genome sequence of Dietzia sp. strain SYD-A1, a bacterium isolated from a terrestrial subsurface coal seam in New South Wales, Australia.

Subsurface Stappia: Success Through Defence, Specialisation and Putative Pressure-Dependent Carbon Fixation.

Diverse microbial communities living in subsurface coal seams are responsible for important geochemical processes including the movement of carbon between the geosphere, biosphere and atmosphere. Microbial conversion of the organic matter in coal to methane involves a complex assemblage of bacteria and archaea working in syntrophic relationships. Despite the importance and value of this microbial process, very few of the microbial taxa have defined metabolic or ecological roles in these environments. Additionally, the genomic features mediating life in this chemically reduced, energy poor, deep subsurface environment are not well characterised. Here we describe the isolation and genomic and catabolic characterisation of three alphaproteobacterial Stappia indica species from three coal basins across Australia. S. indica genomes from coal seams were compared with those from closely related S. indica isolated from diverse surface waters, revealing a coal seam-specific suite of genes associated with life in the subsurface. These genes are linked to processes including viral defence, secondary metabolite production, polyamine metabolism, polypeptide uptake membrane transporters and putative energy neutral pressure-dependent CO2 fixation. This indicates that subsurface Stappia have diverse metabolisms for biomass recycling and pressure-dependent CO2 fixation and require a suite of defensive and competitive strategies relative to their surface-dwelling relatives.

Genomic and phenotypic insights point to diverse ecological strategies by facultative anaerobes obtained from subsurface coal seams.

Microbes in subsurface coal seams are responsible for the conversion of the organic matter in coal to methane, resulting in vast reserves of coal seam gas. This process is important from both environmental and economic perspectives as coal seam gas is rapidly becoming a popular fuel source worldwide and is a less carbon intensive fuel than coal. Despite the importance of this process, little is known about the roles of individual bacterial taxa in the microbial communities carrying out this process. Of particular interest is the role of members of the genus Pseudomonas, a typically aerobic taxa which is ubiquitous in coal seam microbial communities worldwide and which has been shown to be abundant at early time points in studies of ecological succession on coal. The current study performed aerobic isolations of coal seam microbial taxa generating ten facultative anaerobic isolates from three coal seam formation waters across eastern Australia. Subsequent genomic sequencing and phenotypic analysis revealed a range of ecological strategies and roles for these facultative anaerobes in biomass recycling, suggesting that this group of organisms is involved in the degradation of accumulated biomass in coal seams, funnelling nutrients back into the microbial communities degrading coal to methane.

Who eats what? Unravelling microbial conversion of coal to methane.

Microbial communities in subsurface coal seams are responsible for the conversion of coal organic matter to methane. This process has important implications for both energy production and our understanding of global carbon cycling. Despite the environmental and economic importance of this process, little is known about which components of the heterogeneous coal organic matter are biodegradable under methanogenic conditions. Similarly, little is known about which taxa in coal seams carry out the initial stages of coal organics degradation. To identify the biodegradable components of coal and the microorganisms responsible for their breakdown, a subbituminous coal was fractionated into a number of chemical compound classes which were used as the sole carbon source for growth by a coal seam microbial community. This study identifies 65 microbial taxa able to proliferate on specific coal fractions and demonstrates a surprising level of substrate specificity among members of this coal-degrading microbial consortia. Additionally, coal kerogen, the solvent-insoluble organic component of coal often considered recalcitrant to microbial degradation, appeared to be readily converted to methane by microbial degradation. These findings challenge our understanding of coal organic matter catabolism and provide insights into the catabolic roles of individual coal seam bacteria.

Succession Patterns and Physical Niche Partitioning in Microbial Communities from Subsurface Coal Seams.

The subsurface represents a largely unexplored frontier in microbiology. Here, coal seams present something of an oasis for microbial life, providing moisture, warmth, and abundant fossilized organic material. Microbes in coal seams are thought to syntrophically mobilize fossilized carbon from the geosphere to the biosphere. Despite the environmental and economic importance of this process, little is known about the microbial ecology of coal seams. In the current study, ecological succession and spatial niche partitioning are explored in three coal seam microbial communities. Scanning electron microscopic visualization and 16S rRNA sequencing track changes in microbial communities over time, revealing distinct attached and planktonic communities displaying patterns of ecological succession. Attachment to the coal surface is biofilm mediated on Surat coal, whereas microbes on Sydney and Gunnedah coal show different attachment processes. This study demonstrates that coal seam microbial communities undergo spatial niche partitioning during periods of succession as microbes colonize coal environments.