A bet-hedging strategy for denitrifying bacteria curtails their release of N2O

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Transcriptional and Environmental Control of Bacterial Denitrification and N2O Emissions

In oxygen-limited environments, denitrifying bacteria can switch from oxygen-dependent respiration to nitrate (NO3−) respiration in which the NO3− is sequentially reduced via nitrite (NO2−), nitric oxide (NO) and nitrous oxide (N2O) to dinitrogen (N2). However, atmospheric N2O continues to rise, a significant proportion of which is microbial in origin. This implies that the enzyme responsible for N2O reduction, nitrous oxide reductase (NosZ), does not always carry out the final step of denitrification either efficiently, or in synchrony with the rest of the pathway. Despite a solid understanding of the biochemistry underpinning denitrification, there is a relatively poor understanding of how environmental signals and respective transcriptional regulators control expression of the denitrification apparatus. This mini-review will describe the current picture for transcriptional regulation of denitrification in the model bacterium, Paracoccus denitrificans, highlighting differences in other denitrifying bacteria where appropriate, as well as gaps in our understanding. Alongside this, the emerging role of small regulatory RNAs (sRNAs) in regulation of denitrification will be discussed. We will conclude by speculating how this information, aside from providing a better understanding of the denitrification process, can be translated into development of novel greenhouse gas mitigation strategies

The effect of pH on Marinobacter hydrocarbonoclasticus denitrification pathway and nitrous oxide reductase

PTDC/BBB-BQB/0129/2014 (IM). This work was supported by the Applied Molecular Biosciences Unit-UCIBIO, and Associate Laboratory for Green Chemistry-LAQV, which is financed by national funds from FCT (UIDB/04378/2020 and UIDB/50006/2020, respectively).Abstract: Increasing atmospheric concentration of N2O has been a concern, as it is a potent greenhouse gas and promotes ozone layer destruction. In the N-cycle, release of N2O is boosted upon a drop of pH in the environment. Here, Marinobacter hydrocarbonoclasticus was grown in batch mode in the presence of nitrate, to study the effect of pH in the denitrification pathway by gene expression profiling, quantification of nitrate and nitrite, and evaluating the ability of whole cells to reduce NO and N2O. At pH 6.5, accumulation of nitrite in the medium occurs and the cells were unable to reduce N2O. In addition, the biochemical properties of N2O reductase isolated from cells grown at pH 6.5, 7.5 and 8.5 were compared for the first time. The amount of this enzyme at acidic pH was lower than that at pH 7.5 and 8.5, pinpointing to a post-transcriptional regulation, though pH did not affect gene expression of N2O reductase accessory genes. N2O reductase isolated from cells grown at pH 6.5 has its catalytic center mainly as CuZ(4Cu1S), while that from cells grown at pH 7.5 or 8.5 has it as CuZ(4Cu2S). This study evidences that an in vivo secondary level of regulation is required to maintain N2O reductase in an active state. Graphic abstract: [Figure not available: see fulltext.].preprintpublishe

Digestat fra biogassproduksjon som substrat og vektor for introduksjon av N2O-respirerende bakterier til landbuksjord

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. Whilst 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. N2O 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. As 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. A 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. As 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. A 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. Although 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.Menneskeskapte utslipp av drivhusgassen lystgass (N2O) skyldes i stor grad tilførsel av nitrogenholdig gjødsel til landbruksjord. N2O-utslipp fra landbruksjord i Europa er estimert til 0,51 Tg årlig (Fig. I), som utgjør om lag 48% av de totale utslippene av N2O, som igjen representerer 35 % av det totale klimagassfotavtrykket fra europeisk landbruk. Begrensning av disse utslippene har vært utfordrende grunnet mangel på implementerte metoder og teknologier som effektivt reduserer lystgassutslippet fra landbruksjord. Flere biogeokjemiske reaksjoner kan frigjøre N2O (Fig. I), men enzymet lystgassreduktase (Nos) er det eneste kjente enzymet som reduserer N2O til N2. Nos uttrykkes av denitrifiserende prokaryoter og katalyserer reduksjonen av N2O til N2. Denitrifiserende prokaryoter katalyserer den trinnvise reduksjon av NO3- → NO2- → NO → N2O → N2, som katalyseres av enzymene Nar/Nap, Nir, Nor og Nos som er kodet av genene nar/nap, nir, nor og nosZ (Fig. I). Men, en betydelig andel av det denitrifiserende mikrobesamfunnet i jord er trunkert, dvs. en andel av denitrifikantene mangler ett til tre av genene som koder enzymene involvert i reduksjonen av NO3- til N2. En organisme som kun mangler nosZ vil produsere N2O. I motsatt tilfelle vil en organisme som kun er utstyrt med nosZ bare evne å redusere N2O. Organismer utstyrt med et komplett sett av gener for en fullstendig denitrifikasjon kan være både sterke og svake N2O-reduktanter. Dette bestemmes av deres regulatoriske biologi. N2O-utslipp fra jord utgjør en betydelig mengde av det totale klimafotavtrykket fra matproduksjon og en reduksjon av dette utslippet er nødvendig om vi skal nå de målene som er satt i Parisavtalen og begrense global oppvarming til 2 °C. En mulighet for å redusere N2O-utslipp er å modifisere jordmikrobiomet ved å øke andelen N2O-respirerende bakterier (NRB) – noe som vil redusere utslippene av N2O. Men, som ett frittstående tiltak vil en storskala modifisering av mikrobiologien i jordsmonnet være svært ressurskrevende. Som et ledd i overgangen til en lav-karbon sirkulærøkonomi forventes anaerob utråtning (AD) å øke i omfang og rekkevidde de neste årene. Denne utviklingen skaper en unik mulighet for å redusere N2O-utslipp dersom digestater, restproduktet fra AD, som brukes som organisk gjødsel i landbruket, kan anrikes med N2O-reduserende bakterier før disse digestatene benyttes som gjødsel (Fig. I). Her demonstrerer vi at lett tilgjengelige digestater kan benyttes som vekstsubstrat og en vektor for å overføre NRB til jord. En slik modifikasjon være et svært kostnadseffektivt N2O-reduserende tiltak. Det primære målet i denne avhandlingen var å lete etter egnede organismer som 1) kan gro til høy celletetthet i digestater, og 2) redusere N2O-utslipp fra jord. For å oppnå dette ble anrikninger av slike organismer ved bruk av N2O som eneste elektronakseptor gjennomført. Anrikningskulturene ble monitorert ved å måle gasskinetikk og ved overvåking av samfunnsprofiler og bakteriell populasjonsdynamikk ved bruk av DNA- og proteomanalyser. Med basis i den genetiske informasjonen var målet å isolere dominerende organismer fra anrikningskulturene. Som en første tilnærming anriket vi N2O-reduserende bakterier som er naturlig tilstedeværende i digestat i anoksiske inkubasjoner hvor N2O ble tilsatt som eneste elektronakseptor. Gasskinetikk predikerte at NRB vokste til høye celletettheter under inkubasjonen, som ble bekreftet av metagenom- og metaproteomanalyser av det anrikede digestatet. Meta-omikk analysene viste at organismer utstyrt med nosZ Type II (genet for N2O-reduktase), men også med de øvrige genene for et komplett denitrifiseringsspor, dominerte anrikningen. Tre N2O-reduserende bakterier ble isolert hvorav det ene isolatet, en Azonexus sp., samsvarte med et gjenvunnet Dechloromonas-beslektet metagenom som dominerte anrikningen med en aminosyreidentitet på 98,2% delt med det dominerende metagenomet. Metaproteomikk viste at dette metagenomet utrykte brorparten av Nos under anrikningen. Gasskinetikk og meta-omikk avslørte videre at det metanogene konsortiet i digestatet forblir aktivt også under den anaerobe inkubasjonen med N2O, og at dominerende bakterier med en anaerob respiratorisk metabolisme sannsynligvis vokste ved å høste fermenteringsmellomprodukter fra det metanogene samfunnet. Det sistnevnte ble støttet ved karbonkatabolismeprofiler for de isolerte organismene. Den isolerte Azonexus sp. demonstrerte regulatoriske egenskaper som ville forutsi at organismen var en sterk N2O-reduktant, og den reduserte N2O-utslipp fra jord gjødslet med Azonexus anriket digestat. Likevel så var anrikningsvinneren sannsynligvis ikke en ideell N2O-reduserende inokulant i jord fordi dens evne til å overleve i jord-miljøet sannsynligvis var begrenset. Betydningen av et aktivt metanogent bakteriesamfunn, som produsenter av karbonkilder for NRB igjennom anrikningene, gav sannsynligvis en selektiv fordel for organismer med en strømlinjeformet (smal) katabolsk kapasitet, som var tilfelle for Azonexus sp.. Det var tydelig at vi trengte å videreforedle anrikningsprosedyrene våre for å anrike kompetente organismer en bredere metabolsk fleksibilitet. En ny tilnærming for å oppnå mer ideelle isolater som evner å vokse i både jord og i digestat ble designet med utgangspunkt i å selektivt anrike organismer med disse egenskapene. Vi antok at slike organismer kunne anrikes ved en «dobbelt-anrikning»-prosedyre der miljøet ble vekslet mellom jord og digestat. Mao: En sekvens av batch-anrikningskulturer hvor en overfører en fraksjon av anrikningen til en ny batch og vekslet mellom jord og digestat som vekstsubstrat. Med dette utgangspunktet ble logistisk vekst, kun med konkurranse om tilgjengelig karbon, modellert for tre ulike bakteriegrupper; 1) Organismer med konkurransefortrinn i digestat (digestat-spesialister), 2) Organismer med konkurransefortrinn i jord (jordspesialister), og 3) organismer som er i stand til å opprettholde vekst/aktivitet i begge miljøer (generalister). Modelleringen avslørte at generalister teoretisk sett kunne anrikes ved å passere fraksjoner av disse anrikningene mellom digestat og jord, avhengig av generalistenes konkurransefortrinn relativt til spesialistene. Basert på denne modelleringen realiserte vi et nytt anrikningseksperiment med bruk av digestat og jord som initielt inokulum og sterilt digestat og jord som vekstsubstrat og lot populasjonene konkurrere om tilgjengelig karbon med tilsats av N2O. Monitorering av gasskinetikk og populasjonsdynamikk (ved 16S amplikonsekvensering) igjennom syv sammenhengende anrikninger viste en populasjonsutvikling slik predikert fra modelleringen: Gasskinetikken støttet modellprediksjonen om en gradvis ankrikning av organismer som vokste i jord og digestat, og 16S-analysen vist at et fåtall operasjonelle taksonomiske enheter (OTUer) dominerte anrikningen. Isolatene fra disse anrikningskulturene var omsluttet av en dominerende gruppe OTUer som portretterte vekstegenskaper igjennom hele anrikningsserien som representerte de ønskede generalistvinnerne. Ett av isolatene, en Cloacibacterium sp., hvis genom kun kodet for genet for Nos, dominerte anrikningene, og denne reduserte også N2O-utslipp i jord med lav pH. Et annet isolat, en Pseudomonas sp., demonstrert en mer langvarig N2O reduserende aktivitet i jord da aktiviteten var fremtredende selv 30 dager etter gjødsling. Genomanalyse av isolerte organismer kastet noe lys kring hvorfor disse organismer kunne ha et konkurransefortrinn i anrikningene. Selv om det ideelle isolatet ennå ikke er funnet, har vi åpnet en vei for et konsept som, i kontekst av den forventede utviklingen av AD, kan skaleres for å sikre betydelig reduksjon i N2O-utslipp.Vestfjorden Avløpsselskap (VEAS

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

Rapid Succession of Actively Transcribing Denitrifier Populations in Agricultural Soil During an Anoxic Spell

Denitrification allows sustained respiratory metabolism during periods of anoxia, an advantage in soils with frequent anoxic spells. However, the gains may be more than evened out by the energy cost of producing the denitrification machinery, particularly if the anoxic spell is short. This dilemma could explain the evolution of different regulatory phenotypes observed in model strains, such as sequential expression of the four denitrification genes needed for a complete reduction of nitrate to N2, or a “bet hedging” strategy where all four genes are expressed only in a fraction of the cells. In complex environments such strategies would translate into progressive onset of transcription by the members of the denitrifying community. We exposed soil microcosms to anoxia, sampled for amplicon sequencing of napA/narG, nirK/nirS, and nosZ genes and transcripts after 1, 2 and 4 h, and monitored the kinetics of NO, N2O, and N2. The cDNA libraries revealed a succession of transcribed genes from active denitrifier populations, which probably reflects various regulatory phenotypes in combination with cross-talks via intermediates (NO2−, NO) produced by the “early onset” denitrifying populations. This suggests that the regulatory strategies observed in individual isolates are also displayed in complex communities, and pinpoint the importance for successive sampling when identifying active key player organisms

Phylogenetic and functional potential links pH and N2O emissions in pasture soils

This work was funded by the New Zealand Government through the New Zealand Fund for Global Partnerships in Livestock Emissions Research to support the objectives of the Livestock Research Group of the Global Research Alliance on Agricultural Greenhouse Gases (Agreement number: 16084) awarded to SEM and the University of Otago.peer-reviewedDenitrification is mediated by microbial, and physicochemical, processes leading to nitrogen loss via N2O and N2 emissions. Soil pH regulates the reduction of N2O to N2, however, it can also affect microbial community composition and functional potential. Here we simultaneously test the link between pH, community composition, and the N2O emission ratio (N2O/(NO + N2O + N2)) in 13 temperate pasture soils. Physicochemical analysis, gas kinetics, 16S rRNA amplicon sequencing, metagenomic and quantitative PCR (of denitrifier genes: nirS, nirK, nosZI and nosZII) analysis were carried out to characterize each soil. We found strong evidence linking pH to both N2O emission ratio and community changes. Soil pH was negatively associated with N2O emission ratio, while being positively associated with both community diversity and total denitrification gene (nir & nos) abundance. Abundance of nosZII was positively linked to pH, and negatively linked to N2O emissions. Our results confirm that pH imposes a general selective pressure on the entire community and that this results in changes in emission potential. Our data also support the general model that with increased microbial diversity efficiency increases, demonstrated in this study with lowered N2O emission ratio through more efficient conversion of N2O to N2.New Zealand Fund for Global Partnerships in Livestock Emissions Researc

Optimization of Glycerol-driven Denitratation and Dissimilatory Nitrate Reduction to Ammonia

This dissertation aims to expand our knowledge of glycerol-driven engineered biological nitrogen removal processes by elucidating the link between operational controls and the structure and function of the microbial ecology grown under stoichiometrically-limited and excess glycerol conditions. Specific objectives were to: 1. Develop and experimentally evaluate an improved metric for denitratation performance that can be objectively compared across studies; 2. characterize the process kinetics, nitrogen conversion efficiencies, and microbial ecology of a glycerol-driven, stoichiometrically-limited denitratation process; 3. elucidate the impact of kinetic limitation on microbial community structure and function in a glycerol-driven, stoichiometrically-limited denitratation process; 4. explore the biological mechanisms contributing to nitrite (NO2-) accumulation in a glycerol-driven denitratating microbial community; and, 5. characterize the nitrogen conversion efficiencies and microbial ecology that favor dissimilatory nitrate reduction to ammonium (DNRA) in a glycerol-driven denitrification process at stoichiometric excess. Accordingly, a nitrate (NO3-) conversion ratio (NaCR) was first proposed as an improved metric of denitratation performance metric. Previous metrics used throughout literature were deemed insufficient as they provided an incomplete and subjective representation of denitratation performance by not accounting for residual NO3- remaining in the system following the selective reduction of NO3- to NO2-. The NaCR represented a singular metric that better signifies true denitratation performance and can be compared across studies regardless of carbon source or system configuration. Second, a glycerol-driven denitratation process was optimized according to different operational controls. Steady-state reactor operation and in situ and ex situ batch assays indicated that the influent chemical oxygen demand to NO3- (COD:NO3--N) ratio was determined to influence process kinetics and nitrogen conversion efficiencies leading to significant NO2- accumulation. A singular microbial community structure correlated to system performance was identified. Third, the application of kinetic limitation (by imposing different solids retention times [SRTs]) at a given influent COD:NO3--N ratio was demonstrated as an effective mechanism in the selection for a denitratating microbial ecology capable of significant NO2- accumulation. Steady-state reactor operation was used to characterize process kinetics and nitrogen conversion ratios supporting the determination of the optimal SRT for reactor operation. Analysis of the microbial community structure elucidated the impacts of kinetic limitation on the microbial ecology which were correlated to system performance. Functional denitrification gene transcripts were found to be significantly different under kinetic limitation, indicating that NO2- accumulation was driven more by differences in microbial community structure as opposed to differential expression at different operating SRTs. Fourth, ex situ batch assays were used to elucidate the microbial transcriptional response to the presence of varied sequences of electron acceptors. The microbial community was found to be enriched with NO3--respirers, or microorganisms incapable of NO2- reduction, and progressive onset denitrifiers, which express functional denitrification genes in sequence. The presence or re-introduction of NO3- in a NO2--reducing community was found to elicit an immediate transcriptional change and shift of electron flow to NO3- reductase. Electron competition as the primary contribution to NO2- accumulation was confirmed through the artificial inactivation of NO3- reductase. Lastly, an influent COD:NO3--N ratio was applied in stoichiometric excess to create the conditions necessary to support DNRA over denitrification. System performance at steady-state was found to vary under different kinetic regimes. The induction of DNRA was found to be far more complex than simply providing glycerol in stoichiometric excess. Additionally, glycerol does not appear to be an optimal COD source for DNRA under these conditions. In sum, the optimization of engineered biological nitrogen removal processes through the manipulation of process kinetics and the resulting impacts on nitrogen conversion efficiencies and microbial community structure and function was investigated in detail. From an engineering perspective, this knowledge can help guide the design and operation of biological nitrogen removal processes to systematically maximize the accumulation of targeted nitrogenous products or mitigate unintentional and undesired products

N cycling and microbial dynamics in pasture soils

Pasture soils are a significant source of the greenhouse gas, nitrous oxide (N2O) and as such they contribute to global warming. It has been reported that N2O is approx. 300 times more potent than carbon dioxide (CO2) as a greenhouse gas. Thus, understanding the mechanisms for controlling N2O emissions from soil is key to developing new soil management strategies to counter or prevent climate change throughout the world. Despite this, very little is known about the key regulators of production and consumption of N2O in pasture soils, especially under urine patch conditions. To address this, we used pasture soils representing both Northern (Ireland) and Southern (New Zealand) Hemispheres in experiments designed to understand both phenotypic and genotypic characteristics associated with N2O emissions. We used a combination of gas kinetics, soil physicochemical characterization, metagenomics, 16S amplicon sequencing and quantitative PCR (of denitrifier: nirS, nirK, nosZI and nosZII; and nitrifier: bacterial and archaeal amoA genes) to link physical, chemical and biological parameters associated with emissions. This thesis work was able to show how in nitrate-amended pasture soils the rate of carbon mineralization under oxic and anoxic conditions is positively linked to the rate of denitrification. In addition, the emission ratio of N2O is negatively linked to pH. Both pH and N2O emission ratio were significantly associated with 16S microbial community composition as well as microbial richness. This result confirms that pH imposes a general selective pressure on the entire community and that this is associated with changes in emission potentials. This supports the general ecological hypothesis that with increased microbial diversity, efficiency of N2 production increases (i.e. more efficient conversation of N2O to N2). Worked performed in a simulated urine patch (oxic conditions) suggested other pathway (e.g., nitrifier-denitrification) as a source of N2O emissions. No clear trend was observed between emission ratio of N2O under urine patch condition and emission ratio under true denitrification conditions (i.e. under anoxic environment). The urine patch accelerated the rate of C mineralization about 10 times, concurrent with a decrease in prokaryotic richness and a shift in community composition. Community response identified two major groups of responders: negatively affected prokaryotes we hypothesized utilized energy from N-linked redox reaction for maintenance and positively responding populations that use this energy for growth. Overall, this study provides new insights into the N2O emissions and microbial dynamics for reduction of N2O in pasture soils

Integrating molecular-biological data and process-based models of nitrogen cycling

Microorganisms play a key role in the transformation of nutrients and contaminants in the environment, with significant consequences for drinking water quality, eutrophication, or green house gas emissions. Advances in molecular-biological and omics tools have revolutionized microbiology, providing information about the abundance, diversity and function of microorganisms in the environment. Mathematical models of microbial processes present a potential link between molecular-biological data and biogeochemical reaction rates, but it is important to consider whether the added complexity of these models is justified by the information gained from molecular-biological data. In this thesis, I present modeling approaches that integrate gene and transcript data of functional genes, using nitrogen cycling as an example. By comparing an enzyme-based model to a traditional Monod-type model, I assess the value of accounting for enzymatic regulation in the prediction of denitrification rates. Both model formulations perform similarly with respect to nitrogen species, but the enzyme-based model offers a valuable tool for understanding the relationship between biomolecular quantities and reaction rates. Based on the simulations, I examine whether transcript and enzyme concentrations can directly serve as proxies for reaction rates. My analysis shows that under environmental conditions, the prediction of reaction rates from transcript concentrations is impractical due to time delays in enzyme production, and the limitation of reaction rates by substrates and inhibitors. Building on these findings, I propose sampling strategies to improve the integration of molecular-biological data and reactive-transport modeling. Finally, I investigate how functional-gene data affects the uncertainty of nitrogen cycling rates and model parameters in a flow-through column experiment. Using Bayesian parameter estimation, I quantify uncertainty of the model parameters and reaction rates. My results also provide insights on the poor identifiability of the parameters in the standard Monod rate law. While functional gene data do not reduce the uncertainty of nitrogen cycling rates, they influence the estimates and reduce uncertainty of several parameters related to microbial nitrogen cycling