Nitrate (NO3-) is a ubiquitous groundwater contaminant in agricultural and wastewater discharge areas. The prediction of microbial mediated NO3- removal in subsurface environments requires an understanding of the rates at which electron donors are utilized by denitrifying microbes. This study focuses specifically on the following organic carbon compounds as electron donors: glucose, acetate, adenine, cysteine and fulvic acid. Six triplicate series of flow through reactors (FTRs) containing 35 cm3 of natural, organic-poor sediment were supplied for 10 weeks with solutions containing nitrate and the individual carbon compounds, along with a no-carbon added control. The organic carbon compounds were selected to yield a range of different types of organic carbon (sugars, amino acids etc.) as well as a range of Gibbs Free Energy (???G) values when their oxidation is coupled to denitrification. The initial flow rate of the FTRs was 1 ml h-1. Once steady NO3- concentrations were reached in the outflow, the flow rate was increased to 2 ml h-1 and, subsequently, 4 ml h-1. Potential denitrification rates (RD) measured for the different carbon substrates spanned a range of 0 to 114 nmol cm-3 h-1. Fulvic acid did not induce denitrification, while acetate yielded the highest rate. The outflow solutions for FTRs supplied with adenine and cysteine contained ammonia and sulfate, respectively. These results are consistent with the molecular structure of adenine, which contains an amine group, and of cysteine, containing an amine and thiol group. The results show that the addition of C-substrates to the sediment promotes denitrification, and the rate at which it occurs are dependant on which C-substrate is provided. RD results were used to determine if the denitrification rates imposed by the different carbon substrates could be predicted using theoretical approaches such as ???GR or the nominal oxidation state of carbon (NOSC). However, predictions determined by thermodynamics alone were not significantly correlated with the observed trends in denitrification rates.

Agricultural management is essential for achieving optimum crop production and maintaining soil quality. Soil microorganisms are responsible for nutrient cycling and are an important consideration for effective soil management. The overall goal of the present research was to better understand microbial communities in agricultural soils as they relate to soil management practices. For this, we evaluated the differential impact of two contrasting drainage practices on microbial community composition and characterized active denitrifiers from selected agricultural sites. Field drainage is important for crop growth in arable soils. Controlled and uncontrolled tile drainage practices maintain water in the field or fully drain it, respectively. Because soil water content influences nutrient concentration, moisture, and oxygen availability, the effects of these two disparate practices on microbial community composition was compared in paired fields that had diverse land management histories. Libraries of the 16S rRNA gene were generated from DNA from 168 soil samples collected from eight fields during the 2012 growing season. Paired-end sequencing using next-generation sequencing was followed by read assembly and multivariate statistical analyses. Results showed that drainage practice exerted no measureable effect on the bacterial communities. However, bacterial communities were impacted by plant cultivar and applied fertilizer, in addition to sampled soil depth. Indicator species were only recovered for depth; plant cultivar or applied fertilizer type had no strong and specific indicator species. Among indicator species for soil depth (30-90 cm) were Chloroflexi (Anaerolineae), Betaproteobacteria (Janthinobacterium, Herminiimonas, Rhodoferax, Polaromonas), Deltaproteobacteria (Anaeromyxobacter, Geobacter), Alphaproteobacteria (Novosphingobium, Rhodobacter), and Actinobacteria (Promicromonospora). Denitrification in agricultural fields transforms nitrogen applied as fertilizer, reduces crop production, and emits N2O, which is a potent greenhouse gas. Agriculture is the highest anthropogenic source of N2O, which underlines the importance of understanding the microbiology of denitrification for reducing greenhouse gas emissions by altered management practices. Existing denitrifier probes and primers are biased due to their development based mostly on sequence information from cultured denitrifiers. To circumvent this limitation, this study investigated active and uncultivated denitrifiers from two agricultural sites in Ottawa, Ontario. Using DNA stable-isotope probing, we enriched nucleic acids from active soil denitrifiers by exposing intact replicate soil cores to NO3- and 13C6-glucose under anoxic conditions using flow-through reactors, with parallel native substrate controls. Spectrophotometric chemistry assays and gas chromatography confirmed active NO3- depletion and N2O production, respectively. Duplicate flow-through reactors were sacrificed after one and four week incubation periods to assess temporal changes due to food web dynamics. Soil DNA was extracted and processed by density gradient ultracentrifugation, followed by fractionation to separate DNA contributed by active denitrifiers (i.e., “heavy” DNA) from that of the background community (i.e., “light” DNA). Light and heavy DNA samples were analyzed by paired-end sequencing of 16S rRNA genes using next-generation sequencing. Multivariate statistics of assembled 16S rRNA genes confirmed unique taxonomic representation in heavy fractions from flow-through reactors fed 13C6-glucose, which exceeded any site-specific or temporal shifts in putative denitrifiers. Based on high relative abundance in heavy DNA, labelled taxa affiliated with the Betaproteobacteria (71%; Janthinobacterium, Acidovorax, Azoarcus, Dechloromonas), Alphaproteobacteria (8%; Rhizobium), Gammaproteobacteria (4%; Pseudomonas), and Actinobacteria (4%; Streptomycetaceae). Metagenomic DNA from the original soil and recovered heavy fractions were subjected to next-generation sequencing and the results demonstrated enrichment of denitrification genes with taxonomic affiliations to Brucella, Ralstonia, and Chromobacterium in heavy fractions of flow-through reactors fed 13C6-glucose. The vast majority of heavy-DNA-associated nitrite-reductase reads annotated to the copper-containing form (nirK), rather than the heme-containing enzyme (nirS). Analysis of recovered nirK genes demonstrated low sequence identity across common primer-binding sites used for the detection and quantification of soil denitrifiers, indicating that these active denitrifiers would not have been detected in molecular surveys of these same soils.