Bacteria play important roles in the transformation of nutrients in wetlands, but few studies have examined parameters affecting variation in bacterial community composition between wetlands. We compared the composition of eubacterial and denitrifying bacterial biofilm communities in 32 agricultural constructed wetlands in southern Sweden, and the extent to which wetland environmental parameters could explain the observed variation. Structure and richness of the eubacterial 16S rRNA gene and three denitrifying bacterial enzyme genes (nirK, nirS and nosZ), analysed by molecular fingerprinting methods, varied among the constructed wetlands, which could be partly explained by different environmental parameters. Results from the enzyme gene analyses were also compared to determine whether the practice of using a single denitrifying bacterial gene could characterize the overall community composition of denitrifying bacteria. We found that nirK was more diverse than both nirS and the nosZ, and the band structure and richness of the three genes were not related to the sam environmental parameters. This suggests that using a single enzyme gene may not suffice to characterize the community composition of denitrifying bacteria in constructed agricultural wetlands. / <p>Included in doctoral thesis: Milenkovski, Susann. Structure and Function of Microbial Communities in Constructed Wetlands - Influence of environmental parameters and pesticides on denitrifying bacteria. Lund University 2009.</p>

Nitrous oxide (N2O) is a greenhouse gas (GHG) with 300 times the radiative forcing in the atmosphere of carbon dioxide (CO2), and has recently become a subject of great concern because the nitrogen (N) fertilizers which have been necessary to increase agricultural productivity have also dramatically increased N2O emissions from agroecosystems. Many N control practices have been suggested and implemented in agroecosystems, but their ability to simultaneously remove reactive N from the environment and prevent the production of N2O is, at best poorly understood. The goal of this work is to characterize environmental controls on production of N2O in denitrifying bioreactors. The review portion of this work first discusses the geologic history of the N cycle, how its past and present processes differ, and how it is being affected by human activity. It then explores the N cycle's biochemical pathways, reviews the controls for each of its steps, and discusses the environmental drivers of these controls. The review closes with a discussion of environmental N management strategies. The experimental portion of this work further explores these concepts by observing how biochar amendment and the modification of pH affect N2O production in the denitrification pathway in denitrifying bioreactors. Both pH and biochar have previously been shown to affect N2O production and many N management practices utilize biochar or manipulate pH to increase N retention. The objectives of the experiment were to: 1) Examine headspace N2O concentration in sealed, biochar-amended, denitrifying bioreactors; 2) Determine if the effects of pH on N2O production differ in biochar-amended systems versus controls (under acidic, unbuffered, and buffered conditions); 3) Quantify key denitrification genes (nirK, nirS, nosZ) in each treatment combination. Experimental results showed biochar treatment to significantly increase N2O emissions, a result which runs contrary to most, but not all studies regarding its effects on N2O production. Differences between treatments decreased with increasing pH levels. Biochar did not exhibit significant effects on individual denitrification genes, but it did show influence on the ratios of their populations. On the other hand, pH was found to have significant effects on nirS and nosZ populations. Differences in N2O production between biochar and controls were thus explained by biochar's chemical effects, likely its ability to increase denitrification activity. Developing an understanding of the mechanisms behind these differences will require using a combination of isotope tracing, enzyme assays, and mass balance approaches. Future microbial work in biochar-amended systems should attempt to characterize differences in gene expression, overall community structure, and long-term population trends in the genes of interest. The combination of these approaches should allow researchers to better predict where N2O production will occur and develop strategies to mitigate it while simultaneously increasing food production to meet the demands of a growing population. / Master of Science

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.