Knowledge of community structural response to environmental variation is essential for understanding the microbial regulation of nitrogenremoval processes in aquatic systems. In particular, it is important to elucidate the environmental factors that support higher abundance of different denitrifying populations as they significantly contribute to overall denitrification activities. A customized microarray containing 165 nirS gene probes (archetypes) was designed from all available nirS sequences detected in environments worldwide to represent sequences separated by >15% divergence. We conducted nirS gene microarray analysis with sediment samples collected in two seasons (summer and winter) at four sites in the New River Estuary, NC, USA to identify the abundant members of the denitrifying community and examine their responses to changing environmental variables over spatial and temporal scales. In addition, the contribution of specific archetypes to overall denitrifying activities was evaluated. The abundance of specific archetypes associated with marine and estuarine environments and
Biological anaerobic ammonium oxidation (anammox) is a process catalyzed by a group of Bacteria comprising the order of the Brocadiales. Since their discovery in a wastewater treatment plant nearly 20 years ago, these organisms have been detected in anaerobic ecosystems worldwide and it has been shown they play a pivotal role in global nitrogen cycling. On top of this, anammox bacteria have been studied intensely to understand their physiology and biochemistry, and finally they have been applied in a novel, cost-effective and environmentally friendly process to remove ammonium from wastewater. Despite all advances made, a pure culture of anammox bacteria was never obtained. This presents a challenge for genome sequencing, as the total genome information of the culture, called metagenome, will originate from a mix of organisms. To reconstruct the genome of a target organism, the anammox bacterial sequences have to be separated from the sequences originating from other organisms in the culture in a process called binning. Binning is mostly feasible on relatively simple microbialcommunities such as enrichment bioreactors, but gaining wider application as sequencing throughput and analysis methods improve. Before binning was applicable to complex systems, metagenomic sequencing of complex communities relied mostly on characterizing the environment under scrutiny based on the DNA fragments from known microbial clades. In this thesis we have applied metagenomic sequencing to microbialcommunities involved in nitrogen conversions, with a focus on anammox bacteria.
Massive amounts of plastic enter and reside within riverine, estuarine and coastal environments. Although it was once con- sidered completely recalcitrant, we now know that plastic degrades to varying extents in the marine environment over time and that microbialcommunities may play a role in this 9,25,42 . The leaching of chemicals from plastic alone has been shown to potentially contribute to the dissolved organic C pool in marine waters 43 and to the production of greenhouse gases, such as methane and ethylene 44 . It was estimated that between 1.15 and 2.41 million tons of plastic enter the coastal zone and oceans from rivers annually, much of which eventually reaches sediments 45 . These plastics once served a variety of consumer purposes; as such, they are extremely diverse in form and chemistry. Here, we have demonstrated that microplastics generated from four diverse polymers inﬂuenced marsh sediment microbiomes and biogeo- chemical cycling. Although the difference between bioﬁlm com- munities and that of the surrounding sediment cannot be differentiated using our approach, the outcomes between our treatments robustly illustrate the inﬂuence microplastics may have on intact sediment ecosystems. This is foundational for future efforts to assess risks of microplastic pollution in diverse environments. Further, the work presented here demonstrates that microplastics are indeed capable of ecosystem-level effects, including alteration of biogeochemical cycles 3 . Thus, we should evaluate plastic debris as a potential planetary boundary threat 3,4,46 .
Jayakumar et al., 2004). Falk et al. (2007) compare denitri- fying microbialcommunities across environmental gradients within the water column and coastal sediments of the Baltic Sea by employing one of the two nitrite reductase genes, nirS, as a molecular marker for denitrifiers. Phylogenetic analysis of nirS genes from the Baltic Sea and of sequences from other areas indicate distinct denitrifier communities that seem to group mostly according to their habitats. They con- clude that distinct marine nirS-type denitrifier communities developed after selection determined by their habitats, the ambient environmental conditions and isolation by large ge- ographic distances between habitats.
and building more cohesive communities ( Lovell, 2010; Lovell and Taylor, 2013; McPhearson et al., 2013; Specht et al., 2014 ). However, it can be challenging to integrate ecosystem services, if each requires different management strategies. For example, intensive vegetable production and stormwater management are among the ecosystem services emphasized in the studies of rooftop farming ( Ackerman et al., 2013; Specht et al., 2014; Thomaier et al., 2015; Whittinghill et al., 2015; Goldstein et al., 2016 ). Intensive agriculture can require higher nutrient inputs to maintain crop yield and quality, while stormwater management aims to reduce nutrient loads to surface water by managing vegetation with limited supplemental nutrients. Drainage output of nutrients from intensive agriculture, such as nitrogen (N) from application of high amounts of fertilizer, has led to multiple negative consequences, including contamination of groundwater and development of “dead zones,” such as the Chesapeake Bay, Gulf of Mexico, and Long Island Sound ( Howarth et al., 2000; Rabalais et al., 2002; Kemp et al., 2005; Howarth, 2008 ). These nutrient loadings into waterways are regulated by the Clean Water Act through the total maximum daily load and best management practices ( Wainger, 2012 ). Some estuaries are more sensitive to nutrient loads than others. For instance, the longer mean residence time of water in Long Island Sound (1,100 days) compared to Delaware Bay (60 days) and Chesapeake Bay (250 days) makes N management particularly important to New York City, which is a major source of N for Long Island Sound ( Nixon et al., 1996; Howarth et al., 2006 ). Nutrient loadings from urban land uses including urban agriculture are often managed by sewer systems. For example, 60% of NYC’s sewer systems manage stormwater and sanitary sewage together in the same sewers ( NYC EDC, 2013 ), and these combined sewers can bypass treatment plants during storm events, discharging untreated effluent to surface water ( US EPA, 2004 ). The Clean Water Act requires that cities using combined sewers must implement best management practices ( Carter and Fowler, 2008 ). In the infancy of rooftop farming practices, there are opportunities to establish and implement best management practices to limit the nutrient loads from urban farms to surface waterbodies.
A model describing a given system should be as simple as possible – but not simpler. The appropriate level of complexity depends both on the type of system and on the intended use of the model. This chapter addresses the critical question of which purposes justify increased complexity of biofilm (reactor) models. The additional model feature compared to conventional models considered is the inclusion of microbial diversity, distinguishing between different species performing the same function. With a multispecies model considering interspecies diversity, by implementing the growth and endogenous respiration of 10 ammonia-oxidizing and 10 nitrite-oxidizing species, it was demonstrated that a given reactor performance in terms of bulk liquid concentrations does not necessarily reflect microbial steady state conditions. In a second case study, the functional redundancy of the nitrifying community, i.e., the possibility of a changed nitrifying community to function equally as the original one, upon an increased nitrogen loading rate, was verified. It was concluded that increased complexity in biofilm models, concerning microbial diversity, is likely more useful when the focus is on understanding microbial competition and coexistence, but under specific conditions, these additional model features can be critically informative for bulk reactor behaviour prediction and general understanding.
) in the suboxic zone of sediments may be a key
controlling factor for ANAMMOX in constructed wetlands. In addition, wetland vegetation has been suggested to have an important role of ANAMMOX as shown in a mesocosm study of Tao and Wang (2009). Thus, ANAMMOX and denitrifying bacteria in constructed wetlands appear to have either a mutualistic or competitive relationship for available NO x - substrates, as well as being supported by different wetland vegetation types/species. In addition, wetland vegetation may enhance ANAMMOX and denitrification by supplying substrates and niches to both bacterial communities. Particle size of sediment could also influence microbial N removal processes since higher denitrification has been measured with fine textured soils with high contents of silt and clay (Pinay et al., 2000). With the crucial importance of N as a pollutant and major component of stormwater runoff, determining the environmental parameters controlling ANAMMOX and denitrification in constructed wetland sediments is imperative to enhancing its removal. Such information can lead to optimizing the performance of N removal from
ABSTRACT Wetland ecosystems are important reservoirs of biodiversity and signiﬁ- cantly contribute to emissions of the greenhouse gases CO 2 , N 2 O, and CH 4 . High an-
thropogenic nitrogen (N) inputs from agriculture and fossil fuel combustion have been recognized as a severe threat to biodiversity and ecosystem functioning, such as control of greenhouse gas emissions. Therefore, it is important to understand how increased N input into pristine wetlands affects the composition and activity of microorganisms, especially in interaction with dominant wetland plants. In a series of incubations analyzed over 90 days, we disentangled the effects of N fertilization on the microbial community in bulk soil and the rhizosphere of Juncus acutiﬂorus, a common and abundant graminoid wetland plant. We observed an increase in green- house gas emissions when N is increased in incubations with J. acutiﬂorus, changing the system from a greenhouse gas sink to a source. Using 16S rRNA gene amplicon sequencing, we determined that the bacterial orders Opitutales, subgroup 6 Acido- bacteria, and Sphingobacteriales signiﬁcantly responded to high N availability. Based on metagenomic data, we hypothesize that these groups are contributing to the in- creased greenhouse gas emissions. These results indicated that increased N input leads to shifts in microbial activity within the rhizosphere, altering N cycling dynam- ics. Our study provides a framework for connecting environmental conditions of wetland bulk and rhizosphere soil to the structure and metabolic output of micro- bial communities.
Sample collection and experimental setup. Plants and sandy soil were sampled from the Raven- vennen (51.4399°N, 6.1961°E) in Limburg, The Netherlands (August 2015), and returned to the Radboud University greenhouse facilities for conditioning. The Ravenvennen is a protected marshy area consisting of sandy soil rich in vegetation with a high prevalence of Juncus spp. Plants were removed from soil, and rhizomes were cut into eight 2-cm fragments and reconditioned on hydroculture in a nutrient-rich medium as described by Hoagland and Arnon (66). After sufﬁcient root development (to approximately 25 cm after 2 weeks), eight plants and eight bulk soil incubations were randomly assigned to high- or low-nitrogen experimental groups, totaling 16 incubations (see Table S1 and Fig. S1 in the supplemental material). Soil collected from the ﬁeld was homogenized and sieved to remove any contaminating roots and potted. The reconditioned plants were transferred to pots with diameters of 19 cm at the base and 26 cm at the top and a height of 19 cm containing the prepared soil, moved to an indoor water bath set to 15°C with a cryostat (Neslab Thermoﬂex 1400; Thermo Electron Corp., Breda, The Netherlands), and cultivated with a day/night cycle of 16 h of light and 8 h of dark (Master Son-T PiaPlus; Philips, Eindhoven, The Netherlands). Pots were kept waterlogged with a 2-cm water layer on top. A drip-percolation-based system ensured a constant supply of nutrients. The low-N-input nutrient solution contained 12.5 M NH 4 NO 3 , corresponding to an N loading rate of 40 kg N·ha ⫺1 ·year ⫺1 . The high-N-input solution contained
tree and shrub species dominated by Fagus sylvatica, Corylus avellana, Salix caprea and Quercus robur) and exhibiting intermediate dissolved N concentrations com- pared to our experimental N gradient (0.75 mg L −1 on average, based on 15 analyses carried out year-round be- fore the beginning of the experiment). Decomposing leaves collected in the stream were carried to the labora- tory and submerged for 3 days in 3 L of demineralized water with constant shaking and aeration at a temperature of ca. 14 °C. The spore density and species composition of aquatic hyphomycetes in the suspension were deter- mined just before inoculation of the microplate wells by filtering ten replicate 2-mL aliquots of the suspension over a nitrocellulose membrane (5 μm pore size; Whatman International Ltd., Maidstone, UK). The spores were stained with Trypan blue (0.5% in 60% lactic acid) and counted and identified at × 200 magnification. The 2-mL aliquots contained 11,473 ± 1337 (SD) spores be- longing to an average of 18.2 ± 1.7 (SD) species of aquat- ic hyphomycetes (Supplementary Material 2 ). The identi- fied species are among the most common in temperate forest streams [ 31 ], reflecting particularly well the com- position of aquatic hyphomycete communities in streams of the Montagne Noire (south-western France) [ 32 ]. After 48 h, the spore suspensions were replaced by mineral solutions with variable N concentrations.
Rima, I am forever in your debt.
Thanks to Dr. Scott Neubauer for giving me knowledge and insight into the field of biogeochemistry. He has been an invaluable collaborator on this work, and has been ever willing to provide assistance. I am grateful to Dr. Bonnie Brown, for guiding development of my skills in molecular biology and for sharing resources for stable isotope studies. In addition, she has provided excellent advice and has been an unwavering source of support over these last four years. I also thank my other committee members Dr. Roy Sabo and Dr. Leigh McCallister, for their time and assistance. I give special thanks to my past supervisor Dr. Steve Negus: under his guidance I fell in love with science, and his belief in me helped me gain the confidence to pursue a PhD.
Positive in situ acetylene assays indicated nitrogen fixation both in a debris-rich 100 m marginal zone and up to 5.7 km upslope on Leverett Glacier (with rates up to 16.3 µmoles C 2 H 4 m −2 day −1 ). No positive acetylene assays were de- tected > 5.7 km into the ablation zone of the ice sheet. Po- tential nitrogen fixation only occurred when concentrations of dissolved and sediment-bound inorganic nitrogen were un- detectable. Estimates of nitrogen fluxes onto the transect sug- gest that nitrogen fixation is likely of minor importance to the overall nitrogen budget of Leverett Glacier and of negligible importance to the nitrogen budget on the main ice sheet it- self. Nitrogen fixation is however potentially important as a source of nitrogen to microbialcommunities in the debris- rich marginal zone close to the terminus of the glacier, where nitrogen fixation may aid the colonization of subglacial and moraine-derived debris.
105 transformation and to include biological filtrations for the management of the resulting nutrients (DON and NH 4 + ) as the critical steps in secondary treatment.
Secondary treatment - All settlement pond systems facilitate the biological transformation of nutrients, or secondary treatment, through microbial transformation and assimilation into biomass. However, because there is little control over the prevailing abiotic variables in these environments it is difficult to enhance the beneficial microbial pathways of nitrification, denitrification and anammox. These pathways compete with detrimental pathways that recycle nitrogen in the system, such as DNRA and mineralisation, and the latter often prevails (Fig. 5.2), especially in tropical and subtropical sulfidic environments (Castine et al., 2012). However, biological transformation of nutrients by bacteria is an extremely powerful mechanism which has not yet been optimised for LBAS and represents significant opportunities for marine and brackish water LBAWWT.
Denitrification, landfill leachate, microbial culture, nitrification
The landfill leachate is a highly variable and heterogenous mixture of high strength organic and inorganic contaminants including, among others, humic acids, xenobiotic organic compounds (XOCs), ammonia nitrogen, heavy metals and other inorganic salts. 1 Due to the toxicity and potential risk to surrounding soil, ground or surface waters, landfill leachates become a major pollution hazard and must be collected and appropriately treated be- fore being discharged into the environment. 2–8 Many factors affect the quality of leachate, such as age, precipitation, seasonal weather variation, waste type and composition. 1,9 The chemical composition of leachate varies greatly depending on the age and maturity of the landfill site. An immature leachate contains high strength organic compounds and am- monia nitrogen, while a mature leachate contains relatively low concentrations of degradable organic matter but high concentrations of ammonia nitro- gen. 1,9 The ammonia nitrogen constitutes a major long-term pollutant in landfill leachate. 1 Biological nitrogenremoval through the process of nitrifica- tion and denitrification is usually suggested for treatment of landfill leachate with low COD/N ra- tio. 10–13 Ammonia nitrogen present in mature land- fill leachate in high concentrations causes difficul-
There were major differences in ammonia-oxidizing and nitrous oxide-reducing community composition and structure between centralized and decentralized BNR wastewater treatment systems. amoA richness and diversity were similar at the two treatment scales, but nosZ diversity and richness were higher in the WTP than in the OWTS. Ordination analysis of beta diversity showed clear differences in the amoA and nosZ communities between the WTP and the OWTS. Relative abundances of Nitrosomonas and Nitrosospira were different between the WTP and the OWTS. The higher diversity and closer clustering of beta diversity for nosZ in the WTP suggests that the larger scale of treatment supports a wider variety of denitrifiers in sufficiently large numbers to maintain more heterogeneous communities compared to OWTS. We also observed nosZ genera with more diverse metabolic strategies in the WTP. Together, these factors may make the WTP more resilient to environmental changes such as shifts in climate and influent properties. Like nosZ, amoA community composition was more similar within a scale of treatment, but the community of WTP and OWTS had similar alpha diversity metrics, likely because there is a limited number of nitrifying taxa.
*S 0 yield = g of S 0 produced/g of SO 4 2- -S added
*N 2 0 yield = g of N 2 produced/g of NH 4 + -N added
IV. C ONCLUSION
This study showed that sulfate and nitrogen compounds can be removed simultaneously in a single reactor. The level of dissolved oxygen (DO) concentration was an effective process parameter to regulate the specific microbial activities and their metabolic products. The optimal DO concentration for simultaneous removal of sulfur and nitrogen compounds was 0.10-0.15 mg/L. S 0 and N 2 gas were the main metabolic products of this process. Increasing DO concentration only 0.10 mg/L significantly inhibited SRB, leading to a decrease of sulfate removal efficiency and S 0 production. In addition, when decreasing DO to 0.05-0.10 mg/L, nitrifier was inhibited significantly and N 2 production was decreased.
In this three-year, seasonal study of sediment N biogeochemistry, I found that cores taken amongst oysters exhibited significantly higher rates of total denitrification compared to control cores in the summer and fall months when oysters are most active at Site 1 and in the fall at Site 2. Gross nitrification rates, though not significantly different in oyster and control cores, were sufficiently high to support coupled-dominated DNF in all cores. DNRA rates were highest in the summer at both sites, but, contrary to my hypothesis, did not appear to be directly affected by oysters. Additionally, I observed higher rates of both DNF and DNRA at the lower OC, higher salinity Site 2 overall. Carbon quality and organic matter source appear to account for these differences in DNF rates between the sites, while increased salinity and therefore sulfate
Filip (2001) in his review paper stressed the fol- lowing ecologically important soil characteristics:
microbial biomass, composition of microflora (ra- tio bacteria/fungi, microflora of the C-cycle and N-cycle), mineralization processes (CO 2 and NH 4 + release), and synthesising processes. This author underlined that there is no doubt that firm link- ages exist between microbialcommunities, their activities, and ecologically important processes.