Inhibition, gene expression and modeling of ammonia oxidation in biofilms of Nitrosomonas europaea

This dissertation explores the physiology and gene expression of the ammonia-oxidizing bacterium Nitrosomonas europaea in surface-associated bacterial communities, or biofilms. Biofilms of N. europaea were cultivated in drip flow reactors for several weeks and gene expression microarrays were used to detect 240 genes differentially expressed between the mature biofilms and exponential batch cells. Using RT-qPCR, genes up-regulated in the biofilm microarray were detected in continuous cultures of suspended cells, and were increasingly up-regulated with decreasing dilution rates. The observations suggest a correlation between the biofilm-related gene expression and slow growth rates of the cells. N. europaea cells in the biofilms upon exposure to the aromatic hydrocarbons phenol and toluene were more resistant to inhibition of ammonia oxidation than suspended cells. 50% inhibition was observed upon exposure of mature biofilms to 60 μM phenol and 100 μM toluene, compared to 10 μM phenol and 20 μM toluene with exponential batch (suspended) cells. However, the transcriptional response to the hydrocarbons was similar between suspended cells and biofilms, with two genes up-regulated in both growth states in response to phenol (NE1545-NE1546) while no transcriptional response was observed during toluene exposure. To further explore the response to phenol exposure, cells were cultivated in continuous reactors at various growth rates and NH3 oxidation rates and exposed to phenol. The inhibition of ammonia oxidation by 20 μM phenol decreased with slower growth rates and NH₃ oxidation rates and approached the inhibition level of the biofilms. Increasing the dissolved oxygen (DO) concentration in the biofilms resulted in higher NH₃ oxidation rates and greater phenol inhibition, leading to the conclusion that the tolerance of biofilms is likely related to O₂ limitation causing slow NH₃ oxidation rates and slower growth rates. DO and pH were measured in the biofilms with microsensors and the concentration profiles were simulated with 2-D reactive transport that combined advective and diffusive transport. The half-saturation coefficient for suspended cells fit the biofilm profiles, indicating that the kinetics of NH₃ oxidation between the biofilm and suspended cells were the same. The model was also able to simultaneously predict the observed pH and DO biofilm profiles for tests conducted with limited buffering capacity, where pH was lowered by the cells, which resulted in NH₃ limitations and reduced O₂ consumption.Keywords: biofilms, aromatic hydrocarbon inhibition, Ammonia oxidizing bacteria, Nitrosomonas europaea, nitrificatio

Differences Between Heterotrophic and Nitrate-dependent Iron-oxidizing Microbial Communities in Bioreactor Sediment Treating Mine Wastewater

Nitrate-dependent iron oxidation (NDFO) is a novel mechanism for microbial bioremediation of metal and metalloid contaminants. During NDFO, microbes catalyze a redox reaction wherein nitrate is reduced to nitrite and nitrogen gas while Fe(II) is oxidized to solid Fe(III) hydroxide minerals. Metalloid contaminants such as selenium and arsenic have a propensity for adsorption to iron minerals produced during NDFO; some contaminants may also be concurrently bioreduced. A number of bacterial isolates have been shown to be capable of NDFO (e.g., Kappler et al. (2005), Kiskira et al. (2017)), but little work has been done to date characterizing mixed microbial communities performing NDFO. Some autotrophic communities have been characterized, with high relative abundances for strains of Gallionellaceae in both a freshwater sediment enrichment culture and an activated sludge culture (Blöthe and Roden 2009, Tian et al. 2020). In mixotrophic activated sludge cultures, the concentration of Fe(II) amendment was found to significantly impact microbial community composition; these cultures were fed with methanol in addition to Fe(II), and the dominant community members were Methyloversatilis and other methylotrophic strains (Liu et al. 2018). The work presented here examines microbial communities performing NDFO in the context of remediation, and in particular how differences between NDFO and heterotrophic communities may influence remediation effectiveness.This research characterizes and compares microbial communities performing NDFO versus heterotrophic denitrification during removal of selenium and nickel from mining wastewater. Sediment and influent water from a subsurface bioreactor treating mining wastewater were used to construct batch bioreactors, which were amended with selenium and nickel as well as either Fe(II) or methanol to investigate contaminant removal and microbial community composition in NDFO versus heterotrophic microbial communities. Both Fe(II) and methanol reactors removed total aqueous selenium to below the quantification limit, but Fe(II) reactors removed it more rapidly, likely due to adsorption of selenite. For nickel, removal to below the detection limit was achieved with methanol amendment, while Fe(II) amendment resulted in 42-95% removal. This was likely due to precipitation of nickel sulfide during sulfate reduction in methanol-amended reactors.DNA from the batch bioreactors will be sequenced and the results analyzed for differences among communities. Permutational multivariate analysis of variance and non-metric multidimensional scaling will be used to determine significant correlations of community composition with experimental variables, selenium and nickel removal, and NDFO (Roberts 2023, Kruskal 1964). Indicator species analyses (De Cáceres et al. 2010) will be applied to identify taxa found significantly more often (i.e., at a higher relative abundance) in one group of microbial communities than in any other group. The indicator species analysis may reveal whether there are groups of denitrifiers that predominate in NDFO conditions vs. groups that predominate during heterotrophic denitrification. The results of these microbial community analyses, in combination with the geochemical analyses, will improve our understanding of microbial communities performing NDFO in remediation environments

Gel-Entrapped Staphylococcus aureus Bacteria as Models of Biofilm Infection Exhibit Growth in Dense Aggregates, Oxygen Limitation, Antibiotic Tolerance, and Heterogeneous Gene Expression

Cathedral, model of the nave; Gaudí’s masterpiece, and, although only partially completed, it is among the most impressive buildings of the 20th century. He took charge of the works at the age of 31 and continued for the rest of his life; it thus summarizes his evolution as an architect (from Modernisme to a mature individualism) as well as the increasing depth of his spiritual conviction, measured in terms of his increasing dedication to the task. His assistants on the project included Jujol, Berenguer, Rubió, the sculptor Lorenzo Matamala i Pinyol (1856-1927), Gaudí’s friend and site supervisor, and Carlos Mani i Roig (1866-1911). The concept of a church dedicated to the Holy Family was widened to become a cathedral for the new metropolitan areas of Barcelona, and del Villar’s neo-Gothic design had been under construction for more than a year when Gaudí took over in 1883. The towers and the church show Gaudí’s structural aesthetic at work in the idiom of the Gothic cathedral, eliminating the need for flying buttresses. The triple portals (1903) of the Nativity façade, represent Faith, Hope and Charity. The four great facade towers are named after the Apostles: from south to north, Barnabas, Simon, Thaddeus and Matthew. Only Barnabas was completed before Gaudí’s death. The project is scheduled to be completed in 2026, the 100th anniversary of Gaudi's death. Source: Grove Art Online; http://www.oxfordartonline.com/ (accessed 7/16/2010