Development and applications of a high-throughput activity assay, using the ammonia-oxidizing bacteria Nitrosomonas europaea Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/jm214r81k

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  • The rising demand on freshwater resources emphasizes the benefit of biogeochemical processes that maintain clean water by removing pollutants such as excess nitrogen. The most sensitive step in the removal of nitrogen from waterways is the oxidation of ammonia, a part of nitrification, which is carried out by ammonia oxidizing bacteria. To measure nitrification inhibition, a simple High-throughput bioassay has been developed using the model ammonia oxidizing bacteria Nitrosomonas europaea. This High-throughput bioassay provides a rapid and sensitive means of screening for nitrification inhibition and builds upon a previously established Large Bottle bioassay. The High-throughput bioassay introduces the use of a low volume 96-well plate format to perform a 180 minute nitrification activity assay via the amount of nitrite (NO₂⁻) produced. The High-throughput bioassay was shown to have good agreement with results from a previously developed assay utilizing silver ions (Ag⁺) and silver nanoparticles (AgNPs) as nitrification inhibitors. Applications of this method included the exploration of the potential of trace nutrients, growth conditions, ammonium concentration, and the presence of Suwannee River natural organic matter (SRNOM) to confer protection against inhibition of N. europaea nitrification activity when exposed to Ag⁺ or 20 nm citrate-capped AgNPs. The addition of trace nutrients to cells exposed to Ag⁺ or AgNPs demonstrated that the presence of divalent cations, magnesium (Mg⁺²) or calcium (Ca⁺²), showed the greatest effect in reducing nitrification inhibition. The presence of the ligand chloride (Cl⁻) was also observed to reduce inhibition, albeit to a lesser degree. While SRNOM conferred protection against inhibition to AgNPs, little or no protection was seen when cells were exposed to Ag⁺ which indicated protection was achieved by preventing the dissolution of Ag⁺. Results of varying growth conditions, achieved by changing the hydraulic retention time (HRT) of fill-and-draw reactors, demonstrated that cells with slower growth rates had increased resistance to inhibition from Ag⁺ and AgNPs. Other activity assays can also be adapted to the High-throughput method including fluorescent oxygen consumption, live/dead staining, long-term chronic inhibition, and gene expression. In addition, it may be possible to further develop this method to utilize freeze-dried cells to overcome the limitations of using an aqueous culture.
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  • description.provenance : Approved for entry into archive by Julie Kurtz(julie.kurtz@oregonstate.edu) on 2014-01-28T17:39:14Z (GMT) No. of bitstreams: 2 Bartow Thesis Final Draft.pdf: 1050385 bytes, checksum: 9a68f7f5aee790cde11b940009b82dfb (MD5) license_rdf: 1370 bytes, checksum: cd1af5ab51bcc7a5280cf305303530e9 (MD5)
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  • description.provenance : Made available in DSpace on 2014-01-29T18:56:24Z (GMT). No. of bitstreams: 2 Bartow Thesis Final Draft.pdf: 1050385 bytes, checksum: 9a68f7f5aee790cde11b940009b82dfb (MD5) license_rdf: 1370 bytes, checksum: cd1af5ab51bcc7a5280cf305303530e9 (MD5) Previous issue date: 2014-01-07
  • description.provenance : Approved for entry into archive by Laura Wilson(laura.wilson@oregonstate.edu) on 2014-01-29T18:56:24Z (GMT) No. of bitstreams: 2 Bartow Thesis Final Draft.pdf: 1050385 bytes, checksum: 9a68f7f5aee790cde11b940009b82dfb (MD5) license_rdf: 1370 bytes, checksum: cd1af5ab51bcc7a5280cf305303530e9 (MD5)

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