Browsing by Subject "bioremediation"
Now showing 1 - 3 of 3
Results Per Page
Sort Options
Item Genome editing in alfalfa (Medicago sativa) to hyper-accumulate phosphate(2019) Samac, Deborah A; Miller, Susan S; Dornbusch, Melinda R; Curtin, Shaun JRock phosphate, the main source of phosphate (P) for crop fertilizers, is a finite resource that is predicted to be depleted in 50-100 years. P is a critical nutrient in agriculture and its application can dramatically improve plant productivity. However, many soils have excess amounts of P from application of animal manures and runoff of phosphate from agricultural lands is the major source of nonpoint water pollution in the Midwestern US. The goal of this project is to create mutations by gene editing in the ubiquitin E2 conjugating enzyme PHO2, involved in P signaling and P homeostasis in alfalfa so that plants hyper-accumulate phosphate. Such plants could be used to reduce soil P levels and reclaim P for use as a fertilizer. From a draft diploid Medicago sativa genome scaffold sequence and the alfalfa transcriptome database (AGED), three PHO2 genes were identified. The genes, two of which are >99% homologous (a/b), each have seven exons interspersed by six introns. The open reading frames are 912 amino acids except when an alternate splice site is used in a/b gene transcript resulting in a 902 amino acid sequence. Alfalfa plants grown under P limiting conditions expressed low levels of the a/b transcripts with higher levels seen for PHO2c, while application of higher P induced increased expression mainly of the a/b transcripts. Under high P conditions, roots and shoots accumulated 4.1x and 2.5x more P than in low P conditions, respectively. An initial CRISPR/Cas9/Cys4 reagent targeting all three genes was generated and used to transform alfalfa cv. RegenSY. A total of 67 verified transgenic plants were screened by acrylamide gel shift assays, cloning, and sequencing to identify plants with mutations. Mutations ranging from a 1 bp insertion to a 25 bp deletion were identified in a total of 10 plants and some plants had multiple targets hit. Recently, a second attempt at CRISPR/Cas9 mutation utilized a cassette vector system with either the tRNA or Cys4 splicing system and exonuclease components. Initial screening results indicate that the tRNA splicing system may have yielded greater numbers of mutations. TaqMan probes were designed to identify plants with changes in the target sites and were verified by restriction digestions, cloning, and sequencing. Data on inheritance of mutations and phosphate accumulation in edited plants will be presented. The results of these experiments demonstrate that editing of multiple targets can be accomplished in alfalfa, although the tetraploid inheritance of genes complicates analysis.Item Identification of Microorganisms for the Bioremediation of Nitrate and Manganese in Minnesota Water(2018-08) Anderson, EmilyBioremediation is a way to safely and cost-effectively remove contaminants using living organisms. In this thesis, microorganisms capable of remediating two pollutants, nitrate and manganese, were identified using culture-dependent and –independent approaches. Nitrate in agricultural wastewater can lead to algal blooms and eutrophication. Edge-of-field woodchip bioreactors are a promising approach to prevent nitrate in wastewater from reaching surface waters by utilizing microbial denitrification to remove nitrate from the system. However, woodchip bioreactors experience low efficiency under cold temperatures, so one strategy to enhance bioreactors in the early spring involves bioaugmentation, or inoculating the bioreactors with cold-adapted denitrifying microorganisms. In order to identify a cold-adapted denitrifier for bioaugmentation, microorganisms were isolated from field woodchip bioreactors and subjected to denitrification testing under cold temperatures, measuring nitrate, nitrite, ammonium, nitrous oxide and dinitrogen gas, as well as whole genome sequencing to identify the presence of genes involved in denitrification and other important microbial processes. Based off of these results, two strains, Microvirgula sp. BE2.4 and Cellulomonas sp. WB94 were recommended for bioaugmentation. In part two, manganese was addressed. High levels of manganese in drinking water can cause health problems, and common treatment methods require cost-intensive chemicals, conditions and maintenance. In this study, a novel algae bioreactor was established to remove manganese from water. In this bioreactor, the algae provided fixed carbon for manganese-oxidizing microorganisms that oxidized the dissolved manganese, precipitating it out of solution. Using a culture-dependent approach, manganese-oxidizing bacteria and fungi were isolated from an environmental sample, including known oxidizers Bosea, Pseudomonas, Plectosphaerella and Phoma and some not previously known to oxidize manganese such as Aeromonas, Skermanella, Ensifer and Aspergillus. A culture-independent approach was also employed to determine how abundant the isolated manganese-oxidizing bacteria are in an actively oxidizing environmental sample. Using nitrate and manganese as examples, this thesis identified useful microorganisms involved in remediation and demonstrated how microorganisms can be utilized to effectively remove pollutants from the environment.Item Microbial Degradation of Polyfluorinated Chemicals and Detection of Fluoride via a Colorimetric Assay(2022-05) Bygd, MadisonPolyfluorinated compounds have become a popular topic in recent years for their widespread use and prominence as a pollutant in our environment. Their use in thousands of commercial products and chemicals has raised concern due to their resistance to degradation, coining them the “forever chemicals.” To better understand how these compounds are degraded and find microorganisms capable of remediation, biological degradative mechanisms were investigated. It was shown that 2,2-difluoro-1,3-benzodioxole, a common fluorinated moiety found in agricultural chemicals and pharmaceuticals, can be degraded and defluorinated by Pseudomonas putida F1. To better detect and screen for additional microbial related defluorination mechanisms, a color screen using alizarin and lanthanum was developed for use in biological contexts. Compatible with various media and solvents, this assay uncovered an additional 23 defluorination reactions initiated by P. putida F1. Further screening was continued with the E. coli ASKA library in which an additional novel defluorination reaction was found to be catalyzed by the enzyme PgpB. The color screen proved to be a highly effective and efficient method for detecting fluoride release by biological means in a high-throughput manner. The data presented in this thesis show many previously undocumented defluorination reactions and outline an efficient and effective method for detecting microbial based bioremediation of polyfluorinated chemicals.