Browsing by Subject "Microbial engineering"
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Item Characterization of cyanobacterial fatty aldehyde deformylase and biodegradation of hydrocarbons by S. oneidensis MR-1(2012-01) Golwala, DeepPetroleum-like high-energy compounds are biosynthesized by microorganisms. With increasing interest in biofuels, the mechanisms of hydrocarbon biosynthesis are under intense study. One mechanism of hydrocarbon biosynthesis involves the deformylation of fatty aldehyde to produce alkanes/alkenes. The gene encoding the deformylase enzyme from Nostoc punctiforme PCC 73102 was cloned into pET28b+ vector and transformed into E. coli BL21. The protein was expressed and purified using a Ni-NTA column. The purification process was optimized for the production of stable and active enzyme. The enzyme was characterized by ultraviolet-visible (UV-Vis) spectroscopy, electron paramagnetic spectroscopy (EPR) and inductively coupled plasma-mass spectrometry (ICP-MS). ICP-MS showed that 2 iron atoms were present per subunit of protein. In vitro activity assay was demonstrated using NADPH, spinach ferredoxin reductase, spinach ferredoxin and octadecanal (substrate). The activity was low, 4 nmoles of heptadecane (product) formed per 2 mg/ml protein in 2 hours. Octadecanoic acid, octadecanol and butanal were not the substrates. In an effort to increase activity, a crude lysate from Nostoc punctiforme PCC 73102 was added to reaction mixture, but no increase in activity was observed. Further, studies were carried out to study biodegradation of long chain ketone and alkene by S. oneidensis MR-1. This study was done in order to maximize production of fuel-like molecules produced by S. oneidensis MR-1. Resting cell assay was carried out to study biodegradation. There was no evidence for the biodegradation of 14-heptacosanone and cis-9-tricosene, compounds mimicking those produced by recombinant S. oneidensis MR-1.Item Development and application of variable strength expression vectors in Shewanella oneidensis MR-1(2014-12) Harris, Audrey JeanA suite of expression vectors was designed and constructed with modular promoter and ribosome binding site sequences resulting in a wide range of protein expression levels in S. oneidensis MR-1. To allow IPTG induction regardless of host background, a subsequent set of vectors was constructed containing lacIq, the E. coli gene encoding lac operon repressor protein. The practical application of these inducible plasmids in S. oneidensis MR-1 was demonstrated by driving variable expression of genes involved in riboflavin biosynthesis and flavin adenosine dinucleotide transport across the inner membrane in order to increase extracellular flavin levels and thus extracellular respiration rates. This study demonstrates the benefit of being able to temporally control select gene expression and amplitude of expression by using a set of predesigned vectors. The modularity of this system will enable researchers to easily exchange alternative promoters or ribosome binding sites in order to modify protein expression for custom applications.Item Fatty acid metabolic engineering: insights for bacterial hydrocarbon production.(2012-01) Robinson, Lee T.Abstract summary not available.Item Occurrence, species composition, and genetic diversity of enterococci in a Lake Superior Watershed.(2012-07) Ran, QinghongFecal contamination of recreational waters is a widespread problem in the United States, resulting in beach closures, huge economic losses, and increased public health risk. In 2012, the U.S. EPA suggested coastal and Great Lakes states adopt enterococci as an alternative indicator for monitoring recreational water quality. However, little information is known about the occurrence and persistence of enterococci in freshwater environments. The overall objectives of the study were to examine the density, species distribution, and potential persistence of enterococci at two study sites in a Lake Superior watershed from May to September over a 2-year period. We also examined the genetic diversity of E. faecalis isolated in sands, sediments, water, and soils. Results of this study showed that enterococci were detected in 149 out of 159 samples (94%) by the MPN analyses and that their densities ranged from 3 to 5.6 x 105 MPN / 100 g sample. The densities were generally higher in the summers than those in other months examined. A total of 2,441 enterococcal isolates over the 2-year period were subjected to speciation. The composition of Enterococcus species was spatially and temporally dynamic, with the dominant species being: E. hirae, E. faecalis, E. faecium, E. mundtii, and E. casseliflavus, suggesting that enterococci in the study might have originated from environmental and fecal sources. Moreover, the genetic diversity of 536 E. faecalis isolates was assessed by HFERP DNA fingerprinting. Results indicated that the E. faecalis population in the watershed was genetically diverse, in part due to spatial and temporal changes. The recurrence of some E. faecalis fingerprints over multiple sampling events in soils and the occurrence of enterococci in the exclosure boxes suggested that some enterococci were persistent in soils for a prolonged time. However, enterococci did not appear to become naturalized in these environments. Our results also suggested that enterococci were transported between matrices within each site. My studies indicate that further research on the persistence of enterococci in soils and sands needs to be done before enterococci are used as a fecal indicator in freshwater ecosystems.Item The Utilization of Algicidal Bacteria for Improved Lipid Extractions and Insights into Neutral Lipid Production in a Wax Ester Accumulating Bacterium(2013-08) Lenneman, Eric M.Based on finite and diminishing quantities of available fossil fuels and the increased demand by a growing population, the identification and production of biologically derived fuels and alternatives to petroleum based compounds has become increasingly important. Biological fuels hold promise from both an economical and environmental standpoint. Microorganisms may hold the key to producing these compounds as many algae and bacteria have been found to produce high quantities of lipids and other bioproducts similar to those obtained from fossil fuels. The purpose of these studies was to evaluate the potential application of algae-degrading bacteria to aid in lipid extractions from the microalgae Neochloris oleoabundans and Dunaliella tertiolecta. Separate studies evaluated the wax ester biosynthetic pathway in the lipid accumulating bacterium Marinobacter aquaeolei VT8 through the analysis of transcriptional levels within wild-type cells, and finally these studies were complimented by gene deletion efforts for specific enzymes within this biosynthetic pathway.Item Utilization of aqueous product generated by hydrothermal carbonization of waste biomass(2014-10) Vozhdayev, Georgiy VladimirovichHydrothermal carbonization (HTC) is a thermochemical treatment process that allows for the conversion of relatively dilute biomass slurries into value added products which are hydrochar and filtrate. This investigation focuses on the potential for utilization of the filtrate (aqueous by-product) created via HTC. A majority of the research to date has focused on the solid HTC product (hydrochar), however little attention has been paid to the utilization of the HTC filtrate, which makes up the larger mass fraction. Finding value added products is key to making the process a viable treatment option for waste biomass and other organic by-products. The option of using HTC filtrate as a fertilizer replacement for agricultural crop production was evaluated through studies of soil microbial effects and impacts on seed germination and early plant growth. These studies confirmed bio-toxicity effects of HTC filtrate on agricultural soil microbes at high application rates. On the other hand, lower rates of application induced biodegradation of the phytotoxic components of the filtrate and released additional plant nutrients through N-mineralization. These effects are dependent on filtrate type, concentration, and post-treatment of the applied filtrate. Phytotoxicity effects on seed germination and seedling growth of corn (Zea mays L.) also showed a dependence on HTC filtrate source and concentration. Similar to the impacts observed on the soil microbes, high concentration typically inhibited seed germination and growth, but lower concentrations stimulated early corn growth. Characterization of the filtrates via a 2-dimensional gas chromatography (GC) time-of-flight mass spectrometry confirmed a very complex chemical fingerprint of the filtrates. Chemical speciation in the filtrate appeared to be a function of the feedstock. More importantly, the simple storage of filtrate in an open container for 90 days drastically alters the chemical species composition and correspondingly the observed impact on soil microbes and plant growth, leading to the conclusion that there could be chemical inhibitors present in the filtrate that are responsible for the observed effects that are eliminated though simple volatilization or microbial mineralization during storage. This work shows great promise for utilization of HTC filtrates as an agricultural fertilizer and the recycling of critical plant nutrients. Additional work is needed to fully characterize the chemical diversity present in these filtrates prior to the implementation of this renewable and sustainable source of agricultural fertilizers.