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Electro-selective Fermentation of Scenedesmus acutus algae for enhanced lipid extraction

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The microalgae Scenedesmus have been regarded as a potential source for biofuel production, having up to ~30% of dry weight as lipids used for biodiesel fuel production. Electro-selective fermentation (ESF) is a novel approach that can selectively degrade proteins

The microalgae Scenedesmus have been regarded as a potential source for biofuel production, having up to ~30% of dry weight as lipids used for biodiesel fuel production. Electro-selective fermentation (ESF) is a novel approach that can selectively degrade proteins and carbohydrates while conserving lipids within algal cells, while simultaneously enhancing lipid wet-extraction and biohydrogenation. ESF is a combination of SF and Microbial Electrolysis Cell (MEC) technologies. Experiments reported here prove that ESF is an effective means of enhancing lipid wet-extraction by ~50% and achieving 36% higher lipid saturation conversion, compared to SF, over 30 days of semi-continuous operation. Anode-respiring bacteria (ARB) residing on the anode surface produced a current that led to increased rate of organic substrate utilization, protein degradation, and ultimately enhanced lipid extraction and biohydrogenation that converted unsaturated to saturated fatty-acids. Thus, ESF provides a promising method for enhancing lipid extraction for biofuel production.

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2019-05

Algal Fuels: A Future Less Green than the Plant

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The algal fuel industry has existed since the 1980s without fully commercializing a product. Algal fuels are potentially viable replacements for fossil fuels due to their fast cultivation, high oil content, carbon dioxide sequestration during growth, and ability to

The algal fuel industry has existed since the 1980s without fully commercializing a product. Algal fuels are potentially viable replacements for fossil fuels due to their fast cultivation, high oil content, carbon dioxide sequestration during growth, and ability to be grown on non-arable land. For this thesis, six companies from 61 investigated were interviewed about their history with biofuels, technological changes they have gone through, and views for the future of the industry. All companies interviewed have moved away from fuel production largely due to high production costs and have moved primarily toward pharmaceuticals and animal feed production as well as wastewater treatment. While most do not plan to return to the biofuel industry in the near future, a return would likely require additional legislation, increased technological innovation, and coproduction of multiple products.

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2019-05

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Exploring the consequences of permeate recycling in a photobioreactor using multi-component, community-level modelling

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While biodiesel production from photosynthesizing algae is a promising form of alternative energy, the process is water and nutrient intensive. I designed a mathematical model for a photobioreactor system that filters the reactor effluent and returns the permeate to the

While biodiesel production from photosynthesizing algae is a promising form of alternative energy, the process is water and nutrient intensive. I designed a mathematical model for a photobioreactor system that filters the reactor effluent and returns the permeate to the system so that unutilized nutrients are not wasted, addressing these problems. The model tracks soluble and biomass components that govern the rates of the processes within the photobioreactor (PBR). It considers light attenuation and inhibition, nutrient limitation, preference for ammonia consumption over nitrate, production of soluble microbial products (SMP) and extracellular polymeric substance (EPS), and competition with heterotrophic bacteria that predominately consume SMP. I model a continuous photobioreactor + microfiltration system under nine unique operation conditions - three dilution rates and three recycling rates. I also evaluate the health of a PBR under different dilution rates for two values of qpred. I evaluate the success of each run by calculating values such as biomass productivity and specific biomass yield. The model shows that for low dilution rates (D = <0.2 d-1) and high recycling rates (>66%), nutrient limitation can lead to a PBR crash. In balancing biomass productivity with water conservation, the most favorable runs were those in which the dilution rate and the recycling rate were highest. In a second part of my thesis, I developed a model that describes the interactions of phototrophs and their predators. The model also shows that dilution rates corresponding to realistic PBR operation can washout predators from the system, but the simulation outputs depend heavily on the accuracy of parameters that are not well defined.

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2018-05

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Improving Biochemical Production in Escherichia coli through Nutrient Limitation

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Escherichia coli is a bacterium that is used widely in metabolic engineering due to its ability to grow at a fast rate and to be cultured easily. E. coli can be engineered to produce many valuable chemicals, including biofuels and

Escherichia coli is a bacterium that is used widely in metabolic engineering due to its ability to grow at a fast rate and to be cultured easily. E. coli can be engineered to produce many valuable chemicals, including biofuels and L-Phenylalanine—a precursor to many pharmaceuticals. Significant cell growth occurs in parallel to the biosynthesis of the desired biofuel or biochemical product, and limits product concentrations and yields. Stopping cell growth can improve chemical production since more resources will go toward chemical production than toward biomass. The goal of the project is to test different methods of controlling microbial uptake of nutrients, specifically phosphate, to dynamically limit cell growth and improve biochemical production of E. coli, and the research has the potential to promote public health, sustainability, and environment. This can be achieved by targeting phosphate transporter genes using CRISPRi and CRISPR, and they will limit the uptake of phosphate by targeting the phosphate transporter genes in DNA, which will stop transcriptions of the genes. In the experiment, NST74∆crr∆pykAF, a L-Phe overproducer, was used as the base strain, and the pitA phosphate transporter gene was targeted in the CRISPRi and CRISPR systems with the strain with other phosphate transporters knocked out. The tested CRISPRi and CRISPR mechanisms did not stop cell growth or improved L-Phe production. Further research will be conducted to determine the problem of the system. In addition, the CRISPRi and CRISPR systems that target multiple phosphate transporter genes will be tested in the future as well as the other method of stopping transcriptions of the phosphate transporter genes, which is called a tunable toggle switch mechanism.

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2018-05

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Determination of Higher Heating Value for Algal Products

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Hydrothermal Liquefaction of Algae represents one of many pathways for the sustainable replacement of fossil fuels in transportation. When processing and researching algal biofuel, determination of the higher heating value (HHV) is paramount. Bomb calorimetry represents to current method for

Hydrothermal Liquefaction of Algae represents one of many pathways for the sustainable replacement of fossil fuels in transportation. When processing and researching algal biofuel, determination of the higher heating value (HHV) is paramount. Bomb calorimetry represents to current method for direct determination of HHV. When determining HHV’s indirectly, the industry standard is using one of many linear correlations relating elemental composition to HHV. Most of these correlations were developed from coal industry data, meaning that they do not necessarily fit algal product data well. In this study bomb calorimetry data and CHNS/O elemental composition data were collected for Chlorella, Micract, GS 5587.1, Kirchnella, and Gal 87.1 MM8 algae species. This data was added to CHNS/O and HHV values for other algal products in literature, and utilized to test the accuracy of the Dulong, Gumz, Vandralek and Boie correlations for algae products. Several preliminary algae specific correlations were proposed through a linear regression model of the data. Of the 5 samples tested, Kirchnella exhibited the highest HHV (23.2405 ± 0.0216 MJ/kg) and Chlorella exhibited the lowest (20.2055 ± 0.0484 MJ/kg). For both the experimental, and literature CHNS/O vs HHV data, the Vandralek and Boie correlations provided the best approximations in this study. For the totality of the data collected and researched in this study, 6 of 8 proposed correlations outperformed the Vandralek equation for HHV approximation. The most promising proposed correlations incorporated multiple linear regressions for elemental fractions of CHS, CHSO and CHNSO. Being that only 20 distinct algal product samples were regressed to create the proposed correlations, more data should be incorporated before publication of a final correlation. This study should serve as a starting point for the compilation of an exhaustive database for algal product assay and HHV data.

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2017-05

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Backend Construction of a Web Service

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A growing number of stylists \u2014 cosmetologists \u2014 are finding it harder to afford the basic necessities such as rent. However, the ever-increasing presence of smartphones and the increasing need for on-demand services like Uber and Uber Eats creates a

A growing number of stylists \u2014 cosmetologists \u2014 are finding it harder to afford the basic necessities such as rent. However, the ever-increasing presence of smartphones and the increasing need for on-demand services like Uber and Uber Eats creates a unique opportunity for stylists \u2014 Clippr. Clippr is an application that aims to connect individual stylists directly to their customers. The application gives stylists a platform to create and display their own prices, services, and portfolio. Customers get the benefit of finding a stylist that suits them and booking instantly. This project outlines the backend for the Clippr application. It goes over the framework, REST API, and various functionalities of the application. Additionally, the project also covers the work that is still needed to successfully launch the application.

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2017-05

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Engineering a Co-culture System for Co-utilization of Lignocellulose-derived Sugars for Improved Biomass Conversion

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The inability of a single strain of bacteria to simultaneously and completely consume multiple sugars, such as glucose and xylose, hinder industrial microbial processes for ethanol and lactate production. To overcome this limitation, I am engineering an E. coli co-culture

The inability of a single strain of bacteria to simultaneously and completely consume multiple sugars, such as glucose and xylose, hinder industrial microbial processes for ethanol and lactate production. To overcome this limitation, I am engineering an E. coli co-culture system consisting of two ‘specialists'. One has the ability to only consume xylose and the other only glucose. This allows for co-utilization of lignocellulose-derived sugars so both sugars are completely consumed, residence time is reduced and lactate and ethanol titers are maximized.

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2017-05

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Modulating the Heat Shock Response in E. coli to Optimize Membrane Protein Expression

Description

Membrane proteins are essential for cell survival and show potential as pharmacological and therapeutic targets in the field of nanobiotechnology.[1,2] In spite of their promise in these fields, research surrounding membrane proteins lags since their over-expression often leads to cell

Membrane proteins are essential for cell survival and show potential as pharmacological and therapeutic targets in the field of nanobiotechnology.[1,2] In spite of their promise in these fields, research surrounding membrane proteins lags since their over-expression often leads to cell toxicity and death.[3,4] It was hypothesized that membrane protein expression could be regulated and optimized by modifying the heat shock response of Escherichia coli (E. coli). To test this hypothesis, the membrane protein expression pathway was reprogrammed using gene-blocks that were antisense to vital membrane protein DNA and RNA binding-site sequences and included an IbpA-σ32 heat shock promoter. Anti-PBAD and anti-HtdR gene-blocks were designed to have antisense sequences to the DNA of the arabinose PBAD promotor and Haloterrigena turkmenica deltarhodopsin (HtdR) transmembrane protein respectively. These sequences were then employed to be cloned into a pMM102 vector and grown in NEB-5α E. coli cells.

Stable glycerol stocks of the pIbpA-antiPBAD and pIbpA-antiHtdR in BW25113 cells with either a pBLN200 or pHtdR200 plasmid were created. Then after inducing the cells with L-arabinose and 10mM all-trans retinal to allow for membrane protein expression, spectrophotometry was used to test the optical density of the cells at an absorbance of 600nm. Although general trends showed that the pHtdR200-pMM102 and pHtdR200-pIbpA cells had lower optical densities than the pBLN200 cells of all types, the results were determined to be statistically insignificant. Continuing, the pHtdR200 cells of all types showed a purple phenotype when spun down, as expected, while the cells with the pBLN200 plasmid had a colorless phenotype in pellet form. Further work will include cloning a GFP gene-block to test the ability of the anti-PBAD sequence in tuning the transcription of the GFP protein.

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2018-05

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Investigation of the effect of efflux pumps on the toxicity of phenol, 2-phenylethanol, and styrene to E. coli

Description

Depletion of fossil fuel resources has led to the investigation of alternate feedstocks for and methods of chemical synthesis, in particular the use of E. coli biocatalysts to produce fine commodity chemicals from renewable glucose sources. Production of phenol, 2-phenylethanol,

Depletion of fossil fuel resources has led to the investigation of alternate feedstocks for and methods of chemical synthesis, in particular the use of E. coli biocatalysts to produce fine commodity chemicals from renewable glucose sources. Production of phenol, 2-phenylethanol, and styrene was investigated, in particular the limitation in yield and accumulation that results from high product toxicity. This paper examines two methods of product toxicity mitigation: the use of efflux pumps and the separation of pathways which produce less toxic intermediates. A library of 43 efflux pumps from various organisms were screened for their potential to confer resistance to phenol, 2-phenylethanol, and styrene on an E. coli host. A pump sourced from P. putida was found to allow for increased host growth in the presence of styrene as compared to a cell with no efflux pump. The separation of styrene producing pathway was also investigated. Cells capable of performing the first and latter halves of the synthesis were allowed to grow separately and later combined in order to capitalize on the relatively lower toxicity of the intermediate, trans-cinnamate. The styrene production and yield from this separated set of cultures was compared to that resulting from the growth of cells containing the full set of styrene synthesis genes. Results from this experiment were inconclusive.

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2015-05

Process Design and Refinement for Bioproduction of Sustainable Butanol

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This document outlines the research work done by Shona Becwar in the process design and refinement for the production of sustainable butanol from Clostridium, along with the required background knowledge on the subject. The process that the microbiological organisms go

This document outlines the research work done by Shona Becwar in the process design and refinement for the production of sustainable butanol from Clostridium, along with the required background knowledge on the subject. The process that the microbiological organisms go through to produce butanol must be an oxygen free environment for up to 21 days with multiple perforations made into the environment in this period. There was not previously a cost effective method to do this, even in small scale. It was determined that using a butyl rubber septa would allow for the environment to be sustained during the growth process. The pervaporation process was losing butanol product at a rate of approximately 60%, changing the tubing from silicon to stainless steel allowed for a mere 7% loss during the separation process, greatly increasing the prospective of upscaling this process. These improvements to the sustainable butanol production process will allow for a more efficient, therefore more economically competitive product which can be used as a drop in equivalent to the current butanol market.

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2016-05