Biotechnology Commons^™

Metabolite Analysis To Understand Factors Limiting Microbial Fermentation At High Substrate Concentrations, Bishal D. Sharma

Dartmouth College Ph.D Dissertations

Clostridium thermocellum, a thermophilic anaerobic bacterium, holds promise as a biocatalyst for conversion of lignocellulosic biomass into biofuels. This microbe has been subjected to various metabolic engineering strategies, including the deletion of genes encoding secondary fermentation products such as H₂, formate, acetate, and lactate, as well as the expression of heterologous genes to redirect carbon flux toward ethanol production. Despite these efforts, achieving economically viable ethanol titers remains a challenge. We hypothesized that studying how intracellular metabolite concentrations change as fermentation stops could reveal metabolic bottlenecks or regulatory mechanisms limiting ethanol production at high titers. However, metabolomics studies at …

Prospecting For And Isolation Of Microalgae In South Texas, Mauricio Alejandro Peña

Theses and Dissertations

Microalgae is a very important microorganism that can be used for beneficial purposes for humans. One way to show the importance of algae in this planet is the contribution of Oxygen it provides to the atmosphere, which many organisms rely on (Medeiros et al., 2015). Carbon footprint has been one of the greatest causes for global warming and it is due to the use of fossils to create diesel. One of the ways to avoid contamination is to replace those fuels that are used on daily basis, such as for transportation. The microalgae are able to produce lipids that can …

Characterization Of The Overexpression Of The Native H+-Pumping Pyrophosphatase In The Microalga Picochlorum Soloecismus, Kimberly T. Wright

Biology ETDs

Microalgae are of interest for the creation of sustainable and cost competitive alternatives to petroleum-based fuels and chemicals. However, cultivation, extraction and processing of algal biomass requires improved yields to achieve economic feasibility. The advancement of microalgal biotechnology and various genetic engineering techniques allow the improvement of microalgae biomass for this purpose. Here, the characterization of the overexpression of the native vacuolar H+ pumping pyrophosphate (AVP1) in Picochlorum soloecismus was examined. AVP1 overexpression causes biomass increase in relevant plant crops. When overexpressed in this microalga it increases carbon storage in the form of starch in a closed laboratory photobioreactor. However, …

Hexadecane Petroleum, And Biofuel Utilization In Marine Bacteria Isolated From Ballast Tanks, Alex Yashchenko

Graduate Theses

This study characterized the growth of bacteria isolated from ballast tank fluids in hexadecane, petroleum, plant, and algae-derived fuels. The study was performed to explore the capacity of ballast tank isolates to survive and grow within fuels that may be stored within ballast tanks. Results of the hexadecane analysis indicated that most isolates had higher viable cell counts in media supplemented with hexadecane. Members of Alteromonas, Pseudoalteromonas, and a single Brevundimonas species had viable cell counts that were one or several orders ofmagnitude greater than that of controls. Results offuel analysis indicated higher viable cell counts in pure JP-5 and …

Towards The Synthetic Design Of Camelina Oil Enriched In Tailored Acetyl-Triacylglycerols With Medium-Chain Fatty Acids, Sunil Bansal, Hae Jin Kim, Gunnam Na, Megan E. Hamilton, Edgar B. Cahoon, Chaofu Lu, Timothy P. Durrett

Department of Biochemistry: Faculty Publications

The ability to manipulate expression of key biosynthetic enzymes has allowed the development of genetically modified plants that synthesise unusual lipids that are useful for biofuel and industrial applications. By taking advantage of the unique activities of enzymes from different species, tailored lipids with a targeted structure can be conceived. In this study we demonstrate the successful implementation of such an approach by metabolically engineering the oilseed crop Camelina sativa to produce 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) with medium-chain fatty acids (MCFAs). Different transgenic camelina lines that had been genetically modified to produce MCFAs through the expression of MCFA-specific thioesterases and …

Efficient Dehydration And Recovery Of Ionic Liquid After Lignocellulosic Processing Using Pervaporation, Jian Sun, Jian Shi, N. V. S. N. Murthy Konda, Dan Campos, Dajiang Liu, Stuart Nemser, Julia Shamshina, Tanmoy Dutta, Paula Berton, Gabriela Gurau, Robin D. Rogers, Blake A. Simmons, Seema Singh

Biosystems and Agricultural Engineering Faculty Publications

Background: Biomass pretreatment using certain ionic liquids (ILs) is very efficient, generally producing a substrate that is amenable to saccharification with fermentable sugar yields approaching theoretical limits. Although promising, several challenges must be addressed before an IL pretreatment technology can become commercially viable. One of the most significant challenges is the affordable and scalable recovery and recycle of the IL itself. Pervaporation (PV) is a highly selective and scalable membrane separation process for quantitatively recovering volatile solutes or solvents directly from non-volatile solvents that could prove more versatile for IL dehydration.

Results: We evaluated a commercially available PV system for …

Lipid Content And Biomass Analysis In Autotrophic And Heterotrophic Algal Species, Addie M. Lauder, Daniel P. Jones, Thomas E. Walker, Todd Allen

Montview Journal of Research & Scholarship

Biofuels are a form of renewable energy derived from living matter, typically plants. The push for biofuels began in order to decrease the amount of carbon dioxide (CO2) released into the atmosphere, as biofuels are essentially carbon neutral. The idea is the same amount of CO2 the plants took in to perform photosynthesis will then be released in the burning of the biofuels. Algae is an excellent source of biofuels because it grows quickly and is versatile in terms of the type of fuel it can produce. The two most common mechanisms for algae growth are heterotrophic or photoautotrophic. Heterotrophically …

Biological Lignocellulose Solubilization: Comparative Evaluation Of Biocatalysts And Enhancement Via Cotreatment, Julie M. D. Paye, Anna Guseva, Sarah K. Hammer, Erica Gjersing

Dartmouth Scholarship

Feedstock recalcitrance is the most important barrier impeding cost-effective production of cellulosic biofuels. Pioneer commercial cellulosic ethanol facilities employ thermochemical pretreatment and addition of fungal cellulase, reflecting the main research emphasis in the field. However, it has been suggested that it may be possible to process cellulosic biomass without thermochemical pretreatment using thermophilic, cellulolytic bacteria. To further explore this idea, we examine the ability of various biocatalysts to solubilize autoclaved but otherwise unpretreated cellulosic biomass under controlled but not industrial conditions.

Elimination Of Hydrogenase Active Site Assembly Blocks H2 Production And Increases Ethanol Yield In Clostridium Thermocellum, Ranjita Biswas, Tianyong Zheng, Daniel G. Olson, Lee R. Lynd, Adam M. Guss

Dartmouth Scholarship

Background: The native ability of Clostridium thermocellum to rapidly consume cellulose and produce ethanol makes it a leading candidate for a consolidated bioprocessing (CBP) biofuel production strategy. C. thermocellum also synthesizes lactate, formate, acetate, H₂ , and amino acids that compete with ethanol production for carbon and electrons. Elimination of H₂ production could redirect carbon flux towards ethanol production by making more electrons available for acetyl coenzyme A reduction to ethanol. Results: H₂ production in C. thermocellum is encoded by four hydrogenases. Rather than delete each individually, we targeted hydrogenase maturase gene hydG, involved in converting the …

Phenotypic Screening Identifies Brefeldin A/Ascotoxin As An Inducer Of Lipid Storage In The Algae Chlamydomonas Reinhardtii, Nishikant Wase, Boqiang Tu, Paul N. Black, Concetta C. Dirusso

Department of Biochemistry: Faculty Publications

The use of microalgae as a biofuel feedstock is highly desired, but current methods to induce lipid accumulation cause severe stress responses that limit biomass and, thus oil yield. To address these issues, a high throughput screening (HTS) method was devised to identify chemical inducers of growth and lipid accumulation. Optimization was performed to determine the most effective cell density, DMSO and Nile Red (NR) concentrations to monitor growth and lipid accumulation. The method was tested using 1717 compounds from National Cancer Institute (NCI) Diversity Set III and Natural Products Set II in Chlamydomonas reinhardtii. Cells were inoculated at …

Triacylglycerol Synthesis During Nitrogen Stress Involves The Prokaryotic Lipid Synthesis Pathway And Acyl Chain Remodeling In The Microalgae Coccomyxa Subellipsoidea, James W. Allen, Concetta C. Dirusso, Paul N. Black

Department of Biochemistry: Faculty Publications

Triglyceride (TAG) synthesis during nitrogen starvation and recovery was addressed using Coccomyxa subellipsoidea by analyzing acylchain composition and redistribution using a bioreactor-controlled time course. Galactolipids, phospholipids and TAGs were profiled using liquid chromatography tandem mass spectroscopy (LC–MS/MS). TAG levels increased linearly through 10 days of N starvation to a final concentration of 12.6% dry weight (DW), while chloroplast membrane lipids decreased from 5% to 1.5% DW. The relative quantities of TAG molecular species, differing in acyl chain length and glycerol backbone position, remained unchanged from 3 to 10 days of N starvation. Six TAG species comprised approximately half the TAG …

The Exometabolome Of Clostridium Thermocellum Reveals Overflow Metabolism At High Cellulose Loading, Evert K. Holwerda, Philip G. Thorne, Daniel G. Olson, Daniel Amador-Noguez, Nancy L. Engle, Timothy J. Tschaplinski, Johannes P. Van Dijken, Lee R. Lynd

Dartmouth Scholarship

BackgroundClostridium thermocellum is a model thermophilic organism for the production of biofuels from lignocellulosic substrates. The majority of publications studying the physiology of this organism use substrate concentrations of ≤10 g/L. However, industrially relevant concentrations of substrate start at 100 g/L carbohydrate, which corresponds to approximately 150 g/L solids. To gain insight into the physiology of fermentation of high substrate concentrations, we studied the growth on, and utilization of high concentrations of crystalline cellulose varying from 50 to 100 g/L by C. thermocellum. .

Identifying, Monitoring, Quantifying And Converting Algae To Bio-Fuels In Bio-Reactors, Alice C. Jernigan

Graduate Theses and Dissertations

Growing algae as a source for bio-fuels has become an area of interest due to concerns about global warming and the reliability and ecology of the production of fossil fuels. Dried algae harvested from a pilot water quality improvement technology at the Rockaway Wastewater Treatment Facility in New York were examined as a source of carbohydrates and lipids for the production of bio-fuels in bio-reactors. The length of storage time, storage conditions, sugar and lipid extraction processes, and fuel production were studied. The results show that if the algae is stored dry (0.015 g/g algae even after a year in …

Comparative Efficiency And Driving Range Of Light- And Heavy-Duty Vehicles Powered With Biomass Energy Stored In Liquid Fuels Or Batteries, Mark Laser, Lee R. R. Lynd

Dartmouth Scholarship

This study addresses the question, "When using cellulosic biomass for vehicular transportation, which field-to-wheels pathway is more efficient: that using biofuels or that using bioelectricity?" In considering the question, the level of assumed technological maturity significantly affects the comparison, as does the intended transportation application. Results from the analysis indicate that for light-duty vehicles, over ranges typical in the United States today (e.g., 560-820 miles), field-to-wheels performance is similar, with some scenarios showing biofuel to be more efficient, and others indicating the two pathways to be essentially the same. Over the current range of heavy-duty vehicles, the field-to-wheels efficiency is …

Analyzing Environmental Microbes For Genomic Regions Promoting Ionic Liquid Tolerance In E. Coli, Ann Nguyen, Alison Richins, Thomas Rüegg, Steven Singer, Michael Thelen

STAR Program Research Presentations

Ionic liquids (ILs) are promising as solvents to increase the efficiency of biofuel production; however, ILs are toxic to microbes used in the fermentation of liquid fuels. To engineer IL resistant biofuel hosts, environmental bacteria were screened for tolerance, and these were used to create gene libraries to test in E. coli. Future characterization of these libraries using molecular techniques will be used to identify genes that contribute IL-tolerance to transformed microbes.

Dcm Methylation Is Detrimental To Plasmid Transformation In Clostridium Thermocellum, Adam M. Guss, Daniel G. Olson, Nicky C. Caiazza, Lee R. Lynd

Dartmouth Scholarship

Background: Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies. Results: We report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam + dcm + E. coli strain, pAMG206 transforms C. thermocellum 100-fold better …

Potential Role Of Micro-Algae On Global Energy Supply, Sage Callaway-Keeley, Stephanie Huynh

Student Research Posters

The most effective ways to reduce CO2 emissions are to improve the energy efficiency of each economic sector and to reduce the cutting of tropical and temperate forests around the world. These options, however, may not fully reach their technical and economic potential due to various political and socioeconomic. The most practical of these is to increase CO2 sinks through photosynthesis in both standing tree biomass and in ocean primary producers. The use of marine algae as CO2 sinks is for large-scale CO2 mitigation: the use of phytoplankton through Fe fertilization and macro algal (kelp) farms, which can be used …