Plastic recycling is a difficult process because of the variability in starting material and contamination with food and other chemicals. Very little of current plastic production comes from recycled material. In addition, many plastics are sourced from petroleum, which is nonrenewable and releases toxic chemicals into the environment. However, more plastic is produced each year. To solve this problem, it is necessary to devise new methods for recycling plastic. In this thesis, two bacterial isolates are shown to grow using polyurethane foam as their sole carbon source, their genomes sequenced, and proteomics methods used to identify potential urethanase enzymes. Next, enzymes were expressed in E. coli and tested using an Impranil assay. Finally, two novel urethanase assays were developed, a fluorometric one using a synthesized urethane substrate, and a mass-spectrometry one directly using particles of polyurethane foam. Genome sequencing of the two isolates produced assembled draft sequences, one of which was fully polished and circularized, and the other which was polished but did not circularize. Proteomics identified a large number of hydrolase enzymes overexpressed when isolates were grown in polyurethane media, of which nine were selected as likely urethanase candidates. One enzyme was successfully expressed, but it was a slightly truncated variant and only expressed in low quantities. All three enzyme assays were tested and resulted in promising proof-of-concept results.

Recombinant proteins are widely used for industrial purposes including manufacturing, pharmaceuticals, and beauty. Using microalgae as a biomanufacturing platform has piqued research interests due to their photosynthetic or heterotrophic capabilities and cellular machinery that has the ability to correctly fold and secrete many recombinant proteins. However, several obstacles remain, such as low heterologous protein expression and a lack of strong expression and secretion vectors specific to microalgae. Here we made progress toward a molecular biology tool to help overcome these challenges by easing the quantification of a secreted recombinant protein. This was achieved by fusing the recombinant protein to a self-cleaving viral peptide from the Foot and Mouth Disease Virus (FMDV), a secretion peptide, and a GFP fluorescent marker. By establishing the ratio of the GFP to the protein of interest, a quantification can be obtained of the secreted recombinant protein of interest by measuring GFP fluorescence. This work presents the basis for a polycistronic tool that offers a quick method of quantifying recombinant protein in the microalga Chlamydomonas reinhardtii for biotechnological and industrial purposes.

Microalgae are drawing increased attention from a variety of fields ranging from nutrition and health care to energy, and deservedly so. The potential of algae is almost unparalleled as a biomanufacturing platform. Microalgae can be used to produce complex human antibodies to target cancerous cells, or as crops for the production of high quality food, or even as sources of lipids for conversion into biofuels. They can grow to tremendous density in bioreactors, or be cultivated in open ponds where their yield per acre dominates that of higher plants.

Research in microalgae needs to take it to the next step, to transform potential into reality and make production strains of microalgae into designer products that are custom tailored to suit the needs of the industry. The diversity of microalgae is enormous, and as a result, it is unlikely that the strains we have identified today will be the same we see as ideal production strains tomorrow. So in order to continue advancing the field, techniques for ideal cultivation, genetic modification, and metabolic engineering will need to be developed. This thesis work seeks to do just that by covering cultivation technology, metabolic engineering of lipids, and genetic modification of potential production strains.

From a molecular perspective, a broad review of the current state of lipid metabolic engineering in both microalgae and higher plants is provided, covering both the biofuel relevant molecules as well as nutritionally relevant omega-3 fatty acids. New techniques for rapid interrogation of protein, lipid, and dry weight content in microalgae under either indoor or outdoor cultivation techniques are also described herein. These will enable more efficient harvesting and culturing techniques as well as more efficient use of fertilizers and nutrients in cultivation of microalgae. Proof of concept for custom tailoring lipid profile is demonstrated in the manipulation of Chlamydomonas reinhardtii lipid metabolism for the production of very long chain polyunsaturated fatty acids, which also provides insights into how changes in lipid profile can have unexpected effects and yield new insights on lipid metabolism. Finally, attempts to characterize and transform a variety of potential production species are covered and reveal challenges to the transformation of new microalgae species. Taken together, this work represents significant advances in the field with regard to both cultivation and transformation and metabolic engineering of microalgal species.

The Cre/lox-mediated recombination system from the bacteriophage P1 is a fundamental genetic tool for the study of cell biology and genetics, as it allows for easy site-directed manipulation of genomes. Application of this system in the green unicellular model algae organism Chlamydomonas reinhardtii has not been sufficiently explored or applied in vivo. Establishing an efficient method of Cre/lox-mediated recombination will transform the way this organism can be studied and utilized as a bio-factory. A new reporter vector, pLox, was designed to easily screen for both cells with high protein expression—via mClover green fluorescent protein (GFP) detection before Cre recombinase activity—and cells with successful GFP deletion—via Oxyfluorfen herbicide resistance activation after Cre recombinase activity. Experiments were then designed to test if Cre/lox recombination is possible in vivo in C. reinhardtii using two approaches: 1) electroporating in exogenous Cre recombinase and 2) expressing Cre recombinase endogenously. In this thesis, the degrees to which the preliminary experiments and methods showed promise were documented. The design of pLox allows for straightforward verification of deletion, both through observing phenotypic change and PCR genotyping. Commercially supplied Cre recombinase was efficacious in recombining pLox in vitro, but our methods showed no positive results in vivo. However, one method of stably integrating our Cre recombinase expression cassette shows definite Cre excision, verified by genotyping and sequencing. In conclusion, in-vivo Cre/lox-mediated recombination in C. reinhardtii using our methods is promising, but Cre recombinase delivery must be optimized for efficiency and efficacy.

Our initial objectives in this project were three-fold. I. Express functional antibodies in the chloroplast of Chlamydomonas reinhardtii II. Express functional antibodies in the nucleocytoplasmic compartment of C. reinhardtii and III. Define optimal parameters for the large scale culture of antibody producing strains of C. reinhardtii.

In recent years, there has been a surge of interest in replacements for typical petroleum-based products due to the environmental strain of fossil fuels. Renewably-sourced and readily-biodegradable plastics have the potential to make plastic production a more sustainable process. The following study analyzes the biodegradation process of a 52% bio-based polyurethane through the identification of organisms associated with biodegradation, an analysis of the chemical breakdown into starting monomers, and physical analysis of degradation. Experiments involved incubating polyurethane in compost and soil environments, adapting organisms to utilize the polyurethane as a carbon source, as well as employing enzymes to break down the polyurethane structure. Several organisms appeared to successfully use the polyurethane as a carbon source. When reacted with commercial esterases, the starting chemical precursors were seen through GCMS and LCMS, indicating breakdown of the polyurethane structure into starting monomers. Through understanding the biodegradation process of the polyurethane, there is the potential to develop a more renewable plastic production system through the recycling of monomers.

Human need for food and fuel has disturbed the balance of the biosphere thus triggering a catastrophe known as the Anthropocene Extinction. The use of microalgae in the biotechnology field offers multiple solutions that could alleviate the demand human activity imposes on the ecosystem. The most well studied microalga is the model organism Chlamydomonas reinhardtii, resulting in multiple genetic tools available in the alga for recombinant protein expression. However, the yields of recombinant protein expression are not high enough to be commercially viable, therefore this organism is usually not considered as a biotechnological host.To boost the recombinant protein productivity in Chlamydomonas reinhardtii two milestones need to be achieved: increased recombinant protein expression at single cell level, and increased number of cells per unit of culture volume. To accomplish higher transgene expression in C. reinhardtii the GAL4/UAS system was adapted into algal protein expression vectors. This system showed a 10-fold improvement in recombinant mRNA and protein accumulation of a reporter gene under the control of a chimeric promoter 5XUAS-AR1. To accomplish higher number of cells, or biomass, per unit of culture volume an optimized algal fed batch bioreactor was designed. Through media optimization we achieved a 1.67-fold improvement in biomass accumulation which in turn yielded a 3-fold improvement over the highest recombinant protein concentration reported in the literature using C. reinhardtii. Finally, an extremophile green alga from the Chlamydomonas genus was isolated from the wild and used to express recombinant GFP. Said extremophile showed robust growth in open ponds thriving in media at pH 11 while continuing to express recombinant protein for the duration of the experiment. These findings highlight the potential of Chlamydomonas reinhardtii to become a robust biotechnological host at commercial scale.