Macroalgae are important primary producers in the ocean and form expansive underwater forests to support diverse organisms in the ecosystem. They also contribute to a large amount of carbon export below the euphotic zone and carbon sequestration in the deep ocean in the form of detritus. As a result, there is an increasing number of studies suggesting long-term storage of carbon in macroalgae as a carbon dioxide removal strategy to mitigate climate change. However, in contrast to the growing attention of macroalgae as one of the blue carbon ecosystems, the fate of macroalgal-derived carbon after entering the deep ocean remains largely unexplored. Polysaccharides are a major component of both living and detrital macroalgae, and the degradation of these complex polymers is an important process for the turnover of carbon in the ocean. Macroalgal polysaccharides have diverse structures varying significantly in their monomeric saccharides, glycosidic linkages, branching sites, and modifications by chemical groups like sulfate. Therefore, the degradation of macroalgal polysaccharides requires an extensive number of enzymes produced by microorganisms specialized in this process. Carbohydrate-active enzymes (CAZymes) and sulfatases are the key enzymes typically organized in polysaccharide utilization loci (PULs) that mediate a cascade of pathways to degrade polymers to oligosaccharides and eventually to monosaccharides. However, we still have knowledge gaps in the details of the degradation processes and the various enzymes involved. In this dissertation, approaches like cultivation, genomic, and transcriptomic sequencing were applied to study the microbial degradation of macroalgal polysaccharides from cultures grown in the laboratory to marine sediments collected in the deep Santa Barbara Basin. A novel anaerobic bacterial strain NLcol2 was isolated from microbial mats in sediments offshore Santa Barbara, California, USA, and the name Pontiella agarivorans sp. nov. was proposed. It is the fifth cultivated representative in the phylum Kiritimatiellota with the ability to degrade macroalgal polysaccharides including agar and iota-carrageenan and is the first strain reported to be capable of nitrogen fixation. More than 10% of its genome also encodes CAZymes, sulfatases and nitrogenases that facilitate these metabolisms. To further study the enzymes and pathways involved in macroalgae degradation, a transcriptomic study was performed on strain NLcol2 growing with macroalgal polymers of agar and dried seaweeds compared with the monosaccharide D-galactose as control condition. During the growth of strain NLcol2 on polymers, 55% of the genome was expressed differentially, among which many CAZymes and sulfatases involved in the degradation pathways were significantly up-regulated. 12 putative PULs were identified and a selfish mechanism for agar degradation was proposed. The highly expressed genes involved in flagella formation and capsule polysaccharide synthesis with growth on seaweeds could promote motility and biofilm formation for better access and attachment to seaweed particles. To explore the ecological role of P. agarivorans and its relatives in the natural marine environment and to investigate the microbial communities enriched in kelp detritus on the seafloor, marine sediments with and without kelp detritus were sampled in the deep Santa Barbara Basin. The 16S rRNA gene survey showed an enrichment of phyla (Bacteroidota, Spirochaetota, Desulfobacterota, Campylobacterota, Verrucomicrobiota etc.) in kelp-laden sediments with cultivated relatives specializing in polysaccharide degradation, dissimilatory sulfate reduction and nitrogen fixation. Close relatives of P. agarivorans are widely distributed across the basin in low abundance in general but exhibited a high relative abundance in carbon-rich sediments with kelp detritus and at shallower depths. These microbial communities together contribute to the coupled biogeochemical cycling of carbon, sulfur, and nitrogen in the Santa Barbara Basin sediments in low oxygen conditions.

The ocean is a dynamic environment for methane’s biogeochemical processes. Three major processes take center stage: biological methanogenesis, anaerobic methane oxidation, and aerobic methane oxidation. These three key methane biogeochemical processes are intricately balanced within the oceanic environment, resulting in minimal methane escaping into the atmosphere. This dissertation aims to integrate fieldwork with laboratory research, combining investigative tools such as chemical measurements, radiotracer incubations, anaerobic cultivation, and stable isotope probing. The objective is to probe the behaviors of aerobic methane oxidation in relation to the ocean’s oxygen availability and isotopic signatures during methanogenesis from methyl-based compounds.Chapters 1 and 2 of my dissertation delve into the effectiveness and efficiency of aerobic methane oxidation in the ocean’s water column. The Santa Barbara Basin (SBB) is known for experiencing seasonal deoxygenation and reoxygenation cycles, resulting in fluctuating methane concentrations in the deep water column. In chapter 1, my study comprehensively investigated this seasonal cycle through a nine-month period of repeated sampling and measurement of key parameters associated with methane biogeochemistry in the deep waters of the SBB. These parameters include oxygen, nitrate, methane concentrations, and the rate of aerobic methane oxidation. My findings revealed a sequential pattern. First, a decline in oxygen concentration was observed to precede a decrease in nitrate concentration. Second, the accumulation of methane followed, with a marked decline in both oxygen and nitrate levels. Finally, changes in the methane oxidation rate, which reflects the activity of the methanotroph community, occurred subsequently, albeit with a slight time lag. I also discovered that the rate of methane oxidation is primarily dependent on the availability of methane within the water column. Furthermore, my research uncovered that the transient methane pulse accompanying the observed oxygen depletion in the SBB triggered the development of a persistent methanotrophic community, even after methane concentrations had returned to normal levels – an ecological memory effect. In Chapter 2, beyond the analysis of observed trends, I utilized methane concentrations, methane oxidation rates, and vertical methane diffusion to calculate the minimum methane source required for the deep water column. This computation was conducted across a range of contrasting environmental conditions, ultimately revealing that anoxic conditions demand a greater influx of methane into the water column. A comparison to data collected during a 2023 oceanic research expedition revealed an even greater demand for methane input, in the presence of well-established and persistent anoxic conditions. Chapter 3 of my dissertation is dedicated to investigating mechanistic underpinnings of stable carbon isotope fractionations during methylotrophic methanogenesis by marine archaea. Understanding these mechanistic underpinnings enables the inclusion of methylotrophic methanogenesis in isotopically informed biogeochemical reaction networks for anaerobic environments. This study included wild type methylotrophic methanogens as well as a mutant strain in which the reduction of methylated substrates is coupled to hydrogen oxidation or acetate oxidation, providing further mechanistic insight of isotopic variations originating at the reaction branch point.

Hydrocarbons, such as methane or oil, flow to the seafloor from subsurface reservoirs and can induce elevated or depressed bathymetric terrains. They can harbor poorly understood life and induce mass wasting or slumping. Identification of hydrocarbon source and seepage activity is a nested process, involving a combination of multibeam surveys, seismic surveys, and direct observations. Classification of terrains induced by different hydrocarbon source based on bathymetric signatures has not yet been attempted in the literature. In this study, geomorphic analysis is conducted on high-resolution bathymetric data, collected by the autonomous underwater vehicle (AUV) Sentry, of hydrocarbon induced terrains formed by various sources offshore Southern California and in the Gulf of Mexico. Analyses of elevated hydrocarbon induced terrains sourced from gas hydrate, mud, and oil suggest that different phases of hydrocarbons may produce detectable scale-dependent geomorphic and hypsometric signatures. Actively seeping depressed hydrocarbon induced terrains, or pockmarks, may be characterized by a higher number of large coalesced and small unit pockmarks when compared to dormant terrains. The quantitative classification of hydrocarbon induced terrains by source or activity shown here suggest that bathymetric signatures may be representative of hydrocarbon seepage properties. These analyses can be easily implemented into existing automated feature detection techniques.

The creation, movement, and consumption of distinct biomolecules by marine organisms has far reaching implications regarding ecosystem material and energy flow and how we manage the marine environment. Lipids are ubiquitous, energy rich biomolecules that are essential for all life and are used for cell membrane structure, energy storage and serve as useful indicators for ecosystem and food web dynamics. In this dissertation, the flow of specific lipid biomolecules through multiple marine environments is measured, explored, and clarified to better understand biogeochemical cycles, marine food webs and ecosystem connectivity. In the first chapter of my dissertation, I measure, quantify, and close the loop of the open ocean microbial hydrocarbon cycle, with implications for priming effects of the ocean microbiome to oil spills. It is estimated that seeps, spills, and other oil pollution introduce ~ 1.3 million tons (1.3 Tg) of hydrocarbons into the ocean each year. Additionally, it is known that globally abundant marine cyanobacteria Prochlorococcus and Synechococcus which account for ~25% of ocean net primary production also produce hydrocarbons from fatty acids. But little is known about the size, turnover and fate of these cyanobacterial hydrocarbons and the implications for the ocean’s microbiome response to future oil spills. From a research expedition in the North Atlantic, I report that cyanobacteria in an oligotrophic gyre mainly produce n-pentadecane which correlates tightly with fluorescence and Prochlorococcus abundance in oligotrophic waters. Using chemical and isotopic tracing I find that pentadecane production and diel dynamics mainly occurs in the lower euphotic zone at the deep chlorophyll maximum. I estimate the global flux of cyanobacteria-produced pentadecane exceeds total oil input in the ocean by 100 to 500-fold, with cyanobacteria producing ~ 130-650 million tons of pentadecane per year. Analysis of sinking particles at the base of the euphotic zone show that nearly all pentadecane (< 0.001 % remaining) is consumed within the euphotic zone, suggesting near complete consumption of these hydrocarbons by hydrocarbon degrading microbes. These findings characterize a wide-spread microbial hydrocarbon cycle that selectively primes the ocean’s microbiome with long-chain alkanes. In the second chapter of my dissertation, I conduct a large-scale feeding experiment on a symbiotic reef-building coral (Stylophora pistillata) in the Red Sea to clarify fatty acid and isotopic biomarker patterns of coral heterotrophy for use in the field. Coral heterotrophy is an often-overlooked facet of coral nutrition that provides essential nutrients that help corals resist and recover from thermally induced bleaching that is degrading reef ecosystems around the world due to rising global ocean temperatures. Yet, methods for measuring coral mixotrophy, the balance between organic matter contributions to the coral host from autotrophic photo endosymbionts and heterotrophy on particles and plankton have typically been too coarse to elucidate source contributions. Through my experiment I show that fatty acids and isotopic biomarkers reliably separate experimental and reef nutritional source groups (heterotrophic or autotrophic). I show that heterotrophic fatty acid biomarkers are reliably recorded into coral host and symbiont tissues, with a divergent metabolic pattern of autotrophic biomarkers as feeding increases due to positive feedback of heterotrophy on the in hospite photo symbiont population. Additionally, I show that nitrogen and essential fatty acids are preferentially recorded into coral tissue while most heterotrophic carbon is respired or exuded as mucous; this shows that the use of bulk carbon isotopes as a feeding proxy for the last ~ 40 years is largely underestimating the contribution of heterotrophy to the trophic ecology of reef building corals. Overall, this finding underscores a connectivity between oceanic phyto- and zooplankton and reef-building coral. In the third chapter of my dissertation, I explore the mixotrophic differences of divergent bleaching responses of Acropora hyacinthus colonies on the forereef of Mo’orea during the 2019 mass bleaching event. During this bleaching event, all colonies of A. hyacinthus on the deep forereef (14 m) bleached and recovered, while colonies on the shallow forereef (5 m) near the reef crest resisted bleaching entirely, despite the same temperature stress. Using fatty acid and isotopic biomarkers I show through several lines of evidence that bleaching resistant colonies near the reef crest were likely consuming more particulate organic matter than deep forereef colonies. This conclusion is supported by isotopic feeding proxies, less isotopic niche overlap of the host and symbiont of resistant colonies, and larger proportions of putative POM fatty acid biomarkers in the host of resistant colonies relative to recovered colonies. This interpretation is in line with observations that benthic communities on the reef crest are a net sink of oceanic POM and that increased reliance on heterotrophy is associated with bleaching resistance. These data show the vital importance of reef environment, coral heterotrophy, and planktonic subsidies in structuring bleaching response of corals in a warming ocean and ultimately show that the reef crest may serve as a potent zone for reseeding coral populations after marine heat waves.

Natural gases have many environmental consequences if released into the atmosphere due to their high global warming potentials. However, these gases are subject to oxidation by microorganisms, which act as an effective biofilter that limits the atmospheric input. Organisms responsible for this oxidation are present in a wide range of environments and hydrocarbon compositions. To quantify the rate at which a microbial community oxidizes and regulates the flux of hydrocarbons into an environment, a combination of chemical tracer techniques is typically applied. This work applied both stable and radioactive tracers to monitor the oxidation capacity of the microbial community in both water column and marine sediment, with hydrocarbon concentrations generally ranging from low (coastal systems near hydrocarbon reservoirs) to medium (natural seeps) to high (oil spills).

Samples containing low and medium natural gas concentrations were collected from the world's largest natural marine seep field, Coal Oil Point, located offshore of Santa Barbara, California. Natural gas emanating from Coal Oil Point first diffuses into sediment porewater before migrating into the overlying water column to form a dissolved plume. Novel radioactive tracers were developed and applied to assess the timing by which microbes metabolize these gases within both the water column and sediment. A three phase study was employed to track the dissolved hydrocarbon plume within the water column. Phase 1 synthesized tritiated ethane, propane, and butane using Grignard reagents and tritiated water; Phase 2 systematically assessed the experimental conditions, wherein the indigenous microbial community was found to rapidly oxidize ethane, propane, and butane; Phase 3 applied radioactive tracers to track microbial oxidation within a dissolved hydrocarbon plume. Spatial and temporal patterns of ethane, propane, and butane oxidation down current from the hydrocarbon seeps demonstrated that >99% of these gases were metabolized within 1.3 days following initial exposure. Estimates based on the observed metabolic rates and carbon mass balance calculations suggest that ethane-, propane-, and butane-consuming microorganisms may transiently account for a majority of the total microbial community within impacted waters.

Sediment studies focused on the metabolism of ethane oxidation from two individual seeps within the Coal Oil Point field: Campus Point Mounds and Patch Seep. Ethane oxidation was quantified in slurry incubations under anaerobic conditions, which indicated that >97% of the gas was removed after 43 days when incubated near in situ temperature and gas concentrations. This study was the first to quantify anaerobic ethane oxidation within cold seep sediments. Total ethane consumption rates from Campus Point Mounds and Patch Seep were calculated to be 2 µM day-1 and 0.012 µM day-1, respectively, revealing that anaerobic oxidation in sediment is a dynamic process capable of modulating ethane's release into the ocean and atmosphere. Ethane oxidation was also quantified in whole core profiles. These profiles showed elevated ethane oxidation rates to depths of 11 cm beneath the sea floor, but rates below detection in the underlying zone of low sulfate.

Samples containing high natural gas concentrations were collected from the Deepwater Horizon oil spill, both during and following the nearly three-month hydrocarbon release. Microbial oxidation of ethane and propane was tracked using stable isotope (13C) tracers. Within 20 weeks of the onset of hydrocarbon release, the microbial communities had "bloomed" to consume elevated concentrations of ethane and propane, and subsequently "busted" following a prolonged period of starvation. This short time interval for complete natural gas consumption contradicts initial predictions based on previous methane studies, and indicates that oxidation rates can accelerate in environments of high hydrocarbon concentrations.

Our seas, oceans, and coastal zones are under great stress and pollution, particularly by crude oil, which fuels the global economy. Subsurface petroleum reservoirs originate from geo-thermo-chemical reactions on biological debris over millions of years, resulting in a complex heterogeneous mixture of hydrocarbons, with major components consisting of alkanes with different chain lengths and branch points, cycloalkanes, branched cycloalkanes, mono-aromatic, and polycyclic aromatic hydrocarbons. Populations of hydrocarbon-degrading bacteria, including many species that cannot utilize other carbon sources, are present in all marine systems and play an important role in turnover and fate of these compounds. In this dissertation, the microbial response to petroleum components is probed in multiple environments to understand the role different chemical fractions play in eliciting different niches of oil consumers, and to identify factors controlling basal seed populations of hydrocarbon degraders poised to bloom to petroleum disasters. Through study in the subtropical North Atlantic Ocean, a cryptic long-chain alkane cycle has been confirmed, originating from cyanobacteria, dwarfing the quantity of other petroleum inputs to the ocean. In Chapter 1, I demonstrate waters in the mesopelagic underlying the photic zone hosted n-alkane degrading bacteria that bloomed rapidly when fed pentadecane, exhibiting exponential oxygen loss due to respiration within a week. Parallel experiments performed with sinking particles collected in situ from beneath the deep chlorophyll maximum—representing an export flux of particulate-phase pentadecane and its microbial consumers from the euphotic zone—exhibited similarly rapid bloom timing with pentadecane, but with greater oxygen decline. Notably, bloom onset timing for other petroleum compounds with no biogenic origin in the mesopelagic is an order of magnitude slower compared to biogenic alkanes. Metagenomic analyses of pentadecane blooms exposes the metabolic pathways used for pentadecane consumption. Analysis of gene abundance in unaltered seawater from oligotrophic settings reveals long-chain alkane genes are prolific in this setting and highlights the much lower prevalence of genes related to aromatic and short-chain alkane consumption. This work emphasizes the impact of phytoplankton-derived alkanes on the widespread abundance of long-chain alkane degraders in the ocean. The lack of biological hydrocarbon accumulation in the ocean points to their efficient consumption by networks of microorganisms. From analysis of sinking particles out of the photic zone, we know biogenic alkane consumption largely occurs in the sunlit ocean. To gain an understanding of which microbes consume biogenic alkanes we analyze the Tara Oceans global dataset for the presence of the alkane-1-monooxygenase gene (alkB). Stations within the North Atlantic subtropical gyre reveal alkB-related genes are abundant in the surface ocean and deep chlorophyll maximum and these genes are phylogenetically distinct from the ancestrally related delta-9 fatty acid desaturase and xylene monooxygenase. Notably, a dominant clade of alkB-like monooxygenases belongs to the globally abundant Marine Group II (MGII) archaea and is consistently present in all surface and DCM stations. This highly successful group of surface-ocean-dwelling archaea is known for a chemoorganoheterotrophic lifestyle targeting lipids, proteins and amino acids and can utilize photoheterotrophy, but a key role in biohydrocarbon cycling was unexpected as MGII archaea are not among the ~300 genera of bacteria and archaea previously identified as hydrocarbon degraders. Our further analysis of MGII genomes show alkane-1-monooxygenase genes are present in every genus of the MGII taxonomic order, and that other genes required to shunt alkane-derived carboxylic acids into central carbon metabolism are common among MGII. In the Gulf of Mexico (GOM), the biodegradation of n-pentane in the deep ocean was investigated along a gradient of natural seepage influences, illuminating the regional influence natural seeps have on priming petroleum hydrocarbon consumption. This seawater-soluble, volatile, compound is known to partition to the ocean’s interior following release from the seafloor and is abundant in petroleum reservoirs and refined products. The predominant member of the microbial community in n-pentane blooms is Cycloclasticus, and interestingly one Cycloclasticus ecotype favors the seep-ridden region of the GOM, whereas the other favors the open ocean environment far from natural seepage. Metagenomic analysis of the contrasting Cycloclasticus variants indicate the open ocean adapted variant encodes more general pathways for alkane consumption including short-chain alkanes, aromatics, and long-chain alkanes and possess pathways for dissimilatory nitrate reduction and thiosulfate oxidation, whereas the near-seep variant specializes solely on short-chain alkanes and aerobic metabolism. Metagenomic reconstruction and phylogenetic analysis of Cycloclasticus from publicly available sequence data reveals distinct strategies in hydrocarbon generalization and specialization for each major clade within the Cycloclasticus genera. Cycloalkanes are a major component of petroleum and many refined petroleum products. Methylated cycloalkane incubations using methylcyclohexane and methylcyclopentane were conducted in stations spanning a transect across the Gulf of Mexico. In three out of four stations, a single strain belonging to a novel genus within the Poricccaceae family and class Gammaproteobacteria bloomed to consume the methyl-cycloalkane provided within 18-21 days. Metagenomic reconstruction and analysis of the central carbon metabolism reveal a distinct strategy to oxidize cycloalkanes. A larger phylogenomic analysis reveals this methyl-cycloalkane consumer belongs to a monophyletic clade of genomes belonging to the same genera which originate from other environments with petroleum influence at subseafloor aquifers, hydrothermal vents, and petroleum mesocosms from a variety of marine sources. A defining feature of this clade is the presence of a divergent particulate hydrocarbon monooxygenase, which in the phylogenetic tree of the CuMMO enzyme superfamily (of ammonia and hydrocarbon monooxygenases) forms a distinct novel monophyletic clade from all other ammonia, methane, ethane, and ethylene monooxygenases.

Marine sediment microbial communities are significantly influenced by anthropogenic activities, which alter their composition and function. They also play an invaluable role in determining the fate of contaminants in the sediment environment. The San Pedro (SP) Basin in the Southern California Bight is a prime case study to explore this phenomenon, having been subjected to extensive contamination from sewage input to the bordering Palos Verdes (PV) Shelf and offshore dumping. Historical records have confirmed the occurrence of offshore dumping of industrial wastes, and we observe some of these waste products, such as petroleum (tar) and dichlorodiphenyltrichloroethane and its derivatives (DDX), in high abundance in the SP Basin. This study analyzes sediment cores from the SP Basin to assess the structuring of the microbial community and its relationship to contaminants. Using 16S rRNA gene sequencing, this study reveals variations in microbial community composition corresponding to a range of environmental gradients such as sediment depth, water depth, sedimentation rates, and inputs from the PV shelf, with emphasis on variations that correspond to anthropogenic impacts. To my knowledge, this work is the first to characterize microbial community structure of deep coastal basin sediments at high spatial resolution on a regional scale. Taxa such as Proteobacteria and Myxococcota decrease in abundance with sediment depth, while Chloroflexi, Firmicutes, and Archaea tend to increase. A decline in Shannon diversity indices with increasing sediment depth and distance from the shelf suggests that factors such as sediment age and organic matter (OM) characteristics play crucial roles. Non-metric multidimensional scaling (NMDS) analysis highlights correlations between sediment age, water depth, excess carbon load, and microbial community structure. Some taxa like Anaerolineae and Desulfobacterota show increased abundance in regions with high levels of contaminants as indicated by excess carbon or DDX, which may reflect their established roles in processes such as reductive dehalogenation and anaerobic hydrocarbon degradation. This research underscores the profound impact of anthropogenic inputs on sediment microbial communities and their essential roles in biogeochemical processes. Future studies will employ metagenomic analyses alongside additional geochemical analyses to further explore the functional potential of these communities. Understanding the dynamics of microbial communities in contact with toxic contaminants is crucial for developing effective strategies to mitigate pollution and preserve marine ecosystems.

Synthetic biology holds promise for adapting and modifying molecular systems found in nature toward improving human life. This has sparked a modern bioindustry with applications ranging from biomanufacturing pharmaceutical products to engineered microbes for supporting space exploration. In the last decade two biotechnological advances have revolutionized the field of synthetic biology: CRISPR/Cas-mediated genome editing and directed protein evolution. As these principles have matured, advances in automation and artificial intelligence have been quickly adopted as major tools toward the generation of new molecular biotechnologies.

This thesis incorporates the use of specific genetic systems and broad principles of evolution toward engineering new biotechnologies. To adopt novel genetic systems in synthetic biology applications they must first be fully understood. Thus, an important interest underlying the applied aspect of synthetic biology is solving genetic and proteomic mechanisms of action through basic science. One way of doing so is to understand the effects that these genetic systems of interest have had on the natural evolution of the organisms that utilize them.

Toward this interest, my first chapter aims to better understand the impact that diversity generating retroelements (DGRs), may have had on evolving vast morphological, physiological, or adaptive complexity observed within the cyanobacterial phylum. This first chapter develops the hypothesis that, in cyanobacteria, DGRs have evolved to accelerate the evolution of proteins of a distinct functional class, compared to the DGRs of all other prokaryotic organisms currently known. This further sets apart cyanobacteria as an incredibly unique and diverse taxonomic clade within bacteria and sets DGRs apart as an important player in the evolutionary success of cyanobacteria. A paper has been communicated on the work presented in my first chapter, detailed below.

Aside from the potential evolutionary and ecological impact DGRs may have had within the cyanobacterial phylum they are also of interest for biotechnological applications. DGRs are a potential tool for continuous in-vivo, targeted hyper-diversification within a small region of a gene. This is a desirable tool for directed evolution efforts, as the field currently lacks a means for generating highly diverse random mutations in-vivo in targeted genomic loci. As such, in-vivo directed evolution schemes are generally not attempted, though the efficiency of in-vivo natural selection schemes may be much greater when compared to in-vitro screening of individual protein variants as a selection scheme.

The remaining two chapters of my thesis deal with mining the marine environment for other molecular systems that hold significant promise for developing new biotechnologies including products that may aid in space exploration. Marine isolates of Bacillus subtilis have been shown to display a unique propensity for producing highly complex and highly bioactive secondary metabolites. Furthermore, their ability to form endospores that are highly resistant to extreme environmental conditions make them an ideal candidate for engineering toward bioremediation and drug production for space conditions.

The majority of pharmaceuticals are set, conservatively, to expire within two years of the manufacturing date, after which the reported potency and pharmacokinetics are no longer guaranteed. Though there are efforts to study and extend pharmaceutical expiration dates, viability of a product will vary based on manufacturing lot, storage conditions, and packaging. The environs of spaceflight may also affect the decay of pharmaceuticals, though the extent remains unclear. As such, for longer crewed missions, the pharmaceutical supply at launch is unlikely to remain viable for the duration of the mission. However, replenishment becomes complicated on any space mission, and particularly on longer- duration missions, when resupply is difficult, and it is impossible to predict the specific medication needs of the crew. Thus, a platform for on-demand drug synthesis is necessary to ensure the success of long-term space exploration and colonization missions to Mars and beyond.

Thus, my second chapter presents the first step in a three-step process known as an “Astropharmacy”, a peptide drug production platform that uses genetically engineered bacterial spores or a cell-free system to produce drugs on-demand. Not only would this platform allow astronauts to produce drugs according to their needs, but it would also have utility on Earth, with applications including bioterror prevention, orphan drug production, and drug production in remote areas where refrigeration is impossible. Whereas my second chapter deals with mining the marine environment for microbial hosts which have unique and favorable evolutionary characteristics for space, my third chapter seeks to isolate a microalgal marine protein and study its ability to be engineered further to meet different needs we may have on earth.

Microalgae have been considered the most promising biological chemists for the mass production of biologically sourced fuels. Algal organisms may have adapted this ability as a way of regulating cell membrane curvature and hydrophobicity. One goal in biofuel-focused synthetic biology is to reverse-engineer pathways that lead to the production of different hydrocarbon molecules to increase the efficiencies, yields, and culture survivability. One of the newest discoveries that apply to this effort is fatty acid photodecarboxylase (FAP) – an enzyme which uses light to make fuel from acidic fats.

My third chapter details the need for confirming homologs of the originally discovered enzyme. Can mutations that work in the original enzyme be successfully transferred to its homologs? If so, it would allow us to consider homologs for directed evolution strategies including chimeric gene splicing. It would also allow us to add molecular tools found in other organisms to the biocatalytic toolbox of enzymes that enable light-driven decarboxylation of fatty acid substrates.