Publications

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Under robust algae growth conditions, algal carbohydrates and proteins typically comprise up to ~. 80% of the ash-free dry weight of microalgae biomass. Therefore, production of algal biofuel through comprehensive utilization of all algal components and the addition of high energy density fuel compounds with "fit for purpose" properties or high-value bioproducts will both diminish the process cost and improve the overall process feasibility. In this study, we firstly demonstrated the concept of a "one-pot" bioconversion of algal carbohydrate and protein into value-added terpene compounds as advanced biofuel and high value bioproducts to improve the overall process feasibility through the development of engineered microbial consortium. The consortium for caryophyllene production yielded the highest titer of total terpene, up to 507.4 mg/L, including 471 mg/L of sesquiterpene, 36.4 mg/L of monoterpene, and 124.4 mg/L of caryophyllene on algal hydrolysate from Nannochloropsis sp. Additionally, the consortium expressing chamigrene synthase produced 187 mg/L total terpene including 87 mg/L of monoterpene, 100 mg/L of sesquiterpene, and 62 mg/L chamigrene on hydrolysate from benthic polyculture biomass. Compared to the yields of terpene extracted from plant tissue, both consortia increased the terpene yield about 3-40 times, which makes it a promising alternative pathway for terpene production.

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We demonstrate that metal-organic frameworks (MOFs) can catalyze hydrogenolysis of aryl ether bonds under mild conditions. Mg-IRMOF-74(I) and Mg-IRMOF-74(II) are stable under reducing conditions and can cleave phenyl ethers containing β-O-4, α-O-4, and 4-O-5 linkages to the corresponding hydrocarbons and phenols. Reaction occurs at 10 bar H2 and 120 °C without added base. DFT-optimized structures and charge transfer analysis suggest that the MOF orients the substrate near Mg2+ ions on the pore walls. Ti and Ni doping further increase conversions to as high as 82% with 96% selectivity for hydrogenolysis versus ring hydrogenation. Repeated cycling induces no loss of activity, making this a promising route for mild aryl-ether bond scission.

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The suitability of crude and purified struvite (MgNH4PO4), a major precipitate in wastewater streams, was investigated for renewable replacement of conventional nitrogen and phosphate resources for cultivation of microalgae. Bovine effluent wastewater stone, the source of crude struvite, was characterized for soluble N/P, trace metals, and biochemical components and compared to the purified mineral. Cultivation trials using struvite as a major nutrient source were conducted using two microalgae production strains, Nannochloropsis salina and Phaeodactylum tricornutum, in both lab and outdoor pilot-scale raceways in a variety of seasonal conditions. Both crude and purified struvite-based media were found to result in biomass productivities at least as high as established media formulations (maximum outdoor co-culture yield ~20±4gAFDW/m2/day). Analysis of nutrient uptake by the alga suggest that struvite provides increased nutrient utilization efficiency, and that crude struvite satisfies the trace metals requirement and results in increased pigment productivity for both microalgae strains.

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Recent strategies for algae-based biofuels have primarily focused on biodiesel production by exploiting high algal lipid yields under nutrient stress conditions. However, under conditions supporting robust algal biomass accumulation, carbohydrate and proteins typically comprise up to ~80% of the ash-free dry weight of algae biomass. Therefore, comprehensive utilization of algal biomass for production of multipurpose intermediate- to high-value bio-based products will promote scale-up of algae production and processing to commodity volumes. Terpenes are hydrocarbon and hydrocarbon-like (C:O>10:1) compounds with high energy density, and are therefore potentially promising candidates for the next generation of value added bio-based chemicals and “drop-in” replacements for petroleum-based fuels. In this study, we demonstrated the feasibility of bioconversion of proteins into sesquiterpene compounds as well as comprehensive bioconversion of algal carbohydrates and proteins into biofuels. To achieve this, the mevalonate pathway was reconstructed into an E. coli chassis with six different terpene synthases (TSs). Strains containing the various TSs produced a spectrum of sesquiterpene compounds in minimal medium containing amino acids as the sole carbon source. The sesquiterpene production was optimized through three different regulation strategies using chamigrene synthase as an example. The highest total terpene titer reached 166 mg/L, and was achieved by applying a strategy to minimize mevalonate accumulation in vivo. The highest yields of total terpene were produced under reduced IPTG induction levels (0.25 mM), reduced induction temperature (25°C), and elevated substrate concentration (20 g/L amino acid mixture). A synthetic bioconversion consortium consisting of two engineering E. coli strains (DH1-TS and YH40-TS) with reconstructed terpene biosynthetic pathways was designed for comprehensive single-pot conversion of algal carbohydrates and proteins to sesquiterpenes. The consortium yielded the highest total terpene yields (187 mg/L) at an inoculum ratio 2:1 of strain YH40-TS: DH1-TS, corresponding to 31 mg fuel/g algae biomass ash free dry weight. This study therefore demonstrates a feasible process for comprehensive algal biofuel production.

In the early stages of infection, patients develop non-specific or no symptoms at all. While waiting for identification of the infectious agent, precious window of opportunity for early intervention is lost. The standard diagnostics require affinity reagents and sufficient pathogen titers to reach the limit of detection. In the event of a disease outbreak, triaging the at-risk population rapidly and reliably for quarantine and countermeasure is more important than the identification of the pathogen by name. To expand Sandia's portfolio of Biological threat management capabilities, we will utilize Raman spectrometry to analyze immune subsets in whole blood to rapidly distinguish infected from non-infected, and bacterial from viral infection, for the purpose of triage during an emergency outbreak. The goal of this one year LDRD is to determine whether Raman spectroscopy can provide label-free detection of early disease signatures, and define a miniaturized Raman detection system meeting requirements for low- resource settings.

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The potential for producing biofuels from algae has generated much excitement based on projections of large oil yields with relatively little land use. However, numerous technical challenges remain for achieving market parity with conventional non-renewable liquid fuel sources. Among these challenges, the energy intensive requirements of traditional cell rupture, lipid extraction, and residuals fractioning of microalgae biomass have posed significant challenges to the nascent field of algal biotechnology. Our novel approach to address these problems was to employ low cost solution-state methods and biochemical engineering to eliminate the need for extensive hardware and energy intensive methods for cell rupture, carbohydrate and protein solubilization and hydrolysis, and fuel product recovery using consolidated bioprocessing strategies. The outcome of the biochemical deconstruction and conversion process consists of an emulsion of algal lipids and mixed alcohol products from carbohydrate and protein fermentation for co-extraction or in situ transesterification.

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Lignin is a potentially plentiful source of renewable organics, with %7E50Mtons/yr produced by the pulp/paper industry and 200-300 Mtons/yr projected production by a US biofuels industry. This industry must process approximately 1 billion tons of biomass to meet the US Renewable Fuel goals. However, there are currently no efficient processes for converting lignin to value-added chemicals and drop-in fuels. Lignin is therefore an opportunity for production of valuable renewable chemicals, but presents staggering technical and economic challenges due to the quantities of material involved and the strong chemical bonds comprising this polymer. Aggressive chemistries and high temperatures are required to degrade lignin without catalysts. Moreover, chemical non-uniformity among lignins leads to complex product mixtures that tend to repolymerize. Conventional petrochemical approaches (pyrolysis, catalytic cracking, gasification) are energy intensive (400-800 degC), require complicated separations, and remove valuable chemical functionality. Low-temperature (25-200 degC) alternatives are clearly desirable, but enzymes are thermally fragile and incompatible with liquid organic compounds, making them impractical for large-scale biorefining. Alternatively, homogeneous catalysts, such as recently developed vanadium complexes, must be separated from product mixtures, while many heterogenous catalysts involve costly noble metals. The objective of this project is to demonstrate proof of concept that an entirely new class of biomimetic, efficient, and industrially robust synthetic catalysts based on nanoporous Metal- Organic Frameworks (MOFs) can be developed. Although catalytic MOFs are known, catalysis of bond cleavage reactions needed for lignin degradation is completely unexplored. Thus, fundamental research is required that industry and most sponsoring agencies are currently unwilling to undertake. We introduce MOFs infiltrated with titanium and nickel species as catalysts for the C-O bond hydrogenolysis in model compounds, which mimic the b-O-4, a-O-4, and 4-O-5 linkages of natural lignin. The versatile IRMOF-74(n) series is proposed as a platform for creating efficient hydrogenolysis catalysts as it not only displays tunable pore sizes, but also has the required thermal and chemical stability. The catalytic C-O bond cleavage occurs at 10 bar hydrogen pressure and temperatures as low as 120 degC. The conversion efficiency of the aromatic ether substrates into the corresponding hydrocarbons and phenols varies as PhCH 2 CH 2 OPh > PhCH 2 OPh > PhOPh (Ph = phenyl), while the catalytic activity generally follows the following trend Ni@IRMOF-74>Ti@IRMOF-74>IRMOF-74. Conversions as high as 80%, coupled with good selectivity for hydrogenolysis vs. hydrogenation, highlight the potential of MOF-based catalysts for the selective cleavage of recalcitrant aryl-ether bonds found in lignin and other biopolymers. This project supports the DOE Integrated Biorefinery Program goals, the objective of which is to convert biomass to fuels and high-value chemicals, by addressing an important technology gap: the lack of low-temperature catalysts suitable for industrial lignin degradation. Biomass, which is %7E30 wt% lignin, constitutes a potentially major source of platform chemicals that could improve overall profitability and productivity of all energy-related products, thereby benefiting consumers and reducing national dependence on imported oil. Additionally, DoD has a strong interest in low-cost drop-in fuels (Navy Biofuel Initiative) and has signed a Memorandum of Understanding with DOE and USDA to develop a sustainable biofuels industry.

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Pathogenic viruses are a primary threat to our national security and to the health and economy of our world. Effective defense strategies to combat viral infection and spread require the development of understanding of the mechanisms that these pathogens use to invade the host cell. We present in this report results of our research into viral particle recognition and fusion to cell membranes and the role that protein affinity and confinement in lipid domains plays in membrane curvature in cellular fusion and fission events. Herein, we describe 1) the assembly of the G attachment protein of Nipah virus using point mutation studies to define its role in viral particle fusion to the cell membrane, 2) how lateral pressure of membrane bound proteins induce curvature in model membrane systems, and 3) the role of membrane curvature in the selective partitioning of molecular receptors and specific affinity of associated proteins.

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Formation of complex nanomaterials would ideally involve single-pot reaction conditions with one reactive site per nanoparticle, resulting in a high yield of incrementally modified or oriented structures. Many studies in nanoparticle functionalization have sought to generate highly uniform nanoparticles with tailorable surface chemistry necessary to produce such conjugates, with limited success. In order to overcome these limitations, we have modified commercially available nanoparticles with multiple potential reaction sites for conjugation with single ssDNAs, proteins, and small unilamellar vesicles. These approaches combined heterobifunctional and biochemical template chemistries with single molecule optical methods for improved control of nanomaterial functionalization. Several interesting analytical results have been achieved by leveraging techniques unique to SNL, and provide multiple paths for future improvements for multiplex nanoparticle synthesis and characterization. Hyperspectral imaging has proven especially useful for assaying substrate immobilized fluorescent particles. In dynamic environments, temporal correlation spectroscopies have been employed for tracking changes in diffusion/hydrodynamic radii, particle size distributions, and identifying mobile versus immobile sample fractions at unbounded dilution. Finally, Raman fingerprinting of biological conjugates has been enabled by resonant signal enhancement provided by intimate interactions with nanoparticles and composite nanoshells.

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