Investing in Maine Research Infrastructure: Sustainable Forest Bioproducts

The University of Maine, the University of Southern Maine, several baccalaureate institutions in the state, along with other federal, state and local public, private, and non-profit institutions will collaborate to create the Forest Bioproducts Research Institute (FBRI) at the University of Maine. The vision of the FBRI is to advance understanding about the scientific underpinnings, system behavior, and policy implications for the production of forest-based bioproducts that meet societal needs for materials, chemicals, and fuels in an economically and ecologically sustainable manner.The research plans Integrate three themes. They are (1) forest sustainability modeling of life cycle assessment, (2) integrated biopolymer separations and residual solids modifications, and (3) biological and chemical platform conversion technologies.The research capitalizes upon Maine\u27s unique position of having a large natural resource base, existing research capacities in pulp and paper, forestry, and wood products, along with a strong industrial presence. State, national, and global collaborations, including those with Rensselaer Polytechnic Institute and the University of Tennessee-Knoxville, will contribute broader benefits to society as a result of this investment in forestry research.The FBRI will serve as the forest-based carbohydrate economy center of excellence for the region, with a primary goal of transitioning developed science and technology to the state\u27s industrial arena. State, national, and global impacts will be realized as a result of the investment in this research. In addition, a cadre of future engineers and scientists in multidisciplinary disciplines as well as policy-makers will result from the expected collaborations. Support is provided through the NSF Experimental Program To Stimulate Competitive Research (EPSCoR)

Doctor of Philosophy

dissertationMonitoring and remediation of environmental contaminants (biological and chemical) form the crux of global water resource management. There is an extant need to develop point-of-use, low-power, low-cost tools that can address this problem effectively with min­ imal environmental impact. Nanotechnology and microfluidics have made enormous ad­ vances during the past decade in the area of biosensing and environmental remediation. The "marriage" of these two technologies can effectively address some of the above-mentioned needs [1]. In this dissertation, nanomaterials were used in conjunction with microfluidic techniques to detect and degrade biological and chemical pollutants. In the first project, a point-of-use sensor was developed for detection of trichloroethylene (TCE) from water. A self-organizing nanotubular titanium dioxide (TNA) synthesized by electrochemical anodization and functionalized with photocatalytically deposited platinum (Pt/TNA) was applied to the detection. The morphology and crystallinity of the Pt/TNA sensor was characterized using field emission scanning electron microscope, energy dis­ persive x-ray spectroscopy, and X-ray diffraction. The sensor could detect TCE in the concentrations ranging from 10 to 1000 ppm. The room-temperature operation capability of the sensor makes it less power intensive and can potentially be incorporated into a field-based sensor. In the second part, TNA synthesized on a foil was incorporated into a flow-based microfluidic format and applied to degradation of a model pollutant, methylene blue. The system was demonstrated to have enhanced photocatalytic performance at higher flow rates (50-200 ^L/min) over the same microfluidic format with TiO2 nanoparticulate (commercial P25) catalyst. The microfluidic format with TNA catalyst was able to achieve 82% fractional conversion of 18 mM methylene blue in comparison to 55% in the case of the TiO2 nanoparticulate layer at a flow rate of 200 L/min. The microfluidic device was fabricated using non-cleanroom-based methods, making it suitable for economical large-scale manufacture. A computational model of the microfluidic format was developed in COMSOL Multiphysics® finite element software to evaluate the effect of diffusion coefficient and rate constant on the photocatalytic performance. To further enhance the photocatalytic performance of the microfluidic device, TNA synthesized on a mesh was used as the catalyst. The new system was shown to have enhanced photocatalytic performance in comparison to TNA on a foil. The device was then employed in the inactivation of E. coli O157:H7 at different flow rates and light intensities (100, 50, 20, 10 mW/cm2). In the second project, a protocol for ultra-sensitive indirect electrochemical detection of E. coli O157:H7 was reported. The protocol uses antibody functionalized primary (magnetic) beads for capture and polyguanine (polyG) oligonucleotide functionalized sec­ ondary (polystyrene) beads as an electrochemical tag. The method was able to detect concentrations of E. coli O157:H7 down to 3 CFU/100 mL (S/N=3). We also demonstrate the use of the protocol for detection of E. coli O157:H7 seeded in wastewater effluent samples

A high-throughput, combinatorial and robotic approach to catalysis

Abstract: Please refer to full text to view abstract.D.Phil. (Chemistry

Direct Conversion of Wet Microalgae and Oleaginous Yeast to Biodiesel Using Ionic Liquids

This thesis examined the development of an oleaginous yeast or microalgae based biorefinery process. First, improvements were made to the Nile Red assay, a high-throughput method for monitoring lipid accumulation in oleaginous organisms resulting in a significantly more reproducible and accurate assay. This assay was then used to optimize lipid production during heterotrophic cultivation of microalgae on glucose using response surface methodology resulting in microalgae with high lipid content (37.6% wt.). In order to improve the renewability of heterotrophic lipid production, both oleaginous yeast and microalgae were cultivated using pyrolytic sugars, produced via fast pyrolysis of pinewood waste. The effects of inhibitors on glucose consumption and lipid accumulation as well as the quality of the produced fatty acid methyl esters (FAME) were examined. Upon the establishment of cultivation processes for these two organisms, the overall objective of this work shifted towards the development of a fractionation process for producing and recovering multiple end products. Over 20 ionic liquids were screened for the ability to disruption microalgal cell structure. This was the first report of non-imidazolium ionic liquids assessed for algal bioprocessing applications and the first examining room temperature ionic liquids. The leading candidate ionic liquid was further studied for its ability to disrupt fresh microalgal cultures which were dewatered containing up to 82% water content. This was the first in depth report of the effect of process parameters on the use of ionic liquds for algae disruption. The resulting lipid extraction process was minimized to a simple 1.5 h process conducted at ambient temperature with wet algae. This was further extended to include a catalyst in order to directly convert intracellular lipid to biodiesel from whole yeast biomass. The effects of the reagent ratios, reaction temperatures, and reaction time were studied in depth using response surface methodology. Recovery of the ionic liquid and catalyst for reuse was quantified. Finally, the carbohydrates and protein fractions were recovered after the ionic liquid lipid extraction process using microalgae and it was demonstrated that the resulting sugars primarily in the form of starch could be directly fermented to biobutanol, bioethanol, and acetone using a traditional ABE fermentation process

Monolithic separations and analysis of molecules of biological importance

Monolithic stationary phases are attractive tools for chromatographic separations and analyte extraction from complex matrices. Monoliths with porous surfaces can be cast as thin films or with flow channels for monolithic chromatographic columns. Monoliths describe a wide variety of materials, either organic or inorganic in nature, and in several physical forms. Methacrylate-co-ethylene glycol dimethacrylate polymers are especially of importance for separations of biomolecules. These polymers exhibit high biocompatibility and are highly tunable to isolate compounds ranging from small molecules to large molecular weight species, with a wide range or polarities. One way to achieve additional selectivity in these polymers is the introduction of molecular imprinting through the addition of a template molecule during casting. The template is removed after curing, leaving a cavity with high selectivity toward the analytes of interest. In this thesis, both molecularly imprinted and non-imprinted monoliths are employed for isolation and analysis of molecules of importance from complex biological matrices. Mycophenolic acid (MPA) is an anti-rejection drug commonly administered after organ transplant. The interpatient variability of free MPA in blood is very high, due to complex pharmacokinetic and pharmacokinetic properties of the molecule and must be closely monitored during and after therapeutic drug administration. A monolithic thin film molecularly imprinted polymer (TF-MIP) was developed to selectively extract MPA from human plasma in a simple and rapid process. Tyrosine kinase inhibitors (TKIs) are a class of compounds which are commonly used as chemotherapeutic agents for cancer patients. A TF-MIP was designed for the extraction of representative TKIs (imatinib, dasatinib, ponatinib, and nilotinib) in human plasma in a 96-well plate format. The developed extraction regime allowed for highthroughput sample processing with a minimum amount of sample handling using small volumes of plasma. Gene transfer agents (GTAs) are virus-like particles that transport genetic material from one bacterium to another. Using a 2-step monolithic chromatographic approach, a new method for the preparative isolation of functional GTAs from Rhodobacter capsulatus was developed. The isolated particles were intact after isolation and could transfer genetic material. The newly developed process allows for purification and concentration of the particles for downstream use in biochemical, bacterial, and genetic assays, allowing for the advancement of knowledge about GTAs and the discovery of previously undiscovered GTAs. The method also has broad applicability to many small phages which are the focus of phage therapies that are used to fight antibiotic resistant bacterial infections in humans. Coated-blade spray (CBS) mass spectrometry is a technique for direct sample introduction without traditional chromatographic separation. Innovative CBS workflows allow for rapid and simple analyses of a wide variety of compounds. Compared to traditional workflows, CBS methods are particularly attractive for biological matrices due to simplified sample processing. A custom coated-blade spray source was developed for the Xevo TQ-S where data is presented for measurement of mycophenolic acid, cocaine, MDMA, methamphetamine, methadone and methadone-d3 in human biological fluids. A custom source was designed and used to semi-quantitatively measure mycophenolic acid on the MX908 handheld MS. A fibreglass fabric-based MIP mesh was used with the thermal desorption accessory of the MX908 to measure the organophosphorus pesticides malathion and chlorpyrifos in water

Biopharmaceutical Process – Contract Development Organization: Startup

Due to their high specificity and the wide range of treatments they can provide, monoclonal antibodies (MAbs) from mammalian cell cultures have gained increasing popularity in therapeutics. As a result, treatments have become cheaper and easier to manufacture while maintaining their natural effectiveness, further increasing their appeal. Building MAb manufacturing facilities can be costly for biopharmaceutical companies, especially smaller biotech firms, and current production capacities are limited. As a result, there is an everincreasing demand for contract development organizations (CDOs). The CDO being proposed targets demand within this regime specific to MAbs entering clinical trials. It has the capability to screen clones, grow MAb-producing cells up to a 2500 L culture, and purify the MAb to clinical standards. By employing the newest technology available, the facilities will provide flexibility necessary for producing a myriad of different MAb therapeutics in Chinese Hamster Ovary (CHO) cells. Microbioreactors can screen dozens of clones at the millileter scale, saving time and money. Disposable bioreactors in the upstream process allow for variance in the production capacity due to the range of sizes they are available in. Finally, the purification process has been designed to allow for flexibility depending on the size and needs of every client’s product to maximize value to the costumer as well as the company. The current market for MAb production has an astounding worldwide value of approximately $27.5 billion and continues to expand as the number of MAbs entering clinical trials increases (Cowen 2006). It is estimated that within the next four years that the worldwide market value will reach$50 billion (“Preclinical Development”, 2010). The profitability of this proposal is based on running 39 batches a year at 4.326 kg MAb/batch or 168.71 kg MAb/year. By charging a reasonable average of $1,125,000/kg MAb, a profitability profile can be created. Assuming a 70$111,907,800 and 52.96% respectively. However, using a 70% production capacity also leaves room for even higher profit margins. The plant design also has space allotted for future expansion within the mammalian suite as well as room for a future microbial suite

Isolation and evolution of novel nucleoside phosphorylases

Approximately 33.4 million people are living with HIV/AIDS. Of those, 97% live in low and middle income countries, with 22.4 million in sub-Saharan Africa. Only 42% of the people who require anti-retrovirals (ARVs) in low to middle income countries are receiving anti-retroviral therapy (ART). There is a need to develop novel and cost effective methods for producing antiretroviral drugs. Stavudine and azidothymidine (AZT) were identified as potential targets because they could both be produced through a common intermediate – 5 methyluridine (5-MU). It has been established that the biocatalytic production of 5-methyluridine is possible through a reaction known as transglycosylation, in a process which has not previously been demonstrated as commercially viable. A selection of biocatalysts were expressed either in recombinant E. coli strains or in the wild type organisms, purified and then screened for their ability to produce 5-MU. A combination of Bacillus halodurans purine nucleoside phosphorylase 1 (BHPNP1) and E. coli uridine phosphorylase (EcUP) gave the highest 5-MU yield (80%). This result represents the first combination of free enzymes from different organisms, giving high yields of 5-MU under high substrate conditions. Both enzymes were purified and successfully characterised. The established pH optimum was pH 7.0 for both enzymes. Temperature optima and stability data for BHPNP1 (70 C and t1/2 at 60 C of 20.8 h) indicated that the biocatalytic step was operating within the capabilities of this enzyme and would operate well at elevated temperatures (up to 60 C). Conversely, the temperature optimum and stability data for EcUP (optimum of 40 C and t1/2 at 60 C of 9.9 h) indicated that the enzyme remained active at 40 C for the duration of a 25 h biotransformation, but at 60 C would only be operating at 20% of its optimum activity and would lose activity rapidly. BHPNP1 and EcUP were used in a bench scale (650 ml) transglycosylation for the production of 5-MU. A 5-MU yield of 79.1% was obtained at this scale with a reactor productivity of 1.37 g.l-1.h-1. Iterative saturation mutagenesis was used to rapidly evolve EcUP for improved thermostability. A moderately high throughput colorimetric method was developed for screening the mutants based on the release of p-nitrophenol upon phosphorolysis of a pyrimidine nucleoside analogue. By screening under 20 000 clones the mutant UPL8 was isolated. The mutant enzyme showed an optimum temperature of 60 C and improved stability at 60 C (t1/2 = 17.3 h). The increase in stability of UPL8 is due to only 2 mutations (Lys235Arg, Gln236Ala). These mutations may have caused an increase in stability due to interactions with other structural units in the protein, stabilization of the entrance to the binding pocket, or by decreasing the flexibility of the α-helix at the N-terminus. Transglycosylation experiments showed that the mutant enzyme UPL8 is a superior catalyst for the production of 5-MU. A 300% increase in reactor productivity was noted when free enzyme preparations of UPL8 was combined with BHPNP1 at 1.5% m.m-1 substrate loading. The high yield of 5-MU (75-80% mol.mol-1) was maintained at 9% m.m-1 substrate loading. A commercially viable productivity of 31 g.l-1.h-1 was thus realised. Further optimisation of the process could produce still higher productivities. Future work in directed evolution of nucleoside phosphorylases is envisaged for improved stability and enhanced substrate range for application to other commercially relevant transglycosylation reactions

Automated high-throughput approaches for the development and investigation of novel oxidative biocatalytic processes

Oxidative biocatalysts have a vast industrial and biotechnological potential in areas such as fine chemical and antibiotic synthesis. They offer an environmentally compatible and sustainable route to catalysis, often simpler and more specific than chemical alternatives. However, the routine use of biocatalysts in biopharmaceutical manufacture has been hindered by biocatalyst complexity and the experimental burden necessary for implementation. This thesis aims to investigate, using automated microscale technologies, how oxidative biocatalytic bioprocesses can be designed and developed at a reduced cost and timeframe compared to conventional laboratory scale experimentation. A robotic platform was used with 96-Deep square well microtiter plates to develop an effective bioprocess for investigating cyclohexanone monooxygenase (CHMO). E. coli cultivations for CHMO production, bioconversion, liquid-liquid metabolite extraction and analytic techniques were conducted using the developed microscale automated approach. Each step allowed rapid and reproducible collection of quantitative kinetic data over multiples runs achieving ‘walk away operation’. Whole bioprocess evaluation was achieved, whereby linking multiple unit operations enabled rapid assessment of process interactions. Factors influencing CHMO activity and bioconversion yields were investigated along with alternative bioconversion substrates. From identified limitations of the CHMO system an optimised process was developed where the processing time was almost halved and CHMO activity increased 5-fold. Two novel self-sufficient cytochrome P450 systems, P450SU1 and P450SU2 were investigated using an automated approach where factors limiting bioconversion were identified. Implementation of the required improvements resulted in a 5-fold improvement in enzymatic expression and 5-fold and 1.5-fold increase in product formation from cytochrome P450SU1 and P450SU2, respectively. A matched oxygen transfer coefficient approach was used for predictive scale-up. The optimised microscale CHMO and P450 processes were scaled to 75 L and 7.5 L bioreactor scale, respectively. Growth and bioconversion kinetics were found to be identical between scales for the CHMO system whereas differences were observed for the P450 systems. Results described in this thesis have demonstrated the benefits of microscale automated methodologies for the creation, investigation and predictive scale-up of oxidative biocatalytic bioprocesses. The established strategies evaluated in this work contribute to meeting the current demand to decrease developmental costs and timelines