Barbara L. Knutson

Compressed solvents for the extraction of fermentation products within a hollow fiber membrane contactor

Abstract The feasibility of extracting aqueous ethanol and acetone within a hollow fiber membrane contactor (HFC) has been examined using compressed CO 2 (69 bar), ethane (69 bar), and propane (34.5 bar) at ambient temperature. Ethanol and acetone were chosen as ‘model’ fermentation products to further examine the potential for extractive fermentation with compressed fluids. Aqueous and compressed solvent streams were contacted within a single hydrophobic isotactic polypropylene membrane fiber (0.6 mm ID; 106.7 cm in length; 75% porosity), providing a porous barrier between the two immiscible phases. The amount of solute extracted was determined as a function of the aqueous flowrate (tubeside) and molar solvent to feed ratio. The amount of aqueous ethanol (10 wt.%) and acetone (10 wt.%) extracted from binary feed solutions with compressed propane ranged from 6.4 to 14.3% and 21.8 to 90.6%, respectively, as a function of the aqueous flowrate (0.1 to 2 ml/min) and molar solvent to feed ratio ( S/F =1 to 10). Comparatively, ethanol extraction with compressed CO 2 ranged from 4.7 to 31.9% with similar variations in the aqueous flowrate (0.1 to 1 ml/min) and molar solvent to feed ratio (3 and 10). Acetone extracted with CO 2 ranged from 67.9 to 96.1% when varying the aqueous flowrate (0.1 to 1 ml/min) at a molar solvent to feed ratio of 3. Ternary ethanol/acetone/water mixtures were also examined to determine the effect of multi-solute aqueous solutions. The effect of aqueous and compressed fluid flows on extraction are interpreted based on the equilibrium distributions of the solutes between water and the compressed fluid (estimated using a group contribution association equation of state (GCA–EOS)) and the mass transfer characteristics of the compressed fluid.

Analysis of composition and structure of Clostridium thermocellum membranes from wild-type and ethanol-adapted strains

Clostridium thermocellum is a candidate organism for consolidated bioprocessing of lignocellulosic biomass into ethanol. However, commercial use is limited due to growth inhibition at modest ethanol concentrations. Recently, an ethanol-adapted strain of C. thermocellum was produced. Since ethanol adaptation in microorganisms has been linked to modification of membrane lipids, we tested the hypothesis that ethanol adaptation in C. thermocellum involves lipid modification by comparing the fatty acid composition and membrane anisotropy of wild-type and ethanol-adapted strains. Derivatization to fatty acid methyl esters provided quantitative lipid analysis. Compared to wild-type, the ethanol-adapted strain had a larger percentage of fatty acids with chain lengths >16:0 and showed a significant increase in the percentage of 16:0 plasmalogens. Structural identification of fatty acids was confirmed through mass spectral fragmentation patterns of picolinyl esters. Ethanol adaptation did not involve modification at sites of methyl branching or the unsaturation index. Comparison of steady-state fluorescence anisotropy experiments, in the absence and presence of ethanol, provided evidence for the effects of ethanol on membrane fluidity. In the presence of ethanol, both strains displayed increased fluidity by approximately 12%. These data support the model that ethanol adaptation was the result of fatty acid changes that increased membrane rigidity that counter-acted the fluidizing effect of ethanol.

Molecular charisma in supercritical fluids

Abstract The unique properties of supercritical fluids (SCF) solutions near the critical region are often a manifestation of local properties which are significantly different than bulk properties. A variety of spectroscopic measurements, as well as thermodynamic and theoretical treatments, support the existence of local density and local composition augmentations in the vicinity of large solute molecules in SCF solutions. Opportunities to exploit this concept of molecular charisma exist for a variety of engineering application including separations using cosolvent/SCF systems and chemical reactions in SCF solvents. The component of cosolvent effect on solubilities in SCF systems resulting from molecular charisma is discussed and related to specific solute/cosolvent interactions and cosolvent concentration. As a consequence of two effects of molecular charisma, the potential to adjust reaction rates and reaction selectivities in SCF solvents is also enhanced. Reaction rates may be altered through the concentration of reactants at the reaction site. Additionally, modification of local solvent strength in SCF solutions, a property of the local reactant environment, can alter reaction rates and selectivities.

Synthesis and biocompatibility evaluation of partially fluorinated pyridinium bromides

Although cationic surfactants are of general interest for a variety of consumer and biomedical applications, only a limited number of partially fluorinated, single-tailed, cationic surfactants have been synthesized. To study the potential usefulness of fluorinated cationic surfactants for these applications we synthesized a series of partially fluorinated pyridinium bromide surfactants. Three 10-perfluoroalkyldecyl pyridinium surfactants were synthesized by coupling a perfluoroalkyl iodide with 9-decene-1-yl acetate using an AIBN mediated radical reaction. The resulting 9-iodo-10-perfluoroalkyldec-1-yl acetates were deiodinated using HI–Zn–EtOH and hydrolyzed using KOH–EtOH to yield the corresponding 10-perfluoroalkyldecanol. The partially fluorinated alcohol was converted into the bromide using Br2–PPh3. Alkylation of excess pyridine with the bromides gave the desired 10-perfluoroalkyldecyl pyridinium bromides in good yields. Three 10-perfluoroalkylundecyl surfactants were synthesized using a similar approach with 10-undecenoic acid methyl ester as starting material. Based on an initial in vitro toxicity assessment, the toxicity of the partially fluorinated pyridinium surfactants was slightly lower or comparable to benzalkonium chloride, a typically cationic surfactant (with IC50s of tested compounds ranging from 5 to 15 μM). An increase in the length and/or the degree of fluorination of the hydrophobic tail correlated with a mild decrease of cytotoxicity and haemolytic activity. Partially fluorinated pyridinium surfactants may, therefore, be useful for biomedical applications such as components for novel gene and drug delivery systems.

CO2 and Fluorinated Solvent-Based Technologies for Protein Microparticle Precipitation from Aqueous Solutions

Precipitation with a compressed or supercritical fluid antisolvent (PCA) has been used to produce microparticles of biologically active proteins, pharmaceuticals, and polymers. However, the application of PCA to a wider range of proteins is limited by the low mutual solubility of water (necessary to dissolve most proteins) and CO2 (traditionally used as the compressed antisolvent). This investigation extends PCA to proteins in aqueous solutions by utilizing ethanol as a cosolvent to enhance the antisolvent properties of CO2 toward aqueous systems. α‐Chymotrypsin, a model protein, was precipitated from both compressed CO2 and a liquid fluorinated antisolvent, a hydrofluoroether (HFE). The equilibrium phase behavior of the antisolvent/ethanol/water systems was examined to identify a one‐phase region suitable for protein precipitation. Spherical protein microparticles with a primary particle size of approximately 0.2–0.6 μm were recovered using both the compressed CO2 and fluorinated antisolvents. Although the proteins retained significant activity using both antisolvent systems, the HFE‐precipitated chymotrypsin retained higher activity than the CO2‐precipitated protein.

Pore-Size Dependent Protein Adsorption and Protection from Proteolytic Hydrolysis in Tailored Mesoporous Silica Particles

Protein adsorption and interactions with mesoporous silica are of interest for a broad range of applications including drug delivery, chemical synthesis, biosensors, and bioseparations. A major challenge in designing mesoporous silica supports for tailored protein interaction is the differentiation of protein interactions at the surface of the particle from interactions within the pore, important features when considering mesoporous silica as a protective support for active proteins. In this investigation, the location of Enhanced Green Fluorescent Proteins (EGFPs) adsorbed on tailored mesoporous silica particles is examined as a function of pore diameter using proteolytic hydrolysis to distinguish between accessible and inaccessible proteins. Pore size control is achieved by tuning the hydrothermal aging temperature (60-110 °C) during synthesis, where the synthesis results in 5-15 μm diameter spherical particles appropriate for imaging by confocal scanning laser microscopy (CSLM). In low pH environments, EGFP unfolds within pores and on the surface of particles, rendering it susceptible to proteolytic hydrolysis by the protease Pepsin A. Upon return to neutral pH, un-hydrolyzed EGFP regains its fluorescence and can be visualized within the mesoporous particles. The pore-size dependent loading and protection of EGFP (2.4 nm diameter×4.2 nm) from proteolytic attack by Pepsin A (7.3 nm×3.6 nm×5.4 nm) is demonstrated by the retention of fluorescence in 7.3 nm pores. Larger-pored materials (>9 nm) provide diminishing protection for EGFP, and the protection is greatly reduced with increasing pore size and pore size distribution breadth. Proteolytic hydrolysis is used to delineate the activity of pore-loaded versus surface-bound proteins and to establish that there is an optimal pore diameter for loading EGFP while protecting it from attack by a larger proteolytic enzyme.

Synthesis and tuning of bimodal mesoporous silica by combined hydrocarbon/fluorocarbon surfactant templating.

Hydrocarbon and fluorocarbon surfactants show highly nonideal mixing that under some conditions results in demixing of the two types of surfactants into distinct populations of fluorocarbon-rich and hydrocarbon-rich aggregates. This also occurs in materials prepared by cooperative assembly of hydrolyzed tetraethoxysilane with mixtures of cetyltrimethylammonium chloride (CTAC) and 1,1,2,2-tetrahydro-perfluorodecylpyridinium chloride (HFDePC). Here, we report conditions under which demixed micelles lead to bimodal mesoporous materials (including specific concentrations of ammonia and salt in the synthesis solution) and show that the sizes of the hydrocarbon-templated and fluorocarbon-templated pores can be finely and independently controlled by adding lipophilic or fluorophilic oils, respectively. Nitrogen sorption isotherms and transmission electron microscopy provide clear evidence for a single phase of demixed but disordered wormhole-like pores.

Synthesis and biocompatibility evaluation of fluorinated, single-tailed glucopyranoside surfactants

Partially fluorinated non-ionic surfactants are of interest for a range of biomedical applications, such as the pulmonary administration of drugs using reverse water-in-perfluorocarbon microemulsions. We herein report the synthesis and characterization of a series of partially fluorinated β-D-glucopyranoside surfactants from the respective alcohols and peracetylated β-D-glucopyranoside using BF3·Et2O as catalyst. The surfactant packing parameter of the fluorinated surfactants ranged from 0.472 to 0.534 (MOPAC calculations) or 0.562 to 0.585 (calculated from literature values), which is comparable to surfactants with a similar partially fluorinated tail. Based on an initial biocompatibility assessment, the β-D-glucopyranoside surfactants have low toxicities in the B16F10 mouse melanoma cell line and comparatively low haemolytic activities towards rabbit red blood cells. The fluorinated surfactants appear to be less toxic towards cells in culture and to have a lower haemolytic activity compared to their hydrocarbon analogs. Furthermore, an increasing degree of fluorination appears to reduce both the cytotoxicity and the haemolytic activity. Similar structure–activity relationships have been reported for other partially fluorinated surfactants. Overall, these findings suggest that the surfactants may be useful for biomedical applications, such as novel drug delivery systems.

Synthesis, Thermal Properties and Cytotoxicity Evaluation of Hydrocarbon and Fluorocarbon Alkyl β-D-xylopyranoside Surfactants

Alkyl β-d-xylopyranosides are highly surface active, biodegradable surfactants that can be prepared from hemicelluloses and are of interest for use as pharmaceuticals, detergents, agrochemicals, and personal care products. To gain further insights into their structure-property and structure-activity relationships, the present study synthesized a series of hydrocarbon (-C(6)H(13) to -C(16)H(33)) and fluorocarbon (-(CH(2))(2)C(6)F(13)) alkyl β-d-xylopyranosides in four steps from d-xylose by acylation or benzoylation, bromination, Koenigs-Knorr reaction, and hydrolysis, with the benzoyl protecting group giving better yields compared to the acyl group in the Koenigs-Knorr reaction. All alkyl β-d-xylopyranosides formed thermotropic liquid crystals. The phase transition of the solid crystalline phase to a liquid crystalline phase increased linearly with the length of the hydrophobic tail. The clearing points were near constant for alkyl β-d-xylopyranosides with a hydrophobic tail ⩾8, but occurred at a significantly lower temperature for hexyl β-d-xylopyranoside. Short and long-chain alkyl β-d-xylopyranosides displayed no cytotoxicity at concentration below their aqueous solubility limit. Hydrocarbon and fluorocarbon alkyl β-d-xylopyranosides with intermediate chain length displayed some toxicity at millimolar concentrations due to apoptosis.

Cationic–anionic vesicle templating from fluorocarbon/fluorocarbon and hydrocarbon/fluorocarbon surfactants

Spontaneous catanionic vesicle formation is studied in systems comprising fluorinated surfactants, the cationic/anionic fluorinated surfactant system of 1,1,2,2-tetrahydroperfluorododecylpyridinium chloride (HFDPC)/sodium perfluorooctanoate (SPFO) and the analogous mixed hydrocarbon/fluorocarbon surfactant system of cetylpyridinium bromide (CPB)/SPFO. Aggregate formation is explored in the anionic-rich surfactant system (weight fraction of anionic surfactant, gamma=0.66-0.85) and a total surfactant concentration range of 0.1-2% wt/wt for the fluorinated system and 0.4-2.2% wt/wt for the mixed hydrocarbon/fluorocarbon system. Vesicle sizes range from approximately 40 to 200 nm for CPB/SPFO, as determined by negative staining transmission electron microscopy (TEM) and confirmed by dynamic light scattering. The primary vesicle diameter observed by TEM in the catanionic fluorinated/fluorinated surfactant system is smaller (20-50 nm). However, the relatively few larger vesicles (100 nm) in the HFDPC/SPFO system dominate the dynamic light scattering measurements. Successful templating of silica hollow spheres is demonstrated in both HFDPC/SPFO and CPB/SPFO vesicle systems, using tetramethoxysilane (TMOS) as the silica precursor for the acid-catalyzed synthesis. The size of the resulting hollow silica particles is consistent with the templating of vesicles of the size range observed by TEM. Changes in zeta potential are used to monitor colloidal stability. At the conditions investigated (TMOS/surfactant weight ratios of 0.25-1.0, pH 3), the colloidal silica particles templated from fluorinated HFDPC/SPFO vesicles are more stable than the particles templated from the corresponding mixed fluorinated CPB/SPFO system. Further improvement of the stability of the colloidal particles is achieved in the CPB/SPFO systems by titrating the acid synthesis solution with base over the course of the particle synthesis.