Charlene A. Woodward

Solute diffusion in biocatalyst gel beads containing biocatalysis and other additives

Abstract Biocatalyst beads can be produced by the entrapment of microorganisms or enzymes into a stabilized hydrocolloidal gel such as alginate or carrageenan. In order to determine and predict the effectiveness of such catalytic material, it will be necessary to know the mass transport properties of substrates and products within the gel beads, especially where there are high concentrations of the biocatalyst or other additives. Diffusion of several solutes within the beads has been studied by measuring the transport of solutes to and from a well-stirred solution of limited volume. Tests have been performed with biocatalyst beads made from alginate and carrageenan covering a range of bead diameter, gel concentration, concentration of biocatalyst and other additives, and solute molecular weight. At low gel concentration with entrapped biocatalyst, a solute of low molecular weight has a diffusion coefficient approaching that measured in water. However, with increased gel concentration, and especially with high loadings of microorganisms and other additives, the diffusion coefficients are significantly decreased. An empirical relationship was developed for the diffusion coefficient as a function of microbial loading.

Poly(ethylene glycol)‐modified ligninase enhances pentachlorophenol biodegradation in water–solvent mixtures

Polychlorinated hydrocarbons are prevalent environmental contaminants whose rates of biodegradation are limited by their minimal solubilities in aqueous solutions where the biological reactions take place. In this study, ligninase (LiP) from Phanerochaete chrysosporium was modified by poly(ethylene glycol) to enhance its activity and stability for the biodegradation of pentachlorophenol (PCP) in the presence of acetonitrile (MeCN), a water-miscible solvent. The modified enzyme retained 100% of its activity in aqueous solutions and showed enhanced tolerance against the organic solvent. The activity of the modified enzyme was found to be over twice that of the native enzyme in the presence of 10% (v/v) MeCN. The solubility of PCP was enhanced significantly by the addition of MeCN to aqueous solutions, such that it was over 10-fold more soluble in the presence of 15% (v/v) MeCN than in pure aqueous buffer solution (from 0.06 to 0.65 mM). Capitalizing on the enhanced substrate solubility and the increased activity of the modified enzyme, the catalytic efficiency of the modified LiP in solutions containing 15% MeCN was over 11-fold higher than that of the native enzyme in buffer solutions (pH 4.2) in unoptimized reactor systems (from 44 to 480 mol PCP/mol LiP.h). Continued research both in the use of organic solvents to increase the availability of recalcitrant contaminants and in the modification of enzymes to enhance their activity and stability in such solvents promises to dramatically affect our ability to remediate contaminated sites. Published by John Wiley & Sons.

Degradation of organic sulfur compounds by a coal-solubilizing fungus

Paecilomyces sp. TLi, a coal-solubilizing fungus, was shown to degrade organic sulfur-containing coal substructure compounds. Di-benzothiophene was degraded via a sulfur-oxidizing pathway to 2,2′-dihydroxybiphenyl. No further metabolism of that compound was observed. Ethyl phenyl sulfide and diphenyl sulfide were degraded to the corresponding sulfones. A variety of products were formed from dibenzyl sulfide, presumably via free radical intermediates. Diphenyl disulfide and dibenzyl disulfide were cleaved to the corresponding thiols and other single-ring products. It was concluded that degradation of organic sulfur compounds byPaecilomyces involves an oxidative attack localized at the sulfur atom.

Coal solubilization by enhanced enzyme activity in organic solvents

Both oxidative and reductive enzymes can be utilized to enhance coal solubilization in aqueous and organic media. Aerobic solubilization was carried out with oxidases in a relatively polar medium, whereas solubilization in an anaerobic environment was conducted with reducing enzymes (dehydrogenase or hydrogenase) in aqueous and both polar and nonpolar organic media in the presence of hydrogen or a hydrogen donor. Solubilization of some enzymes in organic liquids was enhanced by complexation with polyethylene glycol. Enzyme concentration of 1–20 mg/mL was used, and most of the reactions occurred during the first 4 h, with up to 85% of the coal solubilized. There was some evidence of coal product inhibition and enzyme deactivation at higher temperatures.

Enzymatic catalysis in organic solvents: Polyethylene glycol modified hydrogenase retains sulfhydrogenase activity in toluene

Naturally occurring enzymes may be modified by covalently attaching hydrophobic groups that render the enzyme soluble and active in organic solvents, and have the potential to greatly expand applications of enzymatic catalysis. The reduction of elemental sulfur to hydrogen sulfide by a hydrogenase isolated from Pyrococcus furiosus has been investigated as a model system for organic biocatalysis. While the native hydrogenase catalyzed the reduction of sulfur to H(2)S in aqueous solution, no activity was observed when the aqueous solvent was replaced with anhydrous toluene. Hydrogenase modified with PEG p-nitrophenyl carbonate demonstrated its native biocatalytic ability in toluene when the reducing dye, benzyl viologen, was also present. Neither benzyl viologen nor PEG p-nitrophenyl carbonate alone demonstrated reducing capability. PEG modified cellulase and benzyl viologen were also incapable of reducing sulfur to H(2)S, indicating that the enzyme itself, and not the modification procedure, is responsible for the conversion in the nonpolar organic solvent. Sulfide production in toluene was tenfold higher than that produced in an aqueous system with equal enzyme activity, demonstrating the advantages of organic biocatalysis. Applications of bio-processing in nonaqueous media are expected to provide significant advances in the areas of fossil fuels, renewable feedstocks, organic synthesis, and environmental control technology. (c) 1996 John Wiley & Sons, Inc.

Microbial solubilization of a preoxidized subbituminous coal : product characterization

Preliminary characterization of a microbial coal solubilization product has been performed. Submerged cultures ofPaecilomyces TLi orCandida ML13 were grown in defined minimal media containing preoxidized Wyodak subbituminous coal. Culture supernatants contained high-molecular-weight, acid-precipitable material that was separated by gel permeation chromatography (GPC) in aqueous and polar organic solvents. Organic GPC also separated a low-molecular-weight (<2700 daltons) fraction that was converted to higher-molecular-weight material upon acidification. Elemental analysis of the acid-precipitated material indicated an increased oxygen content as a result of the biological treatment. The biosolubilized product may undergo further microbial modification.

The chemical modification of enzymes to enhance solubilization in organic solvents for interaction with coal

Some enzymes can effectively function as catalysts in contact with organic solvents in a variety of ways; however, it is desirable to utilize the enzyme in a soluble form for interactions with a solid substrate such as coal. Dinitrofluorobenzene (DNFB) has been used as the reagent to add dinitrophenyl groups to enzymes, thus increasing hydrophobicity and solubility (up to 20 mg ml -1 ) in less-polar organic solvents ranging from dioxane to benzene. Mixed reducing enzymes modified by DNFB and used in pyridine or benzene under hydrogen have been shown to enhance significantly the solubility in organic solvents of coals ranging from lignite to bituminous, with up to 35.3 wt% coal conversion. A fluidized-bed bioreactor appears to be the most effective contactor

Use of chemically modified enzymes in organic solvents for conversion of coal to nliquides

Abstract Reductive enzymes can be used to enhance the conversion of coal to liquids. This can be carried out in both aqueous and organic media, but, in the latter case, the enzymes must be chemically modified so that they are soluble and active in the organic environment. The most effective chemical modification of enzymes is achieved by using reagents such as activated polyethylene glycol or dinitrofluorobenzene that add hydrophobic groups. Use of such modified enzymes in organic solvents such as benzene or pyridine has resulted in the conversion of over 40% of bituminous coal.

Use of Dinitrofluorobenzene to Modify Enzymes for Enhanced Solubilization and Activity in Organic Solvents Scientific Note

It is now known that many enzymes can function as biocatalysts in, or in contact with, various organic solvents (1-5). As a result, such enzymes can now be considered for many different applications not previously thought to be possible in organic solvents. Approaches taken with this type of biocatalytic system have included the use of immobilized enzymes (6), enzymes particulates (7), enzymes in micelles (8), or enzymes in an aqueous phase that is in contact with the organic phase (6). These are effective ways of using biocatalysts for some applications providing the reaction is carried out in the liquid phase. However, if the enzyme must interact with a solid substrate, such as coal in the organic phase, it is desirable for the enzyme to be present in the organic media in a soluble form, since direct molecular interactions are a requirement. Some enzymes have minimal solubility in polar organics, especially those that contain water (9). However, there is only a limited solubility of enzymes in nonhydrous, polar organic solvents, since proteinaceous molecules are primarily hydrophilic. It may be possible to modify enzymes chemically to increase hydrophobicity and, thus, increase organic solubilization while maintaining catalytic activity. Several methods have been studied to

Use of modified enzymes for the solubilization/liquefaction of bituminous coal in a fluidized-bed reactor

Biocatalysts allow the solubilization/liquefaction of coal at nearambient temperatures. This research has focused on the chemical modification of enzymes to enhance their solubility and activity in organic media, and on optimal reactor design for a biocatalyst coal liquefaction process. Modification of hydrogenase and cytochrome c using dinitrofluorobenzene (DNFB) or methoxypolyethylene glycolp-nitrophenyl carbonate (PEG-n) has effected increased solubilities up to 20 g/L in organic solvents ranging from dioxane to toluene. Use of these modified enzymes in a small fluidized-bed reactor (with H2 sparge) resulted in >40% conversion of bituminous coal in 24 h. Research using model compounds suggests that the conversion process may be in part owing to splitting at methyl or ethyl bridges, and perhaps saturation of ring structures. A new class of continuous columnar reactors will be necessary to achieve the high throughput and low inventory necessary for biocatalyst processes. The controlling mechanisms of particle transport in fluidized-bed systems using very small coal particulates are being studied. This has included the hydrodynamic modeling of coal segregation in fluidized-bed reactors, with direct microscopic visualization using fluorescence microscopy. A summary of our previously published work on enzyme modification and fluorescence visualization is presented.