Much of the past and current focus of bioremediation has been on laboratory studies of microbial processes. By necessity, early studies have ignored important field properties, parameters, and processes that control the ultimate success of in situ bioremediation of contaminated groundwater. This paper presents a bioengineering systems approach that examines the impact of some of these field variables on common bioremediation practices. Using simple systems, the niche of biostimulation is shown to be aquifers with high contaminant sorption. A novel gas-phase biostimulation filter and a novel resting-state bioaugmentation/biofilter approach which show promise for effective field implementation are discussed.

Biocatalytic systems utilizing either living organisms or modified enzymes have been shown to enhance the liquefaction (products are liquid at ambient conditions) or solubilization of coal under anaerobic conditions. Microbial tests have been carried out in aqueous media with organisms isolated from outcropping of coal or from premium coal samples. Some of these isolates have been shown to grow on coal as the only carbon source and to produce small quantities of oxychemicals such as acetate or ethanol. Reducing enzymes, such as hydrogenase and cytochrome C, can be chemically modified to increase solubilization in organic solvents by attaching less polar chemicals, such as phenyl groups or polyethylene glycol, to the free amino groups on the enzymes. These biocatalysts have been shown to degrade model compounds and enhance the solubilization of coal in organic solvents under a hydrogen atmosphere. The resulting product is a relatively light hydrocarbon mixture with reasonably high volatility. 5 refs., 6 figs., 4 tabs.

Paecilomyces sp. TLi, a coal-solubilizing fungus, was shown to degrade organic sulfur-containing coal substructure compounds. Dibenzothiophene 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 by Paecilomyces involves an oxidative attack localized at the sulfur atom.

The solubilization of low-ranked coals by fungi, such as Paecilomyces, in defined submerged culture systems has been demonstrated. Current efforts focus on the conversion of the aerobically-solubilized coal into less oxidized products. Anaerobic methanogenic consortia have been developed that can remain active and viable in the presence of the aqueous coal product or vanillin, a coal model compound. The results suggest that a methanogenic consortium was able to produce methane and carbon dioxide from the product of coal biosolubilization by Paecilomyces as a sole carbon source. Work continues on the development of cultures able to convert the aqueous coal product and its various fractions into methane or fuel alcohols.

Paecilomyces TLi, a fungus isolated from coal, had previouslt been shown to transform solid coal into a water-miscible liquid during surface growth on coal or on a complex, solid microbiological medium. Coal solubilization has now been demonstrated in submerged cultures grown in defined liquid minimal media, although activity under these conditions is less than that observed in surface culture. Coal solubilization occurs under both alkaline and acidic conditions. Alkaline catalysis by biogenic material(s), produced in specific response to the presence of coal, has been implicated in both systems. A variety of oxidized hydrocarbon substrates, including both aliphatic and aromatic compounds, can be utilized by this organism. Coal solubilization is enhanced by growth on aromatic compounds structurally related to lignin or coal monomers. Preliminary evidence suggests that coal solubilizing activity is self-regulating in this organism.

Resting cells of Micrococcus luteus have been shown to remove strontium (Sr) from dilute aqueous solutions of SrCl 2 at pH 7. Loadings of 25 mg of Sr per g of cell dry weight were achieved by cells exposed to a solution containing 50 ppm (mg/liter) of Sr. Sr binding occurred in the absence of nutrients and did not require metabolic activity. Initial binding was quite rapid (<0.5 h), although a slow, spontaneous release of Sr was observed over time. Sr binding was inhibited in the presence of polyvalent cations but not monovalent cations. Ca and Sr were bound preferentially over all other cations tested. Sr-binding activity was localized on the cell envelope and was sensitive to various chemical and physical pretreatments. Bound Sr was displaced by divalent ions or by H + . Other monovalent ions were less effective. Bound Sr was also removed by various chelating agents. It was concluded that Sr binding by M. luteus is a reversible equilibrium process. Both ion exchange mediated by acidic cell surface components and intracellular uptake may be involved in this activity.

Immobilization of Micrococcus luteus within beads of bone gelatin results in a material which is able to adsorb significant quantities of strontium from dilute aqueous solutions analogous to some nuclear industry wastewaters. The mechanism appears to be principally an ion-exchange phenomenon. Both the bone gelatin and the microbial cells contribute to strontium removal; the principal contribution from the cells appears to be sorption onto cell wall material. This particular biosorbent may not be an immediate replacement for conventional ion-exchange materials currently used to remove strontium from wastewaters. However, the study does indicate that relatively inexpensive biological materials can be incorporated into particulate forms such as gel beads and used for the removal of dissolved metal ions from aqueous solution.

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 solubilization of low-ranked coals by fungi, such as Paecilomyces and Candida, in defined submerged culture systems has been demonstrated. Current efforts focus on the characterization of the aqueous solubilized coal products and the development of technologies for their subsequent utilization. Solubilized coal products have been fractionated, and preliminary characterizations performed. Differences in product composition have been detected with respect to the organism used in culture duration. Prospects for the conversion of the aerobically-solubilized coal into less-oxidized products have been developed which can remain active and viable in the presence of the aqueous coal product or vanillin, a coal model compound. The results suggest that a methanogenic consortium was able to produce methane and carbon dioxide from the product of coal biosolubilization by Paecilomyces as a sole carbon source. Work continues on the development of cultures able to convert the aqueous coal product and its various fractions into methane or fuel alcohols. 17 refs., 8 figs.

The use of isolated enzymes for coal solubilization has been investigated, with an emphasis on enhancing enzyme activity, especially in organic solvents. Possible enzymatic interactions and oxidative processes are discussed. Subbituminous and bituminous coals were studied in two different types of solubilization tests, followed by two analytical methods. (CBS)

Wastewaters from numerous industrial and laboratory operations can contain toxic or undesirable components such as metal ions, which must be removed before discharge to surface waters. Adsorption processes that have high removal efficiencies are attractive methods for removing such contaminants. For economic operations, it is desirable to have an adsorbent that is selective for the metal contaminant of interest, has high capacity for the contaminant, has rapid adsorption kinetics, can be economically produced, and can be regenerated to a concentrated waste product or decomposed to a low-volume waste. Selected microorganisms are potentially useful adsorbents for these applications because they can be inexpensive, have high selectivities, and have high capacities for adsorption of many heavy metals, which are often problems in a variety of industries.

Paecilomyces TLi, a fungus isolated from coal, had previously been shown to transform solid coal into a water-miscible liquid during surface growth on coal or on a complex, solid microbiological medium. Coal solubilization has now been demonstrated in submerged cultures grown in defined liquid minimal media. Activity under these conditions is less than that observed in surface culture, and is affected by both nitrogen and carbon nutrition. Coal solubilization occurs at low pH (≤ 4) but may be associated with pH increases during the assay period. Spectroscopic data suggest a role for alkaline catalysis in coal solubilization by this organism.

A bioprocess can be defined as the directed and controlled use of living organisms or their products to bring about desired chemical and/or physical change. Because of their specificity, bioprocesses can be used to separate a product or contaminant from mixtures or to convert a component of a mixture to a useful product at mild operating conditions. There are an increasing number of concepts that are being considered for use in the processing of fossil fuels, particularly for coal. These include coal preparation technologies such as beneficiation; conversion of coal to useful liquid or gaseous products; product upgrading with removal of detrimental and hazardous components; and environmental control technology for gaseous, liquid, and solid effluents. The conversion of coal to liquids and gases is of specific interest, since the mild operating conditions of biological processes would be very attractive when compared to the rather severe chemical and physical process environment needed for the thermal/chemical conversion of coal. Research results on the biological conversion of coal are summarized and the potential impact of this evolving technology is assessed.

Biocatalytic solubilization of coal can be achieved by microorganisms in aqueous medium or by enzymes in aqueous or organic media. In these systems, coal is converted to a liquid product through a variety of oxidative and/or hydrogenative reactions. The mechanism and control of coal solubilization activity have been studied in organisms associated with coal in nature. The solubilization apparently involves catalysis by alkaline cell metabolites. The aqueous product of this microbial action may be a suitable substrate for other biological interactions such as biogas production by methanogenic organisms. Enzymatic coal solubilization has been demonstrated in both shake flasks and fluidized-bed reactors. The process has been carried out under both anaerobic and aerobic conditions, with aqueous and hydrous organic solvents. The solubilization product of the anaerobic process is much less polar than that from microbial solubilization.

Current investigations into the biological solubilization of coal with microorganisms focus on the production of solubilizing activity in fungi. Test organisms for this work include a species of the yeast Candida previously isolated from a lignite outcrop, and P. chrysosporium, a filamentous higher fungus which has played a major role in lignin biodegradation research. The studies described are primarily exploratory in nature, and are fundamental to the design of more sophisticated inquiries into the physiology of fungal coal solubilization.

Ligninase activity in Phanerochaete chrysosporium is stimulated by incubating cultures with various substrates for the enzyme, including veratryl (3,4-dimethoxybenzyl) alcohol, which is a secondary metabolite of this fungus. This study was designed to provide insight into the mechanism involved in this stimulation. Ligninase activity increased 2 to 4 h after the addition of exogenous veratryl alcohol to ligninolytic cultures. This increase was prevented by inhibitors of protein synthesis. Analysis of the extracellular proteins by high-performance anion-exchange liquid chromatography revealed increases in the amounts of some, but not all, ligninase species. The normal rapid decrease in ligninase activity in aging cultures was not prevented or retarded by veratryl alcohol, indicating that veratryl alcohol does not increase ligninase activity by protecting extant enzyme. We conclude that veratryl alcohol probably functions via an induction type of mechanism, affecting only certain ligninase species. Results with an isolated lignin indicate that lignin (or its biodegradation products) functions in the same way that veratryl alcohol does.

The regulation of an H2O2-dependent ligninolytic activity was examined in the wood decay fungus Phanerochaete chrysosporium. The ligninase appears in cultures upon limitation for nitrogen or carbohydrate and is suppressed by excess nutrients, by cycloheximide, or by culture agitation. Activity is increased by idiophasic exposure of cultures to 100% O2. Elevated levels of ligninase and, in some cases, of extracellular H2O2 production are detected after brief incubation of cultures with lignins or lignin substructure models, with the secondary metabolite veratryl alcohol, or with other related compounds. It is concluded that lignin degradation (lignin → CO2) by this organism is regulated in part at the level of the ligninase, which is apparently inducible by its substrates or their degradation products.