BARR for Decontamination of Soil and Groundwater - Volume 1 This is the REPORT Copyright 1993 by Larry Dieterich All Rights Reserved Duplication and distribution is permitted, but credit must be given to the author and the document must be distributed in its entire form. No fragmentation, editing or deletions are permitted on copies for distribution. This document is part of a 3 volume set. Volume 1 (this document) BARR report Volume 2 BARR Technical Appendix Volume 3 BARR Bibliography The BARR process was developed by Larry Dieterich Larry Dieterich 405 E 7th Street Davis, California 95616 USA voice/fax (916) 758-9260 Internet email- water@well.sf.ca.us This edition of the BARR reports is made available in ASCII format for distribution over the Internet. Paper, disk copies or formatted Macintosh files with graphics are available upon request. Contact the author at the above address. BARR: For Decontamination of Soil and Groundwater B Bio- A Anaerobic R Reduction & R Re-oxidation Bio-Anaerobic Reduction & Re-oxidation BARR: For Decontamination of Soil and Groundwater REPORT ABSTRACT A technique for in situ degradation of organic pollutants in the subsurface is described. The BARR process (Bio Anaerobic Reduction & Reoxidation) is a technique that utilizes a "treatment cocktail" to stimulate microbial activity to form multiple and var iable catalysts and create sequential thermodynamic gradients to induce the transformation and degradation of the target chemicals. The "treatment cocktail" utilizes biodegradable reductants and oxidants with diverse, broad-spectrum inoculum in a buffered mixture that provides for the nutritional needs of the microbial components of the process. Mineralization of the target pollutant is achieved through a combination of direct metabolism, cometabolism and abiotic physical degradation processes, which are used again and again, until complete mineralization of the target pollutant has been achieved. *************************** TABLE OF CONTENTS- The next few pages are a detailed table of contents for the report. ABSTRACT INTRODUCTION THE BARR PROCESS DESCRIBED -Underground Bioreactor The BARR process capitalizes on existing Technology. The BARR process begins with location and description of the contaminated area. The next step in the BARR process is to assess the contaminant subsurface distribution. Approximate 3-dimensional mapping of the subsurface profile (soil and groundwater), including contaminant distribution, soil types and strata will be accomplished as part of the initial site assessment process. The chemical and microbial parameters of the system will be investigated also. An important product of site characterization process using probes is the resulting access to the formation. Contaminant Isolation The contaminated zone may be isolated from the rest of the formation by placement of materials to plug the porous strata at the perimeter of the contaminant zone. Injection of clay solutions may clog the transmissive strata permanently. Injection of substrate and inoculum may be used to clog the porous strata and thereby isolate the treatment zone. Injection of a wax/steam emulsion into the porous strata surrounding the contaminant zone may be used to seal the system. Treatment Cocktail Delivery "Treatment cocktail" delivery techniques and timing are an important part of the overall treatment scheme. Coupled Flow will be used to move the treatment cocktail into the highly polluted formations and strata. Inoculum The "treatment cocktail" will contain inoculum capable of degrading both the contaminant and added surfactant/substrate under reducing and oxidizing conditions. Inoculum provisions are made to insure that suitable organisms are present in the treatment zone. It may be possible to rely on the native microbial population in some sites. In many sites, it will be desirable to provide inoculum in the form of landfill leachate or other material collected from sites where the native microflora has been conditioned to degrade pollutants. Specific Toxicity Specific Toxicity = (growth inhibitory toxin concentration/population density) Treatment Cocktail Composition Based on the chemical, physical and microbial parameters established in the site analysis and characterization step, a first stage "treatment cocktail" is prepared for the target pollutant and system. The treatment cocktail will be miscible with the target pollutant. The "treatment cocktail" will contain mineral nutritional materials. Nitrogen may be provided as ammonia. The "treatment cocktail" will contain biologically active reductant. Catabolite Repression The reduced carbon provision might take the form of sugars. There is a wide variety of fats, oils and waxes that may find application in the BARR process. Simple Lipids Compound Lipids Phospholipids Glycolipids Nonsaponifable Lipids Cellulose Hemicellulose Volatile Fatty Acids Incubation After the introduction of the "treatment cocktail", the system is warmed for anaerobic microbial growth Monitoring The chemical and physical status of the treatment zone will be monitored via in-situ probes accessing the contaminant area, or by solution extraction for lab analysis. REOXIDATION AND REDUNDANCY The repetition of oxidation and reduction cycles will increase the opportunity for degradation to occur. CHANGES RESULTING FROM THE BARR PROCESS Temperature The BARR process creates temperature changes in the contaminated system. Coupled Flow Coupled flow will result from the BARR process pE Induced biological activity in the treatment zone will change the pE which will be accompanied by radical changes in the system chemistry. The changes in redox potential will tend to be buffered by the local system composition. The redox potential can be maintained at some desired level by the presence of inorganic electron acceptors. The electron status of the system, described as the redox potential will flux as the supplies and nature of reductant and oxidant are varied. pH Soil colloids are negatively charged and accumulate hydrogen ions. Bacteria are amphoteric and will acquire positive or negative charge depending on the pH of the system. The dissolution of minerals accompanying changes in pE and pH will also create changes in the makeup of the system solution by increasing the ionic strength of the solution. The increase in the ionic strength of the solution will also cause a collapse in the electrical double layer (multilayer) on charged surfaces. This solid matrix may be stationary aquifer matrix or mobile colloids. Introduction of water of low ionic strength will dislodge those particles which are attached by high-ionic strength conditions. When oxidation occurs, there will occur mineral reprecipitation. Colloids The large surface area associated with these colloids introduces an increased capacity for surface catalysis. The colloids formed from the precipitation of minerals are an important part of the BARR process. The creation of microbial biomass constitutes a biocolloidal phase that represents a separate, semipolar phase in the system. The changes in solution chemistry accompanying the BARR process will cause repeated flux in the metal ions in solution. Solution Chemistry The "treatment cocktail" will serve to increase the solubility of the pollutant by solvent/surfactant properties. See Technical Appendix for a complete discussion of cosolvency The solvent effect of the treatment cocktail will serve to dilute the concentration of the contaminant, a factor which may enhance the biodegradability of the pollutant. Microbes As the redox status of the system varies, different microbial consortia will be favored. The increase in the microbial biomass and the diverse consortia of the system resulting from the BARR process will increase the amount of enzymes in the system and thereby increase the opportunity for both direct metabolism and cometabolism of the target pollutant. Yeasts Fungi Protozoa Changes in the system will influence the dissemination of microbes in the treatment zone. Nitrogen transformations resulting from BARR The BARR method will be useful for treatment of inorganic as well as organic contamination The growth of microbial biomass will tend to diminish the transmissivity of the aquifer. Sealant effects may be very beneficial. The BARR process is repeated until the desired degree of degradation is achieved. The BARR process may be used in conjunction with existing pump-and-treat technology to enhance recovery of the target contaminant. Aspects of the BARR process requiring special attention. BARR requires high-class geotechnical & engineering capabilities. BARR process requires high-class biotechnical capabilities. BARR is undemonstrated CONCLUSION ********************** This begins the actual report BARR: for Decontamination of Soil and Groundwater ABSTRACT A technique for in situ degradation of organic pollutants in the subsurface is described. The BARR process (Bio Anaerobic Reduction & Reoxidation) is a technique that utilizes a "treatment cocktail" to stimulate microbial activity to form multiple and var iable catalysts and create sequential thermodynamic gradients to induce the transformation and degradation of the target chemicals. The "treatment cocktail" utilizes biodegradable reductants and oxidants with diverse, broad-spectrum inoculum in a buffered mixture that provides for the nutritional needs of the microbial components of the process. Mineralization of the target pollutant is achieved through a combination of direct metabolism, cometabolism and abiotic physical degradation processes, which are used again and again, until complete mineralization of the target pollutant has been achieved. INTRODUCTION Objective- This report provides a rationale and a procedure to achieve the in-situ mineralization of organic chemical contamination of the subsurface. BARR (Bio-Anaerobic Reduction and Re-oxidation) creates accelerated degradation of contaminants by creating repetitive redox flux in a high surface-area biocatalytic environment. BARR is not strictly bioremediation, rather it is a biochemically assisted process of stabilizing and degrading contaminants in soil and groundwater. The process can be carried out in-situ. The application of bioremediation to contaminated soil and subsoil has been explored extensively in past years. The promise of bioremedial processes has been fostered by the observed ability of various microbes to degrade a wide variety of contaminants. Microbially mediated aerobic oxidation of organic contaminants has been the objective of nearly all applications of bioremediation. For a number years, evidence has been accumulating that anaerobic transformation of "recalcitrant" organic materials is poss ible. Both aerobic and anaerobic degradation have demonstrated limitations of capability; chiefly the inability to completely mineralize recalcitrant compounds. More recently, there has been a significant body of evidence presented that indicates that sequential oxidation and reduction may hold the key to effective bioremediation. Coupled anaerobic-aerobic process have been reported to degrade recalcitrant organ ics. For example, reductive dechlorination of PCB's produces less-chlorinated congeners which are suitable substrates for oxidative degradation by a wide range of aerobic organisms. (HARKNESS et. al. 1993) Sequential environments are sometimes the best alternative for the detoxification of organic compounds. For example, compounds that degrade through a series of reductive and oxidative steps are most efficiently biodegraded by sequential anaerobic-aerobic processes. Other benefits relate to the detoxification of a broad range of chemicals. Aerobic and anaerobic environments each have limitations in their biodegrading abilities, but they often compliment each other when they are combined. One limitation of aerobic processes involves the recalcitrance of highly chlorinated chemicals such as hexachlorobenzene, tetracholoroethylene and carbon tetrachloride, which appreciably degrade only under anaerobic conditions. In contrast, conventionally cultured aerobes are efficient degraders of aromatic compounds that are anaerobically recalcitrant. Notable exceptions exist to these generalizations. For example, highly chlorinated compounds such as 1,1,1-trichloroethane, trichloroethylene and chloroform will biotransform under aerobic conditions if methane, phenol or toluene is provided as primary source of carbon and energy for growth. However, these reactions are cometabolic rather than direct metabolism. In fact, the majority of highly chlorinated compounds, such as 1,2,4-trichlorobenzene, 1,2,4,5-tetrachlorobenzene, and hexachlorobenzene are recalcitrant under aerobic treatments. In contrast to highly chlorinated aliphatic compounds, aromatic compounds are more successfully degraded under aerobic, rather than anaerobic conditions. Conventionally cultured aerobic microorganisms are considered particularly successful degraders of aromatic compounds because they often produce mixed function oxidase enzymes, which initiate aromatic ring cleavage. This report provides the technical background for this new, emerging technology and describes a process whereby the biological and abiological processes of sequential oxidation and reduction may be utilized to effectively degrade contaminants of the surface and subsurface in-situ. The BARR process turns the subsurface treatment zone into an in-situ bioreactor. By manipulating properties such as pE, pH, temperature, chemical composition of the system and mass flow, BARR creates fundamental changes in the system. The presence of specialized enzymes and extremely high surface areas, along with modifications to the polarity of the aqueous phase, exert strong transformation pressures on contaminants. BARR is a broad spectrum treatment. The overall result of the BARR process is the dissolution and complete degradation of the target contaminants to mineral components. THE BARR PROCESS DESCRIBED The BARR process utilizes thermodynamic gradients and multiple mechanisms of transformation to degrade the target pollutant in a multiple step process. In the first step, the polluted system is characterized and mapped. Where possible and desirable, the contaminated formation may be isolated by injection of different materials to temporarily or permanently plug the transmissive strata surrounding the pol luted zone. A "treatment cocktail" is delivered into the contaminated system which creates biological activity and causes a decrease in the pE and shifts in other fundamental chemical and physical parameters of the system, such as pH, temperature and chem ical composition. The chemical and physical status of the system is monitored and incubated before a second "treatment cocktail" is introduced to re-oxidize the system. The process is repeated. In subsequent steps, the "treatment cocktail" is varied to cr eate sequential reducing and oxidizing conditions in the treatment zone. The sequential environments are repeatedly created until complete degradation is achieved. Coupled, or sequential anaerobic-aerobic process have been reported to completely mineralize a wide range of recalcitrant organics. The BARR process capitalizes on existing Technology. BARR uses existing geotechnical and site characterization methods. Furthermore, the use of existing landfill experience is of great value to the development of this process. Biotechnical expertise and experience with contaminant microbiology are utilized by the BARR process. BARR also makes use of existing steam technology and relies heavily on probe and injection lance technology. (Contact Larry Dieterich (the author) for information about existing lance and probe technology. The author may be contacted via email; his Internet address is water@well.sf.ca.us). The BARR process begins with location and description of the contaminated area. Standard methods are used to determine the depth and lateral extent of the contaminated soil and water. These methods include surface examination and site record analysis, including current and historical records. Where possible, aerial photographs of the site may reveal information useful to the remediation process. In some cases, it may be necessary and practical to remove the contaminated surface soil to reveal the location of the pollutant entry into the subsoil. Where excavation is necessary, the contaminated soil will be stockpiled and treated thermally, biologically or with BARR in an on-site or off-site heap process. The objective of the site analysis is to determine the nature of the contamination and the location of the target pollutant in the subsurface. The next step in the BARR process is to assess the contaminant subsurface distribution. The subsurface investigation is carried out with available geophysical tools. Where possible, the subsurface will be characterized and the contaminant distribution ascertained by utilizing hydraulic probes fitted with soil samplers. Where possible, the BARR process relies on the use of probes for sampling and contaminant location and characterization. The cost of investigation with hydraulic probes is much less than with the use of drilled wells. Where probes are used, soil strata and contaminant distribution may be determined by groundwater and soil core recovery from hollow sampler attachments. Soil core samples are taken to ascertain the subsurface profile as well as contaminant distribution. In addition to the location of the major zone of contamination, the downslope areas are probed and the soil vapor phase is sampled to track the distribution of the contaminant plume. This is practical for volatile contaminants or volatile tracers that may indicate the location and nature of the contaminant. Soil texture is an important parameter in pollutant distribution because hydrophobic contaminants tend to distribute themselves in the finer textured sediments. Sands in the vadose zone will tend to pass liquid contaminant downward. Clay layers (or other confining layers) will tend to stop the material and impede its downward flow. Lateral flow along the top of clay strata may be expected as well. The behavior of the contaminant when it encounters the water table will depend largely on the density of the contaminant. Liquids heavier than water will tend to sink while those lighter than water will tend to float on the water surface. Low-density contaminant liquids that float on top of the water table and will tend to "smear" at phreatic surface. The fringe zone gets coated as the water table rises and falls. The water soluble fraction mixes with the groundwater and the insoluble fraction floats. It floats downgradient as well, downslope under the influence of gravity. If the contaminant liquids are heavier than water, they move down, through the groundwater. They are moved by the mass flow of the moving groundwater as they move downward. If they encounter an impermeable layer, they will flow downslope. Some of the material will dissolve in the water, depending on the intrinsic water solubility and the chemistry of the solution. Approximate 3-dimensional mapping of the subsurface profile (soil and groundwater), including contaminant distribution, soil types and strata will be accomplished as part of the initial site assessment process. The chemical and microbial parameters of the system will be investigated also. In addition to the soil textural distribution, soil and pollutant chemical parameters such as pH, pE and EC (electrical conductivity) of the subsurface soil and groundwater will be ascertained as part of the preliminary assessment in the BARR process. Contaminant location and assessment will be accompanied by sampling to assess the mineral nutritional status of the system as it regards microbial mineral nutritional needs. This will include nitrogen, phosphorous, potassium, etc. As part of the contaminant analysis, the biodegradability or biocidal properties of the contaminant will be estimated. Microbial population assessment is part of the characterization and analysis process as well. Indicators of the resident microbial population will be determined. All of these parameters will be estimated as part of the contaminant distribution assessment which will be accomplished, where possible, by probes and core recovery. The nature of the target pollutant, and the chemical and microbial composition of the subsurface system will help determine the required inoculum provisions, the pH and pE buffering capacity of the system and the composition of the "treatment cocktail". An important product of site characterization process using probes is the resulting access to the formation. The contaminated formation is accessed by the sampling probes, which serve as injection lances into the ground. Many or all of the probes are left in the ground to provide access to the contaminated subsurface. The ground resembles a "pin cushion" after the characterization and investigation process. These probes, fitted with porous sections, serve as conduits which may be used to deliver a variety of materials into the ground to isolate and treat the contaminant. Contaminant Isolation The contaminated zone may be isolated from the rest of the formation by placement of materials to plug the porous strata at the perimeter of the contaminant zone. This presupposes detailed knowledge of the subsurface and the ability to selectively place materials such as biomass, clay or wax to seal the transmissive formation around the outside of the contaminated zone. If the contaminated zone is isolated and contained, any liquid materials added in the treatment cocktail will remain in the treatment zone. In such an event, accumulated liquid in the treatment zone may have to be pumped out. The requirement of liquid removal might necessitate a drilled well. In most cases, or where predominately gas "treatment cocktail" is used, there will be no such requirement for liquid removal. Injection of clay solutions may clog the transmissive strata permanently. This may or may not be desirable. It may also be difficult to effectively plug the formation with clay slurries. Injection of substrate and inoculum may be used to clog the porous strata and thereby isolate the treatment zone. Given that there is a lag time anticipated with microbial response to "treatment cocktail", it may be possible to place a considerable volume of "treatment cocktail" in the contaminated formation before significant microbial growth begins. Biologically active oxidizing conditions tend to cause greater biomass accumulation than biological activity under reducing conditions, because anaerobic metabolism produces less usable energy than aerobic metabolism. The amount of biomass, and hence the effectiveness of the plugging from such a treatment, will largely be a function of the aeration status of the system and will vary with changes in "treatment cocktail". Such a formation-plugging process will be temporary, since the biomass will decrease when the substrate is exhausted by microbial metabolism. In addition to the production of microbial biomass, the oxidizing step will result in the precipitation of iron and manganese compounds, which will also tend to plug the formation under oxidizing conditions. This mineral precipitate will be subject to manipulation in the interior of the treatment zone, where the redox is controllable, but the precipitate will persist at the perimeter of the treatment zone, where an oxic interface exists with the ambient subsurface. Oxygen diffusion from the atmosphere will tend to create oxidizing conditions in the subsoil in the absence of some reducing agent. Formation Sealing and Isolation Injection of a wax/steam emulsion into the porous strata surrounding the contaminant zone may be used to seal the system. Subsurface temperature is relatively constant. In the absence of heat generated by the oxidation steps of the BARR process, or intentional heat injection into the system, the soil and groundwater surrounding the contaminated formation will be relatively constant temperature. A paraffin wax, or hydrogenated vegetable oil can be introduced into the formation as a heated emulsion. Once the formation returns to its natural temperature, the emulsion will form a solid in the transmissive strata. The wax/steam emulsion may be formulated to contain catalysts, nutrients and inoculum to degrade the wax within a known time frame, under predetermined conditions. The isolation step assists in the containment of the pollutant against migration out of the zone of treatment. It may be challenging to effectively isolate a contaminated formation, however partial plugging of connected transmissive strata may serve to minimize pollutant transfer into sensitive groundwater resources nearby. Treatment Cocktail Delivery "Treatment cocktail" delivery techniques and timing are an important part of the overall treatment scheme. Obviously, the ability to deliver treatment cocktail to the pollutant will depend on a number of things, notably the texture and layer structure of the subsurface. As well as the depth and lateral extent of the contamination. Cobbles and stones in the subsurface may also frustrate efforts to place injection lances to deliver the "treatment cocktail". In some situations, in very difficult soils, piledrivers may be used drive injection lance jackets. Drilled wells may be needed, but for reasons of expense, should be minimized where possible. Under good subsurface conditions, this technique is effective for small or large areas of highly or slightly contaminated soil and groundwater. The same probes used for sampling and contaminant distribution assessment can also be used for cocktail delivery. (ascii format does not support this graphic- see original report for graph) (graphic inserted here showing lance installation and cocktail delivery) The "treatment cocktail" delivery method will be largely dependent on the type of formation in the treatment zone. In areas where the cocktail can be delivered right into the contaminated formation, it may be possible to pump "treatment cocktail" directly into the contaminated soil. Areas with accessible transmissive strata directly adjacent to high concentrations of pollutant are ideal for cocktail delivery. For example, a contaminated clay formation overlain by a porous layer could be treated by injection of "treatment cocktail" into the porous layer. The "treatment cocktail" would then migrate to cover and permeate the contaminated clay. A hydrophobic "treatment cocktail" would be driven by hydrophobic forces to diffuse into the finer textured matrix, in response to the same gradient that drives hydrophobic contaminants to partition into fine textured materials. High pressure pumps may be able to deliver "treatment cocktail" into clay formations where high concentrations of contaminants are detected. In such "tight" formations, the use of gaseous "treatment cocktail" should be maximized, applying liquid "treatment cocktail" only for those "treatment cocktail" components having no gaseous phase options (e.g., phosphates & inoculum). In saturated treatment zones, the relative densities of the "treatment cocktail" and the contaminated groundwater should be considered. If it is desired that the "treatment cocktail" migrate downward in the treatment zone after injection, then a "treatment cocktail" with a density greater than water must be formulated. There is a great potential for creative variability in the application of "treatment cocktail". For example, it is possible to use multiple injection points to create desired conditions at some point between the two injection points. One lance might inject inoculum, while another lance, at some distance away, injects steam and ammonia. Both injections may be made into a formation previously injected with volatile fatty acids (VFA's). Such a technique would cause a zone of high bacterial growth at the interface of the two treatments. The nature and composition of the "treatment cocktail" will vary in the different treatment steps. For example, the initial step might involve the injection of an aqueous emulsion of oils, sugars and inoculum with a liquid suspension of cellulose and mineral nutrients. This step could be followed, at some time later by injection of other types of "treatment cocktail", such as ammonia. This step will be followed later by oxygen gas and/or other reactive gasses, such as CO2. It is possible to adjust the redox by injection of "treatment cocktail" as well. Obviously, the injection of oxygen will raise the pE radically. The injection of anhydrous ammonia, for example, will have an effect on the processes taking place in the treatment zone. Injection of a mineral solution such as nitrate or sulfate will have the effect of buffering the redox at the potential where those materials are reduced; assuming adequate reductant (bioavailable energy source) is present. Coupled Flow may be used to move the treatment cocktail into the highly polluted formations and strata. Coupled flow is mass flow that is induced in the absence of a pressure gradient. It may be induced by heat, solute concentration, or electrical gradients. The presence of a temperature gradient can cause groundwater flow as well as heat flow when hydraulic gradients do not exist. Water will tend to flow outward from heated areas. This can be used to advantage by injection of "treatment cocktail" in steam in the area surrounding the concentrated contaminant. Such a "treatment cocktail" delivery strategy may be coupled to a liquid removal process in the center of the contaminated zone, to create an additional gradient for flow of "treatment cocktail" into the contaminated zone. Chemical gradients cause mass flow of water as the osmotic potential varies spatially. Water naturally moves to dilute concentrations of dissolved or suspended materials. The role of chemical gradients in the movement of chemical constituents is of considerable importance in the BARR process, because the process creates dissolution and precipitation of minerals in the subsurface matrix. Additionally, the process introduces solutes into the system, which will cause osmotic gradients. The impact of these osmotic gradients alone on coupled flow is difficult to assess. It is also likely to be variable with different treatment stages and ion flux. The presence of existing probe access to the formation offers the opportunity for electrode placement to induce electrokinetic flow into or out of the contaminated zone. Inoculum The "treatment cocktail" will contain inoculum capable of degrading both the contaminant and added surfactant/substrate under reducing and oxidizing conditions. In natural environments a number of relationships exist between individual microbial species and between individual cells. The interrelations and interactions of the various microbial groups making up the soil community however are in a continual state of change and this dynamic state is maintained at a level characteristic of the flora. The composition of the microflora of any habitat is governed by the biological equilibrium created by the associations and interactions of all individuals found in the community. Members of the microflora rely on one another for certain growth substances, but at the same time they exert detrimental influences so that both beneficial and harmful effects are evident. Inoculum provisions are made to insure that suitable organisms are present in the treatment zone. Many toxic organic chemicals persist at underground waste sites despite being readily biodegradable under laboratory conditions. When this occurs, bacteria selected for their capacity to degrade the contaminants, and to proliferate after injection in the aquifer, may be added to enhance biodegradation. It is apparent from the literature that microbial consortia, rather that pure cultures, are most likely to degrade organic mixtures in contaminated soil. Multiple-step degradation processes are undoubtedly the rule, where the product of one reaction becomes the input reagent for succeeding reactions. The literature commonly reports the involvement of a consortium of organisms in the ultimate degradation of contaminants. It is commonly found that biodegradation processes are characterized by a "lag period" or an "acclimation period", where there is no observed degradation taking place. This "lag period" is followed by active degradation of the target pollutant. The "lag period" is most frequently explained as a period of time when organisms capable of utilizing the pollutant (direct metabolism) are selectively multiplying. Degradation of the target is not observed until sufficient numbers of the degrading organisms are present. It is often reported that the "lag period" is shortened or eliminated by the provision of inoculum from "acclimated populations" of inoculum taken from sites that have been previously contaminated with the target pollutant. The explanation offered is that such an "acclimated population" has sufficiently high numbers of adapted organisms capable of degrading the target pollutant, so the lag period is not observed. It may be possible to rely on the native microbial population in some sites. Conditioned, or acclimated organisms are frequently found in environments where the target contaminant has been present for some time. Old contaminated sites, therefore, may not need inoculum. It is doubtful if introduction of inoculum would be harmful, however, since well-adapted microbes would probably persist in spite of any introduced inocula. In many sites, it will be desirable to provide inoculum in the form of landfill leachate or other material collected from sites where the native microflora has been conditioned to degrade pollutants. The variability and heterogeneity of soil and the discreteness of the microhabitats is very great. Even over small distances, <1mm, the composition and size of the particulates, the amounts and types of water, nutrients and gases and the pH, pE, ionic strength and other physicochemical characteristics can vary widely. This variability of abiotic factors is reflected in simultaneous occurrence in the same soil sample of autotrophs and heterotrophs, aerobes and anaerobes, vegetative cells and spores, procaryotes and eucaryotes, cells with different requirements for and tolerances to osmotic pressure, pH, pE, temperature etc. It may prove desirable to collect a wide range of inocula from diverse environments, such as other contaminated sites, landfill leachate, healthy soils, sewage sludge, mucks, sediments, etc to guarantee a diverse inoculum base for the BARR process. There are reports of diverse inocula being beneficial to degradation of xenobiotics. The source of the culture greatly influences transformation ability of the microbial consortia. For instance, 6% of 3,5- dichlorobenzoate disappeared in sewage sludge, whereas 100% disappeared in methanogenic aquifer seed from a site bordering a municipal landfill. (ZITOMER & SPENCE 1993). Lotter, et al. 1990 report a significant enhancement of the biological degradation of oil contaminated soils can be achieved if compost of separately collected household, yard and garden waste (biowaste) is mixed with the soil. This undoubtedly is the result of diverse inocula, as well as diverse substrate. While it is commonly reported that bacteria are the most prevalent organisms involved in biodegradation, there are reports of protozoans living under methanogenic conditions as well. Fungal spores are also likely to be present in the inoculum. Most fungus are obligate aerobes. As the BARR process becomes more developed, new sources of inoculum may be developed. It will probably arise that the BARR process itself will develop inoculum for future treatments, i.e., the application of BARR to a particular site will produce well-adapted inocula for future applications of BARR to other contaminated sites. Adequate care must be taken to collect and maintain the inoculum in a form that preserves the viability of the inoculum. Anaerobic inoculum should be handled anaerobically so as not to be inactivated by oxidizing conditions. Certainly, some microbes are either facultative (able to live in oxic or anoxic conditions) and many others are able to form resistant "resting structures" to withstand toxic concentrations of oxygen. There are undoubtedly others, some of which may be very useful to the BARR process, which are unable to withstand oxygen at atmospheric concentrations. Careful sample recovery techniques, utilizing air-tight samplers and inert atmosphere storage and analysis conditions may be important to the survival of some inocula. Additionally, inoculum must be successfully delivered. For such an approach to bioremediation to be successful, the introduced species must be able to reach the contaminated zone and to move through the porous material along a possible contaminant plume in a viable state. Hence the importance of pE buffers in the "treatment cocktail". Bacterial adhesion to the solid matrix may be enhanced by groundwater of high ionic strength. Since this adhesion may limit the desired dispersal of the microbes in the treatment zone, it may be of value to counteract the adhesion, at least initially. Such attachment can be reversed by injection of deionized water, or by changing the pE to cause dissolution of the mineral matrix or precipitation of supersaturated solution. All of these measures are easily accomplished in the BARR process by varying the treatment cocktail. Specific Toxicity Specific toxicity is a descriptive parameter that explains the ability of the BARR process to degrade high concentrations of toxic organic materials. The use of high levels of varied inoculum and supplemental carbon substrates can enhance absolute degradation rates of organic compounds at concentration levels of pollutant that would be otherwise toxic to the degrading organisms. In order to objectively express the ability of such a system to perform its function of degrading toxic pollutants, a quantifiable parameter characterizing how much toxin the system can withstand is generated by a parameter called Specific Toxicity. This is a division of lethal concentration of contaminant, by the biomass concentration. The use of a biomass-based metric for toxicity assessment is not uncommon in toxicology. Toxicants are often evaluated and expressed in terms of the quantity toxic to the unit mass of the organism in question. For example, the lethal dose of a substance for 50 percent of a given population (LD50) is typically expressed in units of mg toxin per kg biomass. The specific toxicity of an organic compound can be calculated if one knows both the inhibitory concentration and the population density at which that toxin concentration proves fatal to the system. While the amount of toxic compound necessary to kill an individual cell may remain unchanged, upon addition to the supplemental carbon source, the population increase effected by the supplement allows the population to survive and metabolize concentrations of toxin which would kill a lower amount of cells. The increase in cell number due to the supplemental carbon decreases the amount of toxin available to each cell. Increased absolute biodegradation rates are primarily the result of augmented biomass generated through accelerated growth rates in the presence of the supplemental carbon source. Specific toxicity is second order; in other words, it depends not only on the concentration of the toxic compound, but on the population density of degraders as well. Analogous to specific degradation rate providing predictive information on absolute degradation rates in carbon supplemented systems, specific toxicity allows prediction of absolute levels of toxin which would prove fatal to the overall system of degraders. Specific Toxicity = (growth inhibitory toxin concentration/population density) This quantity may be determined for organisms who have no known ability to degrade the toxin. For example, algae in a bioreactor may be fed toluene, causing a die off. The use of a supplementary carbon source to stabilize a toxin-degrading population at high toxin concentrations is based on the premise that a higher population density of microbes can withstand higher concentrations of toxin because the amount of toxin per cell is lower. In the case of low organic toxin concentrations, the specific degradation rate (i.e. mg toxin degraded per mg biomass) may remain unchanged, but the secondary carbon source provides for more biomass and hence elevated absolute biodegradation rates. Treatment Cocktail Composition Based on the chemical, physical and microbial parameters established in the site analysis and characterization step, a first stage "treatment cocktail" is prepared for the target pollutant and system. The BARR process utilizes a "treatment cocktail", the composition of which must be considered on a case-by-case basis, at least initially, until enough experience has been gained to allow for generalizations. In general, adding combinations of nutrient materials will result in greater mineralization than the addition of any single nutrient. Since several types of organisms may be required to degrade any single compound, the concept of a single limiting nutrient may not be applicable to heterogenous subsurface populations. The metabolic abilities and nutrient requirements of subsurface microbes can vary substantially within a single system. This variability may be a result of the existence of a variety of micro habitats, the patchy distributionof microbes in the subsurface, differences in microbial community structure over short distances, and differences in nutrient requirements and availability in different microhabitats within superficially uniform material. This "treatment cocktail" will consist of: reduced carbon substrate, microbial nutritional provisions, inoculum, and buffers to maintain eH and pH within a desired range. The "treatment cocktail" will be provided as a sequence of different materials delivered at different times to create the desired conditions in the treatment zone. The treatment cocktail will be miscible with the target pollutant. Part of the objective of the "treatment cocktail" is the stimulation of biological activity in the zone of the target pollutant. As far as is possible, the "treatment cocktail", or some substrate component of it, will be miscible with the target pollutant. Since many petrochemically derived pollutants are hydrophobic (lipophilic), the polarity of the "treatment cocktail", will be close to that of the target pollutant and so may contain lipids. Lipids are organic compounds that are soluble in organic solvents such as ether, benzene, acetone, chloroform, hexane, carbon tetrachloride and petroleum ether, etc.; and are only sparingly soluble in water. Many of the degradation products of high molecular weight organochlorine compounds are lipophilic. The lipids used in the BARR process are also chosen for their biodegradability. The injected "treatment cocktail" will serve to modify the properties of the solution. The "treatment cocktail" may contain surfactants capable of forming micelles. Humic acids have been observed to have surfactant properties at concentrations well below any critical micelle concentration. The "treatment cocktail" may therefore contain humic materials or other biodegradable compounds capable of forming micelles. The "treatment cocktail" will contain mineral nutritional materials. Nutritional provisions are necessary to establish a favorable environment for microbial growth. Adequate primary, secondary and micronutrients must be available for the desired increase in biological activity. These provisions for mineral nutrition will include nitrogen, phosphorous, potassium, vitamins, as well as other micronutrients and microbial growth agents such as amino acids, purines, pyrimidines, etc. The assessment of mineral nutrient requirements is part of the initial site assessment and characterization process described above. Nitrogen may be provided as ammonia. Ammonia is the most readily utilized of the inorganic forms of nitrogen. Its utilization requires no oxidation or reduction, since the nitrogen in cellular constituents is at the same oxidation state as ammonia; the valence being -3. The ability of an organism to utilize ammonia as a source of nitrogen, i.e. to assimilate ammonia, depends on its ability to undergo amination, the addition of an amino group to the molecule. Extracellular ammonia will be oxidized to nitrate during the oxidative phase of BARR. It may be desirable to provide nitrate in the "treatment cocktail", both as a biological nitrogen source and as a pE buffer, since nitrate serves as an electron acceptor at moderately reducing conditions. The nitrogen metabolism of the BARR process creates N2 gas from oxidized forms of nitrogen during the reducing phase of BARR, in a process known commonly as denitrification. The reduction of NO3- (denitrification) produces N2, which is stable and inert. The "treatment cocktail" will contain biologically active reductant. The injection of an electron-rich biodegradable material into a soil environment that is biologically active will create reducing conditions. Reduced carbon material serves as an electron donor in biological processes. The result of the addition of readily degradable reduced carbon is the increase in the amount of biological activity and biomass and thereby increases in the opportunity for metabolic and cometabolic transformation of the target contaminant from biologically influenced catalysts. Highly chlorinated compounds, which are some of the most commonly found "recalcitrant" organic contaminants, are partially degraded by anaerobic cometabolic transformation in tests utilizing an electron donor "treatment cocktail". Reductive dehalogenation is a commonly reported process in anaerobic cultures. Secondary carbon sources are reported to increase biomass levels and catalyze rates of pollutant transformation. Secondary carbon sources provide both energy and cellular carbon for microbial growth. The compound or compounds from which the carbon of the cellular material is derived is the carbon source. Prototrophs can utilize as a sole carbon source either an inorganic (i.e. carbon dioxide (autotrophic prototrophs) or an organic compound (heterotrophic prototrophs). The ability of any species to use any specific carbon source depends on its genetic information. Some prototrophs, such as pseudomonas, are capable of utilizing any one of approximately 100 different types of organic molecules. The biological activity resulting from the introduction of a food source will be a function of several variables, such as the inoculum and the ambient conditions of pE, pH, specific toxicity, electron supply (biodegradable reductant), and electron acceptors. If the inoculum or resident organisms are not able use the introduced carbon source, there will be no increase in the population. Broadly usable carbon energy sources will have the most obvious application in the BARR process. In general; lower aliphatic hydrocarbons, gases from methane to pentane, are commonly used by microbial organisms as a sole source of energy. Aliphatic hydrocarbons from C5 to C16 are readily degraded by microbes. In general, the longer the C chain, slower the rate of decomposition. Extensively branched chains are more slowly degraded than linear chains. High molecular weight compounds are degraded the slowest. Multiple substrate metabolism will provide the opportunity to effect biodegradation of toxic organic compounds at rates well above those possible in systems where toxic substrates are the sole carbon and energy sources available. Catabolite Repression Catabolite repression is the favored use of one substrate over another. When a target pollutant is the intended substrate for the microbial population, the presence of a more easily degradable substrate will inhibit the degradation of the target, as the microbes preferentially degrade the easier substrate. High concentrations of easily metabolized carbon substrates have been shown to repress the catabolic enzyme systems needed by bacteria to degrade less desirable organic substrates until no easily metabolizable substrate remains. Most of the work describing simultaneous multiple substrate utilization involves growth-rate limiting substrate concentrations. It is reported (LINDSTROM AND BROWN, 1989) that the addition of secondary carbon sources was found to inhibit mineralization of toxic substrates, presumably due to preferential utilization of the more easily degraded carbon supplements (catabolite repression). Maintenance of carbon-limited conditions in culture prevents catabolite repression and allows simultaneous multiple carbon substrate metabolism by microbial populations that directly metabolize toxic materials. Simultaneous direct metabolism of the toxic and supplemental substrates requires maintenance of growth limiting carbon concentrations in culture to avoid catabolite repression. Catabolite repression is not a limitation to BARR because the process relies on heavily on cometabolism and abiotic degradation processes as well as direct metabolism. Catabolite repression is only a problem if direct metabolism is the sole mechanism of pollutant breakdown. Cometabolic degradation will be enhanced by biological activity in the general sense. Only direct metabolism is influenced by catabolite repression. BARR utilizes cometabolism, which does not yield energy for the organisms involved. And abiotic degradation mechanisms. Hence, catabolite repression has diminished impact on the overall effectiveness of the BARR process. The reduced carbon provision might take the form of sugars. Sugar is a readily oxidizable substrate. Fermentation is a common event when sugars are present in a biologically active environment lacking adequate oxygen. In the fermentation process, the substrate molecule itself serves as both reducing agent and oxidizing agent. The injection of some sugar, or mixture of sugars, will be accompanied by an increase in the populations of organisms that have the ability to ferment or otherwise utilize the chemical energy contained in the sugar, under the ambient conditions Fermentation products such as alcohol will effectively increase the solubility of hydrophobic pollutants in the aqueous system by a phenomenon known as cosolvency. See Tecnhical Appendix (a separated document) for a complete explanation of cosolvency. For reasons of polarity and bioreductant value, oils and fats will probably find great utility in the BARR process. There is a wide variety of fats, oils and waxes that may find application in the BARR process. A general discussion of paramaters and properties of fats and oils is provided to assist in making intelligent choices about which oil, or mixture of oils, to use. Fats and oils are esters of higher fat acids and the trihydric alcohol, glycerol. Esters of glycerol frequently are called glycerides. The difference between fats and oils is merely that fats are solid or semisolid at room temperature, whereas oils are liquids. Vegetable fats and oils usually occur in the fruits and seeds of plants and are extracted by pressing or solvent extraction. The oils may be hydrogenated to provide a fat. The extent of hydrogenation will influence the properties of the fat; more extensive hydrogenation increases the melting point. Unsaturation lowers the melting point of the fat. Hence saturated acyl groups predominate in fats and unsaturated acyl groups predominate in oils. Another factor that affects the melting point is the molecular weight. The acids obtained from low melting fats such as coconut oil, palm oil and butter contain relatively small amounts of unsaturated acids but considerable amounts of lower fat acids. Although classified as fats because they are solid in temperate zones, coconut oil and palm oil are called oils because they are liquids in the tropics where they are produced. Oils autooxidize sometimes via peroxides that degrade the molecule to a complex mixture of volatile aldehydes, ketones and acids. Rancidity is microbially mediated. Vegetable oils are more resistant to autooxidation than animal oils. Oils can be very unstable in an oxidizing environment. Waste or rags containing unsaturated oils are subject to spontaneous combustion if air is not excluded, or if there is not enough ventilation to prevent a rise in temperature as the oil oxidizes. Any rise in temperature increases the rate of oxidation, and the process is accelerated until the material bursts into flame. The classification of vegetable oils into nondrying, semidrying and drying oils depends on the ease of autooxidation and polymerization, which increases with increasing unsaturation. The chief unsaturated fat acids from the nondrying oils contain only one double bond; those from semidrying oils contain a higher percentage of the doubly unsaturated acid, linoleic acid; the acids from drying oils contain very little oleic acid but chiefly linoleic and the triply unsaturated linolenic, eleostearic and licanic acids. However there is no distinct line of demarcation between the various types of oils. The characteristic chemical features of the fats are the ester linkages and the unsaturation. As esters they may be hydrolyzed in the presence of acids, enzymes or alkali to free fat acids, or their salts, and glycerol. The saponification value defined as the number of milligrams of potassium hydroxide required to saponify one gram of fat. The extent of unsaturation is likewise characteristic of a fat and may be determined by the amount of halogen that the fat can add. Drying oils include safflower, hemp, linseed oil or tung oil. Oiticica oil is also a drying, high iodine value oil. Castor oil has a high percentage of ricinoleic acid, which contains a hydroxyl group. The acetyl value measures degree of hydroxylation. Castor oil has an acetyl value of 142-150, but other common fats and oils range from 2 to 20. Castor oil has a low degree of unsaturation and it is a nondrying oil. Simple and compound lipids contain fatty acids and alcohols and, for the most part, these are bonded with ester linkages. The fats and oils are the most commercially important lipids. Glycerol is a fairly small molecule and contributes little to the average weight of fat and oil molecules. Generally, the fatty acids are even numbered, straight-chain molecules containing 12 or more carbon atoms and one carboxyl group. It might be expected then that the physical and chemical behavior of the lipids is determined primarily by the fatty acids. Because of this and the fact that they are metabolically important compounds in the aqueous environment, they warrant some closer attention. The fatty acids exist in nature as products of the microbial metabolism of various compounds or as partial products of the breakdown of decaying animal and vegetable fats. When an ester linkage is made, a hydrogen atom from the alcohol and a hydroxyl group from the carboxyl group of the acid are removed as water. When the reverse reaction occurs, i.e., when the fat is broken down into its constituent parts of hydrolyzed, water is added. Although lipids include a highly heterogeneous mix of organic compounds, there is a common chemical characteristic: simple and compound lipids contain fatty acids and alcohols and, for the most part, these are bonded with ester linkages. Simple Lipids Fats and oils are esters of various fatty acids and the alcohol glycerol. Waxes are esters of various fatty acids and various long-chain monohydroxyl alcohols. These are simple lipids. Glycerol is easily oxidized to the carbonyl level (ketone or aldehyde) and is readily incorporated into metabolic schemes. The fatty acids are also metabolized, but their orderly utilization is somewhat more involved, and the process is complicated by the fact that all but the very short-chain fatty acids are insoluble in water. Water solubility decreases sharply with chain length. Beta-hydroxybutyric is soluble, but a macromolecular polymer consisting of chains of this compound, called poly-beta-hydroxybutyric acid (PHB) is insoluble. Some microorganisms store PHB as a source of reserve carbon as animals store fats. The low molecular weight acids, particularly acetic, propionic, and butyric are collectively known as volatile acids because they can be vaporized at atmospheric pressure. They are also sources of food for aerobic and anaerobic microorganisms. The higher molecular weight fatty acids occur in microorganisms and in higher plants and animals largely as constituents of simple and compound lipids. Compound Lipids The compound lipids are of two general types: phospholipids and glycolipids. The phospholipids are found in all microorganisms, plants and animals, but the glycolipids are found primarily in animals and occur in only a few types of microorganisms. Phospholipids are esters of various fatty acids and glycerol; they also contain phosphorous and nitrogen compounds. The phospholipids commonly found in microorganisms are derivatives of glycerol phosphate. They contain many different fatty acids, most frequently with 16 or 18 carbons, saturated or unsaturated. In bacteria, the substituent attached to phosphate by an ester bond is most frequently ethanolamine. Choline, serine, and inositol are attached to phosphate in other types of phospholipids. Glycolipids are esters of various fatty acids and alcohols; they also contain a carbohydrate. Glycolipids are a class of lipids that contain sugars and fatty acids. One type of glycolipid contains no glycerol but may contain a long chain alcohol. Glycolipids are suggested as enhancing biodegradation by functioning as a biosurfactant which acts to emulsify contaminant and facilitate biodegradation under oxidizing conditions (HURTIG et al., 1991). A species of bacteria produces rhamnolipids. These accelerate hydrocarbon degradation by solubility enhancement under oxidizing conditions. Nonsaponifable lipids are compounds that are soluble in organic solvents but do not contain fatty acids (do not yield soaps when subjected to hydrolysis with strong alkali. Hence, nonsaponifiable). Sterols, fat soluble vitamins, and plant pigments are included in this subclass. Cellulose may be a useful component of the "treatment cocktail". Cellulose is a polysaccharide that makes up the cell membranes of the higher plants. The insolubility of cellulose in water and alcohols despite the presence of five oxygen atoms for six carbon atoms results from the strong hydrogen bonding between the chains, while the long unbranched molecules account for its fibrous nature and high tensile strength. Cellulose is soluble in sulfuric acid and concentrated solutions of zinc chloride, but since a scission of glycosidic links takes place in the presence of acids, the product regenerated by dilution in water has a much lower molecular weight than the original cellulose. There are numerous organisms, both aerobic and anaerobic that are capable of degrading cellulose. The products of cellulose degradation (glucose or cellobiose) are highly degradable by a number of microorganisms. Aerobic decomposition of cellulose is markedly increased by the availability of inorganic nitrogen. The production of large quantities of ethanol and organic acids such as acetic, formic, lactic and butyric acid is typical of the anaerobic cleavage of the cellulose molecule. Anaerobic degradation of cellulose is frequently associated with the creation of very strongly reducing conditions. Hemicelluloses may be a useful component of the "treatment cocktail". Hemicelluloses are heteroglycans containing one to four different sugar units. Xylans, which are polymers of xylose, are among the most prominent polysaccharides of seed plants. Hemicelluloses are subject to decomposition by a wide range of microorganisms and their breakdown products include uronic acids, as well as hexose and pentose monosaccharides. Mixed, short chain organic acids may be ideal energy yielding components of the "treatment cocktail" Volatile fatty acids, (VFA's) such as acetic or butyric acid may be ideal to stimulate biological activity in the treatment zone. Volatile fatty acids are organic acids with short chain hydrocarbon moieites. Common VFA's include methanoic, ethanoic, propanoic and butanoic acids. These are readily degradable by numerous organisms and may be vaporized at atmospheric pressure. The advantage of being volatile, and therefore easily deliverable into the treatment zone makes the utility of these reductants in the BARR process potentially very great. This may be especially important in a situation where the transmissive strata of the contaminated formation is plugged by biomass or precipitated minerals. Acetic acid is directly utilizable by methanogens. Other anaerobic substrates must be broken down via a multiple-step pathway. Incubation The ability to inject steam may be a very important component of the BARR process. Partial sterilization can be brought about by use of heat. Partial sterilization of soil may be compared with fertilization, because there is a proliferation of organisms in the wake of the treatment. Steam injection may also be used to incubate the treatment zone. After the introduction of the "treatment cocktail", the system is warmed to an optimum temperature for anaerobic bacterial growth. Anaerobic degradation can occur over a wide range of temperatures which has generally been subdivided into three separate ranges, psychrophilic (5-25 C), mesophilic (25-38 C) and thermophilic (50-70 C). The rate of methanogenic metabolism is temperature dependent, with optimum gas production at the higher temperature ranges. In wastewater sludge applications at ambient temperatures in temperate climates, the rate of digestion is so low that the residence time of a bacteria needs to be of the order of 3-12 months. Suitable bacteria for seeding such systems, acclimatized to these low temperatures, can be obtained from marshlands. Most of the literature reports indicate that anaerobic incubation is carried out at room temperature or higher. Some work is reported at actual groundwater temperatures. The importance of temperature in chemical reactions is utilized and manipulated by the BARR process. Injection lances deliver steam to increase the temperature in the treatment zone. Electric heating elements may also be used to generate heat to stimulate transformation reactions, but the ready presence of injection lances makes the use of steam more likely. It is likely that the oxidation step will generate heat from exothermic reactions. Therefore, the oxidation step of the BARR process may be self-heating and it is possible that steam may not be required for incubation of the oxidation step. Monitoring The chemical and physical status of the treatment zone will be monitored via in-situ probes accessing the contaminant area, or by water extraction for lab analysis. For purposes of monitoring and progress evaluation, additional core probes may be taken as the treatment progresses. Dissolved oxygen, redox potential (pE), pH, electrical conductivity, the presence of specific ions are some of the parameters which can be monitored in-situ by sensors placed in the probe holes. The pE measurement is of limited value A meaningful redox measurement can be derived if and only if chemical and electrical equilibrium exists within the solution and all redox reactions between the various redox couples occur reversibly upon the surface of the redox electrode. Unfortunately, these conditions are seldom, if ever met within natural aqueous systems. This is due in part to the irreversibility of biologically mediated reactions. For reasons of physical environmental variability in the subsurface environment, kinetic limitations prevent the establishment of equilibrium in the BARR process. Given the spatial variability of the treatment zone with respect to chemical parameters that may be measured, multiple measuring points will be very useful. REOXIDATION AND REDUNDANCY The repetition of oxidation and reduction cycles will increase the opportunity for degradation to occur. After the reduction step has been accomplished and suitable time for incubation has been allowed, treatment with an electron acceptor such as peroxide, oxygen or ozone, nitrate, etc., will re-oxidize the reduced system and create a proliferation of aerobes who will have ample reduced carbon available from the decomposing anaerobes. This oxidation step will take place in a mixture of elaborated enzymes, bacterial slimes, sugars, proteins, etc. in an amorphous, dynamic colloidal suspension. The buildup of biomass in the form of a large colony of anaerobic bacteria will constitute a substantial number of enzymes. When the electron donor is exhausted and the population of the primary digesters has peaked, the ensuing decline in population will be accompanied by the lysing of bacterial cell walls and the release of elaborated proteins, sugars and other biochemically significant compounds. These elaborated constituents will serve as substrate for another bacterial bloom which will grow and then decline and another following and so on and so on until the utilizable substrate is depleted or some other limiting endpoint is reached. At this point, more "treatment cocktail" is introduced and another pE shift in the system is created. The second round of reduction of the treatment zone may be accomplished by injection of vfa's which are gasses and will be easier to deliver into a plugged formation than a liquid "treatment cocktail". The creation of reducing conditions will cause dissolution of the iron and manganese precipitate, as the pE drops again in response to the addition of reductant. Since the reduction step will deplete the available nitrogen, a subsequent oxidation step may be induced by the introduction of oxygen gas and ammonia. The process may be carried on and on until complete degradation of the target pollutant is achieved. CHANGES IN THE SUBSURFACE ENVIRONMENT AS A RESULT OF THE BARR PROCESS. There will be a number of changes in the environment of the treatment zone resulting from the BARR process. Fundamental forces such as temperature, pH, pE and mass flow are manipulated by the BARR process. Changes in system features including surface area, chemical composition, enzyme activity, ionic strength, oxidation state of reactants, ion exchange characteristics, and velocity of flow are subject to manipulation by BARR. The addition of electron donors (biological reducing agents) to the treatment zone will lower the pE of the system. This represents a fundamental shift in thermodynamic gradients. Initially, the treatment cocktail creates reducing conditions, which alters the thermodynamic mineral stability of the system. The reducing zone will spread, but will be kinetically limited. Mineral dissolution ensues as inorganic and organic compounds both undergo reduction. There is an increase in the biomass as the treatment cocktail is utilized as an energy source. Different microbial consortia will develop; there will be considerable variability both spatially and temporally as the BARR process proceeds. Ultimately, the system will stabilize at a very low pE (approximately the point at which water is reduced). This is a theoretical limit in the pE of aqueous systems. The pE may be maintained at some higher level, if it is desired, by the application of a buffer or oxidizing agent. After the pE has stabilized, an oxidizing "treatment cocktail" is introduced into the system and the system is reoxidized. The oxidation step will be accompanied by the release of heat from the exothermic oxidation reactions. Depending on the rate of oxidation reactions, the heat generated may not increase the temperature of the system by very much, because the ground has a large thermal mass, and so will resist increase in the sensible heat. The amount of heat released can be controlled by the volume and type of oxidant introduced into the reduced system, as well as the stage in the process at which oxidant is introduced. Coupled flow may be electrically, or thermally induced to provide bulk flow and thus tend to diminish the kinetic limitations to the process. It has been reported that transformation in dechlorinating cultures appears to be associated with the transition from oxidizing to reducing conditions. (KAESTNER 1991.) (graphic inserted here, showing the carbon and nitrogen flux in the BARR process ) (ascii format does not support this graphic- see original report for graph) The microbial and inorganic and contaminant will all be subject to reduction over an area determined by the reductant and bulk flow of reduced species. At some distance away from the reductant, the pE will return to equilibrium. The reoxidation process will cause precipitation of biocatalytic colloidal mixture with a very high surface area. The minerals which were soluble in the reduced environment will tend to reprecipitate as the pE rises. Because the BARR method is biologically coupled, the mineral dissolution and reprecipitation takes place in an enzyme-rich, cell wall rich, fertilized, contaminated area. When minerals reprecipitate, they frequently form amorphous solids, rather than crystalline forms which form slowly, under more gradual conditions. These high-surface area colloids will have surface catalytic capacity, as well as cometabolic and metabolic catalytic capacity imparted from the enzymes, enzyme fragments and organisms associated with the precipitate. The formation of a separate, lipophilic phase will cosolve with hydrophobic contaminants and the treatment cocktail, when lipids are included in the "treatment cocktail". The formation of micelles will create a nonpolar pseudophase in the system, which will sorb hydrophobic contaminants into biodegradable micelles, thereby bringing the target contaminant into intimate contact with active organism that are consuming the micelles as substrate. When sugar, or other fermentable substrate is used, the fermentation step will generate alcohols which will diminish the polarity of the aqueous phase, imparting a greater solvent capacity for hydrophobic contaminants. The reduction process is repeated, and another round of mineral dissolution occurs, as well as a shift in the microbial character of the system. There will be turnover of the varied microbial consortia as the pE drops. The reoxidation step carried out again and then the reduction step again and so on. The BARR process is repeated as often as is necessary to completely mineralize the target pollutant. (graphic inserted here showing shifts in microbial consortia as redox fluxes) (ascii format does not support this graphic- see original report for graph) Temperature The BARR process creates temperature changes in the contaminated system. Temperature is a fundamental parameter influencing reaction rates. The effect of the temperature on the rate of the reaction is most often described by the Arrhenius equation. k=Ae-Ea/RT where Ea is the activation energy for the reaction, R is the gas constant T is the absolute temperature and A is a constant. Temperatures in natural water systems range from 0C in temperate zones in winter to as high as 45C in shallow ponds and stagnant waters in summer time. For reactions with an activation energy of as little as 10kcal/mol, this temperature range corresponds to a 14-fold difference of the half life of the pollutant. For a reaction with an activation energy of around 30 kcal/mol, this corresponds to a factor of about 2500-fold difference in half life. (WOLFE et al, 1990). Enzymatically catalyzed reactions proceed at a higher rate at elevated temperatures. The rate of biological reactions approximately doubles for every increase in temperature of 10C, within limits of enzyme and membrane stability. Anaerobic metabolic reactions yield less energy than oxidative metabolism. The introduction of an oxidant into a strongly reduced formation will give rise to exothermic reactions that will increase the temperature of the treatment zone and the surrounding formation. The subsurface has a high heat capacity (i.e. it can absorb a substantial input of heat without increasing in temperature). This will be especially true if the treatment zone is below the water table. Coupled Flow Coupled flow will result from the BARR process There may be mass flow (of water) induced by the heat of the BARR process. Because of differences in density and kinetic energy (energy of motion), coupled flow will cause water to flow out of the heated region. It is difficult to estimate the magnitude and importance of such mass flow at this time, but it may be significant and useful. In addition to the coupled flow resulting from heat, the changes in solution chemistry will create coupled flow as water moves in response to concentration gradients. BARR creates increases in concentration of dissolved salts, and water from the surrounding aquifer will flow toward the concentrated solution and serve to dilute the treatment zone. Coupled flow induced by electro-osmosis is easily accomplished in the BARR process. Coupled flow can serve to move inoculum within the contaminated system. Movement of even a single viable cell into an area suited for growth will greatly influence the overall process of biologically mediated reactions and degradation processes. pE Induced biological activity in the treatment zone will change the pE which will be accompanied by radical changes in the system chemistry. The initial introduction of "treatment cocktail" will cause a drop in the pE as the biologically active system responds to the presence of readily degradable substrate. The demand for oxygen as an electron acceptor will exceed the supply, since diffusion rates cannot keep up with the consumption of molecular oxygen. The resulting increase in electron activity will create shifts in the thermodynamic equilibrium as the demand for electron acceptors creates fundamental changes in the system. Mineral stability is a function of equilibrium, including pE and pH. As the pE drops, solubility equilibria will shift and soil minerals will dissolve in response to the lowering of pE. The dissolution of minerals will, in turn, create other changes in the system, such as increases in ionic strength, particle flocculation and dispersion of sorbed bacteria. Oxides of metals are present in soils, usually in crystalline forms and are insoluble in water. Organic compounds in the "treatment cocktail" will have a significant effect on the solubility and mobility of the metal oxides due to chemical and microbiological action. Under anaerobic conditions, organic compunds can bring about reductivce dissolution of metal oxides from a higher to a lower oxidation state. The reduction has a dramatic impact on the solubility and speciation of metals. For example, oxides of Mn(III, IV) Fe (III), Co(III) and Ni(III), when reduced to divalent ions under anoxic conditions, show an increase in solubility by several orders of magnitude. Microorganisms play an important role in dissolution of metal oxides by direct and indirect action. Direct action involves enzymatic reductive dissolution of the metal oxide, wherein the oxide is used as the terminal electron acceptor, whereas indirect action involves dissolution due to production of metabolites, such as organic acids and chelating agents and lowering the pH of the medium. The changes in redox potential will tend to be buffered by the local system composition. Change in oxidation state of the aquifer solid materials will change the solubility of the minerals. The use of the term aerobic or anaerobic is important only in a relative sense. The utility of different electron acceptors depends on the pE of the system. If the system is initially oxidizing (Eh approximately 500mV), as the pE drops, any nitrate present in the system will be denitrified to N2. Following this, as the pE continues to drop, Mn(IV) will be reduced to Mn(II) with a concomitant increase in solubility of manganese compounds. As the pE continues to drop, there will be an increase in iron solubility as Fe(III) is reduced to Fe(II). (three- graphic sequence inserted here, showing the flux of Nitrogen, Manganese and Iron in the BARR process) (ascii format does not support this graphic- see original report for graph) This process constitutes a buffering effect. Redox buffering, the tendency of a solution to maintain redox stability, is conceptually similar to pH buffering. The pE will tend to poise as an available electron acceptor is reduced. Redox buffering occurs as electron acceptors (oxidizing agents) are consumed and their concentration diminishes. When the concentration of the most easily reduced compound decreases, the electron concentration increases (pE drops) until the threshold of reduction is reached for the next most easily reduced compound. Redox, then, becomes a descriptive measure of the system. Change in the redox potential is the result of actual chemical reactions that have kinetic considerations. A measured redox at one point does not necessarily reveal the redox potential of a larger area. It is commonly accepted that a measured redox potential is not an accurate description of the system. Kinetic limitations and spatial variability preclude the use of a measured redox couple as a meaningful descriptor of the overall status of the system. Because it is so convenient, it is likely that pE measurements will be taken for tracking and development, but in actual BARR practice, significant chemical species (e.g., CH4, CO2, NO3-, SO4-2, H2S, etc.) that are detectable at multiple sampling points will provide the best measure of the overall system status. There is a fairly predictable sequence of reactions that will occur as inorganic compounds are consumed as electron acceptors (reduction step), or electron donors (oxidation step). The treatment cocktail provides the reducing and oxidizing agent for these steps. The sequence of electron acceptors consumed under increasingly reducing conditions is most commonly suggested as O2, NO3-, Fe+3, Mn+4, SO4-2, CO2, H2O. Hence, the pE is governed by the available concentration of reducible/oxidizable compound. The chemical may be naturally present as part of the system or may be introduced in the "treatment cocktail". The redox potential can be maintained at some desired level by the presence of inorganic electron acceptors. The mineralogy of the aquifer or subsurface matrix will have a definite effect on the course of the biological and chemical reactions of the BARR process. The aquifer (or vadose zone) solids will become reduced as the pE drops. The abundance of electron acceptor (oxidizing agent) will determine the amount of reductant (carbon source) which is required for a given level of redox achieved. Such concerns may become relevant as specific organisms or consortia are identified which have the ability to metabolize the contaminant (or a metabolite) and require a certain redox potential to thrive. For example, if methanogenic conditions (very strongly reducing) are required to host an organism with the direct or cometabolic ability to degrade a chemical of concern, an aquifer with a lot of sulfate minerals would be difficult to reduce to a low enough potential for methanogenic conditions to manifest. Such a site would require more reductant than a site where sulfate was in less abundant supply. Similarly, an efficient degrading consortia that requires sulfidogenic, but not methanogenic conditions, may be fostered by a treatment cocktail which contains a lower amount of reductant in a sulfate pE buffer. The solution chemistry will be a function of the geochemistry of the solid matrix and the regional geohydrology. This means that some of the most important governing parameters are relatively quantifiable and may be determined as part of the initial investigation step. The mineralogy of the aquifer, and thus the ionic composition of the groundwater, will greatly affect the behavior of the system under the influence of an introduced reducing or oxidizing agent. There is much information available which may be applied to the BARR process which has been gained from experience with landfill leachate. Leachate from landfills containing organic matter has a content of dissolved organic carbon (DOC) in the range of thousands of mg/l C. The DOC, including fatty acids, and humic compounds, will act as a substrate for microbial processes in the aquifer, potentially inducing major changes in the governing redox environment. Close to the landfill, the aquifer will be methanogenic, similar to the methanogenic conditions that exist in most landfills. Depending on the interaction between migrating leachate rich in organic carbon, the kinetics of the actual redox processes and the availability of the electron acceptors, a sequence of redox zones will develop downgradient from the landfill, ranging from methanogenic, sulfidogenic (sulfate reduction), ferrogenic (Fe(III) reduction), manganogenic (Mn(IV) reduction), nitrate-reducing to aerobic conditions in the most diluted part of the plume farthest away from the landfill. This sequence assumes that the oxidized species are present in the aquifer in significant quantities: free oxygen, nitrate and sulfate in the groundwater and oxidized Fe and Mn compounds in solids associated with the aquifer sediment. If some of these electron acceptors are missing, the corresponding redox zones, of course, are also missing. This redox zone sequence is believed to be key in controlling the fate of reactive pollutants leached from the landfill. Aerobic conditions are identified by free oxygen (O2) concentrations in excess of 1.0 mg/l and very low concentrations all reduced species. The value of 1.0 mg/l is defined in order to minimize the presence of nitrate-reducing microenvironments in the aerobic aquifer. The possibility exists for wide fluctuations in redox potential within microenvironments. In the nitrate reducing zone, the concentration levels must be low for oxygen as well as for the more reduced species, but no criteria are associated with the nitrogen compounds. Nitrate concentrations could be high in the nitrate reducing zone, if the organic carbon is the limiting factor, but low if nitrate is the limiting factor. The manganogenic and ferrogenic zones are defined by the presence of substantial concentrations of dissolved Mn and/or Fe. In their oxidized state, Mn and Fe are practically insoluble at neutral pH and dissolved concentrations are considered to represent reduced species. The sulfidogenic zone contains some sulfide. However, the presence of reduced Fe and Mn may cause precipitation of metal sulfides and may lead to very low concentrations of sulfide (sulfides tend to be insoluble). At the same time sulfate may still be high if the reduction process is limited by organic carbon. The methanogenic zones and the sulfidogenic zones may be very closely related, due to their similar redox potentials and a strict distinction may be difficult. However, sulfate concentrations should not be too high and methane should be present at significant levels. The applied criteria are consistent with thermodynamic principals, but concentration-based criteria are established since a meaningful calculation of the redox potential from the Nernst equation is not warranted in such a complex system. The actual criterion concentrations may be operationally defined by determining the water chemistry of the compounds, and experiences on sampling and water sample analysis. Electrochemical determination of redox potentials is mechanically very simple, but according to experience often yields results difficult to interpret, or in some cases even misleading. Identifying redox zones is assumed to be a key to understand biological and mineral behavior associated with BARR. These realizations, and the fact that determination of an exact redox potential is not needed but rather an identification of the dominating redox processes as expressed by the electron acceptor being reduced by the reductant, lead to the choice of assigning redox status based on analysis of groundwater samples for redox-sensitive compounds. It may be possible to use ion-specific electrodes in-situ in some situations to monitor the BARR process. Approximate criteria for redox parameters used for assigning redox status to groundwater samples (all values in mg/l). Parameters O2 NO3- NO2- NH4+ Mn(II) Fe(II) SO4+2 S(II) CH4 Aerobic >1.0 n.a. <0.1 <1.0 <0.2 <1.5 n.a. <0.1 <0.1 Nitrate Redn. 1.0 n.a. n.a. n.a. <0.2 <1.5 n.a. <0.1 <0.1 Manganogenic <1.0 <0.2 <0.1 n.a. >0.2 <1.5 n.a. <0.1 <1.0 Ferrogenic <1.0 <0.2 <0.1 n.a. n.a. >1.5 n.a. <0.1 <1.0 Sulfidogenic <1.0 <0.2 <0.1 n.a. n.a n.a. n.a. >0.2 <1.0 Methanogenic <1.0 <0.2 <0.1 n.a. n.a. n.a. <40 n.a. >1.0 Organic compounds also function as electron acceptors, but the potential variety makes assumptions and projections difficult without actual experience. There can be widely varying redox subenvironments distributed in the system. Due to kinetic limitations and heterogeneity of "treatment cocktail" distribution, substrate, inoculum, soil mineral composition, temperature, contaminant distribution and induced mass flow, there will be spatial variation in the pE. The presence of water in the system has a buffering effect, or damping effect by virtue of its "reducibility" and "oxidizability"(i.e., water itself can be oxidized and reduced). It is questionable if biological activity or even uncoupled enzyme activity would continue beyond the pE stability ranges of water. It is an interesting exercise to consider the behavior of a system in the vadose zone, above the water table, where a strongly reducing "treatment cocktail" is introduced into a vigorous reducing inoculum. Would the pE drop below the stability limits of water? Such an event would likely cause coupled flow of water in the vapor phase or any capillary network of the soil. The electron status of the system, described as the redox potential will flux as the supplies and nature of reductant and oxidant are varied. The initial reducing treatment cocktail is followed by different treatments, at different times, and potentially in different zones, to manipulate the pE, pH, ionic strength, inoculum composition, temperature, etc. There is potential for application of different "treatment cocktail" to become somewhat of an art as well as a science. pH The values of the pE and pH parameters are strongly dependent on each other. Degradation of the "treatment cocktail" will likely result in the formation of organic acids as well, which will tend to lower the pH. The pH of the solution will change as the chemical reactions (both biological and abiological) consume or produce hydrogen ions. Hydrogen ion activity affects transformation kinetics in two distinct ways. The most common is in acid-base mediated hydrolysis reactions. The rate of the reaction is directly proportional to the proton and hydroxide concentration. In the case of base-catalyzed hydrolysis, the rate equation is (-dP/dT) = k[P][OH-] where k = second order rate constant (mol-1s-1), [P] = pollutant concentration and [OH-] = hydroxide ion concentration. For compounds that have acidic or basic functional groups, the pH of the water also governs the relative ratios of the associated and disassociated species. Because associated and disassociated species react differently, small changes in pH can effect large changes in half-lives of contaminants that are susceptible to hydrolytic transformations. Additionally, the changes in pH will alter the solubility of soil minerals, causing dissolution and reprecipitation as the pH varies spatially and temporally in the system. Denitrification, organic nitrogen breakdown and sulfate reduction are examples of biological reactions that can cause in increase in pH. A decrease in pH may be caused by sulfate oxidation, nitrification, and organic carbon oxidation. However, the actual changes in pH are influenced and determined by the buffer capacity of the system and the amount of substrate utilized by the microorganisms. The presence of surface functional groups which are capable of exchanging a proton creates pH dependent charge, whereby the ionic character of the surface increases with pH. The molecular configuration of polyelectrolytes may be influenced by pH as the molecules coil and uncoil as the pH decreases or increases. In such a situation, charged sites such as acidic hydroxyl groups or amines can lose or acquire charge as a result of changes in solution pH. In a large, flexible, polyfunctional molecule, intramolecular self-association is thought to occur in the absence of electrostatic repulsion. The tendency to form such intramolecular bonds will vary as charged sites are created or satisfied by pH changes. In such a situation, decreases in pH will satisfy the charge on the surface of the molecule, thereby lowering the hydrophilicity of the surface and also decreasing the coulombic repulsion of the molecular chain for itself and permitting intramolecular bonding. The evolution of CO2, the most common end product of the reduction of O2, has considerable influence on the system pH. When a system that was previously under reduced conditions becomes oxidized, its pH may drop drastically due to the oxidation of iron to Fe(III) and the subsequent hydrolysis of the iron or the oxidation of sulfite to sulfate, which is accompanied by the release of protons. Lowering the pE of the system often will result in a rise of pH because many reduction reactions (such as the reduction of sulfate to sulfite) involve the uptake of protons or the release of hydroxyls. When the reaction of a couple that controls the redox potential of a given system involves protons or hydroxyls, a change in pH of the solution will directly cause a change in its pE. The pH may affect the rate and direction of a redox couple in the soil solution. Acidification of the soil, for example, is likely to strongly increase the solubility of trivalent iron and of other oxidized transition metal species, but will have a smaller effect on the solubilities of the reduced species of these metals. Elevated concentrations of calcium, magnesium, and bicarbonate have been identified in a landfill leachate contaminant plume, indicating that carbonate mineral dissolution is an important pH buffering mechanism in a carbonate mineral aquifer. The following reactions illustrate the dissolution of calcite and dolomite by reaction with acid. CaCO3 + H+ = Ca+2 + HCO-3 CaMg(CO3)2 + 2H+ = Ca+2 + Mg+2 + 2HCO-3 Soil colloids are negatively charged and accumulate hydrogen ions. Because of isomorphic substitution, clay particles tend to have a negative charge. This charge may be neutralized by protons. At low pH, the effect of the electrostatic charge on clay particles will be minimized. Bacteria are amphoteric and will acquire positive or negative charge depending on the pH of the system. Bacteria have a variable surface charge. They can be neutral, positive or negative, depending on the nature of the cell and the pH of the solution. At solution pH values equal to the pHZPC (zero point of charge), the surface has no net electrostatic charge. At solution pH-values lower than the pHZPC, the surface is positively charged and becomes increasingly positive at lower pH-values. At solution pH-values higher than the pHZPC, the surface is negatively charged and likewise becomes more negative at higher pH-values. The precipitate coating on the mineral grains affects not only the electrostatic charge expressed at the interface, but the physical surface as well. Bacterial adhesion to the mineral matrix will be affected by the chemical surface of mineral grains. The surface of mineral grains is commonly coated with ferric hydroxides and oxyhydroxides. Such a surface will change as dropping redox reduces ferric iron compounds to form the more soluble ferrous form. When mineral grain surfaces undergo such dissolution, any adsorbed bacteria will be released into the solution and allowed to migrate. The mobility of microbes is very important to the success of the BARR technique. If the bacteria and reducing agent are not transported to the extent of the contamination, the desired direct and cometabolic degradation cannot occur. Extensive advective transport to depth in the subsurface may be limited to macropores or fractures. Similarly, in zones of low permeability, non-advective processes such as growth or motility may play the most important role in widespread dispersal of degrading bacteria. The dissolution of minerals accompanying changes in pE and pH will also create changes in the makeup of the system solution by increasing the ionic strength of the solution. The ionic strength will increase as minerals dissolve, which will cause a further increase in the solubility of slightly soluble mineral components by changes in the activity coefficients of the processes. The common ion effect will cause precipitation of some salts as the solubility is exceeded. The increase in the ionic strength of the solution will also cause a collapse in the electrical double layer (multilayer) on charged surfaces. This collapse of the double layer will allow flocculation of charged species via van der Waals forces. There is greater tendency for dissolved and suspended particles to sorb and flocculate at higher ionic strength, because the high ion concentration compresses the electrostatic field on the charged surfaces, and allows for closer approach of charged solution components. This closer approach allows van der Waals attractive force to overcome electrostatic repulsion and flocculation results. Bacterial adhesion to charged surfaces increases with increasing ionic strength. Theoretical explanation is the close approach resulting from the collapse of the electric multilayer, allowing close enough approach for van der Waals forces to take over. This solid matrix may be stationary aquifer matrix or mobile colloids. The attachment and detachment of bacteria for the solid matrix will occur in repeated cycles as a result of BARR. The dissolution and reprecipitation caused by the fluctuations in the pE and pH will cause the ionic strength and chemical makeup of the solution to flux. In addition to growth as a mechanism of bacterial spread throughout the contaminated formation, bacterial attachment to mobile colloids in solution will also spread bacteria through the formation with mass flow. The bacterial cells will attach to solid materials and then detach as the surface charge of the solid changes, or the solid dissolves in response to changes in ambient pE/pH. Thus, even strongly sorbed bacteria will be spread through the formation as a result of the BARR process. Filtration theory contains both physical and chemical components to describe retention of colloids in porous media. The physical controls of filtration are based upon particle size characteristics of the porous media and of the advected colloid. Chemical filtration theory is described by the Derjaguin-Landau and Verwey-Overbeek (DLVO) theory, which states that initial contact of colloidal particles with surfaces is determined by the additive effect of the attractive and repulsive forces at the interface (van der Waals and electrostatic forces). The balance of these forces may result in adhesion of particles at some distance (a few nm) from the surface. In addition to the forces mentioned, hydrophobic and steric forces can also contribute to binding of cells to solid surfaces. In some cases, the precipitation and dissolution may entrain bacterial cells in mineral ion precipitate. These colloids may be held in solution and moved with bulk flow. Introduction of water of low ionic strength will dislodge those particles which are attached by high-ionic strength conditions. This may be used as a step in the BARR process to stimulate the spread of degrading bacteria. When oxidation occurs, there will occur mineral reprecipitation. Low pE will cause dissolution of soil mineral grains containing Fe(III) and Mn (IV). At some point in the system, either spatially or temporally, the pE will increase to the point of reprecipitation of these minerals as insoluble compounds. They will certainly reform as the pE raises at the oxic interface, or upon the implementation of an oxidative cycle in the BARR process. When it does occur, precipitation can sometimes result in different allotropic modifications, ranging from amorphous to crystalline and can have variations within each form . Coprecipitation can occur when materials in solution get trapped or caught-up in a precipitation event. This can cause scavenging of solution constituents when a precipitate forms. Scavenging can occur on different scales. On the molecular level, dissolved species can become entrapped or bonded in the crystalline lattice, if it forms. This may result in phenomena such as isomorphic substitution in clay minerals or simply the existence of "impurities" in the resulting solid. On the macroscopic level, scavenging can occur when dissolved or suspended solution components are taken out of solution by becoming entrapped in a precipitate. This sort of coprecipitation or scavenging event is intentionally created in the use of coagulants in water treatment operations where a slightly soluble salt is rapidly added to water in sufficient amounts to create a saturated solution. When solubility is exceeded, the precipitation event scavenges materials in solution which are responsible for undesirable turbidity. These are trapped in the amorphous matrix and settled out by gravity, thus removing them from solution. Colloids Since these amorphous precipitates will be forming and dissolving in a rich and varied, biochemically active solution, the precipitate suspension will contain extracellular enzymes and enzyme fragments and whole bacteria. The creation of such lipophilic colloidal particles wil tend to sorb free enzymes and thus increase the cometabolic reactivity of the colloids. Proteins (such as enzymes) adsorb more to hydrophobic surfaces than to hydrophilic surfaces. Adsorption to hydrophobic surfaces initially inhibits protein degradation, which results in low bacterial growth rates. During long incubations, surface bacterial growth rates increase with surface hydrophobicity because of increasing amounts of adsorbed protein, which serves as substrate. Such a colloidal phase will be catalytic, both from the properties of the enzymes as well as from the large mineral surface area. The large surface area associated with these colloids introduces an increased capacity for surface catalysis. Colloid particles interact strongly with the fluid, but the individual particles retain their structural integrity, so they cannot be said to dissolve. The colloidal mixture behaves so distinctively because of the large surface area of interaction between the two phases. Amorphous precipitates with a very large surface area will enhance the apparent solubility of hydrophobic organics. Hydrophobic bonding will tend to drive insoluble components onto the colloidal phase as well as onto the solid matrix. The colloids formed from the precipitation of minerals are an important part of the BARR process. The dissolution and precipitation of soil mineral components resulting from BARR will tend to create a colloidal phase in the system. See Technical Appendix (a separate document) for a complete discussion of colloids. The creation of microbial biomass constitutes a biocolloidal phase that represents a separate, semipolar phase in the system. The lipid content of bacteria varies from 10-15% of the dry weight, it is slightly higher in fungi. Cell walls contain both hydrophobic and hydrophilic surface groups. This presents the opportunity for partitioning of hydrophobic organic contaminants into this nonpolar phase, which is also active catalytically from the enzymes in the organisms. In addition to the lipid content of the organisms, many bacteria produce extracellular semipolar slimes that will tend to sorb slightly soluble organic materials. These slimes will act as surfactants in the system when dissociated from the bacteria which formed them. In addition, these slimes are biodegradable and will serve as substrate for other organisms. In actual practice, these two colloidal phases (mineral colloids and biocolloids) will be intermingled to form a variable composition biomineral colloidal phase with a highly active catalytic surface and a substantial nonpolar phase which will tend to solve and sorb hydrophobic contaminants in a reactive milieu. This will accelerate the degradation of the target contaminants and is really at the heart of the BARR process. The changes in solution chemistry accompanying the BARR process will cause repeated flux in the metal ions in solution. The metals most often proposed to catalyze the hydrolysis of pesticides in natural waters are Cu+2, Fe+3, Mn+2, Mg+2 and Ca+2. These ions will be active in redox couples in the BARR process; repeatedly dissolving and reprecipitating, forming various compounds with components of the contaminated system. Solution Chemistry The "treatment cocktail" will serve to increase the solubility of the pollutant by solvent/surfactant properties. Lack of water solubility is frequently cited as a reason for low degradation rates of degradable pollutants. The increase in the concentration of polyatomic ions in solution resulting from the BARR process will tend to increase the solubility of hydrophobic organics. Van der Waals forces will create associations between hydrophobic species and large ions. Such an association will have the effect of increasing the solubility of hydrophobic system components. The presence of biodegradable surfactants will increase the water solubility of insoluble pollutants and thereby serve to increase the degradation of the target. Surfactants modify the interfacial behavior in liquid systems. The use of surfactants has been observed to increase the biodegradation in toxic organic contaminants. This is related to the increased solubility of the target. The mobility and degradation rate of hydrophobic organics will be enhanced by the BARR process through the creation of colloids and micelles. The use of nonpolar components of the "treatment cocktail" will create a nonpolar micellar phase to enhance the solubility of the target, as well as dilute it with biodegradable substrate and nutrient materials. Hydrophobic bonding will be diminished by the treatment cocktail The "treatment cocktail" will decrease the polarity of the solution in a contaminated aquifer, which will diminish hydrophobic forces which keep insoluble organic pollutants out of solution. The presence of a miscible or partially miscible organic solvent will give rise to a phenomenon known as cosolvency. Partially miscible organic solutes modify the solvent properties of the solution to decrease the interfacial tension and give rise to an enhanced solubility of organic chemicals in a phenomenon often called "cosolvency". The interfacial tension of a solution decreases logarithmically as the concentration of cosolvent increases linearly. Cosolvent effect is especially apparent when both cosolvents are very hydrophobic. Cosolvent effects differ depending upon the nature of the cosolvent. Nonpolar cosolvents (such a lipids) have a very large effect on the sorption of hydrophobic material to the aquifer matrix. While polar cosolvents only have cosolvent effect (i.e. log-linear solubility increase) when the concentration is high (>10%). Increase in hydrophobic organic chemical solubility in the presence of cosolvents is reflected by decreased sorption by the solid matrix and increased mobility of hydrophobic organic chemicals. The cosolvency of partially miscible organic solvents is expected to be most pronounced in systems where a variety of cosolvents is present in high concentrations. For miscible cosolvents, such as alcohols, the log/linear solubility relationship only applies at cosolvent concentrations above about 10%. At cosolvent concentrations below 10%, the solubility enhancement is linear. See Technical Appendix for a complete discussion of cosolvency The solvent effect of the treatment cocktail will serve to dilute the concentration of the contaminant, a factor which may enhance the biodegradability of the pollutant. The effective concentration of toxic pollutants is diminished by dilution. A cosolvent "treatment cocktail" will dilute and disperse the contaminant in a biologically active milieu, thereby enhancing the degradation rates. Solubilization can increase the amount of sparingly soluble hydrocarbons or other organic material taken up by aqueous phases by orders of magnitude. Evidence exists that surfactants can influence the uptake and consumption of insoluble substrates. Many microorganisms produce extracellular surface active compounds. The generally accepted purpose of these compounds is to enhance biodegradation. The three mechanisms traditionally proposed to explain how bacteria take up sparingly soluble substrates are as follows: 1) interaction of cells with hydrocarbon dissolved in the aqueous phase; 2) direct contact of cells with hydrocarbon drops considerably larger than the cells 3) interaction of cells with "solubilized", "pseudosolubilized" or "accommodated" hydrocarbon in entities much smaller than the cells. Note that pseudosolubilization or accommodation need not be put into a classical surfactant micelle, but instead into the lipophilic regions of proteins or other polymeric molecules produced and excreted by cells. Adding surfactants not only facilitates emulsification, with a resulting increase in interfacial area, but also provides micelles for solubilization. When hydrocarbon is solubilized in small micelles of surfactants, its rate of biodegradation can substantially increase. Microbes As the redox status of the system varies, different microbial consortia will be favored. There will be a variety of redox environments created by the BARR process, ranging from strongly oxidizing to strongly reducing. These redox environments will variably favor oxidizing, reducing and fermentative organisms, thereby increasing the pool of enzyme catalysts and the potential for catalytic degradation of the contaminant. The surface texture changes in the soil/mineral matrix accompanying the changes in pE and pH will affect microbial microhabitat. In addition to the dissolution and dispersal mechanisms mentioned above, the surface texture of a mineral grain determines the amount of surface area to which cells can attach and can also affect colonization of grain surfaces. If the surface irregularities are on a scale similar to bacterial size, a larger area of the cell wall may be able to interact with the surface during initial adhesion. Larger surface irregularities may provide protection from shear forces associated with water flow, grazing by protozoa, etc. The increase in the microbial biomass and the diverse consortia of the system resulting from the BARR process will increase the amount of enzymes in the system and thereby increase the opportunity for both direct metabolism and cometabolism of the target pollutant. It is important to remember the role of enzymes in this process. There are a lot of organisms with the ability to degrade a wide array of materials. The BARR process stimulates the development of a wide array of enzyme capabilities and cometabolic opportunities. Since the BARR process is carried out in-situ in the contaminated formation, the microbial community is constantly under pressure to accommodate the presence of the contaminant either by resistance or by utilization. The presence of the treatment cocktail creates an strong driving force for the bacterial biomass to increase. The wide diversity of organisms created by the BARR process will provide a great variety of metabolic capability for the treatment system. Microbial communities behave differently from pure strains. Waste products and exudates from a mixed consortia will provide substrate and habitat for the succeeding consortia. Local conditions may be more or less unique, depending upon such factors as inoculum, temperature, mineral composition of the system, etc.. But the types of organisms will mainly be limited to the availability of viable inoculum. Viability becomes an issue when temperature, pH and pE are variables. Many microbes have the ability to form resting structures, or resistant spores that may enable them to withstand adverse chemical conditions. In addition to the bacteria, there are also a number of other organisms involved in the BARR process. Yeasts Yeasts are commonly responsible for anaerobic fermentation. It is very likely that native or introduced inoculum will contain yeast spores. Fungi Fungi are almost all obligate aerobes. Many fungi are involved in degradation of toxic materials. The BARR process provides wide spectrum inoculum which contains fungal spores. Protozoa Protozoa are important to the microbial ecology. Protozoa are mobile and they eat bacteria. They are said to "graze" on bacteria. Protozoan grazing of bacteria is also likely to influence aquifer transmissivity, by diminishing bacterial populations when formation-plugging populations occur (SINCLAIR et. al. 1993). Protozoans have been found in both oxidizing and strongly reducing environments and are able to form resting structures to persist when conditions become unfavorable. Some protozoans are also host to bacteria. They are reported to have methanogen symbionts living inside (FINLAY and FENCHEL.1991). The ciliated protozoa encyst in response to shortages of food or water, and the methanogens remain viable within the cysts. Unlike free living methanogens, these are not particularly sensitive to oxygen, the symbiotic methanogens remain viable following exposure of the consortium to atmospheric oxygen. Dispersal of methanogen-bearing protozoan cysts through oxygenated environments is a potential mechanism of transfer between different anaerobic environments. Anaerobic protozoan consortia are theoretically capable of making a significant contribution to methane generation under anaerobic conditions. Changes in the system will influence the dissemination of microbes in the treatment zone. Microbial dissemination in the system will occur through several mechanisms; chiefly through growth, motility, symbiotic association with mobile protozoans, attachment to mobile colloids, or mass flow as free cells. All of these processes are stimulated by the BARR process. Nitrogen transformations resulting from BARR The nitrogen transformations resulting from the BARR process are an interesting and important part of the process. Nitrogen is a necessary component of biometabolism necessary for the overall process. Nitrate is an undesirable contaminant of groundwater. BARR utilizes denitrification processes to generate N2 from the nitrogen in the system. The BARR process thereby effects a net removal of available nitrogen. Bioavailable nitrogen in the form of ammonia, will be necessary as a component of the treatment process. Ammonia, in the gaseous form, will also act as a strong base and may be used to adjust the pH of the system, as well as provide nitrogen for the desired biological activity. Cellular or inorganic nitrogen will be oxidized to nitrate during the oxidative phase of the BARR process. Added nitrogen, that is not sequestered in biological organisms, will be subsequently lost upon the implementation of reducing conditions in the reduction cycles of the process. The BARR method will be useful for treatment of inorganic as well as organic contamination. While it is not possible to degrade inorganic contaminants such as heavy metals, it is possible to form and unform stable compounds with them (such as chelates) and it is also possible to utilize changes in pE to influence the solubility and mobility of heavy metals in aqueous subsurface environments. The behavior of heavy metals in soil depends on many factors such as molecular status of the metal, pH of the soil solution, organic matter content, soil cation exchange capacity (CEC) and pE. Organic matter is often regarded as a major factor in the sorptive behavior of metals. There is firm evidence that organic materials have relatively high stability constants for metals. Additionally, considerable evidence has been accumulated indicating that the hydrous metal oxides play a major role in the sorption of heavy metals in mineral soils. These oxides, particularly those of Fe, Mn, and Al are common in soils. They may occur as discrete crystalline minerals or as coatings on other soil minerals. Many are of indefinite structure (amorphous) and composition. They have high surface areas in relation to their weight, are highly reactive, and the Fe and Mn oxides are quite labile since they are formed and dissolved in oxidizing and reducing conditions, respectively, in the soil. The latter is of importance since the BARR process manipulates the formation of these compounds and thus the mobility of heavy metals in soils. Where Cd, Pb and Zn are fixed and immobilized in a carbonate phase, significant mobilization is caused by solution of iron hydroxides and their heavy metal coprecipitates under moderately reducing conditions. In systems rich in organic matter, the role of Fe/Mn oxides is much less important because of competition from the more reactive humic acids, organo-clays, and oxides coated with organic matter. Of prime importance, however, is whether or not the heavy metals have been introduced into the soil as inorganic salts or in forms bonded to organic matter. Both inorganic and organic matter constitute ligands present in the soil solution. In this respect, humic substances, may play the most important role in the behavior of heavy metals in the soil. Copper forms strong complexes with humic substances. Metals complexed by humic substances become unavailable to form sulfides, hydroxides, and carbonates and thus prevent the formation of insoluble salts. The stability of humic/metal associations is as follows: Cu+2>> Pb+2>> Ni+2>> Zn+2> Cd+2. The behavior of Cr(III) in soil is strongly correlated with pH because at neutral and basic pH, Cr(III) forms insoluble oxides and hydroxides. The generalized sequence of the capacity of solids to sorb heavy metals is as follows; MnO2>humic acid> Iron oxide> clay minerals.(GUY and CHARKRABARTI 1975): The solubility of metals in waters is principally controlled by: (1) pH, (2) type and concentration of ligands and chelating agents, (3) oxidation state of the mineral components and the redox environment of the system. Redox affects metal speciation in two ways: (1) by direct changes in the oxidation state of the metal ions and (2) by redox changes in available and competing ligands or chelates. It is reported that upon waterlogging of soils containing sufficient organic matter to effect a decrease in pE (i.e. bioavailable electron donor), the availability of many metals is increased. Increased mobilization of Cr, Ni, Cu, Zn, and Co have been reported, while waterlogging of low organic matter content soils has shown little effect on increased mobilization of metals. Since BARR manipulates the redox of the system, changes in oxidation state may be utilized to increase mobility of heavy metals in conjunction with pump and treat schemes to effect enhanced recovery of heavy metals in the subsurface. The growth of microbial biomass will tend to diminish the transmissivity of the aquifer. The buildup of biomass will affect aquifer permeability as bacterial cells plug the pores of the saturated soil. Biofouling is a commonly reported problem with groundwater injection wells. The ability of the bacteria to plug the aquifer and modify transmissivity will be governed by the pore size of the media. This pore size is frequently spatially variable as a function of soil deposition and morphogenesis. Such changes in diffusivity of the solution components will also alter the reaction kinetics. Because of the microbial energy metabolism, the oxidative step will result in a greater accumulation of biomass than will the reductive metabolic phases of BARR, hence the problem of biofouling will tend to be greatest during the oxidative treatment. The problem of biofouling will be minimized by the fact that the BARR process utilizes gas phase "treatment cocktail" under most steps. In the event that biofouling is a problem which restricts introduction of "treatment cocktail" or removal of recovery leachate, it is possible that steam injection or other biocidal treatment will temporarily alleviate the blockage to enable treatment to proceed. Sealant techniques may be very beneficial. The BARR process will greatly advance the ability to place materials into and around contaminant zones. Where BARR is used in conjunction with a pump-and-treat scheme, the intentional placement of a plugging treatment at the perimeter of the contaminated formation may be used to contain the contaminant. Accurate sealing of the transmissive formation may help to protect sensitive water sources as a preliminary step to the BARR process. The BARR process is repeated until the desired degree of degradation is achieved. The redundant nature of the BARR process is key to the success of the technique. Direct and cometabolic transformation is fostered by the wide diversity of microbes and enzymes induced by BARR. Additionally, the wide swings in pE and pH, and the variations in temperature and the opportunity for surface catalysis all contribute the degradative capabilities of this process. Sequential reduction-oxidation-reduction etc. is carried out by manipulating the chemical balance of the treatment zone. The creation of repetitive reduction and oxidation increases the opportunity for cometabolism to transform the pollutant. Neither the reduction nor the oxidation step is to be overdone. Too much reducing substrate will create too much growth and result in excessive biofouling of the transmissive strata and difficulty in subsequent "treatment cocktail" delivery. Likewise, over oxidizing a reduced treatment step may generate excessive heat or excessive biomass. The process may be controlled by the amount and type of "treatment cocktail" used. Declining levels of hydrocarbon in an biologically oxidative environment will place increased populations of microbes in proximity to the pollutant, thus increasing the likelihood of transformation. The dynamic nature of the environmental forces created by repeated cycles of oxidation and reduction greatly increase the opportunity for non-enzymatic degradation to occur. The BARR process may be used in conjunction with existing pump-and-treat technology to enhance recovery of the target contaminant. Pump-and-treat technology aims to remove the pollutant from the soil matrix in the aqueous phase which is then treated to destroy (or recover) the contaminant after the aqueous phase is pumped out. The limitation to this is the attraction of the contaminant for the solid phase. The contaminant is reluctant to enter the solution phase where it can be removed and recovered via mass flow toward a recovery well. A further limitation exists in that the contaminant is typically concentrated in the finer textured materials of the solid phase where solution transport is extremely low. The BARR process influences both of these limitations to enhance the recovery of the contaminant. The changes in the solution chemistry accompanying the BARR process will tend to diminish the solvophobic force that drives nonpolar contaminants from solution and into the solid phase. In fact, the aqueous phase will acquire sufficient cosolvent to create a logarithmic increase in the solubility of the contaminant in the soil solution. The introduction of a nonpolar treatment cocktail will tend to permeate the finer textured soils and dilute the nonpolar contaminant with a readily biodegradable substrate. Reduction of the native soil minerals will create an increase in the ionic strength of the soil and thereby collapse the diffuse electrical multilayer on the clay surfaces. This will allow the closer approach of negatively charged bacterial particles which can attach to the colloidal precipitate. This will allow greater attachment of bacterial biomass to the coilloidal & solid phases and enhance biological activity in the treatment zone, as well as pollutant recovery in the pumped solution. Aspects of the BARR process requiring special attention. There are limitations to the effectiveness of this process. These limitations are not unique to the BARR process, but are commonly shared by other degradation processes as well. Some of the limitations are in the realm of knowledge, while others are in the realm of technology. This technique is not proposed for amateurs. BARR requires high-class geotechnical & engineering capabilities. The ability to assess the location of the contaminant and to deliver treatment cocktail into the proximity of the contaminant is key to the BARR process. There may be limitations to the BARR process in very deep contaminated formations, due to the limited ability to deliver treatment cocktail into the proximity of the contaminant. Injection lance delivery techniques are limited usefulness in cobbly soil. It may be likewise be difficult to deliver treatment cocktail into clays or tight soils. BARR process requires high-class biochemical and biotechnical capabilities. The location and recovery of viable inoculum from diverse sources and the ability to assess the activities of microbes in the treatment zone are a challenge that must be met by high-class biochemical and biotechnical skills. The transformations that will occur in the BARR process will challenge the best biochemist. Additionally, the formulation of the "treatment cocktail" requires better than average biochemical knowledge and a great deal of creativity. This process is undemonstrated and it is unlikely that complete mechanisms are known. It is of concern that the transformation of the pollutant may not result in complete mineralization. Most of the literature which describes sequential environments indicates that complete mineralization of organic contaminants is the product of such a treatment scheme. Conclusion BARR is a remediation technique to treat subsurface contamination resulting from organic chemicals. The technique is in-situ, bio-assisted remediation. This technique, is a multiple stage process utilizing direct metabolism, cometabolism and abiotic degradation to transform contaminants. The BARR process has significant advantages over existing bioremedial techniques, chiefly effectiveness and broad applicability to a wide array of contaminants. The BARR process utilizes a potentially numerous array of tools to bring about transformation Enzymes, free radicals, and surfaces catalyze transformations over a relatively short time period. This is a multiple-stage process which consists of injection of a specially formulated "treatment cocktail" which contains substrate, inoculum and mineral nutritional provisions to create biological activity in the proximity of the target pollutant. The treated system responds to create variable and controllable reducing conditions along with an increase in microbial biomass. The second-stage treatment creates oxidizing conditions and more biomass. The entire process is repeated as often as is desired, by injection of treatment cocktail to sequentially and redundantly reduce and oxidize the contaminated area. Contaminant mineralization occurs because of thermodynamic conditions favor it. The impact on the target pollutant is subject to considerable manipulation in terms of solution chemistry, temperature, pH, pE, biological activity, etc. This process offers a great tool for decontamination of soil. ----------------------------------------------------------------------------- This document is from the WELL's gopher server, gopher.well.sf.ca.us in the Science and Environment menus. For info e-mail to gopher@well.sf.ca.us .