REDUCING LEVELS OF CONTAMINANTS

TECHNICAL FIELD

This invention relates to reducing levels of contaminants, and more particularly to reducing levels of contaminants by bioremediation and/or using catalyzed reactions that produce contaminant ions.

BACKGROUND

Many industries produce contaminants (e.g. species of arsenic, mercury, lead, boron, cadmium, selenium, chromium, nickel, thallium, vanadium, zinc, nitrogen, chlorine, bromine, iron, copper and aluminum) as byproducts that are expelled as pollutants into our environment. The US Environmental Protection Agency and other regulatory groups consider these contaminants as pollutants because either their explicit presence or excess presence is detrimental to our ecosystems in general and human health specifically. These agencies heavily regulate these contaminants under federal and state statutes in the United States as well as through international agreements.

In the United States, the Clean Air Act of 1970 and subsequent amendments in 1977 and 1990 provide the statutory basis for the regulation of the discharge of these contaminants into air. Similarly, the 1972 amendments to the Federal Water Pollution Control Act (known as the Clean Water Act or CWA) provide the statutory basis for the regulation of the discharge of these contaminants into waterways. The EPA has a news release dated April 19, 2013 announcing new initiatives to reduce discharge of contaminants into waterways. Ironically, the EPA cites recent increases in the contamination of waterways as attributable to industry's increased use of clean air technology. For example, coal-fired power plant technology has improved to remove in excess of 90% of contaminants from stack gases. These same technologies, however, generate wastewaters containing these contaminates.

Contaminant resistance is the ability for microbes to capture contaminants and convert those contaminants from toxic forms to elemental forms and benign compounds. The science and engineering communities have studied contaminant resistant traits for more than half a century and shared a great wealth of knowledge about these traits through refereed scientific literature. One common metric for contaminant resistance is the highest contaminant concentration at which microbes remain viable.

SUMMARY

This disclosure describes the production of and the use of enhanced microbes that have specific contaminant resistance(s). This disclosure also describes the process for removing contaminants from air or gaseous effluents using catalyst(s) in combination with a wet scrubber containing an aqueous solvent.

This disclosure describes cultivation and enhancement processes used to produce living enhanced microbes that very efficiently capture, convert and eliminate contaminants (e.g., species of arsenic, mercury, lead, boron, cadmium, selenium, chromium, nickel, thallium, vanadium, zinc, nitrogen, chlorine, bromine, iron, copper and aluminum) from sediment-laden aqueous environments. Enhanced microbes in the context of this document are the products of embodiments of the cultivation and enhancement processes disclosed. The disclosed process uses native microbes to produce the enhanced microbes. Native microbes refer to microbes indigenous to a specific sediment-laden aqueous environment or microbes from a similar sediment- laden aqueous environment. The enhanced microbes exhibit cellular processes that selectively bind to a specific contaminant or specific contaminants even in the presence of other contaminants or chemicals, which are possibly higher in concentrations, reduce species (e.g., all species) of the contaminant to its elemental form and benign compounds, and expel the elemental form to the extracellular medium. Collectively, these cellular processes are commonly referred to as contaminant resistive properties; i.e., the enhanced microbes are specific- contaminants) resistant. Enhanced microbes not only possess the well-documented properties of specific-contaminant(s) resistance, they also possess properties of resilience (e.g. viability in relatively broad ranges of temperature, excess acidity and/or excess alkalinity) that uniquely distinguish them from other specific- contaminants) resistant microbes discussed in the literature.

Resistant microbes' selectivity, efficiency and overall efficacy in capture and conversion of contaminants have placed them at the forefront of consideration in the development of new remediation technologies since these resistive properties were discovered. The harsh environmental conditions of industrial waste streams have precluded the practical industrial application of resistant microbes. We have developed a process that incorporates resistant microbes that can not only produce the desired remediation results but also produce those results in the harsh environments typically encountered in industrial waste streams. Embodiments of this process provide means to produce enhanced microbes with not only the resistance properties sought for their remediation potential but also resilience properties that allow them to sustain harsh industrial waste stream conditions.

This disclosure describes methods for production of enhanced microbes in sediment -laden aqueous environments that convert

i. most or all compounds of a specific element to the elemental form of that element and other compounds that exclude that element or

ii. most or all compounds of two or more specific elements to the elemental forms of those elements and other compounds that exclude those elements.

The compounds of these elements typically are considered contaminants in aqueous environments because their explicit presence or the concentration of their presence has a detrimental impact on the environment as defined by the US

Environmental Protection Agency. Contaminants include but are not limited to compounds of arsenic, mercury, lead, boron, cadmium, selenium, chromium, nickel, thallium, vanadium, zinc, nitrogen, chlorine, bromine, iron, copper and aluminum. The relative concentration of other elements or compounds of other elements in the aqueous environment do not affect the efficacy of the microbes' abilities to transform compounds of targeted element(s) or contaminant(s). These enhanced microbes have resilience properties contributing to the efficacy and derived from the method of production. These resilience properties allow the enhanced microbes to survive in contaminated aqueous environments and distinguish the enhanced microbes from microbes produced using other techniques and/or processes and used to treat similarly contaminated aqueous environments.

The disclosure identifies the unique action of using microbes indigenous to or native to a contaminated area as a precursor in the production of the previously described enhanced microbes. The combination and relative number of microbial species and the relative spatial structure of those microbial species can be important in the process used to produce the enhanced microbes. The enhanced microbes resulting from this process using the native microbial communities do not necessarily have the same combination and relative number of microbial species as the native microbial communities or the relative spatial structure of microbial species as the native microbial communities.

The enhanced microbes can be used within a bioreactor for the reduction of contaminants from different environments. The enhanced microbes can be produced to reduce contaminants such as, for example, species of arsenic, mercury, lead, boron, cadmium, selenium, chromium, nickel, thallium, vanadium, zinc, nitrogen, chlorine, bromine, iron, copper and aluminum.

The methods described have produced enhanced microbes for use in systems like a bioreactor for the reduction of mercury from different environments. For example, some approaches include using a mixed population of many native bacteria strains. The systems and methods described can provide a cost-effective process to reduce the mercury content of mercury containing solutions including industrial process effluent(s). The system and methods are especially effective in the removal of mercury from coal impoundment ponds that can specifically use enhanced microbes that only remove mercury. The resulting cost of contaminant removal by the enhanced microbes is significantly less than with standard reagents. In several tests, the enhanced microbes were used to treat solutions (e.g., water from coal waste impoundment ponds) initially containing high levels of mercury to reduce mercury levels in the bioreactor effluent below method detection limits of 1 part per billion (ppb). These systems and methods can provide the ability to convert forms of contaminant mercury that are present to elemental mercury, Hg^, for subsequent practical use. In addition, the present disclosure details a method of enhancement to produce competent enhanced microbes with more efficient mercury binding and reduction potential than native colonies in adverse chemical environments.

These systems and methods can provide a resilient process for treating the highly variable waste streams that result from design variations between individual facilities (e.g., power plants) and from changing operating conditions and the types of coal burned. These systems and methods can make the capture of mercury and the reduction of mercuric ion, Hg ²⁺ , and/or highly toxic organomercury compounds to non-toxic Hg° more feasible than other applicable technologies. In particular, these systems and methods can provide the ability to remove Hg ^{2 +} (as opposed to Hg ⁰⁾ ).

In some implementations, these systems and methods exclusively bind, for example, to mercury. These enhanced microbes are not bound to other metals so fewer enhanced microbes may be used to provide the same effect as that of a greater quantity of non-specific complexing agents or other non-specific binding material. A series of naturally occurring (native) microbes can capture mercury exclusively if allowed to appropriately evolve in a toxic mercury environment as described in the disclosed method, then reduce the mercury from an Hg ²⁺ to Hg° in a mixed microbial community. Several colonies have been isolated from coal impoundment ponds. Laboratory work has established that low levels of mercury can be captured by these colonies. In contrast, technologies that utilize expensive non-specific complexing agents also capture numerous other metals requiring an excessive amount of the complexing agent. In these non-specific processes, an irreversible binding complex is formed, eliminating any chance to use the complexing agents multiple times.

In some implementations, these systems and methods exclusively bind, for example, to all forms of selenium.

These systems and methods are well adapted to provide custom generation and adaptation of the microbes to site-specific / process-specific waste streams and conditions. This feature can be particularly advantageous in treating waste streams, for example, from coal-fired power plants, which have varied designs that result in process streams unique to that particular plant. In addition, changing operating conditions and the types of coal burned can further compound the variability of process streams. These parameters provide large variability from plant to plant.

These systems and methods are also well adapted to provide microbes with sufficient resilience to reduce heavy metal concentrations in waste streams that contain other constituents that typically interfere with bioremediation. For example, the systems and methods develop microbes effective in the removal of mercury from aqueous sources under adverse chemical conditions.

These systems and methods also provide a process for removing contaminants from gaseous or airborne waste-streams (gas-process) like but not limited to gaseous waste-streams present at coal-fired power plants. The gas-process includes oxidation of contaminants with a catalyst, which makes them water-soluble. The gas-process includes use of an appropriate wet scrubber to dissolve the contaminant ions in an aqueous solution.

A potential processes for use sequentially after the gas-process includes but is not limited to

i. no subsequent process,

ii. a chemical process to reduce and precipitate the contaminants, or iii. the previously described microbial process of the invention.

The details of one or more embodiments the systems and methods associated with the enhanced microbes and the gas-process are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

Figure 1 is a flow chart of a method for developing enhanced microbes for bioremediation.

Figure 2 is a chart illustrating the systematic increase of mercury

concentration in microbial cultures used in the microbe enhancement process to produce enhanced microbes specifically resistant to mercury.

Figure 3 is a schematic of a plug- flow bioreactor (PFR) that illustrates one embodiment of reactor that incorporates enhanced microbes.

Figure 4 is a process diagram of a treatment system, which equilibrates a first part of a reactor with a later portion of the reactor through a common vent to the atmosphere.

Figures 5 A and 5B are schematics for incorporation of the described systems into a coal-fired power plant and an illustration of the fate of mercury across key waste streams. Figure 5A illustrates the potential placement of mercury removal units using enhanced microbes before placement of waste in an ash pond. Figure 5B illustrates the potential placement of the gas-process to eliminate mercury emissions.

Figure 6 illustrates a typical example of the incorporation of redundant 300 systems with other essentially linearly-scalable equipment. Figure 7 is an illustration of an exemplary application of the gas-process systems at a coal-fired power plant.

Figure 8 is an illustration of an exemplary process for concurrent use of the gas-process and bioreactor with enhanced microbes to treat all sources of air and water pollution at a coal-fired power plant (The Mercury Plus Process).

DETAILED DESCRIPTION

This disclosure describes means for the production of microbes (enhanced microbes) that capture and convert contaminants (e.g. species of arsenic, mercury, lead, boron, cadmium, selenium, chromium, nickel, thallium, vanadium, zinc, nitrogen, chlorine, bromine, iron, copper and aluminum) in environments that include water to their elemental forms and benign compounds. The enhanced microbes exhibit cellular processes that first bind to specific contaminant material(s) even in the presence of other materials which are possibly higher in concentrations. Following the binding, the enhanced microbes very efficiently convert the contaminant material(s) to a non-toxic form. The capture and conversion properties of these microbes are commonly called resistive properties.

This microbial process facilitated through resistive properties can be more efficient than current standard chemical treatments for removing, for example, heavy- metal contaminants and/or toxic heavy-metal species from a contaminated environment. Current standard chemical techniques use complexing agents for chemically and non-specifically binding to, for example, heavy-metal contaminants and/or toxic heavy-metal species, in a contaminated environment subsequently to remove them from that environment. The environments including these toxic heavy- metal species also typically include nontoxic heavy-metal species. These chemical treatments nonspecifically facilitate the binding of complexing agents to both the toxic and the nontoxic heavy metal species. The resulting volume of agent-heavy- metal-species complexes therefore includes both toxic and nontoxic heavy-metal species. Given the agent-heavy-metal-species, complex mixture is a waste requiring disposal and the concentration of non-toxic heavy-metal species are typically much higher than the toxic heavy-metal species, large volumes of what is considered a secondary waste is produced to remove a small amount of contaminant. As a specific example, this disclosure describes a method of mercury removal considerably more sophisticated than chemical treatment. The mercury-resistive enhanced microbes exhibit cellular processes that selectively bind mercury. This binding activates other unique biological processes, which transfers the mercury to other sites within the microbe and subsequently reduces the organic and inorganic forms of mercury to the less harmful elemental form of mercury, Hg°, and other benign compounds. The elemental form of mercury diffuses out of the microbe and out of the microbial community to the bulk media, which allows for collection through precipitation or volatilization techniques.

The enhanced microbes derived from native microbe populations as disclosed are significantly more resilient than resistive microbe populations derived from isolated microbial species. This significant resilience is the enhanced microbes' ability to survive, for example, in broader temperature ranges, broader pH ranges, broader ranges of mechanically perturbed conditions than the counterpart resistive microbe populations derived from isolated microbial species. Collectively, the resistive microbes and their microbial community constitute the enhanced microbes. These enhanced microbes can reduce the problems associated with the pollution of groundwater by contaminants in, for example, waste impoundment ponds and/or the industrial systems feeding these ponds. For example, enhanced microbes developed to reduce mercury concentrations have demonstrated a reduction in mercury species from contaminated waters from coal waste impoundment ponds to a limit for which mercury species are no longer detectable.

Furthermore, the previously described microbial process is contained in the multiple components of the enhanced microbes' microbial community. The presence of the resistive microbes, produced by the process described herein, in an appropriate microbial community imparts an enhanced robustness to the resistive microbes. This robustness is evident when process wastewater from a mercury cell industrial site with highly variable mercury levels, several highly toxic chloro-organic compounds, and highly toxic organomercury compounds was treated using enhanced microbes. The desired reduction of organic mercury and inorganic mercury contaminants to elemental mercury and benign compounds demonstrated the robustness and resilience of the enhanced microbes. Figure 1 illustrates a method 100 of developing living enhanced microbes based on the selection of initial source microbial communities and amplification of resistance to contaminants without isolating individual microbial species. The resulting enhanced microbes exhibit surprisingly high levels of resistance to the toxic effects of contaminants in wastewater being treated. These microbes can be developed so they can respond to a single contaminant or multiple contaminants in environments where the other materials have similar chemical properties to the contaminants and may be present in higher concentration.

The initial step in developing enhanced microbes to treat aqueous solutions associated with a specific site includes identifying dormant bodies of water on the site that have contaminant concentrations representative of the overall site contamination (step 110). In the context of this description, starter-microbe source means an accumulation of water that satisfies the criteria for including microbes with properties that can be used to produce enhanced microbes using the process described herein. The location of a starter-microbe source has standing water continuously present. For example, an area within a wastewater pond that has a continuous head of water throughout the year can be a starter-microbe source. In another example, a starter- microbe source includes wastewater ponds that have standing water present throughout the year such as a coal waste impoundment pond with an area that always has a puddle or pool above the mixture of coal dust and other sediments. Locations that have puddles or pools that temporarily occur after storm events or other events that produce water that eventually runs off or evaporates are not sufficient to produce the requisite precursor microbes to produce enhanced microbes and are not starter- microbe sources. Contaminants in a starter-microbe source are between the average concentration and the highest concentration of contaminants observed on the polluted site. A waterbody or a portion of a waterbody can be considered dormant for the purposes of identifying a starter-microbe source if it has been at least six months (e.g., at least nine months, at least one year, at least 15 months, at least 18 months, etc.) since waste containing contaminants was added to the waterbody or a specific discrete portion of the waterbody.

After identifying a dormant waterbody that has contaminant concentrations representative of site contamination, take samples from the identified waterbody (step 112). Take samples at the interface between sediments and standing water such that the samples contain water that is present, for example, in coal fines and other sediment particles as well as water sitting above these materials in puddles or pools. In addition, if agglomerations of microbial life suspended in water are present, it is desirable that the samples include such agglomerations. The method can include different sampling techniques. For example, the basis for developing desired enhanced microbes can be grab samples. Other sampling techniques include gathering composite samples from multiple locations in an individual waterbody or from multiple waterbodies across a site.

Place samples in a cultivation/enhancement process that exposes the microbes in the sample to gradually increasing concentrations of the target contaminant(s) (e.g., mercury, selenium, arsenic, etc.) in the cultivation medium (step 114). For example, culturing native site microbes in a growth media, nutritive substance, while increasing the mercury content in coordination with successive generations of microbial growth produces living enhanced microbes of mixed native, transformed microbes resistant to mercury toxicity and with enhanced mercury removal activity. Competent microbes are transferred into a conventional growth media with varying levels of mercury added such as three different medias with 0.001, 0.005, and O.OlnM mercuric chloride, respectively. The cultures, mixtures of microbes from samples and medium, are incubated for approximately 24 hours and checked for microbial growth. The microbial colony with the highest mercury concentration but sustained microbial growth can be used as a baseline. From that baseline, fresh growth media with the baseline level of mercury can be inoculated every 24 hours for a week to create stock microbes able to grow using the baseline conditions. Figure 2 illustrates a sequence of using this procedure to raise the mercury concentration to successively higher levels: 0.003mM, O.OlmM, 0.05mM, 0.1 mM, 0.2mM, 0.4mM, 0.6mM, and finally lmM Hg ²⁺ . Each step produces stock samples for subsequent steps with portions of the products at each step stored by freezing for subsequent use. Freezing the microbial colonies facilitates effectively indefinite storage of enhanced microbes for use at will.

Rather than isolating specific microbial species, this approach develops a multi-species microbial community that is able to adapt to a wide range of contaminant concentrations as well as survive in the presence of other chemical and biological constituents and environmental conditions like temperature and turbulence found in waste streams at a particular site. Accordingly, the cultivation medium should not contain any substances that tend to induce speciation of the microbes or selective elimination of a particular species.

The contaminant concentration is gradually increased until the desired contaminant resistance level is acquired. In some instances, developing microbial communities with a contaminant resistance higher than intrinsic contamination levels at a site facilitate the opportunity for incorporating contaminant concentration processes, like reverse osmosis, in the overall remediation process thus reducing the total volume of water treated in the contaminant reduction/removal component of the overall process.

Appendix A includes additional details about the method.

Figure 3 illustrates a contaminant removal technique using a continuous plug- flow bioreactor with living enhanced microbes developed from a mixture of native microbes. System 300 includes a plug-flow reactor 310 receiving a wastewater stream 312 to be treated, an inoculant 314 containing enhanced microbes, and a chemical feed stream 316 to provide nutrients to the microbes.

In the illustrated embodiment, the reactor 310 is a bead-packed column. Live enhanced microbes adhere to beads and act to remove contaminants as wastewater passes through the reactor 310. The system 300 includes pumps (not shown) used to transfer wastewater, inoculant, and chemical feed.

A pump transfers wastewater 312 to the reactor 310 on an ongoing basis during operation of the system. A second pump transfers inoculant 314 containing enhanced microbes from a supply source like a holding tank to the reactor 310 prior to introduction of wastewater to the reactor. After an adequate amount of enhanced microbe, colonies are established, pumping of the wastewater 312 into the reactor 310 initiates normal operating conditions. The second pump also can be used to introduce additional enhanced microbes 314 to the reactor 310 during normal operation of the system 300 to supplement enhanced microbe colonies already present in the reactor 310. A third pump provides means to add the chemical feed 316 to the reactor 310. The chemical feed mixture can include, for example, nutrients such as Luria-Bertani medium to encourage microbial growth activity and caustic/acid additions to control pH. Additional pumps can be used to introduce specific components of the previously described chemical feed mixture through unused ports in the port-manifold of the reactor 310 from pure component sources if a specific application requires variable control of the addition of a specific component of the chemical feed. The composition and magnitude of the chemical feed is application-specific. For example, in a large coal impoundment pond, the pH does not vary much and caustic/acid additions to control pH may not be necessary. In contrast, pH control and/or other types of pretreatment may be necessary before introducing wastewater from, for example, an industrial waste stream directly emanating from a plant process into the reactor 310. For example, the system 300 may include screening and/or filtration of influent wastewater, temperature control, and the like to address issues such as variable pH from chemical treatment of waste streams, variable temperature, intermittent high concentration of organic compounds, various oils, trash such as leaves, sticks, and other miscellaneous materials. For stable operation, the stream to be processed can include pretreatment such as: a trash rake and/or screens to remove solid debris, and/or a pH control loop that can maintain a specified pH, and/or a preheat or precool step to maintain a specified operating temperature that is typically between 75 to 80 degrees Fahrenheit (F), and/or an oil /organic skimmer if the waste stream includes a mixture of immiscible components. If the waste stream includes a mixture of immiscible components, it may be difficult to get the needed concentration of organics below the needed level to avoid a "sheen" on the effluent using a skimmer, a permit violation in many cases. If this is the case, organic control at the source is usually the most economical method of control via an isolation tank or other equipment.

In some embodiments, the system 300 uses other mechanisms for transferring fluids (e.g., gravity feed, etc.) within the system.

Other systems include, for example, a support column, a column packed with the enhanced microbes, simply enhanced microbes suspended in the influent as floes, floccules, or other types of suspensions. Optionally, sensors such as, for example, inline monitor(s), can be used to detect contaminants in the effluent stream as an indicator of when the enhanced microbes efficiency is negatively impacted. The system 300 can also include other sensors such as, for example, a standard pH meter for aqueous solutions, thermocouples to monitor temperature. We expect the simple mechanical design of system 300 to be easy to maintain with limited equipment malfunctions.

Port 320 is optionally provided to facilitate sampling of the effluent stream 318. The system 300 is designed to provide the residence time necessary for inoculated enhanced microbes to react with and remove the specific contaminant from the wastewater being treated. The volume of the wastewater being treated, flow velocity, and residence time can be increased or decreased by changing the cross section and/or volume of the removal apparatus as well as the flow rates of the various materials being introduced into the reactor 310.

In some embodiments, the system 300 includes temperature control mechanisms. For example, the system 300 can include a heat exchanger in which the warm, contaminant- free effluent heats the incoming influent. Electric heaters, natural gas heaters, bottled propane heaters, or similar heaters can provide additional heat as necessary. The ability to heat or cool fluids within the system 300 can be important, especially for portable systems that are deployed outdoors during the winter. For example, skid mounted units can be deployed on-site and adjacent to coal waste impoundments or ash ponds. This feature may be less important for systems that are installed indoors (e.g., to treat industrial wastewater as part of a process stream). Regeneration of the system 300 may be necessary after the significant buildup of elemental forms of contaminants (e.g., elemental mercury, elemental selenium, elemental arsenic) but this is anticipated to take months under normal operation. The elemental forms of contaminants will accumulate in the well (drain port) in the center of the device, which is at the lowest point. Depending on the volume and dispersion of accumulated elemental forms of contaminants, the elemental forms of

contaminants can be collected, for example, by phase separation or centrifugation. In the illustrated embodiment, the reactor 310 is a bead-packed column. Other systems can be implemented using, for example, a support column, a column packed with the enhanced microbes, or simply enhanced microbes suspended in the influent.

Optionally, system 300 can include sensors such as, for example, in-line monitor(s)to detect contaminants in the effluent stream as an indicator of when the enhanced microbes efficiency is negatively impacted. The systems 300 can be provided as independent modules such that multiple systems 300 can be installed and operated in parallel in order to increase throughput and/or provide treatment redundancy. For example, if a first system 300 must be taken off-line because of enhanced microbe exhaustion or a high level of contaminant product collection, the contaminant containing influent can be switched to a second system 300. The simple mechanical design of system 300 is easy to maintain with limited equipment malfunctions and provides significant operational flexibility. For example, multiple columns can be used in parallel (e.g., in a skid-mounted unit) to provide a portable treatment unit. Alternatively, a large unit (40,000 gallons) can process approximately 100,000 gallons per day (gpd). It is feasible to mix and match from different scale processing equipment to fit almost any set of requirements given scale-up for this type of equipment is almost linear. Figure 6 illustrates a typical example of the incorporation of a system 300 with other essentially linearly scalable equipment.

Figure 6 illustrates but one of many different configurations possible for treating contaminants using embodiment of previously described reactor units in conjunction with contaminant concentrating units like reverse osmosis units. For example, a million gallon per day influent to a mercury removal system may require six exemplary units for proper abatement. However, with the flexibility of using enhanced microbes capable of treating higher concentrations of mercury contaminants than present in the influent, the mercury contaminants in the influent can be concentrated with the aid of reverse osmosis so that two exemplary units can accomplish the removal.

Small (column) reactors containing media (e.g., lava rock, etc.) of various sizes can have a tendency to develop gas bubbles, resulting in backpressure in the column, which can cause disruption of continuous operation. The reactor is easily vented through the sampling port valve 300 or similar valves placed in areas to release the gases from the reactor. The occurrence of these backpressure events can cause a loss of residence time and can lead to higher mercury concentrations in the effluent if the residence time is set to a minimum.

Figure 4 illustrates a large scale system 400, e.g., for treatment of mercury contaminants, that addresses the potential issues of gas development resulting in back pressures by equilibrating a first part of a reactor 410 with a later portion of the reactor 410 through a common vent 411 to the atmosphere. The atmospheric vent 41 1 has a material (e.g., Hydrar(R), hopcalite, or sodium sulfide impregnated filter) which can capture the incidental contaminant vapor. For example, the metallic mercury pulled out of solution in the reactor 410 being used to treat waste from coal mining processes usually aggregates due to surface tension effects and then settles to the bottom of the reactor, but the metallic mercury does have a finite, yet low, vapor pressure. In particular, the very high surface tension of mercury can enable the system to trap it in the drain well which can be equipped with a draw-off. The system 400 is similar to the system 300 in having wastewater 412, inoculant 416, and chemical feed streams 414 introduced into the reactor 410 which discharges a treated effluent stream 418. The wastewater feed stream 412 optionally includes pretreatment modules.

In the illustrated embodiment, the wastewater feed stream 412 includes a coarse screening module 420 and a fine filtration module 422. These modules can be included in systems where it is desirable to remove particulate matter (e.g., leaves, sediments, etc.) that particularly can be an issue in treating wastewater from sources such as coal-waste impoundments and ash ponds.

The system 400 includes a process-to-process heat exchanger 424 and an electric heater 426 to warm influent wastewater to approximately 75-80 degrees Fahrenheit before the wastewater is introduced into the reactor 410. The process-to- process heat exchanger 424 uses warm effluent from the reactor 410 to heat the incoming wastewater. The process-to-process heat exchanger 424 reduces the amount of energy required by the electric heater 426 to raise the wastewater temperature to the desired range while also cooling the water being discharged by the system 400 (e.g., to a surface water body, a municipal sewer system, etc.).

The system 400 also includes optional pH adjustment module 428 with an inline pH monitor(s), pumps operable to dispense caustic/acid additions into the influent stream, and a controller receiving signals from the pH monitor(s) and sending control signals to the pumps.

The reactor 410 is horizontally oriented and includes baffles 430 extending from the floor and ceiling of the reactor 410. Some embodiments include baffles that are oriented side-to-side which could completely eliminate the need for any lava rock or other potential obstruction which might end up in the filter after operation. A drain port 432 installed in a low spot in the floor of the reactor 410 facilitates removal of precipitates from the reactor. Figure 4 only shows one port but other embodiments of system 400 include multiple drain ports 432.

Enhanced microbes developed using the approach described herein have been observed to effectively remove contaminants from solution even in the absence of support media (e.g. beads, pumice stone, activated carbon, semi-permeable membranes) used to pack columns in the system 300 described with reference to Figure 3. Bioreactors systems conventionally rely on microbes attached to support media and it is surprising that these enhanced microbes produced as described herein effectively function in suspension. The reactor 410 is generally free of media but includes lava rock chips piled between the bottom of downward extending baffles 430 and the floor of the reactor. The limited use of media in the reactor 410 can provide advantages including but not limited to increased flow capacity, reduced cleaning requirements, lower media costs, lower total organic carbon (TOC) loads and controlled growth of microbes relative to media filled reactors of similar dimensions. The lava rock chips are anticipated to prevent solids build up in a situation where an unusually large amount of microbes is formed that otherwise might load against a baffle. The media can create a zone away from the baffle where the liquid velocity is reduced such that solids are likely to settle away from a baffle. As described previously, atmospheric vent 41 1 equilibrates a first part of a reactor 410 with a later portion of the reactor 410. Other embodiments of system 400 include more than 2 linked atmospheric vents.

Example for Potential Scaling of a Bundled Columns Embodiment of Reactor 410

The reactor 410 discharges to a separation module consisting of a filter 436 and a settling tank 442 that separates water with relatively low levels of solids, recycled slurry, 444 from the filtered slurry. The recycled slurry 444 returns to the inlet end of the reactor 410 to help maintain appropriate levels of the enhanced microbes in the reactor 410. Excess solids can be removed from a slurry, for example, by settling in the settling tank 446. In general, the enhanced microbes act to convert soluble heavy metals into their insoluble elemental form. Removal of solids from the slurry will also tend to remove insoluble contaminants, in particular, as the agglomerated contaminants will tend to be denser and settle faster than enhanced microbes present in the slurry.

The warm water discharged 418 from the separation module consisting of the filter 436 and settling tank 442 is routed to heat exchanger 424 before being discharged from the system 400. The illustrated system 400 includes a total organic carbon (TOC) treatment module 448 and a reverse osmosis module 450 operable to remove salts and/or nutrients added to the influent to support enhanced microbes but not desirable in the effluent. Additional nutrient 414 may be added to increase enhanced microbe populations to capture and convert high concentrations of contaminants. These optional secondary treatment modules can be operated or bypassed as appropriate for conditions in a particular wastewater stream. Some embodiments of system 400 do not include any secondary treatment modules.

Embodiments of the systems described above can be used to treat

contaminants in various ways adapted to particular site conditions and processes. For example, there are multiple ways the systems and methods described can been implemented at a coal-fired power plant for treatment of mercury contaminants.

Figures 5A and 5B illustrates the fate of mercury across key waste streams and a flue gas path at a coal-fired power plant.

The systems can be installed in the byproduct exit stream of either a bag house or an electrostatic precipitator or a flue gas desulfurizer scrubber unit at a coal-fired power plant. With some stream modification (e.g. to control temperature, to address excess acidity and/or excess alkalinity, etc.) to reduce the likelihood that the living enhanced microbes are destroyed, the systems could also be effectively used to remove mercury contaminants from waste streams so mercury contaminants do not jeopardize the continued use of these waste streams in the manufacture of gypsum or concrete products. A conventional approach to prevent mercury escape to the atmosphere through stack gases at coal-fired power plants includes enhancing the trapping of flue-gas mercury by injecting activated carbon at various locations in the process stream and adding halogen salts to the coal being used as fuel. The coal is burned and the flue gas is passed to a selective catalytic reduction unit (SCR). The SCR's primary purpose is to remove nitrogen oxides, but it also oxidizes mercury to the mercuric ion. Based on the mercury oxidation potential, not all of the mercury is oxidized in the selective catalytic reduction unit (SCR) unless halogen salts are added to the flue before the flue gas enters the SCR to complete the mercury oxidation. The flue gas from the SCR subsequently passes through a precipitation unit followed by a desulfurization unit producing fly ash and gypsum, respectively. The mercuric ion produced in the SCR therefore is ultimately included in the fly ash and gypsum if it is not emitted from the stack. While this flue-gas treatment process effectively removes mercury from the flue gas, it indirectly increases the mercury content in the waste streams of ash and gypsum that are used in products like concrete and wallboard. This increased mercury content jeopardizes the continued use of fly ash and gypsum for use in concrete and wallboard, respectively, and similar products. Further, the halogenated compounds produced through use of the halogen salts can cause serious corrosion issues in ducts and carbon silos. The flue gas runs at 230 Fahrenheit so there is no condensation of halogen compounds while in operation. However, when the flue or other equipment is taken off-line, the halogen compounds can rapidly react with the equipment during cool-down, which leads to pitting and general overall corrosion. Similar degradation can occur during the reheating of the equipment at start-up. Our system uses cold liquid solutions and runs near neutrality which slows the corrosion process down considerably. Excluding the addition of mercury salts and capturing the mercury with an embodiment of systems described herein and located as illustrated in Figure 5A after the SCR and flue gas desulfurization unit (FGD) could avoid all corrosion issues, which can have a positive impact on unit uptime, maintenance costs, and the like.

In addition, the systems could be further modified so that they may be installed on the flue gas stream after various units (e.g., electrostatic precipitator, ESP, FGD) have performed their tasks and preferably just prior to the exit stack as illustrated in Figure 5B. The mercury can be oxidized to mercuric ion using existing technologies such as plate-type catalysts, quantitatively captured by a standard wet scrubber and subsequently processed using an embodiment of the systems described herein. That is, the wet scrubber solution can be treated easily using processes incorporating the enhanced microbes produced as previously described.

Figure 6 illustrates but one of many different configurations possible for treating contaminants using embodiment of previously described reactor units in conjunction with contaminant concentrating units like reverse osmosis units, the variance of which are dependent on the size and concentration of contaminants in the influent to the overall process. For example, a million gallon per day influent to a mercury removal system may require six exemplary units for proper abatement. However, with the flexibility of using enhanced microbes capable of treating higher concentrations of mercury contaminants than present in the influent, the mercury contaminants in the influent can be concentrated with the aid of reverse osmosis so that two exemplary units can accomplish the removal.

Other arrangements or embodiments of previously described reactor units in combination with contaminant concentration units can also realize practical process advantages from the treatment capacity of enhanced microbes.

In addition to the production of and the application of enhanced microbes, we also developed a gas-process for removal of contaminants from air or gaseous emissions from industrial plants. Our efforts to find specific applications for our enhanced microbe technology at coal-fired power plant led to development of the gas- process for removal of contaminants from the stack-gases of coal fired power plants. Our gas-process facilitates the increased capture of contaminants in a self-contained unit in the overall flue-gas treatment system in contrast to the current trend of modifying existing operating conditions for existing components of the system.

For example, while coal-fired power plants have dramatically reduced their gaseous emissions in recent years, further reduction is necessary to meet US EPA Mercury and Air Toxic Standards (MATS). In contrast to our gas-process, most coal- fired power plants plan to reduce further mercury and other trace emissions through adjustment of operating conditions for existing air-pollution equipment designed to remove nitrogen oxides (NOx) and sulfur oxides (SOx). The change in operating conditions primarily relies on the injection of activated carbon and an oxidant in the existing air-pollution control process-streams. The result is an improved overall capture of gaseous mercury, but not necessarily to levels set forth in MATS. Another result of the modified conditions is an increased rate of general degradation of process stream equipment. The modified conditions may also cause reduction in the efficiency for removing NOx and SOx to achieve an increase in removal of mercury.

Thus, although the current trend to increase the capture of contaminants by injection of activated carbon and an oxidant in the existing air-pollution control process-streams increases the amounts of contaminated material captured, this approach may not satisfy US EPA Mercury and Air Toxic Standards (MATS) and may create new problems in the flue gas system.

Figure 7 illustrates the gas-process for removing contaminants from gaseous or airborne waste-streams (gas-process) including, for example, gaseous waste- streams present at coal-fired power plants. While the process can be used in several embodiments of gaseous waste streams, Figure 5B includes exemplary placement of the gaseous process in a coal-fired power plant, specifically after the flue gas desulfurization equipment and before the emission stack. Figure 7 illustrates exemplary use of the gas-process to remove mercury and other air toxics from a coal- fired power plant's emissions 700. The polluted gaseous effluent 702 enters a duct 704 that includes a low temperature catalyst. The catalyst facilitates

oxidation/reduction of contaminants including, for example, mercury, arsenic, lead, boron, cadmium, selenium, chromium, nickel, thallium, vanadium, zinc, chlorine, bromine, iron, copper, and aluminum. The gaseous stream that includes the contaminant ions enters the bottom of a wet scrubber 706. The pressure on the gases that exit the area containing the catalyst 704 and enter the wet scrubber 706 forces the gases up the scrubber 706. The rising gases encounter a downward-flowing scrubbing-liquid fluid. While other chemical constituents may be included, the primary component of the scrubbing fluid is water. Contaminant ions are transferred from the rising gas to the scrubbing fluid as the gas rises to the top of the scrubber 706 and the scrubbing fluid flows to the bottom of the scrubber 706.

The aqueous stream that includes entrained contaminant ions from the scrubber 706 flows to a process for extracting and/or filtering and/or precipitating the ions from the aqueous stream 708 (e.g., processing through a bioreactor with enhanced microbes for specific contaminant ions, processing through a reactor that adds lime to the aqueous solution to cause precipitation of hydroxides of the contaminant ions followed by filtration, and processing through a reactor that increases the pH to a point that causes precipitation of carbonates followed by filtration ) to produce a collection of condensed contaminants 710. In effect, the gas- process transfers gas phase materials to the aqueous phase where they can be treated by techniques like chemical precipitation, flotation, adsorption, ion exchange, and electrochemical deposition as well as a bioremediation process using enhanced microbes. The resulting aqueous stream 712 contains very low or non-detectable metals, no biological oxygen demand (BOD) or total organic carbon (TOC). The decontaminated gaseous stream 714 from the wet scrubber exhausts through the conventional emission stack 716 of the plant. In addition, the aforementioned gas- process facilitates the efficient use of the selective catalytic reduction unit (SCR) and the flue-gas desulfurization (FGD) unit for removal of the pollutants for which they originally were designed, nitrogen oxides and sulfur oxides, respectively.

The gas-process facilitates the use of operating conditions for nitrogen oxide and sulfur oxide scrubbers for their intended purposes. The gas-process allows sufficient capture of contaminants so that MATS can be satisfied. Specifically, the gas-process removes 98+% of the air contaminant emissions compared to the MATS requirement of 90%. As a result of these two direct advantages of the gas-process, the indirect effects of reducing the amount of contaminants present in the waste streams emerging from the scrubbers and products formed from components of these waste streams (e.g. gypsum and concrete additives from the nitrogen oxide and sulfur oxide scrubber systems) are also advantageous.

Figure 8 illustrates a process 800 that includes the gas-process and bioreactor with enhanced microbes to treat sources of air and water pollution at a coal-fired power plant. The process 800 is an application of the gas-process 700 using a bioreactor with enhanced microbes 824 as a component of the process for extracting and/or filtering and/or precipitating contaminate ions 820 & 824. The combination of the gas-process with a bioreactor using enhanced microbes produces a feasible scenario for treating all sources of a targeted contaminant at a coal-fired power plant. The process 800 is discussed with respect to the treatment of mercury but can also be used to treat other contaminants. Polluted flue gas 802 and effluents from the scrubbers as well as material already collected in the ash ponds from the scrubbers 810 are concurrently treated in process 800.

Polluted flue gas 802 enters a duct 804 that includes a low temperature catalyst. The catalyst facilitates oxidation/reduction of contaminants including, for example, mercury, arsenic, lead, boron, cadmium, selenium, chromium, nickel, thallium, vanadium, zinc, chlorine, bromine, iron, copper, and aluminum. The gaseous effluent of the duct 804 includes ions of the contaminants formed by the

oxidation/reduction reactions in the duct 804. The gaseous effluent form the duct 804 enters the bottom of a wet scrubber 806. The pressure on the gases that exit the area containing the catalyst 804 and subsequently enter the wet scrubber 806 forces the gases up the scrubber 806. The rising gases encounter a downward-flowing scrubbing-liquid fluid. While other chemical constituents (e.g. acids, bases) may be included, the primary component of the scrubbing fluid is water. Contaminant ions migrate from the rising gas to the scrubbing fluid as the gas rises to the top of the scrubber 806 and the scrubbing fluid flows to the bottom of the scrubber 806.

The gaseous effluent from the scrubber 806 constitutes pollutant- free flue gas from the plant 816. The pollutant free gas 816 is expelled into the atmosphere through the plant stack 818.

The aqueous stream that includes entrained contaminant ions from the scrubber 806 flows to a unit composed of a pump and a flow divider 808. The unit 808 facilitates addition of ash pond water 810 that has undergone pretreatment 812 to the aqueous effluent of the scrubber 806 that enters the first phase of the contaminant removal process 820. The pretreatment process 812 primarily removes trash, sticks, leaves, and other water-insoluble materials from the ash pond water 810 before the effluent of the pretreatment process 812 enters the unit 808. These devices can include but are not limited to traveling water screens and/or trash rakes and/or settlers and/or concentrators and/or clarifiers. Furthermore, the unit 808 can recirculate a portion of the aqueous stream 814 from the pump and flow divider unit 808 to the aqueous influent port of the scrubber 806. Typically, multiple passes of the aqueous solution 814 through the scrubber 806 are required to saturate the aqueous solution with the contaminant ions entrained in or captured by the solution 814. The recirculation 814 allows a net decrease in the flow of aqueous solutions through the process 800. The pump and flow divider unit 808 is equipped with sensory equipment that determines if recirculation of a portion of the effluent from the unit 808 can sustain the desired level of contaminant removal in the wet scrubber 806. If sensory equipment does not indicate that recirculation will sustain the appropriate level of contaminant removal, the unit 808 can circulate pollutant- free water 828 through the recirculation loop 814.

The effluent from the unit 808 excluding effluent recirculated 814 enters a process for extracting and/or filtering and/or precipitating the contaminant ions 820. This process 820 produces a collection of condensed contaminants 822. These condensed contaminants do not include any products derived from organomercury compounds like methyl mercury that were present in the effluent of the unit 808. Organomercury compounds and possibly some inorganic mercury compounds will persist in the effluent of the process for extracting and/or filtering and/or precipitating the contaminant ions 820. Subsequent treatment of the effluent from the process for extracting and/or filtering and/or precipitating the contaminant ions 820 in a bioreactor with enhanced microbes 824 will facilitate production of elemental mercury 826 and innocuous organic compounds. Much, if not all, of the innocuous organic compounds are consumed by the aerobic enhanced microbes in the bioreactor 824 through a metabolic process that produces primarily carbon dioxide and water, which are expelled into the atmosphere. The aqueous effluent of the bioreactor 828 is pollutant-free water, which is allowed to enter local waterways 830 or discharged to sewer systems as appropriate.

Multiple embodiments of the gas-process for removing contaminants from the emissions of other industrial plants are possible. These embodiments include but are not limited to combing the gas-process with a bioreactor that uses enhanced microbes to remove contaminants (e.g. species of arsenic, mercury, lead, boron, cadmium, selenium, chromium, nickel, thallium, vanadium, zinc, nitrogen, chlorine, bromine, iron, copper and aluminum) not removed or not adequately removed by conventional extracting and/or filtering and/or precipitating processes. Example 1

The methods described above were used to develop resilient enhanced microbes effective to treat mercury contamination in water from native microbial populations present in a coal waste impoundment. The research team performed a preliminary site study of a coal waste impoundment and observed color and odor consistent with the presence of live microbes and in particular, bacteria in water at the coal waste impoundment pond. The ponds were known to be contaminated with heavy-metal contaminants and other chemical contaminants that impair water quality. Some microbes present in the ponds would have to be composed of a strain or strains resistant to the heavy metals and other chemicals. Mechanisms for heavy-metal resistance in bacteria include processes that reduced the heavy metals to their elemental form, which, in turn, made the heavy metal contaminants insoluble in water. This physical change in the predominant forms of heavy-metal contaminants from water-soluble inorganic salts and water-soluble organic compounds to water-insoluble elemental forms facilitate the use of a host of conventional means for separating these heavy-metal contaminants from the water and, in turn, facilitate separation of the heavy-metal contaminants from the coal dust and other sediments in contact with the water.

Research team members subsequently returned to the site and collected eight grab samples from each of three areas distinguishable by the relative water content and the extent to which the coal fines and other sediments were covered with water:

Area- 1 samples were taken from the area surrounding the ponds but containing coal fines and other sediments. These samples appeared to be a collection of solid particles devoid of water. They, however, released water when compressed in a standard lab pellet press.

Area-2 samples were taken from the edge of the ponds. These samples were sponge-like and included coal fines and other sediments as the previous samples. These sponge-like samples released water when compressed by hand.

Area-3 samples were taken from areas of the pond that had continuous exposure to a pool of water resting above the coal fines and other sediments. These samples therefore contained not only water that was entrained in the coal fines and other sediment particles, but water sitting above these materials in puddles or pools. Twenty-four samples were collected in pre-cleaned EPA bottles, certified to contain no heavy metals. The samples were immediately refrigerated to a temperature of 4 degrees Celsius. Subsequently, the total mercury content for each sample was measured using cold vapor atomic absorption spectrometry according to EPA Method 245.1. Each sample included detectable mercury. The total mercury content of the samples varied from the detection limit of 0.2 ppb to 40 ppb.

Within 24 hours of obtaining the 24 samples, microbes from each of the 24 samples were cultivated in standard culture mediums, Luria-Bertani (LB) broth, for 24 hours using standard techniques to increase the amount of each sample available for subsequent mercury resistance tests. Escherichia coli (E. Coli) strain MM294 purchased from Carolina Biological Supply Company was concurrently cultivated for use as a blank reference for subsequent mercury resistance studies. Ethylene glycol was placed in the microbe cultures originating from each of the 24 samples and the blank to create 20% ethylene glycol solutions. These stock microbe mixtures were stored at negative 85 degrees Celsius.

An inoculum from each of the 24 stock microbes and blank mixtures was cultivated in standard liquid culture mediums in the presence of 0.001 mM (200 ppb) mercury for 24 hours using standard techniques to create a 0.001-mM-Assay-Sample- Set. The extent of microbe growth in each assay-sample of the set was determined using a growth correlation with the difference of turbidity measurements at the start and end of the growth period. The percent change by mass in the concentration of mercury in each assay-sample was determined using EPA method 245.1. Ethylene glycol was placed in each of the assay-samples of the 0.001-mM-Assay-Sample-Set to create a 20% glycerol solution. These assay samples were stored at negative 85 degrees Celsius.

All assay-samples that showed the presence of live microbes at the end of the growth period were used as sources for inoculums to create the assay-samples of the 0.003 -mM-Assay-Sample-Set. The same procedure used to create the 0.001-mM- Assay-Sample-Set was used to create the 0.003 -mM-Assay-Sample-Set. The only difference in the procedure was the concentration of the mercury. In turn, the 0.003- mM-Assay-Sample-Set was used as a source of inoculums to create the 0.005-mM- Assay-Sample-Set. Sequential application of this series of steps resulted in Assay- Sample-Sets developed from mediums that included 0.001 mM (200 ppb), 0.003 mM, 0.005 mM, 0.010 mM, 0.050 mM, 0.100 mM, 0.200 mM, 0.400 mM, 0.600 mM, 0.800 mM, 1.00 mM, 1.30 mM, and 1.50 mM (300,000 ppb) as shown in Figure 2.

Evaluation of the native microbe colony growth and mercury reduction data clearly indicated that the Area-3 type bacteria could be stimulated to produce the highest level of mercury resistance of microbes from all the areas. 99% reduction of mercury in concentrations as high as 1.5000 mM (300,000 ppb) can be accomplished with enhanced microbes produced from these native, multispecies, Area-3 -type colonies. The research team's review of literature including review articles involving mercury resistance indicates this level of mercury resistance is 4-orders of magnitude larger than mercury resistances recorded in the literature as illustrated in Table 1.

Table 1

Maximum Mercury Resistance Levels

In addition to greater mercury resistance, the microbes produced and used showed greater resilience to physical perturbations like bubbles and agitation, and greater resilience to chemical perturbations like those present in highly organo- chlorinated waste streams. The resilience exceeded our expectations since the microbes survived and grew in a mixture of organo-chlorine components from a mercury-cell chlorine unit.

Subsequent tests demonstrated the Area-3 -type colonies had to be gradually exposed to higher and higher concentrations of mercury for them to develop the high levels of resistance shown by Assay-Sample-Sets originating from the first Area-3 - type colonies. In other words, sudden exposure of the Area-3 type colonies to the higher concentrations of mercury, like 1.5000 mM, resulted in death of the microbes.

The gradual exposure to increasingly higher concentrations of mercury as previously described results in development of very resistant microbe colonies, enhanced microbes. The team hypothesizes the gradual increase of mercury in the sequential culturing process allows microbes in the multi-species community to effectively evolve in a manner and at a rate that allows them to resist a highly mercury- contaminated environment. In other words, the gradual exposure to increasingly higher levels of mercury stimulates the microbes to express formerly dormant genes that generate the mercury resistant characteristics.

Example 2

Further characterization of the type of area necessary to produce the starting colonies for use in the cultivation/enhancement process within a contaminated site was completed after attempts to reproduce the extraordinary results were completed.

Given the unique qualities of the resistant colonies produced from the Area-3 type colonies, simple criteria for obtaining the starting bacterial colony for the cultivation/enhancement process was established. Samples used to provide the starting microbial colony have to be collected from an area that not only has water entrained in the coal dust and other sediments, but water sitting above these materials in puddles or pools. Subsequent studies to reproduce the results after the original site was perturbed by storm events and the like resulted in the following two additional characteristics for the type of area:

1. The area sampled must be an area that continuously has puddles or pools of water sitting above the saturated coal dust and other sediments. Areas that have puddles or pools that temporarily occur after storm events or other activities that produce water that eventually runs off or evaporates are not sufficient to produce the requisite microbial colonies.

2. The area sampled cannot have had waste added within the past year. Henceforth within this document, ponds meeting this criterion will be referenced as dormant ponds.

The team hypothesizes that the continuous exposure to the head of water produced by the puddles or pools provides a continuous source of water-soluble mercury stress on the microbes in these water laden areas. Thus, by natural selection, only microbial colonies with appropriate mercury resistant and other resilience properties will survive in the area.

The team also hypothesizes that the perturbations of the site produced by addition of new waste disrupts the natural selection process. For example, samples taken from ponds that were created within a year of collection of the sample did not produce microbes that could be cultivated for use in subsequent cultivation and enhancement processes. Similar results were found with older ponds to which new waste was being added at the time of sample collection. This disruption prevents the requisite colonies for the cultivation/enhancement process from being established. It is worth noting, however, that the treatment colonies produced by ponds that are not perturbed by the addition of new waste can be used in the treatment of waters in nearby active ponds, ponds to which new waste is being added. The applicability of a treatment colony from a dormant pond in a remediation process for an active pond has only been correlated with two factors thus far. The ponds are within the same valley and the waste added to the newer pond is similar to the waste added to the dormant pond. The applicability of the treatment colonies originating from a dormant pond on an active pond most likely stems from the great similarity of the overall ecosystems in each pond.

Example 3

The team subsequently conducted efforts to isolate the individual strains of bacteria within the highly -resistant multi-species colony with the hope of identifying the specific specie or the best specie for subsequent use in a reduction/separation process. Two of eight species in these colonies were isolated. The level of mercury resistance demonstrated by each of these isolated species in the conventional tests was similar to the level of mercury resistance demonstrated by their parent colony. The conventional tests were conducted using mercuric chloride as the source of mercury, Luria-Bertani (LB) broth as a source of nutrients and growth factors, and distilled water as a source of water. When, however, the distilled water was replaced with water from the site of the original samples, the isolated species demonstrated reduced resistance and resilience compared to the resistance and resilience demonstrated in the conventional, distilled-water tests. The parent colony of the isolated species, however, demonstrated the same resistance with water from the site as with distilled water.

The colony's mercury resistance and viability in the mercury-contaminated water from a polluted site compared to the individual strains' resistance and viability in the same, led the team to conclude that at least a particular mixture of species in a colony was important to producing the resistance and viability for treatment of water from a coal waste impoundment pond. The collective resilience to varying conditions exhibited by sessile bacterial colonies has been associated with unique structural matrices formed by the bacteria and extracellular polymeric substances that they produce. While these matrices are attached to some type of substrate in the sessile bacterial colonies, similar collective resilience has been and is expected for the planktonic (floating) counterparts. The team hypothesizes that the collective properties of the mercury-resistant planktonic colonies produced from the bacteria indigenous to the contaminated water of the coal waste impoundment pond are facilitated through some sort of structural organization of the colony species and, possibly, extracellular polymeric substances they produce. Previous approaches have tried to reduce mercury bioremediation systems to their individual components and optimize these individual components. In contrast, the approach described in this disclosure counter- intuitively relies on a collective effect of the individual components that is not achieved when individual components are separately improved and, subsequently, combined. The team hypothesizes floes (aggregates), floccules (small aggregates), granules or some other type of superstructure with a unique structural matrix of microbial species and extracellular polymeric substances form to maintain certain individual species mercury-resistant characteristics and to collectively exhibit resilience to the range of environmental changes expected in the polluted site. These superstructures have sufficiently low densities to maintain the planktonic characteristics of the colony. While studies about the formation of mercury resistant bacterial colonies in the Aussa River in Italy have properties consistent with the enhanced microbes described, the enhanced microbes in associated planktonic superstructures produced as described in this disclosure have demonstrated increased resistance and resilience.

Previous studies have shown the utility of mercury-resistant bacteria in the treatment of waste-waters contaminated with mercury. These studies have included concurrent use of multiple species of resistant bacteria. However, these bacteria have been unable to survive the conditional variations in a real wastewater stream and rendered the utility of these specific species and multi-species applications for the removal of mercury as impractical. When mixtures of bacteria species have been used or tested in activities described to date in the literature, the mixtures are developed from "isolated" strains albeit the source of the bacteria is sometimes indigenous to the wastewater treated. The methodology described herein allows the indigenous ratio of species and very likely the indigenous matrix structure of those species and extracellular polymeric substances they produce to be reflected in the enhanced mercury-resistant colonies that are referenced herein as enhanced microbes. In addition, the enhanced microbes developed using the methods described herein have been used to convert the most toxic organomercury compound, methylmercury, to elemental mercury and benign compounds. While the team has not conducted studies to verify the retention of the matrix characteristics, we hypothesize that this explanation for the unique resilience of the colonies to the variable conditions of the waste stream is very plausible. This retention of matrix structure could account for the extraordinary mercury resistance and the extraordinary resilience of the colonies produced using the process described herein.

Example 4

The research team has applied the same procedure used to develop the treatment bacteria colony for mercury to produce a treatment bacteria colony for selenium. The results of these studies have shown selenium resistance for

concentrations from 55 ppm to 920 ppm. Studies for concentrations less than 55 ppm have not been completed but are expected to show similar resistive action. In these lower concentrations, the microbes could possibly use selenium in their nutritional systems. Given the selenium reduction studied is an aerobic process, the potential for use of this process on an industrial scale shows great promise compared to anaerobic counterparts due to its comparative simplicity and, thus, concurrent lower cost.

Conclusion

The findings from the work with mercury and selenium support the conclusion that it is plausible to create a treatment microbial colony, enhanced microbes, which can resist specific contaminants. The findings also indicate it is plausible to create enhanced microbes that will resist multiple contaminants concurrently using essentially the same procedure. In addition, the findings indicate the enhanced- microbes have resilience properties that allow them to survive the expected fluctuations in environmental conditions like pH and temperature at the polluted site. Findings also indicate the presence of metal ions in addition to the target contaminants of mercury and/or selenium does not affect the efficacy of the enhanced microbes. The contaminants referenced in this document are detrimental to ecosystems if their soluble concentration levels exceed limits unique to each of the contaminants as reported by the EPA. If the contaminants exist in an area at concentrations considered pollutant levels, microbes cannot survive in the area unless some members of the microbial community have a mechanism for eliminating the contaminant from the aqueous environment. Unless some microbes exposed to conditions that include pollutant levels of contaminants have resistant characteristics, the microbial communities cannot survive in areas that include pollutant levels of the contaminants.

The resistant properties of the enhanced microbes almost exclusively results in the reduction of the contaminant to its elemental form that facilitates a phase separation of the contaminant from the water-laden environment. In other words, the resistant processes invoked by the microbes remove the pollutant from water. The elemental forms of the contaminants are not toxic. (Please note that some elemental forms of contaminants including mercury are referenced as toxic not because the elemental form in itself is toxic but because the element can be easily oxidized/reduced into a toxic form.)

A number of embodiments of the production and application of enhanced microbes and of the gas-process for removal of contaminants from air or gaseous emissions from industrial plants have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of this disclosure.

For example, although the examples discussed above focus on treatment of coal-waste impoundment ponds or coal-fired power plant ash ponds or effluent from an embodiment of the gas-process, the same techniques can be applied to coal ash ponds, soils, and other polluted material that remains in constant contact with water that is used as the sample source for obtaining the starting microbial colony, the precursor to the enhanced microbes. Moreover, these techniques have also been effective in developing enhanced microbes that are highly effective in treating wastewater with high levels of selenium and are anticipated to be able to develop enhanced microbes that provide similar results in treating wastewater containing other contaminants. Accordingly, other embodiments are within the scope of the following claims. Embodiments of the gas-process of this invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of this disclosure. For example, although the examples focus on implementation in a coal-fired power plant and the particular contaminant of mercury, the same gas-process can be used within any process that produces air or gaseous emissions that include contaminants and those contaminants are not limited to mercury. The contaminants can include but are not limited to mercury, arsenic, lead, boron, cadmium, selenium, chromium, nickel, thallium, vanadium, zinc, chlorine, bromine, iron, copper, and aluminum. Accordingly, other embodiments are within the scope of the following claims.

APPENDIX A

Definitions;

Water-Laden Environment - An environment in which all non-water components of the environment have sufficient exposure to water that heavy metal species present in the water are partially if not totally borne from the contact of the water with the non-water component.

Bacterial Colonies - Numerous bacterial cells either derived from one parent or having the individual characteristics as if they were derived from one parent.

Bacterial Communities - Numerous bacterial cells either derived from more than one parent or having the individual characteristics as if they were derived from more than one parent.

Bacterial Microbes - Aggregate structures that are formed by bacterial colonies or bacterial communities. These aggregates have planktonic character in they can be suspended in solution or precipitate from solution but are not affixed to a substrate. The solvent for the solution is water in the context of this document.

Species included in these aggregates can include planktonic and/or sessile bacteria. In addition to the bacteria, these aggregates may be embedded in a matrix of extracellular polymeric substances which these cells have produced.

Bacterial Biofilms - Aggregate structures that are formed by bacterial colonies or bacterial communities. These aggregates have sessile character in they are permanently affixed to a substrate in contact with the water-laden environments. Species included in these aggregates can include planktonic and/or sessile bacteria. In addition to the bacteria, these aggregates may be embedded in a matrix of extracellular polymeric substances which these cells have produced.

Bacterial Superstructures - Microbes, biofilms and other structures formed by colonies and/or communities of bacteria. Spatial structures formed by colonies and/or communities of bacteria.

Bacterial Group - A term used herein to collectively refer to bacterial colonies, bacterial communities, bacterial microbes, bacterial biofilms, other bacterial superstructures, and other collections of bacteria that naturally exist in water-laden environments. The italicized term "Bacterial Group" will be used specifically in reference to a bacterial group developed using the METHOD described herein. Source-bacterial-group - A term used herein to refer to the bacterial group harvested from a site and subsequently used to create the harvested bacterial group's corresponding Bacterial Group using the METHOD described herein.

Heavy Metals - Elemental forms of Al, Mg, As, Cd, Cr, Cu, Pb, Hg, Ni, Se, Zn, and any other elements that can be converted from compounds of that element to compounds that exclude that element and the naturally occurring form of the element using a Bacterial Group produced using the METHOD described herein.

Species of a Heavy Metal - All organic and inorganic compounds that include the heavy metal in a covalent, ionic, or ligand bound form. Any form of the heavy metal excluding its naturally occurring elemental form.

Heavy Metal Reduction - Conversion of all species of a heavy metal to species that do not contain the heavy metal and the heavy metal in its elemental form.

Targeted (Species of) Heavy Metal(s) - Specific (species of) heavy metal(s) among group of (species of) heavy metals present in the environment.

Waste- Water - Water from a site that includes heavy metals or species of heavy metals at concentrations that exceed acceptable standards. These concentration can be reduced using the appropriate Bacterial Group produced using the METHOD described herein and an appropriate application technology, APPLICATION, described herein.

INTRODUCTION

This document describes methods for production of Bacterial Groups (METHOD) including but not limited to bacterial colonies, bacterial communities, bacterial microbes, bacterial biofilms, and other bacterial superstructures that convert all targeted species of a specific heavy metal (HM) or all targeted species of a specific combination of heavy metals (CHM) in water-laden environments to species that exclude heavy metals and elemental forms of the heavy metals. These unique Bacterial Groups provide means for facilitating this conversion in a water-laden environment that includes other, non-targeted heavy metals without any interference by or conversion of those other, non-targeted species of heavy metals; the Bacterial Groups specifically convert HM or CHM in the presence of other heavy metals and species of other heavy metals irrespective of the relative concentration of the HM or CHM to the other heavy metals and/or species of other heavy metals present. BACATERIAL GROUPS

The bacterial property that describes bacterial reduction of species of a heavy metal to species that exclude the heavy metal and the elemental form of the heavy metal is called heavy-metal resistance. The heavy-metal resistance of a Bacterial Group is consistent with many documented observations and mechanistic

developments for bacteria that possess heavy-metal resistance. The mercury resistant Bacterial Group is illustrative of all the heavy-metal resistant Bacterial Groups. The mercury resistant Bacterial Group exhibits cellular processes that selectively bind to mercury even in the presence of other heavy-metals which are possibly higher in concentrations, reduces all species of mercury to elemental mercury and species that exclude mercury, and expel elemental mercury to the extracellular medium. Barkay et al. provide a thorough historical review of bacterial mercury resistance as well as a summary of the accepted molecular mechanisms that facilitate this resistance which is consistent with the observed properties of the mercury resistant Bacterial Group.

A Bacterial Group not only possesses the well-documented properties of heavy-metal-resistance, it possesses resilient properties that uniquely distinguish it from the heavy-metal resistant bacteria predominantly if not exclusively discussed in the literature. The literature shows many examples of the use of colonies of bacteria and communities of bacteria that are heavy-metal resistant. These colonies and communities of bacteria have limited practical use in industrial settings given the very restrictive environmental conditions that must be maintained for the bacteria to survive and exhibit the desirable heavy-metal resistance property.

A Bacterial Group produced using the METHOD described herein has not only heavy-metal resistive properties but high resilience properties with respect to the range of environmental conditions in which it is expected to survive. The ranges of environmental conditions include but are not limited to the range of pH, the range of ionic strength, the range of temperatures and the range of nascent microorganisms that can compete with the mercury resistant bacteria.

The Bacterial Group is prepared from a source-bacterial-group that is known to have the resilience properties required for subsequent industrial applications. The METHOD used to create the Bacterial Group maintains a distribution of species, structural relationship among species, and other important collective characteristics which are necessary to sustain the resilience properties of the source-bacterial-group. The METHOD used to create the Bacterial Group also stimulates the source bacterial group to exhibit enhanced heavy-metal resistance compared to the resistance exhibited in their natural environment. The Bacterial Groups developed using the METHOD described herein have appropriate collective properties to provide them with resilience to real-world application environments and stimulated properties of heavy-metal resistance for them to be used in conventional bioreactor systems for removal of specific heavy metals from water.

The relationship between the Bacterial Group required for treatment of wastewater at a particular site and the site used for collection of the source-bacterial- group gives rise to three categories of Bacterial Groups: Same-site Bacterial Groups (Tl -Bacterial Groups), similar-site Bacterial Groups (T2-Bacterial Groups), and pretreatment-with-similar-site Bacterial Groups (T3-Bacterial Groups). While the METHOD used to produce the Bacterial Group required to treat wastewater at each type of site is the same, additional controls and pretreatments to maximize the efficiency of the treatment may differ among the categories.

The same-site Bacterial Group (Tl -Bacterial Group) is developed from a source-bacterial-group collected from sites that include the wastewater that will be ultimately treated using the Tl -Bacterial Group prepared. The Tl -Bacterial Group is used to treat wastewater from the same site used to collect the source-bacterial-group. Little or no pretreatment of the waste-water treated with the Tl -Bacterial Group is expected given the resilience properties associated with the expected variations in pH, temperature or other environmental properties of the waste-water are inherited from the source-bacterial-group.

The similar-site Bacterial Group (T2-Bacterial Group) is developed from a source-bacterial-group collected from a site that have the same characteristics as the intended-use site when the intended-use site has not had appropriate time to mature as required by the METHOD discussed herein. For a waste-water site to be sufficiently mature, it must remain "dormant" for a sufficient period of time for an appropriate source-bacterial-group to develop. If, however, the environmental conditions of the waste-water site are essentially the same as a "dormant" site, a source-bacterial-group collected from the "dormant" site can be used to develop an applicable T2-Bacterial Group. For example, a coal waste impoundment pond to which additional coal waste has not been added (first pond) for a sufficient period to allow development of a source-bacterial-group can be considered a "dormant" site. A source-bacterial-group may not be present in a second coal-waste impoundment pond to which coal waste is regularly added (second pond). This second pond being continuously perturbed by the addition of coal waste would not constitute a "dormant" site. Nevertheless, the environmental conditions of the second pond sufficiently resemble the first "dormant" pond such that a source-bacterial-group from the first pond can be used to produce T2 -Bacterial Groups that not only have the heavy-metal resistance properties but the resilience properties required to make the T2-Bacterial Groups effective for treatment of the second pond wastewater with little or no pre-treatment.

A pretreatment-with-similar-site Bacterial Groups (T3-Bacterial Group) is developed from a source-bacterial-group that has similar characteristics as the intended-use site but certain resilience properties of the T3 -Bacterial Groups produced are expected to be inadequate. For example, the development of a T3-Bacterial Groups from a source-bacterial-group collected from a "dormant" fly ash pond adjacent to a coal-fired power plant may not be applicable to treatment of wastewater coming directly from the plant scrubber without some pretreatment of the wastewater. This pretreatment of the wastewater includes adjustments to the properties of the wastewater to fall within the range defined by the resilience properties of the T3- Bacterial Group produced from the fly-ash-pond source-bacterial-group. Specific pretreatments will vary depending on the extent to which the waste-water properties deviate from the range allowed by the resilience properties of the T3 -Bacterial Group. An example of potential pretreatment is adjustment of the pH of the wastewater by passing the wastewater across a sufficient amount of limestone to adjust the pH to be within the range defined by the resilience properties of the T3 -Bacterial Group.

While pretreatment of wastewater may be required for the T3 -Bacterial Group to be applicable, the resilience properties of the T3 -Bacterial Group allow enough variation in the environmental conditions or wastewater properties that conventional technologies for easily adjusting the wastewater properties exist. METHOD

Figure 1 illustrates a method (100) for producing a specific Bacterial Group based on the selection of a source location to harvest the source-bacterial-group, collection of the heterogeneous sample of condensed material and supernatant liquids in which the source-bacterial-group is entrained, and cultivation of the source- bacterial-group in a manner that enhances the desired heavy-metal resistance for HM or CHM to form the Bacterial Group while maintaining the resilience properties of the source-bacterial-group. All categories of Bacterial Groups are developed using the illustrated method. The specific category is based on the relationship of the source- bacterial-group and thus Bacterial Group to the wastewater treated as previously described.

The method for producing Bacterial Groups to treat wastewater associated with a specific site includes three steps: identification of the source location, collection of the requisite sample from the source location, and preparation of the Bacterial Group from the cultivation and enhancement of the source-bacterial-groups entrained in the source location sample.

I. Identification of the Source Location

Identifying a source location is the initial step in developing a Bacterial Group to treat wastewater associated with a specific site is identification of a source location within the specific site that has HM or CHM representative of the HM or CHM of concern in the wastewater, (step 1 10). A source location has a body of water continuously present. For example, an area within a waste impoundment pond that has a continuous head of water throughout the year can be considered a source location. Puddles or pools that temporarily occur after storm events or other events that produce water that eventually runs off or evaporates are not sufficient to produce the requisite source-bacterial-group and are not considered a source location.

Typically, the site that includes the body of water or a portion of a body of water that satisfy the continuous head criteria must be dormant to produce a reasonable expectation for identifying a source location. A dormant site has not been exposed to any man-made perturbations including but not limited to addition of waste or treatment chemicals for at least six months (e.g., at least nine months, at least one year, at least 15 months, at least 18 months, etc.). A dormant site can have experienced natural changes to the site including but not limited to addition of water through storms or streams. The area that has the continuous head of water also must contain HM or CHM between the average concentration and the highest concentration of HM or CHM observed within the polluted site to be considered a source location.

II. Collection of the Requisite Sample

After identifying a source location representative of HM or CHM

contamination, samples are taken from the source location (step 1 12). The samples of the sediments and supernatant liquids in the source location are collected such that the samples contain water that is present in sediment particles as well as the solution immediately above the sediment particles. In addition, if agglomerations of microbial life are present, it is desirable that the samples taken from the source location include such agglomerations.

Different sampling techniques can be used. The sampling technique must produce a heterogeneous mixture of sediment and supernatant liquid or a composite sample. Typically, a sample includes approximately one-third by volume wetted sediment and two-thirds by volume supernatant liquid. Composite sampling is typically not used for bacterial examination given the composites will not facilitate the display of the range and variation of bacteria suspended in solution separate from the range and variation of bacteria included in the wetted sediment. Composite sampling, however, is the preferred sampling technique for the METHOD given the objective of the sample is obtain a collection of all potential source-bacterial-groups without concern for the relative proportions of those source-bacterial-groups representing the distribution in the source location. Other sampling techniques that include the composite characteristics including gathering composite samples from multiple locations in an individual body of water or from multiple bodies of water across a site can also be used. Other than the atypical component of composite sampling, conventional sampling protocols for microbiological sampling should be followed. III. Cultivation/Enhancement of the Source-Bacterial-Group to Produce the Bacterial Group

After the requisite sample is collected, a Bacterial Group is produced from the source-bacterial-groups present in the sample using a cultivation/enhancement process that includes a gradual increase of a representative specie of the HM or representative species of the CHM in the cultivation medium (Step 114). The cultivation medium must not contain any substances that could potentially induce speciation of the bacteria or selective elimination of a particular species. The representative HM or CHM species concentration is gradually increased until the desired HM or CHM resistance level is acquired. The METHOD has been used successfully to produce HM or CHM resistances orders of magnitude higher than the contamination levels in the wastewater. This increased level of resistance facilitates the opportunity for incorporating HM or CHM contaminate concentration processes, like reverse osmosis, in the overall remediation process. A process for concentrating the contaminates reduces the total volume of wastewater for treatment in the bioreactor that incorporates the Bacterial Group. The potential for treating wastewater with higher concentrations of HM or CHM provides opportunity for treating larger flow rates of wastewater through a waste-water treatment facility with a relatively low flow rate bioreactor. a. Cultivation

Standard protocols for cultivating bacteria in a broth are followed with the exceptions duly noted in the following.

The sample is thoroughly mixed to maximize the concentration of source- bacterial-groups suspended in the solution. This mixing can be accomplished by hand through capping the sample then shaking and inverting the sample container several times, placing the sample container in a vortex mixer, or using other mixing techniques that agitate the sample to near homogeneity while mixing.

After mixing, the heavy granular components of the composite sample will fall to the bottom of the container. An inoculation loop is placed in the sample container and forced into the granular components until the loop is completely covered by the granular components. The loop is extracted from the granular components, through the supernatant liquid, and out of the sample container. If solid particulates from the granular portion of the sample adhere to the loop, they should not be removed from the loop. These granules can be transferred to the growth medium without affecting the desired results of the METHOD.

The loop is subsequently immersed in a complex medium that will support all types of bacteria indigenous to the site from which the sample was obtained. An example complex medium is Luria-Bertani (LB) broth. After the loop is immersed in the medium, gently swirl the loop to transfer the source-bacterial-groups and any particulates that adhere to the loop.

The source-bacterial-groups are allowed to grow in the inoculated broth for 24 hours. The bacteria and broth after the 24-hour period are considered the stock solution for the initial step of the subsequent enhancement component of the process. b. Enhancement

The following steps provide a general guideline for enhancing the heavy metal resistive properties of the source-bacterial-groups produced using the cultivation component of this process to ultimately produce the desired Bacterial Group. The essential component of the enhancement is increasing the concentration of the contaminants at a slow enough rate that the source-bacterial-groups can naturally evolve to resist the contaminants at the desired concentration. Successive generations of the bacteria will successively express higher levels of heavy metal resistance when those heavy metals are stress factors for previous generations. It is essential not to increase the stress level at a rate faster than the resistance level can increase through this generational evolution.

Step 1

A complex medium broth (e.g. Luria-Bertani (LB) broth) is prepared and a sufficient volume of a solution containing representative species of the HM (CHM) contaminate(s) in the wastewater is (are) added to the broth to create a solution with concentration(s) ten times the highest concentration(s) of HM (CHM) observed within the polluted site. For example, mercuric chloride could be used as a representative species of all the possible species of mercury in the wastewater. This initial broth with ten times the highest concentration of contaminates present in the polluted site is referenced as EB10. A sufficient volume of EB 10 must be produced to allow for sequential dilutions of the EB10 to form a broth that includes 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0.5 times the highest concentration(s) of HM (CHM) observed within the polluted site. These broth mixtures are referenced as EB9, EB8, EB7, EB6, EB5, EB4, EB3, EB2, EB 1, EB0.5. Each of these broth solutions are inoculated with the stock solution produced in the initial cultivation step.

Step 2

After 24 hours, the extent of bacteria growth in each of the eleven inoculated broth mixtures is determined using a growth correlation with the difference of turbidity measurements at the start and end of the growth period. The percent change in the concentration(s) of representative HM (CHM) in each inoculated broth mixture is determined through use of the appropriate EPA method(s). The inoculated broth mixture that has the highest initial concentration(s) of representative HM or CHM, shows maximum growth, and maximum conversion of the HM or HM to their elemental form is used as the inoculant in subsequent enrichment steps. For example, EB0.5, EB 1, EB2, and EB3 show comparable growth while there is diminished or no growth in the other assays. The percentage decrease in the concentration(s) of HM or CHM is approximately 90% for EB0.5, EB 1, and EB2 while the decrease is approximately 50% for EB3. Using the criteria listed, EB2 would be the inoculant for the subsequent enhancement steps.

Step 3

A broth solution that includes ten times the concentration of representative species as present in the inoculant identified in step 2 is created. Dilutions of this broth solution with 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0.5 times the concentration of the representative species in the inoculant identified in step 2 are created. Each of these solutions is inoculated using the inoculant identified in step 2. Step 2 is repeated using the assays developed in this step for EB 10, EB9, EB8, EB7, EB6, EB5, EB4, EB3, EB2, EB1, EB0.5.

Additional Intermediate Steps

Repeat the sequence of step 2 followed by step 3 until an assay that resists the desired concentration(s) of HM or CHM is (are) produced or an assay that demonstrates maximum resistance is obtained. The maximum resistance is identifiable when subsequent steps to increase the concentration of the contaminants in the growth medium result in diminished or no growth.