WO1998051786A1 - Reaction sites for microorganisms used to biodegrade contaminants and methods of use - Google Patents

Abstract

The present invention relates to a novel reaction site with selected microorganisms capable of biodegrading organic or inorganic contaminants. Contaminants are present in soils (solids), liquids or gases, wherein said contaminants include Federal Priority Pollutants, highly volatile organic compounds (VOCs), recalcitrant organic contaminants or inorganic contaminants such as ammonia or sulfides. The invention particularly relates to biodegradation of organic contaminants by microorganisms from the groups fungus, Nocardioforms/Actinomycetes and Pseudomonadacae, either alone or in combination, which biodegrade the contamination in either the solid/soil, gas/vapor or liquid/water phases; and the transformation of inorganic contaminants, such as ammonia by Nitrosomonas spp. and Nitrobacter spp. to nitrate with optional denitrification, and hydrogen sulfide to elemental sulfur by Beggiatoa spp. and/or Thiosphera pantotropha. Porous media and cord media are used to construct the reaction sites.

Description

REACTION SITES FOR MICROORGANISMS USED TO BIODEGRADE CONTAMINANTS AND METHODS OF USE Inventors: George Robert Whiteman and George Harry Whiteman _

BACKGROUND OF THE INVENTION

The present invention relates to novel reaction sites for microorganisms used to biodegrade contaminants. Fungus, and in particular, white rot fungus, is used at the reaction sites for the biodegradation of contaminants, such as contaminants that have been identified by the federal government as Federal Priority Pollutants, highly volatile organic compounds (VOCs) or recalcitrant organic contaminants. Fungus and bacteria are used in combination for some applications. The invention provides methods and compositions to biodegrade contaminants regardless of the environment in which they exist, namely in solids (soils), liquids or gases (atmosphere).

Inorganic contaminants such as ammonia or sulfides can be stabilized using the same reaction site apparatus with different microorganisms.

Protection of the environment has been a top priority issue in the United States for more than 30 years. Federal priority pollutants are contaminants which have been identified by the Environmental Protection

Agency as the most harmful contaminants to the environment. The magnitude of the problem caused by these most harmful contaminants led the EPA to research and make a Public Data Release called "Toxics Release Inventory" which is an annual summary of all toxic releases reported within certain guidelines. Many of these contaminants are either VOCs (highly volatile) and/or highly recalcitrant (that is contaminants resistant to biodegradation) organic compounds resulting in serious air pollution, and soil, drinking water, and groundwater contamination, as well as worker exposure problems. Contaminants have been associated with increased rates of mortality and morbidity, in particular due to carcinogenic exposure. In the last decade, the EPA has focused on certain toxic releases more than others, including solvents used as degreasers, cleaners, fabric scourers, diluents, extractants, and reaction and synthesis media. Much of the focus has been on reduction of contaminants as emphasized by the EPA Seminar Publication "Solvent Waste Reduction Alternatives" and the TRI 33/50 voluntary reduction program in which companies were asked to voluntarily subscribe to reducing toxic releases by 33% by 1992 and 50% by 1995. -

Unfortunately, reductions in toxic chemical releases have been brought about in response to the fact that the environment has already become grossly contaminated by toxic releases. Consequently, the task of cleaning up contaminated groundwater and soil, and reducing vapor emissions, in addition to finding satisfactory, economic alternative processes and cleanup techniques, is formidable.

Typical approaches to cleanup have depended upon the phase which is polluted [i.e., gas/vapor, liquid/groundwater or solid/soil]. Some methods for remediation of contaminated environments are listed in Table 1. The more reliable methods of remediation result in more complete removal or destruction of contaminants from the environment.

TABLE 1

Norris et al., state that:

[c]hlorinated solvents and their natural transformation products represent the most prevalent organic groundwater contaminants in the country. These solvents consisting primarily of chlorinated aliphatic hydrocarbons (CAHs), have been used widely for degreasing of aircraft engines, automobile parts, ~ electronic components and clothing the major chlorinated solvents used in the past are carbon tetrachloride (CT), tetrachloroethene (PCE), trichloroethene (TCE) and 1 ,1 ,1-trichloroethane (TCA). These compounds can be transformed by chemical and biological processes in soils to form a variety of other CAHs, including chloroform (CF), methylene chloride (MC), cis and trans-1 ,2,-dichloroethane (cis-DCE and trans-DCE), 1 ,1-dichloroethene (1 ,1 -DCE), vinyl chloride (VC), 1 ,1-dichloroethane (DCA) and chloroethane (CA).

Handbook of Bioremediation, CPC Press (1993).

Mineral spirits and oxygenated aliphatics are the most common non-chlorinated cleaning solvents and are often associated with groundwater and soil contamination. A potential solution to contaminant reduction is bioremediation, the use of life forms to convert contaminants to non-toxic or less toxic products. Just over a decade ago, most of these contaminants were considered unbiodegradable, that is, not amenable to degradation by means of life forms. HAZMAT World magazine, June 1992, reported that bioremediation was being used for remediating petroleum-contaminated soil and groundwater, and that bioremediation had been used at 22% of all Superfund Records of Decision. However, stripping, for example by vacuum extraction, is still by far the most popular and reliable method recorded because bioremediation is inapplicable for most clean-up sites. The present bioremediation technology is also expensive in many situations, such as where groundwater requires pumping before treatment or where soil has to be excavated for ex-situ treatment.

Moreover, while petroleum-based hydrocarbons have been shown to be biodegradable in groundwater by indigenous bacteria, more common recalcitrant contaminants, such as chloro-organics and solvents, are often toxic to the bacteria. As an example of toxicity, trichloroethene (TCE) inhibits anaerobes at a concentration of 150-50 mg/L, while aerobes are inhibited at 3 mg/L in air phase.

According to the Handbook of Bioremediation microorganisms can transform these contaminants by co-metabolism. This means other chemicals must be present to satisfy the energy needs of these microorganisms because the microorganisms cannot utilize the contaminants as the energy source. Often, however, the added energy source is inherently toxic or hazardous as well. Another problem facing bioremediation is that successful cleanup requires achieving low concentrations of contaminants, which is prohibitive for bioremediation using bacteria without continual supplementation of a carbon source as a co-metabolite to increase the concentration of microorganisms. In addition, the carbon sources are often hazardous in themselves because microorganisms used may require induction of enzymes by a substance structurally similar to the targeted contaminants called a homologue, which is often inherently toxic or hazardous. As a result, bioremediation to remove the targeted contaminant has resulted in some instances merely in the substitution of another contaminant into the environment. U.S. Patent No. 4,554,075 (the "'075 patent") entitled "Process of

Degrading Chloro-organics by White Rot Fungi" (Chang et al., 1985) claims the use of white rot fungus in the secondary phase of metabolism to biodegrade chloro-organics. In this secondary phase, nitrogen or carbon starvation is used to force the fungus to produce ligninase. This secondary phase requires an aerobic environment.

The reactor described in the '075 patent is a rotating biological contactor which allows the fungus to become immobilized on the surface of a plastic media. In practice, the reactor has failed because other indigenous microorganisms caused anaerobic areas to build up between the fungus and the medium causing the fungus to "slough-off which means that insufficient fungus was able to be maintained in a secondary metabolic state to accomplish bioremediation. Although the reactor of the '075 patent has not worked well, the use of white rot fungus metabolism in the secondary phase partially biodegrades chloro-organics, in particular chloro-phenols, by converting the aromatic chemicals to aliphatics. However, complete biodegradation to carbon dioxide, water and a new biomass has not been achieved.

Other biological approaches investigated to remediate various contaminants, including the use of anaerobic microorganisms, have failed-or have resulted in the undesirable build-up of toxic intermediates. These approaches have included the use of methanotrophs which use methane as a cometabolite resulting in expensive and problematic applications. Still other approaches require the input of nutrients to fuel the reactions which can lead to groundwater nutrient contamination. Yet another approach, bacterial metabolism, requires the presence of the contaminant in relatively high concentrations for biodegradation to occur. Biofilms, which use a mature and broad spectrum population of organisms, have been used for bioremediation. Problems include the need to supply nutrients and inefficient and non-reproducible combinations of microorganisms.

Because of the large number of contaminated sites and high costs of cleanup using conventional technology, government and industry must seek new, more effective, less expensive technologies. Despite every effort, economic cleanup has been an elusive goal. For example, National Defense magazine in March 1996 reported that a five-year "Superfund" program designed to cleanup 400 sites at a cost of $1.6 billion has mushroomed into a $30 billion project spread across 1 ,300 facilities. In addition, government and industry are hampered from selling property and assets due to the overwhelming liability of cleanup. For example, a company (Aerojet) in California sits on 20,000 acres of TCE contaminated land and is unable to sell the company, while another (Lockheed Martin) was recently fined $60 million to be applied towards cleanup and the City of Los Angeles is currently plagued with groundwater contamination of methyl butyl alcohol (a fuel additive). Bioremediation has become an increasingly important driving force in the search for a less expensive technology, but has been hampered by (a) high pollutant concentrations causing toxicity to microorganisms, (b) low pollutant concentrations resulting in insufficient food to establish a biological population, (c) pollutants being recalcitrant, and (d) the need to excavate contaminated soils or pump contaminated water. Alternative biological processes which overcome these problems and treat more contamination ~ in-situ are desired. Such processes would be useful in treating contaminated soils, drinking water, groundwater or waste process streams or as a retrofit to existing wastewater treatment plants.

SUMMARY OF THE INVENTION

The present invention relates to novel reaction sites for microorganisms to biodegrade contaminants. Reaction sites include supports for microorganisms such as a porous medium or a cord media on frames. Microorganisms such as white rot fungus are used for the treatment of contaminants, particularly for the treatment of certain contaminated soils (solids), liquids or gases where the contamination is caused by contaminants identified as Federal Priority Pollutants, highly volatile organic compounds (VOCs), or recalcitrant organic contaminants. For some applications both fungus and bacteria are used. Inorganic contaminants such as ammonia or sulfides are biodegraded by other types of microorganisms. These processes are useful in treating contaminated soils, drinking water, groundwater or waste process streams or as a retrofit to existing wastewater treatment plants. Contrary to previous approaches using bacterial metabolism alone, the present invention using fungus does not require a co-metabolite or homologue for induction of the non-specific enzyme systems responsible for biodegradation of the contaminants, and where contaminant concentrations are low, such as in groundwater, which requires a higher mass of microorganisms to achieve clean-up, a simple non-hazardous substrate other than the contaminant can be used to increase the mass of microorganisms, referred to as the biomass. Another aspect is that previous problems, such as the expensive procedure of pumping out water from a contaminated site to be biodegraded, are avoided by locating reaction sites with microorganisms directly in the material to be cleaned up. In an embodiment, reaction sites in the form of tubes to which microorganisms attached, are lowered through a monitoring well, thereby immersing the microorganisms into the contaminated groundwater and bringing the microorganisms and contaminants into contact at the reaction site. Advantages of the invention are that sites are removable and may be upgraded with new microorganisms. In another embodiment, reaction sites with microorganisms are situated to trap and bioremediate contaminated vapors. Reaction sites in the form of looped cord media which immobilize microorganisms are also suitable. Care must be taken to avoid competition from indigenous microorganisms. Looped cord media may be particularly suitable for applications that are low competitive environments in terms of indigenous bacteria, e.g. single pass lagoons and groundwater.

Also, the controlled inoculation of microorganisms of the present invention onto reaction sites minimizes competition.

In particular the invention relates to biodegradation of organic contaminants by microorganisms from the groups of fungus, used alone or in combination with each other or with bacteria, such as Nocardioforms/

Actinomycetes and Pseudomonadacae to biodegrade pollutants in either the solid/soil, gas/vapor or liquid/water phases. Target contaminants for this invention include chlorinated solvents, surface cleaning compounds; volatile organics; and all organic Federal Priority Pollutants. Preferred fungus include White Rot Fungus, Brown Rot Fungus, Black Rot Fungus, Candida,

Aspergillus or Mucor. The synergistic Actinomycete/Nocardioform composition comprises aerobic microorganisms, including at least one of Nocardia, Rhodococcus, Actinomycete or Streptomycete. The synergistic bacteria suitable for invention are aerobic microorganisms from the Pseudomonadacae, such as a Pseudomonad.

The invention also relates to transformation of inorganic contaminants, such as ammonia to nitrate and nitrate, by using reaction sites with Nitrosomonas spp. and Nitrobacter spp. with optional denitrification, and the transformation of hydrogen sulfide to elemental sulfur by, for example, Beggiatoa spp. and/or Thiosphera jz)antotropha.

The reaction sites include fungus and/or bacteria in contact with them. Biodegradation is accomplished generally proximal to the sites. Preferably, the microorganisms may be removed from the water or vapor area by means of the reaction sites.

This invention also relates to a process for improving the - environmental characteristics of certain solids/soils, liquids or gases contaminated by Federal Priority Pollutants, VOCs, recalcitrant contaminants, or inorganic contaminants such as ammonia and hydrogen sulfide. The invention relates to methods and compositions for treatment of gas phases from soil vapor extraction, groundwater air sparging or process waste streams or wastewater treatment systems; liquids from drinking water, groundwater, soil washing or process waste streams or wastewater treatment systems; and solids for regeneration of contaminated activated carbon or decontamination of soils.

For the reaction sites, for example, a porous reactor is optionally incorporated in an aquifer by using lengths of silicone tubing, and/or in the monitoring well (using Vyon tubes) and also to transfer VOCs into the gas phase for biological treatment thereby minimizing/eliminating pumping of groundwater. It may be desirable to heat contaminated air stripped from the groundwater phase in a gas phase reactor. For soil treatments, the gas phase from soil vapor extraction cleanup techniques may be heated. A soil bank of the type used in conventional cleanup techniques is also a target of the invention, but unlike conventional techniques, this invention treats both the contaminated air and soil together at the soil bank sites.

Yet another aspect of this invention is to immobilize a specific fungus and/or other microorganisms inside porous reaction sites, which can be sealed, if required, to prevent or minimize entry of indigenous microorganisms from the treatment environment while allowing entry of contaminants.

Minimizing pore size is another means to minimize indigenous bacterial growth. The looped cord media manufactured by BioMatrix Technologies is suitable for use as an immobilization media for various specific microorganisms, including White Rot Fungus as described herein. The applications of this reaction site include BOD, COD, TOC, TC, specific organics or removal in papermills or other plants producing organic color pollutants. In addition, nitrifiers may also be immobilized to confer nitrification properties to a waste treatment plant. There are broad general applications- including the possibility of groundwater remediation of organic solvents either in situ by lowering the medium with microbes immobilized on the medium, or by pumping the water out of the ground into a reactor to treat the liquid phase. Air stripping the organics out of the ground and using the looped cord media for immobilization of the microorganisms (including WRF) for treatment of volatile organics in the gas phase, is another aspect of the invention.

The methods of the invention involve degrading organic contaminants either in soils, liquids or gases, and comprise the steps of producing a hyphal mat in the primary phase of growth, where the concentration of fungus is increasing due to rapid growth or metabolic state (initially without secondary metabolism) from a specific fungus spore form, alone or in combination with a specific synergistic actinomycetes or specific bacteria, or collectively in a consortium or mixed group of microorganisms; providing an electron acceptor for aerobic metabolism and degrading the organic contaminants. The hyphal mat may be produced either on site or in a laboratory and transported to a treatment system, or some combination thereof. At a treatment system this forms a reaction site. Suitable electron acceptors include oxygen, either from air or as pure oxygen, or derived from H₂0₂, CaH₂0₂ or nitrate.

The methods and compositions of the present invention are useful in treating contaminated environments, such as where the VOC has contaminated drinking water, groundwater, a process waste (air or water) stream or a wastewater treatment system. An aspect of the invention is to use a specific microorganism from a group of fungi in primary metabolism (producing hyphae) alone and/or in combination with other microorganisms, to completely biodegrade solvents, such as mineral spirits, or chlorinated or oxygenated aliphatics, under aerobic conditions in both the liquid and, perhaps more importantly, the gas phase without the build-up of harmful intermediates.

In degradation of some contaminants, e.g. those of high molecular weight, fungus provides transformed products which are used by complementary bacteria such as an Actinomycete/Nocardioform and/or

Pseudomonadacae family, which uses the transformed products under aerobic conditions. "Complementary" means bacteria interacting with fungus to achieve or improve biodegrading bacteria with products allowing fungal reactions to proceed. Induction by cometabolites or homologues is not required in fungus, but enzyme production can be initiated by increasing cell mass. Allowing the process to occur naturally in mixed contaminant environments or where higher biomass concentrations are required is a faster reaction. Simple carbohydrates are used to increase biomass density which will result in induction because of phase of growth to a C-N starvation state. Another aspect of the invention is to use primary substrates for a fungus in the primary phase of growth and/or for a fungus in the secondary phase of growth.

Still another aspect of this invention is to use Nitrosomonas for the oxidation of ammonia in gaseous or liquid phases to nitrite, and, to subsequently use Nitrobacter for the oxidation of nitrite to nitrate, where retention of sufficient mass of nitrifiers is a problem used alone or in combination with a denitrifying microorganism to convert nitrate to nitrogen gas, such as Pseudomonas spp. or Thiosphera pantotropha.

Another aspect of this invention is to use fungus which produce non-specific enzymes without the need for induction by a similar toxic substance and therefore can use a non-hazardous carbon source during initialization of growth. In addition, as the contaminant concentration falls, a fungus will continue to bioremediate as the non-contaminant induced enzyme systems continue to work. In summary, aspects of the invention include:

(i) No toxic intermediates are formed (anaerobic reactions result in toxic intermediates such as vinyl chloride from the breakdown of chlorinated solvents). (ii) Essentially complete destruction of contaminants such as TCE is achieved.

(iii) Reactions are aerobic not anaerobic therefore, reaction times are faster. (iv) White Rot Fungus is resistant to high levels of (TCE) pollutants which are usually toxic, therefore no dilution is required.

(v) Treatment results in lower concentrations of contaminants than normal bioremediation processes.

(vi) No nutrients (e.g. nitrogen or phosphorus) need to be added to the aquifer. Usually groundwater has to have nutrients added, which is an environmental concern. White Rot Fungus microorganisms are pregrown before being added to the aquifer.

(vii) Microorganisms do not contaminate the aquifer because they are recovered in the receptacles (reaction sites such as tubes or cord media).

(viii) Non-specific enzymes, such as ligninase are used, which means (a) other pollutants will be degraded, (b) an easily metabolized carbohydrate can be used to establish a large biomass for treatment without loss of the functional ability to biodegrade the contaminants. (ix) White Rot Fungus may be resistant to metals or other compounds which allows clean-up of situations where organics and metals are mixed, for example, Occidental Chemical carbon tetrachloride and mercury.

(x) Easy to implement in groundwater with two in-situ options to avoid pumping of water out of the aquifer.

(xi) Useful to retrofit existing waste water treatment systems and confer a broad capability to biodegrade pollutants, such as absorbable organic halides (AOX), chemical oxygen demand (COD), biochemical oxygen demand (BOD), total organic carbon (TOC), total carbon (TC) or color in papermill effluents or a specific capability such as nitrification in which ammonia would be oxidized to nitrate, then to nitrate. This could avoid millions of dollars in capital expenditures by making use of existing facilities. The process is cost effective and easy to implement in groundwater because this is an in-situ treatment that does not require pumping of water out of the aquifer (tubes are placed in the well or water is blown out and treated in the gas phase).

The invention may be applied to a gas phase, e.g. air stack, groundwater air sparging, soil vapor extraction, and activated carbon; a liquid phase, e.g. groundwater (retrofit an air stripper), wastewater, and activated carbon; and soil. Soil may be inoculated with WRF/microbes in a soil bank by means of an injection.

A closed loop system is preferred over reinjection of treated gases or return of treated water into the environment until complete destruction is achieved.

The reaction sites of the present invention overcome many of the problems in bioremediation.

Definitions: Microorganism as used herein includes fungus, bacteria and other biodegrading small unicellular organisms.

Retrofit is a modification of an existing waste treatment facility or system.

A System is a part of a facility.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a gas chromatography (GC) graph of TCE without fungus. FIG. 2 is a gas chromatography (GC) graph of the level of TCE in an untreated control after 10 days.

FIG. 3 is a gas chromatography (GC) graph of TCE 5 days after adding TCE to the mature fungus.

FIG. 4 is a gas chromatography (GC) graph of TCE in a sample grown in the presence of fungus for 15 days.

For FIGS. 1-4, gas chromatography (GC) is on the Y axis; the initial level of TCE is on the X axis. FIG. 5 is a looped cord media. DESCRIPTION OF THE PREFERRED EMBODIMENT

In one embodiment of this invention, specific microorganisms are immobilized on or inside a reaction site, such as a porous medium, wherein the specific microorganisms can be blended into a microcosm or maintained separately at a single reaction site or in a series of sequential reaction sites.

The porous medium should contain the microorganisms of the present invention while barring others. ~

The microorganisms are grown under environmental conditions promoting rapid growth of a fungal hyphal mat and biodegradation of the organic contaminants. The production of the hyphal mat and the degradation of the organic contaminants use cellulosic materials or a simple carbohydrate, such as molasses, fructose, corn starch, beer rejects or glucose to provide an energy source for the fungus. A key advantage of this invention over earlier bioremediation efforts is the use of a simple carbohydrate as the energy source for microbe production. Earlier approaches required the use of carbon sources of a molecular structure similar to the targeted contaminants e.g., homologues. Use of these carbon sources or co-metabolites contaminated the environment often as badly as the targeted contaminant. The process of this invention can be used to degrade even the most toxic organic contaminants, such as volatile organic compounds (VOCs), recalcitrant pollutants, or Federal Priority Pollutants, or more broad measures of pollution such as biochemical oxygen demand (BOD), chemical oxygen demand (COD), total and absorbable organic halides (TOX/AOX) and color.

In the present invention, the action of the fungus in combination with an Actinomycete/ Nocardioform or bacteria, or collectively, to degrade the organic pollutant occurs in either the primary or secondary phase of growth of the fungus.

Identified fungi, alone or in combination with the bacteria that are suitable for the biodegradation of organic Federal Priority Pollutants, VOCs, and other recalcitrant contaminants include: Fungi Actinomycetesl Pseudomonadacae

White Rot Fungus Nocardia Pseudomonas

Brown Rot Fungus Rhodococcus Xanthomonas

Black Rot Fungus Actinomycete Fraturia

Candida Streptomyces Zoogleoa

Penicillium

Mucor -

Aspergillus

A preferred fungus is White Rot Fungus Phanerochaete chrysosporium.

A preferred Actinomycete is Rhodococcus spp. A preferred Pseudomonadacae is Pseudomonas spp. The process is performed by immobilizing the microorganisms inside a porous medium, on a porous medium, or on activated carbon. Other media suitable for use in the air phase/vapor systems are bark, wood chips, peat, chicken manure, rye or wheat husks, soil or commercially available plastic media or polyurethane foams. The porous reaction site is shaped to maximize the surface area. Suitable shapes include tubular, concentric tubes or in sheets. The porous reaction site may be made from an extruded high density polyethylene, such as Vyon, or silicone. A preferred media is BioMatrix Looped Cord Media. As used, the cord media systems require "an adequate supply of biodegradable COD ... a consistent supply of nutrients," BioMatrix, page 16.

Porous medium pore size ranges from about 0.25-100μ. For total exclusions of microorganisms the pore size should generally be <0.5μ; moderate exclusion, the pore size should be about 0.5-20μ, and for gross exclusion, the pore size should are about 20-100μ. The reaction sites may be used as in situ fermenters so as to release some of the microorganisms into the bulk waste-water, i.e. not at the reaction site, in which case pore size may be from about 20-1000μ to allow leaching.

The microorganisms are grown on or inside a porous medium in the presence of a carbon source, such as molasses or a cellulosic material and certain nutrients, such as e.g. nitrogen. The microorganisms are immobilized on or inside the porous medium using at least one or more of the following steps: i) sterilizing or sanitizing the porous medium; ii) lining one side of the porous medium by pouring liquid enrichment media inoculated with the specific microorganisms, alone or in consortia, with agar, which on cooling sets into an agar gel; iii) lining one side of the porous medium by pouring liquid- enrichment media inoculated with the specific microorganisms, alone or in consortia, which on cooling remains a liquid; iv) filling the porous medium with commercially available microorganisms or a cellulosic material on which the fungus has been inoculated; v) incubating the inoculated porous medium at a temperature and humidity required to obtain desired growth of the microorganisms; vi) immersing the inoculated porous medium into a liquid fermentation system to obtain desired growth of the microorganisms; vii) combining activated carbon in the immobilization process to enhance initial removal of the contaminant. The microorganisms grown inside the porous medium are introduced into an environment containing organic contaminants, such as a substance for surface cleaning of metal objects, VOC, a chlorinated aliphatic compound such as ethyl chloride, CT, PCE, TCE, TCA, CF, DCE, DCA, VC and CA, or an oxygenated aliphatic compound, such as acetone, ethyl acetate, ethyl alcohol, isopropyl alcohol, methyl alcohol, methyl ethyl ketone and methyl isobutyl ketone or mineral spirits. The organic contaminants are exposed to the microorganisms and the microorganisms degrade said organic contaminants.

A preferred immobilization media is the BioMatrix Looped Cord Media. Looped cord media is constructed of braided linear composite threads. A strong cord backbone made of "space age" materials provides the base for integral biofilm fixing loops. Thousands of feet of media are strung on aluminum, PVC or stainless steel support frames in a system referred to as a media frame. Groups of frames or platforms are placed in a wastewater treatment basin where waste digesting microbes become fixed to looped cord media. Fixed bacteria create a stable biomass that provide enhanced BOD removal and an established source of nitrifiers for ammonia reduction. "Biofilms consist of living cells, dead cells, and cell debris in a matrix of extracellular polysaccharide (glycocalyx) attached to looped cord." (Bishop, P.L. 1995)

Structural characteristics of looped cord media influence the rate of biomass complexing and the initial microbial population distribution. Loop size and "textile" characteristics such as roughness and the free energy of cord thread chemistry are factors affecting biomass adhesion. (P.A. Wilderer 1989).

Looped cord media frames are economical, compared to many other hybrid technologies, and they are simple to install and maintain. Installations of looped cord media platforms require little or no change to wastewater treatment basins into which they are retrofitted and in most cases installation does not require plant operation to be interrupted.

BioMatrix, page 3. Indigenous bacteria in a waste treatment plant will attach to looped cord media and become fixed. The disadvantages of this type of fixation system are:

(i) the microorganisms are non-specific and may not provide the desired treatment; and (ii) selected microorganisms cannot be separated from competition by indigenous microorganisms.

Although tubed media somewhat overcomes the competition aspects of the non-selective, non-specific properties of the looped cord media, there are certain circumstances, where competition between microorganisms may be low, such as in single pass lagoons or aerated stabilization basins. Unlike activated sludge, biomass/microorganism concentrations are extremely low in these type of systems. In such cases, looped cord media is inoculated with specific microorganisms and used to confer specific treatment compatabilities.

A culture of microorganisms, preferably a pure culture, is placed in a lab scale fermentation facility and grown without conventional fermentation, preferably in a porous medium so the desired microorganisms can't get out, concentrating them; at the same time, extraneous bacteria do not get in. The microorganisms are then transported to the location where they are to be used. Retrofitting existing facilities with the reactions sites of the present invention avoids building a reactor vessel which is usually where conventional engineering starts: i.e. other methods of immobilization are used such a fixed film reactors. In retrofitting of the present invention, an existing reactor is used and the immobilization process is added to it. A new reactor is not required. -

Cord can be used on frames or with a pulley system applied to a range of treatment systems including aerobic, anaerobic or facultative systems. Such systems include activated sludge, single pass lagoons, stabilization or polishing ponds. The cord can be inoculated with specific microorganisms to provide specific treatment characteristics to the treatment system. Sections of cord may be dropped inside a site from a roll, then wound up again. Removal of the media from time-to-time is an important aspect of ensuring competition from indigenous microbes does not cause loss of the desired treatment characteristics. Such characteristics may include BOD, COD, TOC/TC, color or nitrification in a single pass lagoon, or removal of phosphorous.

Specific applications include use of the invention for pulp and papermill waste, for BOD, COD, AOX and/or color treatment systems. Polymers

(polyamine) may be used to pretreat such effluents to remove high MW fractions, e.g. >10,000 daltons while lower MW e.g. <10,000 daltons are removed biologically. Effluents, such as bleach plant effluents, may be treated at the source. Retrofitting systems for nitrification, recalcitrants and treatment of Federal Priority Pollutants is an aspect of the invention.

Table 2 shows applicability of the methods and compositions of the present invention to three phases of contaminants.

Treatment of Air/Gases

More specifically, a method for the treatment of organic and inorganic contaminants in gas phases from soil vapor extraction, groundwater air sparging, process waste streams or wastewater treatment systems, includes the following steps: 1. Bringing the contaminated material into contact with a reaction site, for example by passing the contaminants, combined with air or pure oxygen as an electron acceptor, over the surface of immobilized microorganisms, resulting in biodegradation of the majority of organic pollutants or transformation of the inorganic contaminants;

2a. Recycling the gaseous contaminants alone or combined with air or pure oxygen as an electron acceptor to replenish the aerobic electrons in gases, back into the contaminated site making a closed loop system; b. Further reducing or minimizing the level of gaseous contaminants by passing the gases through an activated carbon bed prior to final discharge of the contaminant into the atmosphere, to substantially eliminate residual contamination, or c. Reducing or minimizing the level of contaminants present by introducing the contaminants into a soil bank.

Organic contaminants are biodegraded by immobilizing fungus alone or in combination with microorganisms belonging to the families Nocardioform/Actinomycete and/or Pseudomonadacae on or inside a medium through which the pollutants can pass, but indigenous microorganisms are prevented or minimized from entering, while the medium also acts to concentrate the microorganisms and prevent their loss into the treated effluent. Suitable media include porous or cord media, for example looped cord media. The fungus can be used for treating VOCs in the primary phase of growth and for other contaminants requiring secondary metabolism in conjunction with one or more other microorganisms from the

Nocardioform/Actinomycete and/or Pseudomonadacae families. The fungus may be selected from White Rot Fungus, Brown Rot Fungus, Black Rot Fungus, Mucor, Penicillium and Aspergillus. The Nocardioform/Actinomycete are selected from Nocardia, Rhodococcus, Actinomycetes or Streptomycetes and the like. The Pseudomonadacae are selected from Pseudomonas,

Xanthomonas or Zoogleoa, and the like. Inorganic contaminants, such as ammonia are transformed into nitrite and/or nitrate by immobilizing Nitrosomonas spp. and Nitrobacter spp., respectively, and passing the gases over the surface, or by quenching the gases with water to dissolve the ammonia and subsequently passing this liquid over the surface.

Inorganic contaminants such as sulfides are transformed to elemental sulfur by immobilizing one or more of Beggiatoa and/or Thiosphera ^~~ pantotropha on or inside the medium.

Treatment of Water/Liquids In addition, the invention comprises a method for treatment of organic and inorganic contaminants in liquid phases from drinking water, groundwater, soil washing, process waste streams or wastewater treatment systems wherein the method includes the following steps:

1. Bringing contaminated liquids and the biological reaction site into contact by putting the liquid over the reaction site or the microorganisms over the liquid, or having both flow directions in a countercurrent system, for example by passing the contaminants, combined with oxygen or nitrate as an electron acceptor, over the surface of the specific immobilized microorganisms, resulting in biodegradation of the majority of organic contaminants or transformation of the inorganic contaminants;

2a. Recycling the liquid contaminants alone or combined with oxygen or nitrate as an electron acceptor to replenish the aerobic electron acceptors back into the contaminated site, making a closed-loop system; b. Discharging the biodegraded liquid directly into the environment; or c. Further reducing or minimizing the level of contaminants present by passing the liquid through an activated carbon bed prior to final discharge.

The selected immobilized microorganisms are applied to a medium, preferably a porous or cord medium, designed to maximize surface area, such as tubular, concentric tubes, looped cord in a frame or sheet shape forms. The preferred medium will depend on the application, for example in-situ groundwater remediation may use silicone tubing filled with the specific microorganisms and lowered through a monitoring well into an aquifer. A second approach is to have tubes made of extruded high density polyethylene, such as Vyon, or other porous material, containing the immobilized microorganisms placed inside the well housing . This second approach can also be used to retrofit existing wastewater treatment systems and confer broad (BOD, COD, TOX, AOX, color) or specific biodegradation or transformation capabilities by suspending such tubes at the optimum angle to the vertical and/or horizontal plane of flow to obtain maximum contact between the liquid and reaction site in the secondary treatment system, including where appropriate, putting the reaction sites in a secondary clarifier. Suspension of the tubes can be done for example on a reel or fish and bait type system with floats and weights to obtain the optimum angle of suspension. The capital cost of such a system is estimated to be less than one tenth that compared to a conventional extension of the treatment facility involving additional tanks.

A third approach involves sparging of air to increase aeration, turbulence and to encourage volatilization of organics which can be treated in a gas-phase reactor or reaction site at the surface where oxygen can be introduced to maintain aerobic conditions with at least 2-5% oxygen in the gas phase. The gas can be recirculated to create a closed loop system thereby generating no waste stream. The gas can be recirculated with the addition of oxygen as an electron acceptor to encourage further aerobic activity. Oxygen and/or nutrients such as nitrogen and phosphorus may also be introduced into the tubing and thereby to the microorganisms. This process avoids pumping water and creates a closed-loop with three points of biological treatment e.g., silicone tubing down well, tubes in well, gas into reaction. Unlike other bioremediation processes, the majority of microorganisms of the present invention can be removed after treatment by means of the reaction site without further contaminating the water system.

Inorganic pollutants, such as ammonia, are transformed by immobilizing on the reaction site nitrifying microorganisms such as Nitrosomonas spp. and Nitrobacter spp., which are both slow growing bacteria, and concentrating them on a porous medium, thus preventing or minimizing their loss into the treated effluent. More specifically, immobilization reduces loss into the environment. As a result, growth of the microorganisms exceeds their loss and their concentration increases and hence the efficiency and capacity to remove the contaminant.

The immobilized nitrifying microorganisms are placed into an existing- wastewater treatment system, in a cooling tower process loop, fish farm holding tank, or fish tank used in the home in order to achieve nitrification. This nitrification process can be carried out in conjunction with denitrification using a Pseudomonas, such as P. dentrificans, or another denitrifying microorganism such as Thiosphera pantotropha. Obtaining rapid nitrification in fish farming and in the home market during transfer of fish is vital because ammonia is highly toxic to fish. Obtaining nitrification in municipal and industrial wastewater treatment systems often involves the expenditure of large amounts of capital because nitrifiers are slow growing and do not compete well with indigenous bacteria. Immobilization using a porous or cord or other suitable medium overcomes these problems and, unlike other approaches, can be used to retrofit existing systems without expansion of the facilities. Such capital costs can be at least twice the capital cost of a facility not requiring nitrification. The efficiency of a waste treatment system depends on the concentration of biomass/microbes and residence time, therefore slower growing microbes which may be washed out or require longer residence time in order to remove specific contaminants can be concentrated in the tubes to combat wash-out. Thus by increasing concentration of microbes the residence time does not have to be increased in an existing waste treatment facility, which is traditionally accomplished by expansion of the facilities by building more treatment tanks or reactors. The latter traditional option is extremely capital intensive. In Japan where more and more people are moving into cities the quantity of sewage flowing into rivers and receiving waters has increased. As in many countries, Japanese engineers are faced with costly expansion of their overloaded metropolitan wastewater treatment facilities. Land costs and problems associated with uninterrupted service have pressured Japanese engineers to find treatment plant retrofit solutions. One retrofit solution that has been put to use in more than 500 facilities is the installation of biomass fixing looped cord media placed in the aeration basin(s) of a treatment facility. The looped cord biomass fixing process was discovered by a

Japanese aquaculturist in the 1970's. An observant fish farmer noticed that water quality had dramatically improved in a fish holding pond where a fish net had been placed and forgotten. Increased surface area created by the fish net provided a holdfast for wastewater digesting microbes and subsequently more biomass for wastewater digestion. Japanese scientists perfected the technology by expanding the surface area of a netlike structure and looped cord media was developed.

In 1994, BioMatrix Technologies (BMT) a US based company, began development of advanced looped cord and now offers looped cord media in a variety of constructions for a number of wastewater treatment applications. BMT looped cord has been extensively tested in the laboratory and in full scale installations. BMT looped cord frames, on-site fabrication, installation and maintenance have been developed with the facility operator in mind.

BioMatrix, page 4.

Inorganic contaminants, such as sulfides, are transformed by immobilizing one or more of Beggiatoa and/or Thiosphera pantotropha on or inside a reaction site formed by a porous medium acting as described above.

Treatment of Soil/Solids

This invention further encompasses a process for the treatment of organic pollutants on solids for prolonging the life or the regeneration of contaminated activated carbon or decontamination of soils by using specific microorganisms (members of the Fungus, Actinomycete/Nocardioform,

Pseudomonad families, for example) immobilized on activated carbon. The life of the carbon is extended as bioremediation degrades the contamination. Bio-regeneration is achieved by spraying a liquid suspension of the microorganisms onto the activated carbon and providing conducive environmental conditions for biodegradation. Such environmental conditions include at least adequate oxygen to maintain an aerobic environment (2-5% at least), neutral pH range, and temperatures in the range of 40-95°F. Examples

Example 1 : In vitro Demonstration of Biodegradation of TCE Contamination

1. In a liquid phase study the initial concentration of TCE was 1 ppm TCE in liquid phase. The gas chromatography traces show degradation without buildup of intermediates. Complete degradation was achieved.

2. A gas phase study was done. 10mL pure TCE in 40mL water was allowed to evaporate at 25°C in a closed chamber and reach equilibrium. Plates containing minimal media (no carbon, only non-carbon containing nutrients) were inoculated with WRF. After 10 days, growth of several cultures was noted, showing that White Rot Fungus was biodegrading TCE in the primary phase of growth to be a carbon source for cell growth.

Example 2: Gas Phase Biodegradation Using Technology in the Field Reaction sites may be put in a reactor, i.e. a combination of sites, vessels, and support structures, as follows: a) Forcing gas past WRF and/or bacteria using a plug flow design. b) Selecting microorganisms, such as white rot fungus. c) Preparing and initiating growth of homogenous, or a mixture, of selected microorganisms. d) Selecting one or more tubes made of extruded, high density polyethylene, such as called Vyon manufactured by Porvair in the United Kingdom, silicone, or other porous medium. The tube(s) should have diffusion characteristics which allow flow of contaminants to pass in and out of the tube, while immobilizing

White Rot Fungus with or without bacteria in high concentrations. e) Alternatively, selecting a cord medium. f) Growing microorganisms on or into a hyphal mat on agar inside a tube or on cord media, under sterile/semi-clean conditions, thereby immobilizing them within the tube or cord media with little ability of non-target/indigenous microorganisms to penetrate the tube. A disadvantage of cord media is that non-target microorganisms could penetrate and replace the inoculated links. To void this problem, the frames are designed (at the site) so that each frame consists of sections of (e.g., 8-10 a 1-4/R.) which can be removed/replaced in the field. g) Removing and/or replacing the tubes or cord media to achieve biomass wastage, h) Preferably using the wasted biomass as inoculum for soil - remediation or another reactor. Additionally, the tubes or other reaction sites can be used independently of a dedicated reactor for application to existing wastewater treatment systems where the tubes or other reaction sites are laid out in rows or suspended vertically. Other types of microorganisms such as nitrifiers can also be immobilized in this manner and thereby convey their metabolic capabilities to a wastewater treatment system. This process can be used to remove a number of contaminants including sulfur compounds, ammonia as well as general other specific contaminants and broad measures of contaminants such as BOD, COD, TOX, TC/TOC, AOX and color. Such a process for the broad measures of contaminants would have application to the treatment of pulp and papermill wastewaters where new legislation referred to as the "Cluster Rules" combining air and water regulations will be promulgated in 1997 forcing mills to spend 10s of millions of dollars in the process side using conventional technologies, such as chlorine dioxide bleaching or oxygen delignification, to reach the same treatment objectives which can be achieved in some cases alone and in others in combination with a lesser capital expenditure in process, but with overall a fraction of the capital costs using the reaction site technology — perhaps one tenth the capital costs.

Example 3: Liquid Phase Biodegradation Using Reactor Technology a) Preparing a plug flow design for tubes where flow goes over single faced tubes or through the middle or both sides of concentric tubes. b) Selecting WRF with or without bacteria. c) Preparing and initiating growth of the homogenous microorganisms, or a mixture of selected microorganisms where diffusion is from the outside into the tube. d) Selecting one or more tubes made of extruded high density polyethylene, such as Vyon manufactured by Porvair in the United Kingdom, silicone, or other porous medium. The tube(s-) should have diffusion characteristics which allow flow of contaminants to pass in and out of the tube, while thereby immobilizing microorganisms in high concentrations. e) Growing microorganisms into a hyphal mat on agar inside a tube under sterile/semi-clean conditions, thereby immobilizing them within the tube with little ability of non-target/indigenous microorganisms to penetrate the tube. f) Removing and/or replacing the tubes to achieve biomass wastage, g) Preferably using the wasted biomass as inoculum for soil remediation or another reactor. Additionally, the tubes can be used independently of a dedicated reactor for application to wastewater treatment systems where the tubes are laid out in rows or suspended vertically. Instead of tubes, frames of looped cord media are used. Other types of microorganisms such as nitrifiers can also be immobilized in this manner and thereby convey their metabolic capabilities to a wastewater treatment system. Preventing competition from indigenous microorganisms is not an insurmountable problem. Frames of looped cord media are used. Such frames can be modified to consist of removable sections every 3-12 inches apart. These are replaceable by new sections containing specific microorganisms in order to renew the inoculation reaction site or confer different treatment capabilities by adding new microorganisms.

These types of systems where reaction sites are used and replaceable allow continued upgrading of the system with more effective microorganisms or different microorganisms to meet the changing nature of conservation from a production facility.

Example 4: Solid Phase Biodegradation Using Soil Bank Technology Contamination of soil usually results in groundwater contamination and will therefore be required whenever groundwater is contaminated. The method of soil treatment using bioremediation will depend upon the soil type and other factors. In certain cases, the VOC fraction can be removed by soil vapor extraction and treated in a vapor phase reactor described herein. In other cases, in-situ treatment may be accomplished by introducing fungal spores into the soil and using the soil as the reaction site. The soil may be extracted if highly compacted to expedite remediation and mounded into a soil bank as per traditional methods. The soil can be inoculated with the microorganisms during preparation of the soil bank. Such soil banks often require leachate capture and recirculation systems, as well as forced aeration. Unlike traditional bioremediation, when using fungi to bioremediate, the temperature of the soil bank may tend to rise beyond the mesophilic optimum range of 90-105°F and require a cooling system to prevent thermophilic composting occurring by passing air or extracted vapor through the soil bank or cooling with groundwater. Such heated air, vapor or groundwater can then be used to heat gas or liquid phase reactors on the same site or preheat the influent prior to treatment in these systems in order to optimize the operating temperatures of these processes.

Depending upon the indigenous populations of fungus and other microorganisms, such as bacteria, the site can be supplemented with the missing component microorganisms or stimulated by introducing an easily metabolizable carbon source along with nutrients, such as nitrogen and phosphorus, to simulate the development of the hyphal mat and initiate breakdown of contaminants. Where the contaminants are lower carbon length chlorinated or oxygenated alcohol's such as less than 10 carbon atoms, nutrient stimulation may be sufficient to establish the hyphal mat of an indigenous population and subsequent contaminant biodegradation. In cases where the soil structure is compact, such as with a clay, the soil may be inoculated with pre-inoculated reaction sites containing fungus alone or combined with other microorganisms, on a reaction site such as bark chips, hay, straw, grass, spent barley husks from brewing, chicken manure or other inexpensive cellulosic materials.

In all such applications described for soil decontamination using bioremediation, the use of fungus in combination with other microorganisms- or alone, whether deliberately introduced or through stimulation of the indigenous population, are all useful for biodegradation chlorinated or oxygenated solvents, mineral spirits or other Federal Priority pollutants, such as poly-chlorinated biphenyls which are extremely recalcitrant and expensive to remediate conventionally.

Example 5: Preliminary Experiments in the Degradation of Trichloroethylene by the Wood-Degrading Fungus Phanerochaete chrysosporium

INTRODUCTION

Organic solvents can be useful for a multitude of purposes. An example is trichloroethylene (TCE), which is used as a cleaning solvent in any number of processes. However, these compounds also have a negative aspect in that they are quite toxic and can be very persistent in the environment. This is especially true of organic halides. Removal of organic solvents from waste streams may be difficult and expensive, and disposal of these contaminants requires special facilities and precautions.

In the 1970's and 80's, studies of biological organisms capable of degrading a component in wood- lignin- were accelerated in hopes these organisms could assist in the chemical- and energy-intensive processes required to produce paper from wood. During these studies, it was determined that certain wood-degrading fungi — most notably Phanerochaete chrysosporium — were also capable of degrading the wastewater effluents from pulp mill bleach plants. These effluents contain byproducts of lignin degradation which, like organic solvents, are toxic and difficult to treat. In fact, the basic repeating unit in the chemical structure of lignin is very similar to the basic structures found in many of the ore commonly used organic solvents. Subsequent evaluation of this fungus revealed that it is also capable of degrading a number of the more recalcitrant organic waste products including munitions waste such as TNT, pesticides including pentachlorophenol and DDT, and various phenolic compounds when immobilized in biological reactors.

The purpose of this study was to obtain a preliminary assessment of- the ability of P. chrysosporium to degrade TCE in aqueous solutions. As stated, this solvent is commonly used in various cleaning functions, including as a dry cleaning solvent for clothes, and handling the waste residual is a problem with no apparent immediate solution.

RESULTS

The experiments were conducted in stoppered glass flasks. Although this is not a very efficient method of bringing about fungal degradation of chemical substrates, it is a very safe method of carrying out preliminary investigations since the release of fumes is prevented. Therefore, special equipment and procedures are not required in carrying the process to completion. Two procedures were used in these tests. One involved growing the fungus to maturity in a growth medium, then introducing 1 ppm TCE, and analyzing the solution contents after several days. The second involved growing the fungus in the presence of 1 ppm TCE, and then analyzing the solution contents once the fungus had reached maturity.

Analysis of the degradation progress was monitored by gas chromatography (GC). The data show that very little loss of TCE was noted in the absence of the fungus; the loss noted was certainly due to the establishment of equilibrium between the gas and liquid phases in the liquid and air-spaces of the flasks (FIGS. 1 and 2). However, in both of the sample types containing P. chrysosporium, substantial levels of TCE degradation had occurred. In samples in which TCE was introduced after the fungus was already mature, approximately a 75% reduction in the levels of the compound present were observed (FIG. 3). Although the rate of growth of the fungus was inhibited by the presence of TCE, complete degradation of the compound was noted in samples that had been grown in the presence of TCE (FIG. 4).

CONCLUSION

These data show that P. chrysosporium is capable of completely degrading TCE in aqueous wastewater streams.

DESIGN

Fungal cultures — Fungal cultures (P. chrysosporium BKM-1767) were grown in Bill medium both with and without TCE. Cultures that did not include TCE contained 1% glucose, 2.2 mM nitrogen, 20 mM buffer, 0.2% MgS0₄, 0.04% KH₂P0₄, and a mineral solution containing trace levels of Fe,

Co, Mn, Bo, Al, Cu, Ca, S, and Cl. To induce and optimize enzyme production, 0.2 mM veratryl alcohol and 0.05% Tween 80 were added. Fungal spores were added, and cultures were flushed with 0₂ prior to incubation in an orbital shaker at 30° C and 160 rpm. Cultures incubated with TCE contained the same ingredients except that glucose was supplied at 0.1 or 0.2%.

Analysis — TCE analysis was done on a Perkin-Elmer gas chromatograph equipped with a column capable of detecting TCE directly in aqueous solution. Sample volume analyzed was 1 μL per injection.

Table 2

CROSS-MEDIA APPLICATIONS

X = used

MATERIALS AND METHODS

BioMatrix Looped Cord Media

BMT sells a complete line of looped cord media {and support framing}

(FIG. 5) designed to act as holdfast substrate for municipal and industrial wastewater digesting microbes. BMT looped cord products are patented constructions available only from BMT.

BMT-1014 a looped cord media composite cord construction which is chemically resistant and durable.

• Construction: Polyvinylidene chloride loops interwoven within a linear composite backbone. The number of twists per inch (application dependent). Chemical resistance Resistant to concentrated acids and alkalis, oils and fats as well as most organic solvents. No deterioration with concentrated Sulfuric and Nitric acid. No deterioration with concentrated caustic soda

Water absorption: Less than 0.1%. The material is free from deterioration in water, retains strength, elongation properties and_ holds dimensional stability.

Fungus formation: Remains free of excessive fungus and mold growth. A wide variety of organisms grows on the media without preference to a specific organism.

Elastic recovery: The construction is characterized by high elastic recovery.

Denier: Monofilaments = 320 (0.162mm) - 3100 (0.508mm) Flattened Monofilaments and multifiliments are also available.

• Breakage strength: 100Lb+/ft. Available - standard strength 65Lb/ft.

• Solvent resistivity: Unaffected generally - some dissolving and/or swelling with Cyclohexanone, Dichloro Benzene.

• Softening point: 300+ degrees F

• Specific gravity: 1.70 (loops)

• Strength difference Dry/Wet: No change

• Stretch: Less than 1/32" per foot with 15 Lb. load. 100% retraction with 8% extension.

• Fiber life expectancy: Greater than 12 years when used in wastewater exhibiting less than 70% concentrations of above mentioned chemicals and when used in temperatures less than 200 deg. F.

Biological affinity: High degree of biofilm attraction and fixing degree of media twisting has some relation to % of biomass formation . BMT has proprietary information regarding biological affinity to media constructions. BioMatrix, page 7.

DOCUMENTS CITED

1. BioMatrix Catalogue, 1995.

2. Handbook of Bioremediation, Norris et al., CRC Press, (1993).

3. Hazmat World (June 1992), pp. 50-54. 4. National Defense (March 1996), "Environmental Rule Reform Providing

Prospect of Persuasive, Cheaper Cleanup", Sandra Meadows pp. 22-27. 5. U.S. Pat. No. 4,554,075, "Process of biodegrading Chloro-organics by -

White-Rot Fungi", Houmin Chang, Thomas W. Joyce, Thomas K. Kirk and

Van-Ba Huynh, issued Nov., 1985. 6. "Solvent Waste Reduction Alternatives" (September 1989), Seminar

Publication, U.S. E.P.A., Cincinnati, OH 445268. E.P.A7625/4-89/021. 7. Toxics Release Inventory" (March, 1995), U.S. E.P.A., Office of Pollution

Prevention and Toxics (7408), Washington, DC 20460. E.P.A.

745-R95-010.

Claims

WE CLAIM:

1. A method of biodegrading contaminants comprising the steps of: a) producing a reaction site and microorganisms; and b) providing conditions for said microorganisms to degrade the contaminants.

2. The method of claim 1 : a) wherein said reaction site facilitates formation of a hyphal mat of fungus; and b) wherein said steps of forming the hyphal mat and degrading contaminants derive energy from a simple carbohydrate.

3. The method of claim 2, wherein said simple carbohydrate is selected from the group consisting of molasses, fructose, corn starch, beer rejects and glucose black liquor and bleach plant effluent.

4. The method of claim 1 , wherein the contaminants are selected from the group of organic contaminants consisting of volatile organic compounds (VOCs), BOD, COD, TOC, TC, recalcitrant pollutants, and Federal Priority Pollutants.

5. The method of claim 1 , wherein the contaminants are selected from the group of inorganic contaminants consisting of ammonia, hydrogen sulfide.

6. The method of claim 2, wherein a bacteria is added to said hyphal mat of fungus.

7. The method of claim 2, wherein said fungus is selected from the group consisting of White Rot Fungus, Brown Rot Fungus, Black Rot Fungus,

Candida, Aspergillus and Mucor.

8. The method of claim 6, wherein said bacteria is an Actinomycete/Nocardioform.

9. The method of claim 6, wherein said bacteria is selected from the group consisting of Nocardia, Rhodococcus, Actinomycete or Streptomycetes.

10. The method of claim 6, wherein said bacteria is a Pseudomonadacae.

11. The method of claim 1 , wherein said reaction site is a porous ~ medium.

12. The method of claim 11 , wherein said porous medium is shaped to maximize the surface area.

13. The method of claim 11 , wherein the contaminant to be degraded is in a liquid or gas environment.

14. The method of claim 11 , wherein said porous medium is tubular.

15. The method of claim 11 , wherein said porous medium is made of an extruded, high density polyethylene.

16. The method of claim 11 , wherein the fungus is grown on or inside said porous medium in the presence of a carbon source and nutrients.

17. The method of claim 1 , wherein said reactor site is a cord media.

18. A method of immobilizing microorganisms on or inside a porous medium, comprising the steps of: a) selecting a porous medium, wherein said porous medium has a pore size of about 50-100╬╝; b) sterilizing said porous medium; c) lining one side of said porous medium by pouring a liquid enriched media inoculated with said selected microorganisms, which on cooling sets into an agar gel; d) lining of the porous medium by pouring liquid enriched media inoculated with the selected microorganisms, which on cooling remains a liquid; e) incubating said inoculated porous medium at a temperature and humidity suitable to obtain desired growth of said microorganisms; and f) immersing said inoculated porous medium into a liquid - fermentation system to obtain growth said microorganisms.

19. The method of claim 17, wherein activated carbon is added to the porous medium to remove residual contaminants.

20. The method of claim 17, wherein said microorganisms grown inside said porous medium are introduced into an environment containing organic contaminants, said organic contaminants are exposed to the microorganisms and said microorganisms degrade said organic contaminants.

21. The method of claim 1 , wherein the contaminant is used in surface cleaning of metal objects.

22. The method of claim 20, wherein the contaminant is a VOC.

23. The method of claim 21 , wherein the contaminant is selected from the group of chlorinated aliphatic compounds consisting of CT, PCE,

TCE, TCA, CF, DCE, DCA, VC and CA.

24. The method of claim 21 , wherein the contaminant is selected from the group of oxygenated aliphatic compounds consisting of acetone, ethyl acetate, ethyl alcohol, isopropyl alcohol, methyl alcohol, methyl ethyl ketone, ethyl chloride and methyl isobutyl ketone.

25. The method of claim 21 , wherein the VOC is in an environmental liquid comprising drinking water, groundwater, a process waste stream or a wastewater treatment system.

26. A method for biodegrading contaminants present in the environment, comprising the steps of: a) selecting a porous reaction site, wherein fungus in the primary phase of growth have been immobilized; and b) contacting said contaminated environment with said porous reaction site, whereby said contaminated environment is biodegraded by said immobilized fungus. -

27. A reaction site comprising a porous medium with White Rot Fungus.

28. A reaction site comprising a porous medium with nitrifying bacteria.

29. A reaction site comprising a porous medium with bacteria.

30. A method for retrofitting a treatment system with a reaction site containing microorganisms, said method comprising: a) preparing the microorganisms outside of the system in a laboratory scale fermentor, said microorganisms in contact with a porous medium; and b) transferring the microorganisms in contact with the porous medium to the system.

31. The method of claim 29, wherein the treatment system is a pulp or papermill treatment system.

32. A cord inoculated with microorganisms specific for an application.

33. The cord of claim 32, wherein the cord is looped.

34. The cord of claim 32, wherein the cord is frayed.

35. The cord of claim 32, wherein said cord is assembled on a frame comprising individually removable sections.

36. The cord of claim 32, wherein said cord is assembled on a pulley/spool system.

37. The method of claim 1 , wherein the contaminants are in a single pass lagoon.