TECHNICAL DISCUSSION OF BIOREMEDIATION PROCESS FOR SOIL

I.

Upon arrival, the contaminated soil will be treated to produce the smallest size particle possible with either a shredding device or a vibrating screen. Tightly compacted soil may need additives to increase their permeability. The microbial product, PDM-7 HC and nutrients will be added to the soil by spray method to insure inoculation of the maximum surface area. In most soil treatment, faster remediation is obtained with nitrogen and phosphorous concentrations of 100 PPM.

In our opinion, bioremediation is best accomplished with Bio-augmentation, the addition of large numbers of selected microorganisms grown in the production laboratory. In addition to augmentation, several physical and chemical parameters must be controlled in order to obtain optimal growth and maximum degradation in soil contaminants.

The major factors that PHase III, Inc. control during Bioremediation are:

1) Microbial population

2) Nutrient concentrations

3) Oxygen supply

4) Temperature and moisture content

1) Microbial Population

The synergistic group of microorganisms in PDM-7 HC digest short and long chain hydrocarbons, heavy tarry types of oil and grease, coal tars, phenolic compounds, chlorinated organic solvents and many toxic chemicals.

The organisms in PDM-7 HC are non-pathogenic and use petroleum products or other chemicals in the soil for both a carbon source and an energy source. They covert the toxic chemicals to cell mass and to carbon dioxide and water.

With Bio-augmentation, a sufficient amount of PDM-7 HC product is applied to the soil to give an initial population of one million organisms per gram of soil. Our studies have shown that this concentration insures that a sufficient population will be provided. It is not the intent to provide a ready-made population to immediately digest the contaminants, but rather to provide an inoculum that will insure rapid growth. As the project progresses the population of petrophillic organisms will show up to a one thousand fold increase as they digest the contaminants.

In comparing Bio-augmentation with natural degradation, the former greatly increases the rate of degradation. The microorganisms naturally present, that can degrade petroleum products are usually present only in the upper few inches of soil and in low concentrations. These organisms are greatly diluted when mixed with a large amount of excavated soil. The dilution factor can be so large that it takes an excessively long time, if ever to establish a working population of correct organisms.

2) Nutrient Concentrations

Nitrogen

Nitrogen is needed for amino acid, purine and pyrimidine biosynthesis and can be obtained by microorganisms from either inorganic or organic sources. The most commonly used nitrogen sources in bioremediation are ammonia and nitrate. Many of the bacteria in PDM-7 HC have two pathways for ammonia assimilation and which one functions depends on the ammonia concentration. We purposely keep the ammonia concentration high in order to allow the bacteria to utilize the most simple process.

When nitrate is utilized as the nitrogen source it is reduced to ammonia by a stepwise process. Nitrate is first reduced to nitrite by a molybdenum containing enzyme and then to ammonia.

Phosphorus (in the form of phosphate) is utilized by microorganisms primarily to synthesize phospholipids and nucleic acids (DNA and RNA).

In addition to nitrogen and phosphorous, a variety of minerals is universally required, such as potassium, calcium, magnesium and iron. Many other elements are required only in trace elements. These include zinc, copper, cobalt, manganese and molybdenum. These metals function in enzymes or coenzymes.

Although none of the microorganisms in PDM-7 HC require growth factors, it has been observed that organic growth factors such as B vitamins greatly stimulate growth and Bio-Degradation.

3) Oxygen Supply

Utilization of aliphatic hydrocarbons by microorganisms is strictly an aerobic process. The initial oxidation step of aliphatic hydrocarbons involves molecular oxygen as a reactant and one of the oxygen molecules is actually incorporated into the oxidized product.

The aromatic group of hydrocarbons can be viewed as derivatives of benzene. The breakdown od aromatic hydrocarbons involves the action of either oxygenases or mixed function oxygenases. These two reaction sequences both form catechol which can be degraded in a number of ways leading to either acetyl CoA or TCA cycle intermediates. If the benzene ring contains added methyl groups or other constituents (as in toluene or xylene), these substituents may be attacked before or after the ring is oxidized.

Very importantly, the oxygen concentration is the rate limiting factor in the Bio-degradation of petroleum based products. Microbial activity is most frequently limited by insufficient oxygen due to slow rates of diffusion into the interior of the soil layers or piles and into the center of soil aggregates. Generally, the greater the mass of oxygen that can be distributed the more rapid and complete the cleanup. Effective application can result in greatly accelerated cleanups.

Hydrogen peroxide may be utilized in addition to the aeration process. Hydrogen peroxide in addition to simply providing oxygen for microbial use has been shown in our laboratory to greatly stimulate the degradation of recalcitrant components of diesel fuel such as benzopyrene.

4) Temperature and Moisture Control

The bacteria in PDM-7 HC that were all isolated from the soil and laboratory studies have shown that the optimum temperature of the soil should be maintained as close to 28 Celsius (82 Fahrenheit) as possible. Similar studies have shown that soil moisture should be maintained at 15-20% moisture.

II. ADVANTAGES OVER OTHER REMEDIATION METHODS

Treatment with our product offers numerous advantages over the more conventional means of remediation, which are excavation/relocation or incineration. Both previous methods are very costly. For instance, on site incineration costs approximately $500.00 per ton, while offsite disposal runs as high as $300.00 per ton. Actual bio-remediation costs will vary according to:

1. Location of site to be remediated

2. Size of site to be remediated

3. Equipment rental or purchase cost

4. Labor cost

5. Product cost

III. PRODUCT USAGE

Product usage varies depending on the extent of the contamination. However, the following are estimated figures:

A. One gallon of PDM-7 HC per 1000 gallons of wastewater

B. One gallon of PDM-7 HC per one to three cubic yards depending upon site specifics.

IV. APPLICATION

Application of the product can vary from site to site depending on various environmental factors, as well as the extent of the contamination. Some of the more typical bio-remediation processes are:

A. Top Soil Bio-remediation (up to 18 inch depth)

The product and the proper nutrients can be applied by merely spraying the mixture onto the surface of the soil while simultaneously rototilling the mixture into the soil. This application method provides an even distribution of microbes throughout the soil, as well as providing aeration for the microbes. Repeated rototilling accelerates the biological degradation of the soil contaminants.

B. Sub-surface Soil Bio-remediation (over 18 inches in depth)

Sub-surface bio-remediation procedures require that the product and appropriate nutrients be injected under pressure to a depth to be determined by the depth of the contamination. The interval between injection sites needs to be determined onsite in accordance to soil composition and consistency. If necessary, nutrients or hydrogen peroxide or both can be added at a later date, pending laboratory analysis of soil samples obtained from the site.

Both of the treatments mentioned provide onsite treatment in which the entire bio-remediation process can be completed without any excavation or soil removal. This form of remediation has significant environmental and economic advantages when compared to other means of remediation.

C. Hazardous Waste Lagoon Bio-remediation

Hazardous waste lagoons can be treated by merely spraying the product and the proper nutrients on the surface of the lagoon. Circulating and aerating the lagoon with the use of a direct drive mixer will greatly increase the rate of biological degradation.

D. Railroad Track/Ballast Bio-remediation

Railroad track ties and ballast can be cleaned and remediated by using the following summarized treatment process:

1. Spray railroad ties and ballast with Eliminator Microbial Cleaner/Degreaser. Eliminator loosens the extensive oil and grease built up on the ties, ballast and rock.

2. The pretreatment is then followed by a high pressure wash, which removes the oil and grease from the ties and ballast and deposits this material on the soil below.

3. The ballast and soil is further inoculated with PDM-7 HC and proper nutrients. These petrophilic bacteria will digest the hydrocarbons which were previously deposited on the soil below the ballast.

This discussion is presented as a general outline for the process for soil bio-remediation. Each potential clean-up is site specific and should be treated as such.