Enhanced In Situ Aerobic Hydrocarbon Bioremediation

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					               Enhanced In Situ
      Aerobic Hydrocarbon Bioremediation

                                                                            Kevin Sharfe, President
                                                                          CleanEARTH Solutions Ltd.
                                                                       178 Pennsylvania Ave. Unit 4
                                                                             Concord, ON L4K 4B1
                                                                               Tel: (905) 482-2149
                                                                               Fax: (416) 913-1610

CleanEARTH Solutions Ltd. Concord, ON Canada L4K 4B1 Tel: 905) 482-2149 Fax: (416) 913-1610
An Effective Concept in Understanding and Speeding Bioremediation
Though not obvious, everything happens at the atomic/molecular level. Whether cutting
with a sharp knife, where the fewest number of inter-atomic (intermolecular) attraction
forces are overcome by the concentrated force of the fine edge, or as with the utilization
of nutrients through a series of complex biochemical reactions, atomic/molecular forces
are at work. By learning to think on an atomic/molecular level, many mechanisms and
processes of life and the living can be much more easily understood.

The dissimilation of nutrients and the synthesis of cell constituents is not a single-step
process. Indeed, each step involves numerous chemical reactions, each catalyzed by a
specific enzyme. Exergonic reactions are associated with the dissimilation of a nutrient
or chemical substrate outside the cell, while energonic reactions are associated with
synthesis within the cell. In the living organism, the exergonic reactions provide the fuel
for energonic reactions.

Although some microbes (protozoa) can swallow food particles, most microbes do not
ingest food but absorb dissolved nutrients from the environment. They are
chemoheterotrophic by absorption of soluble nutrients. By exuding enzymes into their
surroundings they break the nearby substrate into absorbable nutrients to power their
metabolism and reproduction. The greater the surface area available to the microbes
and their enzymes, the more rapidly the food can be assimilated.

All microbes require water to survive, metabolize food, and to reproduce. The speed of
microbial activity is often limited by the amount of water present. This can be easily
seen if one notes how wood that is wet (such as in a swamp) is rapidly decomposed by
microbial activity while dry desert wood is relatively long lasting.

The rate of reproduction is determined by the same factors that determine growth:
crowding, nutrient availability, and presence of waste products. If adequate nutrients are
available and crowding and waste accumulation attenuated, the speed of growth and
reproduction progresses exponentially.

The time required for each microbe to divide, or for the population to double, is known as
the generation time. Not all species of micro-organisms have the same generation time.
For Escherichia coli the generation time in a rich medium may be as short as 12.5 min...
Nor is generation time the same for a particular organism under all conditions. E. coli, for
example, will take much longer to divide in a nutritionally poor medium. If a bacterium
the size of E. coli cell divided only once per hour, in six days it would produce progeny
having a total volume 10,000 times that of the earth. It is quite obvious then that the
controlling factors such as crowding, inadequate nutrition, buildup of waste products, etc.
prevent the continued exponential growth and reproduction of the microbes.

There is an initial period in which there appears to be no growth in terms of increase in
cell numbers (the lag phase). Although cells are not dividing (reproducing) during the
lag phase, they are still metabolically active, repairing cellular damage, and synthesizing
enzymes. The lag phase is followed by a period of rapid balanced growth (the
logarithmic, or exponential growth phase, commonly called the log phase).

CleanEARTH Solutions Ltd. Concord, ON Canada L4K 4B1 Tel: 905) 482-2149 Fax: (416) 913-1610
Food (hydrocarbon) Availability
CleanEARTH Solutions Ltd. microbe supporting emulsification
Bioavailability of the substrate is directly related to the accessible substrate surface area.
The more surface area that is exposed to the microbes the more efficiently they can
subject the substrate to their secreted enzymes. It is much like comparing the efficiency
of feeding 10,000 people out of one room filled with food or feeding that same 10,000
from individual servings already set at tables.

When the substrate (pollutant-contaminant) is emulsified into colloidal sized droplets
smaller than the microorganisms, it can be more efficiently used thus enhancing and
greatly speeding degradation. Whether referring to bioremediation performed by
indigenous (on site) or added microorganisms, any acclimatized microbe will use the
pollutant more efficiently as food when it is "served" to them individually. Utilizing
microbe supporting emulsifying agents to break up the contaminant into tiny droplets
thereby exposing more surface area to the enzymatic action of the microbes present
greatly speeds bioremediation.

Bioavailability enhancing mixtures exponentially augment all microbes by making their
food more available to the microbes and their multitudes of rapidly produced
descendants. This is best accomplished by breaking the substrate into tiny droplets
(colloids) which are suspended in water thereby increasing the surface area available to
the microbes while at the same time providing the necessary oil-water interface. This
provides for an extremely rapid microbial population explosion and more rapid and
efficient bioremediation. Hydrocarbonoclastic microbes must be present and adequate
moisture and minerals supplied in petroleum bioremediation. The major consideration
then must be the bioavailability of the substrate.

The use of any chemicals detrimental to the microbes will diminish or destroy
subsequent microbial activity and bioremediation. High pH is the basis for many
bactericidal, viricidal, and fungicidal cleaners and disinfectants commonly used in
hospitals and food establishments. Prior to the use of organic emulsifiers, adding living
microbes to the high pH high shear concentrated cleaners (or dispersants) would mean
virtual instant death for the microbes. Bioavailability enhancing agents should not only
more rapidly prepare the food (substrate) for the microbes but at the same time act as a
food source for them. By adding the microbes to the enhancing agents prior to use, the
microbes can begin to repair their tissues and enzyme systems and thus more rapidly
initiate the bioremediation response.

When the substrate is acted upon by surface active agents, the result is a distribution of
its particles in the other material, e.g. tiny droplets of milk fat (cream) in milk. In this size
range, the relative surface area of the colloidal particle (droplet) is so much greater than
its volume that unusual phenomena occur. For example the particles do not settle out of
suspension by gravity, and are small enough to pass through filter membranes.
Molecules and atoms sometimes swarm together under the influence of intermolecular
forces, and the large conglomerates behave like macro-molecules: these are the
colloids. Colloids can be distinguished from true solutions by the presence of particles
that were too small to be observed with a normal [light] microscope yet were much larger
than normal molecules. Colloids are now regarded as systems in which there are two or
more phases, with one (the dispersed phase) distributed in the other (the continuous
phase). Colloids are thermo-dynamically unstable with respect to the continuous phase;
surface tension favors small surface areas. Moreover, at least one of the phases has

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small dimensions (in the range of 10-9-10-6m). Emulsions are colloidal systems in which
the dispersed and continuous phases are both liquids, e.g. oil-in water or water-in-oil.
Such systems require an emulsifying agent to stabilize the dispersed particles.

Dispersions and emulsions are similar, and very different.
Dispersion. (1) A two-phase system of which one phase consists of finely divided
particles (often in the colloidal size range) distributed throughout a bulk substance, the
particles being the dispersed or internal phase and the bulk substance the continuous or
external phase. Under natural conditions the distribution is seldom uniform, but under
controlled conditions the uniformity can be increased by addition of wetting or dispersing
agents (surfactants) such as a fatty acid. The various possible systems are: gas/liquid
(foam), solid/gas (aerosol), gas/solid (foamed plastic), liquid/gas (fog), liquid/liquid
(emulsions), solid/liquid (paint), and solid/solid (carbon black in rubber). Some types,
such as milk and rubber latex, are stabilized by a protective colloid which prevents
agglomeration of the dispersed particles by an adherent coating. Solid-in-liquid colloidal
dispersions (loosely called solutions) can be precipitated by adding electrolytes which
neutralize the electrical charges on the particles. Larger particles will either gradually
coalesce and rise to the top or settle out, depending on their specific gravity.

Emulsion. A stable mixture of two or more immiscible liquids held in suspension by
small percentages of substances called emulsifiers. These are of two types: (1) Proteins
or carbohydrate polymers, which act by coating the surfaces of the dispersed fat or oil
particles, thus preventing them from coalescing; these are sometimes called protective
colloids. (2) Long-chain alcohols and fatty acids, which are able to reduce the surface
tension at the interface of the suspended particles because of the solubility properties of
their molecules. Soaps behave in this manner; they exert cleaning action by emulsifying
the oily components of soils. All such substances, both natural and synthetic, are known
collectively as detergents.

Note that “dispersion” is a more general term. Dispersions are seldom uniform and the
dispersed material is not necessarily of colloidal size. Emulsions are colloidal systems in
which the dispersed and continuous phases are both liquids. Such systems require an
emulsifying agent to stabilize the dispersed particles. Without the emulsifying agent, the
order will collapse and the dispersed phase will coalesce and reform into a liquid phase
which will separate from the other liquid. This is the process that causes milk fat to
separate from milk and oil to float on water.

By compromising the intermolecular forces within the substrate, the substrate surface
molecules can be more easily separated from the substrate proper to form the dispersed
phase of the emulsion. When this is done with organic chemicals rather than high pH
and high shear stress inorganic materials which could damage the microbes,
bioremediation can proceed rapidly. The microbes not only survive in the mixture, they
multiply exponentially thus increasing the efficiency of the mixture. The organic
chemicals utilized in the emulsifying mixture must not only be non-toxic to
hydrocarbonoclastic microbes, the chemicals actually must serve as food (carbon
source) to the microbes. By the use of the organic chemicals, a stable emulsion of the
substrate is formed which provides for continuous bioremediation of the substrate by
microorganisms until the substrate and the organic chemicals themselves are
consumed. The colloidal emulsion remains stable and bioremediation progresses
rapidly and continuously until biodegradation is complete.

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A sugar cube contains about one and one-half square inches of available surface area
for reaction with its environment (six one cm square faces). If that cube were to be cut
into 10 nanometer cubes, the available surface area of that same sugar cube increases
100,000 times. Such is the effect of using the organic chemicals to make more
substrate surface area available to any microbes which can utilize that substrate. No
matter how small the microbes (bacteria and yeasts are large enough to be seen with an
ordinary light microscope), limited numbers can crowd to a finite amount of surface.
Were that relative amount of surface to become virtually infinite, almost limitless
numbers of microbes would have food available without competition for them and their
abundant offspring produced as a result of the feeding frenzy. There would be little
competition for the food and extensive dilution of waste that might otherwise
compromise them.

Colloids are not only readily available to the microbes but are also immediately available
to the descendants of the microbes. With this doubling many times per hour there is an
exponential explosion of microbe population to efficiently utilize the food source and thus
obliterate the contamination.

Soil presents complex problems primarily because of the extreme variations in soil
characteristics from location to location as well as specific areas within locations.
Although limited permeability will restrain contaminate penetration, desired infiltration by
the remediation procedure will also be constrained.

Physical Considerations and Flow
In a remedial action plan that includes bioremediation, there are a number of physical
(abiotic) limitations to the effectiveness of using microorganisms to speed cleanup even
if the microbes present are quite capable of producing enzymes that can degrade the
containment. Even the most voracious microorganism, like any animal, will be unable to
consume its food if that food and water are not available to it. Also oxygen or some
other electron donor must be available, and the microbes’ wastes must not accumulate
enough to limit its activity. Properly designed emulsifiers will minimize most physical
limitations to bioremediation this increasing speed, efficiency, and completeness.

A lack of consideration of the kinetics of diffusion and adsorption may contribute to
bioremediation failure. If water carrying microbes, their enzymes and nutrients cannot
penetrate an impervious soil particle, it will become adsorbed to the particle surface and
diffuse between the particles. Rates of adsorption and diffusion to inaccessible sites
may be similar to many rates of bioremediation, and these abiotic processes may be
effectively competing with microorganisms for the substrate (containment). Unless
organisms and their enzymes reach the substrate, little or no bioremediation will take
place. Using emulsifiers that will support microbes will significantly speed
bioremediation by making the substrate more available. This is achieved by increasing
the wetting action of the available water thereby increasing its ability to penetrate
between the soil particles, and also by increasing the available surface area of the
substrate by breaking it into tiny droplets.

Unless water is available, bioremediation will at least pause and at most stop altogether
(this can be easily seen by the rapidity of the rotting of wood and plants in a swamp
versus on a desert). The ability of water to penetrate between soil particles is limited by
particle and pore size and the wetting ability of the water itself. If the surface tension of
the water is decreased, the water can more easily penetrate between soil particles and

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marry microbes, oxygen, and nutrients to the containment while at the same time diluting
waste products produced. Efficient buffering non-toxic emulsifiers are a key to speeding
this process.

Sorption (clinging to the surface or penetration) by a chemical has a major impact on the
biodegradation of a compound. Spreading of a substrate can be greatly affected by the
size of soil particles. For instance, tiny soil particles in clay will greatly limit penetration
of a contaminating substrate as well as the later penetration of water and microbes when
bioremediation is attempted. Physical limiting of exposure of a substrate to microbes
and their enzymes will obviously compromise biodegradation of the substrate. Utilizing
effective surface active agents in the conjunction with microbial enzymes stimulates
more rapid penetration of the soil and substrate by water, microbes, inorganic nutrients,

Diffusion of the substrate between soil particles limits the availability of many organic
substrates to microbes and lessens the rate of degradation of these chemicals. They are
protected from microbial attack by being sequestered within small pores or voids
between particles. Soil consists of particles of various sizes and shapes and therefore
the voids and interstitial spaces will vary accordingly. The diameter of interstitial pores is
typically about one-tenth the average size of the soil particles. Porosity of the individual
grains as well as actual grain geometry and composition can greatly affect fluid

Measurements of moisture tension in a silt loam show that as much as 50% of the total
pore volume consists of pores with radii estimated to be <1 um. Most soil bacteria range
in size from 0.5 to 0.8 um and studies indicate that the mean diameter of soil pores
occupied by bacteria is even larger, approximately 2 um. Since bacteria require space
around them, this suggests that in many soils a significant portion of the soil moisture (or
contaminant) retained in pores is inaccessible to most bacteria. Microbes do not have to
actually penetrate every pore for microbes secrete enzymes into their immediate
surroundings that reach some sequestered materials. Enzymes can be carried by the
water into any space water can penetrate. By using microbe-supporting emulsifiers, the
bioavailability of sequestered substrate is greatly increased. The emulsifiers break the
substrate sequestered within the minute pore spaces into very tiny droplets. These
small droplets of “boiled-off” substrate will flow out of the pore spaces as they are being
displaced by low-surface-tension water containing the emulsifier and thus become
readily available to the microbes to use as food.

Although bacterial may not readily penetrate the pores of fine pored soils, e.g. clay &
silty loams, neither are water immiscible contaminates, e.g. perchlorethylene, PCBs, etc.
The soils most likely to be contaminated by water-immiscible contaminants are coarse-
grained soils (grain size 100-1000 um diameter). These soils will have pore diameters
ranging from 10-1000 um, large enough to accommodate bacteria.

Diffusion rates of water into soil can also limit the rate of biodegradation of high
concentrations of organic material. The rate of diffusion of waterborne oxygen or
inorganic nutrients may be limited, and such limitations may be especially important for
bacteria growing in micro colonies. The penetration of waterborne microbes, their
nutrients and necessary oxygen, into any soil will be significantly limited especially if the
soil particles are saturated or coated with oily contaminants thereby functioning as a
water repellent. The use of emulsifiers that are capable of lifting the oily contaminants

CleanEARTH Solutions Ltd. Concord, ON Canada L4K 4B1 Tel: 905) 482-2149 Fax: (416) 913-1610
from the particle surfaces will allow more rapid penetration of water. As the substrate is
broken into tiny droplets, the microbes have more surface area available to their
enzymes and thus consume the contaminant more efficiently. Also, accelerating the
diffusion of toxic products away from the active organisms trough the use of emulsifiers
speeds bioremediation.

The bioavailability of many pollutants that repel water (such as oils) is markedly affected
if the molecule is in a non-aqueous phase liquid (NAPL) at the site. This problem is
increased dramatically by the presence of the space-occupying particles of the soil. The
interfacial (contact) area between the substrate and water will vary in proportion to the
sizes of the soil particles. In a given volume, as the number of soil particles increases,
spaces between them decrease. By using emulsifiers that are capable of breaking the
substrate out of the voids, the speed of bioremediation is enhanced because the
available surface between the NAPL and the aqueous phase is increased. Microbial
growth and therefore bioremediation takes place at this interface, in the aqueous phase
only, and at both the interface, in the aqueous solution. Emulsification of the
contaminant increases available surface area and speeds microbial activity in all
locations. Breaking colloidal sized droplets completely free from the surface of the
substrate so that they are actually within the aqueous phases greatly speeds

Gravity causes water to flow downward in soil. As the moisture content of the soil
decreases, the pressure within the soil water itself decreases because it tends to be held
up by capillary action. The surface tension of water is very high due to intermolecular
hydrogen bonding and is responsible for the rise of water in a capillary tube. Just as sap
climbs a tree, water attempts to climb between soil particles. This decrease in soil water
pressure is termed the soil suction or matric potential. As the space between soil
particles decreases, the higher the soil suction and the higher capillary column of water.
Thus matric suction varies for soils according to grain size. Water will flow from sand to
a loam and from a loam to a clay. Levels of ground water will be higher in clay soils but
it will not flow efficiently because of the tiny particle sizes. Since the reason for surface
tension in water is that its molecules tend to prefer to adhere to themselves,
compromising that bonding will allow the water flow more easily to penetrate other
materials more efficiently but only if the surfactant displaces oily material from a soil
surface or from interstitial pores. If oil is displaced, the size of the conduit available to
carry water is enlarged. However, many surface active agents undergo chromatographic
adsorption on fine grained soils so they do not move very far into the soil body just as
oily materials do not penetrate such soils.

Microbial elimination of soil contaminants is also dependent on the presence of microbes
capable of producing enzymes proficient in degrading the contaminant and its
breakdown products. As a contaminant is degraded, toxic by-products may be produced
that must then be degraded. Breaking the contaminating substrate into carbon dioxide
and water will require many different steps by many different microbial enzymes. The
waste products produced by any one microbe may be toxic to itself (and others) if
allowed to accumulate in high enough concentrations although these waste products
become food for different microbes with different enzyme systems. If microbe
supporting emulsifiers are utilized, the accumulation of any one microbe’s waste
products are dispersed away from the microbe and diluted for use by other microbes.
This speeds bioremediation.

CleanEARTH Solutions Ltd. Concord, ON Canada L4K 4B1 Tel: 905) 482-2149 Fax: (416) 913-1610
A liquid cannot completely discharge another material in soil unless it is miscible
(capable of mixing) with the material it is displacing. An irreducible minimum
concentration of the immiscible material will always remain. For instance, water will not
replace all of the oil in soil, this cannot be done even by using an oil solvent, (10-50% of
soil pore space oil will remain).

This is the basis of much research in secondary oil recovery. The immiscible matter
sequestered in the soil pores is removable only by something capable of dissolving, or
destroying it. Efficient surface active agents and microbial enzymes in water can
effectively reach water-immiscible materials that cannot be displaced otherwise.

Many of the most efficient microbes used in bioremediation are flagellated, that is, they
have tiny hair-like cilia protruding from them that they use as propellers and thus they
have the ability to move. Movement occurs only in water. In addition, they possess
chemotaxis (the ability to move toward a food source). They do this not by the ability to
choose the direction they want to go but rather to eliminate the direction that they do not
want to go. When they detect molecules of food in their environment that they can use
for energy and reproduction they begin a search for the food source and propel
themselves in whatever direction they are capable of moving if they detect a decreasing
number of food molecules they change to some arbitrary direction and repeat the
process until they find themselves moving toward higher concentrations of the food.
This is much like the bird dog homing in on a covey of quail by detecting their scent.
Motile microbes will allow their food and utilize it until the food source is exhausted and
then they will die from the lack of nutrition.

The detection of food and enhancement of the microbial action is greatly increased by
the use of emulsifiers capable of supporting the microbes. Properly designed emulsifiers
themselves can be used by the microbes as food and thus support the microbes while
the emulsifiers are speeding the breakup of the contaminant which releases attracting
food molecules into the aqueous phase (water). This magnifies the lure that greatly
increases the number of biodegrading microbes at the actual site of the degradation of
the contamination, the water- contaminant interface. Efficient bioremediating microbes
produce their own surface active enzymes, which they use to assimilate food. This is
especially desirable for additional surfactants are constantly being produced to offset
that which is decomposed or adsorbed into the surface of soil particles.

BioActivate, a cytokinetic cell building solution
The possibility of forming helpful biologically active molecules through fermentation
processes is opening vast possibilities to produce metabolic enhancement and
normalization. BioActivate, a proprietary biologically enhanced processed water that
speeds anabolic responses, is proving to enhance anabolic activity and tissue

BioActivate by CleanEARTH Solutions Ltd. is a biowater manufactured in a fermentation
process. Anecdotal and empirical evidence indicates that proprietary fermentation will
result in the production of water which contains a broad spectrum of bioactive cytokines.
The organisms used in the fermentation are equipped with numerous mechanisms that
allow them to survive under conditions of nutrient depravation. When grown under strict
conditions, the organisms apparently produce a myriad of microbe stimulating cytokines,
small molecules (8-30 KDa) which are the basis of intercellular communications. These
cytokines trigger both general and specific responses which are capable of triggering

CleanEARTH Solutions Ltd. Concord, ON Canada L4K 4B1 Tel: 905) 482-2149 Fax: (416) 913-1610
anabolic responses in microbes, stimulating microbes to great activity. Their metabolism
is based on oxygenic photosynthesis, similar to that of eukaryotic algae and plants. This
process provides ATP and reducing equivalents from the splitting of the water molecule
in BioActivate. This water splitting enables the bacteria to assimilate simple inorganic
nutrients for their anabolic demands.

The communicating entities that orchestrate this concert of activity are very specialized
tiny molecules. Hormones have long been known. However, there is a vast array of
additional signal carriers. They are referred to as cytokines, molecules capable of
stimulating cellular response and activity. It is derived from two Greek words, cyt- [cell]
+ kine [move or action, “kinetic”]. Many are enzymes, referred to by the more general
term kinase, derived from kine [move or action] + ase [enzyme].

Cellular metabolism and intercellular communication were originally thought to be solely
protein molecule dependent. Current research indicates that these highly effective
reaction-mediating protein molecules have great metabolic responsibilities; they are
often modified, supported and enhanced by specific sugars attached to them at precise
spots. They may be simple to complex proteins, saccharides (sugars) or combinations

The sugars may be simple or quite complex and the possibilities for vast metabolic
support are greatly enhanced by their presence. The four nucleotides that make up
DNA and the 20 common amino acids that form proteins link together in a linear fashion
like beads on a string, always joined by the same chemical connection. In contrast,
roughly 10 simple sugars in mammalian carbohydrates can join together with one
another at many different points and can form intricate branching structures. Moreover,
two linked units do not always orient in the same way: sometimes a building block will
point up relative to another unit and sometimes it will point down. The four nucleotides
in the DNA “alphabet” can combine to produce 256 four-unit configurations and the 20
amino acids in proteins can yield about 16,000 four-unit configurations. But the simplest
sugars in the body can theoretically assemble into more than 15 million four component
arrangements. Although not all of these combinations occur in nature, the possibilities
remain overwhelming. Massachusetts Institute of Technology points out that a mere six-
unit sugar of a kind called a glycosaminoglycan has a staggering 12 billion possible

Cytokines are small secreted molecules which mediate and regulate many mechanisms.
They are produced in response to specific stimuli. They generally (although not always)
act over short distances and short time spans and at extremely low concentration. They
act by binding to specific membrane receptors, which then signal the cell via second
messengers, often tyrosine kinases, to alter the cell’s behavior (gene expression).
Responses to cytokines include increasing or decreasing expression of membrane
proteins (including cytokine receptors themselves), proliferation, and secretion of other
molecules that initiate further activities.

Apparently this same cross-talk mechanism is used by microbes and BioActivate
effectively supplies sufficient quantities of these proteins and glycoproteins to be
effective in stimulating microbial growth and reproduction. This greatly enhances
bioremediation speed and effectiveness.

CleanEARTH Solutions Ltd. Concord, ON Canada L4K 4B1 Tel: 905) 482-2149 Fax: (416) 913-1610
How and why does a cell do what it is supposed to do? In scientific terminology, gene
expression is regulated via proteins called transcription factors and nuclear receptors
that bind to specific regulatory sequences in DNA, thereby modulating the transcription
of genes into RNA. These proteins are often present in the cell in an inactive form, and
only become activated to bind to their cognate recognition sequences as a result of a
specific signal transduction event. In other words, cells
do what they are told to do in response to external or internal messenger

Exhaustive independent laboratory analysis searching for and comparing inorganic
molecules has found, at best, only minute differences between fermentation processed
BioActivate and the source tap water from which it is made.

Compare BioActivate and tap water using standard tests for oxygen present and there is
little difference. Test the same BioActivate against the same tap water in a dynamic
sealed closed system containing living cells and the difference is dramatic. Such tests
on BioActivate were confirmed by independent testing at the Institute For Research, Inc.,
8330 Westglen Dr., Houston, Texas 77063. The tests were performed using Hach
BODTrac analytical equipment which was designed to determine biological oxygen

                   Test Headspace Gasses End of Test vs. Dry Air Based On
                          Analysis by the Institute For Research, Inc.

                                                   Dry Air               End BioActivate Test Vessel
                                                 % by Volume              Headspace % by Volume
Nitrogen, N2                                         78.08                            22.0601
Oxygen, O2                                           20.95                           < 1.0192,3
Argon, Ar2                                            0.93                           < 1.0192
Carbon dioxide, CO2                                  0.033                            27.5634
Hydrogen, H2                                        0.00005                           49.3585

Plus other gasses (Neon, Helium,
                                                    0.00367                       Not Measured6
Methane, Nitrogen Oxide, Krypton)

 Nitrogen as a gas is essentially biologically inert. It is still present in the system but on a mass-balance
partial pressure basis - limited in the total headspace gas aggregate by the expanded quantities of the other
gasses released in the closed system.
    Total for both Argon/Oxygen as quantified by Institute For Research, Inc.
  Oxygen level in the closed test vessel is depleted (converted to energy, cell mass and CO2) by the
metabolic activity of the living cells utilizing the available oxygen for maintenance, growth and reproduction.
 Carbon dioxide is a metabolic waste - high concentration will be produced in a closed system with high
cellular activity (food carbon chains are broken by oxidation releasing CO2 thereby producing the energy
necessary for the metabolic processes).
  High concentration of enclosed headspace free hydrogen remaining apparently as a result of the oxygen
portion of water (H2O) breakdown being utilized by cellular metabolism.

CleanEARTH Solutions Ltd. Concord, ON Canada L4K 4B1 Tel: 905) 482-2149 Fax: (416) 913-1610
    Not biologically significant at these concentrations.

In BioActivate, the stimulation of bioactivity and oxygen availability plus the by-
production of hydrogen are well demonstrated in the above laboratory findings. As with
many observations of the complexities of dynamic living systems, the exact mechanisms
are first speculation and only much later proven. Seeking such, BioActivate plus living
cells and nutrients were placed in a sealed container and tested. Additional
simultaneous tests were done using tap water plus like living cells and nutrients.

When biologically templated, BioActivate was added to a closed test chamber test data
indicate the release of vast quantities of oxygen and hydrogen resulting in greatly
accelerated cellular activity. Both magnetic resonance imaging and living system data
show that such enhanced metabolic activity could be expected to result from the
presence of vast quantities of available oxygen that are rapidly mobilized so that more
efficient metabolism takes place. This could also result from increased translocation of
molecules within the cellular cytoplasm. In the sealed enclosed living-system test,
available oxygen is rapidly used up by cellular growth and reproduction leaving the
metabolic waste (primarily carbon dioxide) and metabolically inert gasses.

The experimental test data show high levels of free hydrogen and carbon dioxide in the
final headspace as compared to dry air. Through the rapid metabolic activity of aerobic
respiration for energy production and cell growth and reproduction, there is virtually no
headspace oxygen left in the sealed test vessel at the end of the test. The metabolically
inactive gasses and metabolic waste (CO2) in the head space remain as indicators of
what transpired.

Dr. Mosier, from the Institute For Research, Inc. concluded the following based his

       1. “The possible breaking of water molecules by enzyme induced catalysis.”
       2. “The enzymatic electrolysis whereby the molecular hydrogen is separated from
          the water molecule thus liberating the oxygen molecule that was attached

This enhanced uptake and utilization of released usable oxygen from water is a vast
improvement over systems that merely bubble oxygen through water to make
“oxygenated water.” Furthermore, oxygen liberation and cytokinetic cell building signals
are activated solely through enzyme activity. BioActivate is not a time release chemical
oxidant which may result in unwanted environmental impact or impede biological

BioActivate has proven to greatly speed the microbial activity that is necessary for them
to rapidly and efficiently utilize their food (in this case the carbon and hydrocarbon) so
they could grow most efficiently.

Pure BioActivate is clear and indistinguishable from pure water unless tested biologically
as described above. It has no odour, coloration or other grossly distinguishing
characteristics other than its ability to greatly accelerate metabolic activity.

CleanEARTH Solutions Ltd. Concord, ON Canada L4K 4B1 Tel: 905) 482-2149 Fax: (416) 913-1610
Testing for inorganic molecules may be of little value when attempting to monitor the
efficacy of BioActivate. Strict laboratory microbiological studies to determine relative
growth rates comparing BioActivate, and distilled water showed virtually no growth rate
difference at 8 hours. At 24 hours the BioActivate plate growth rates of P. aeruginosa
was similar with distilled water colonies developed. The number of colony forming units
in the BioActivate dramatically increased when compared to distilled water as more time
passed. At 72 hours the count was: distilled water, 46cfu/ml, BioActivate, 1200 cfu/ml.

CleanEARTH Solutions Ltd. works to enhance hydrocarbon bioremediation processes by
using advanced science and applicable technologies. Using BioActivate as a stimulating
carrier for microbe supporting effective substrate emulsifiers as found in Hydrocarbon
EM by CleanEARTH Solutions Ltd., is a process that is becoming accepted and tested at a
growing rate.

CleanEARTH Solutions Ltd. Concord, ON Canada L4K 4B1 Tel: 905) 482-2149 Fax: (416) 913-1610

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