Biostimulants for Soil and Groundwater
Bioremediation
JapanOctober 25, 2010

Company Business Made of engineers and scientists and concentrates ondeveloping technologies for environmental market.
 Business: Environmental remediation, Bioinoculants
for agriculture, recycling of industrial effluents, and EHSconsultant.

 Some of the clients are manufacturers of automobile,electronic equipment, electrical apparatus, chemicalindustries, metal industries, semiconductor industries, andgovernment bodies.
 Technological partnership with more than 30 Japaneseengineering companies, and GZA Geoenvironmental, USA.
-2-Potential of EcoCycle Technical expertise in site investigation and remediation ofvarious sites (large-small, running facilities, differenthydrogeology, contamination levels, etc.)
 Pioneers in bioremediation of chlorinated solvents,

hexavalent chromium, cyanide, petroleum hydrocarbons, etc.
applied over 170 sites in Japan, the US, India, and Taiwan
 Good knowledge in other remedial technologies
 Partnership with engineering companies and net work of

research institutes world-wide

Holds several Japanese and international patents-3-Field Experience
Chlorinated Solvents Hexavalent Chromium Contaminations: PCE, TCE, DCE, TCA, DCA, CT,DCM, Cyanide, Benzene, Cr (VI), etc.
 Japan, Thailand, the US, Taiwan, India……..
 High concentration: 750mg/L
 Low K hydrogeology: ~ 10-6cm/sec
 Largest site: 7 million m3

Japan EPA Funded Site (Demonstration oflow cost, environmentally friendly technology)-4-Comparison of Different Leading Remediation Technologies
Technology

Remediation
Cost(per 1m3)

EcoCleanTime span

ActiveFacilities

Low KSoilHigh
Concentration

VadoseZone

GroundwaterLimitations

5~15K円

3 ~ 12months○○○： ~ 150mg/L▲ ： 150mg/L▲○

Concentrations above
500mg/L and extreme pH

Dig andDispose

40 ~ 60K円

Weeks×○○○×

High cost

Pump andTreat(NA)Tens of years

○

×

○

Excavat-ion○

Very long time

Fe(0)

30K円

Months

×○

▲

○

○

Not for active facility/Affecthydrogeology

Oxidants

5~20K円Few months ~years

○×

▲

▲

○

Cr(VI) may release/Not
good under high OC/Kill
useful organisms

Hot soil

15 ~ 40K円

Weeks ~months

×○

○

○

×

High cost/Not for activefacility

Venting

5~15K円Months ~ fewyears

○×○○

×

Not for groundwater

Choose the technology based on site conditions, budget and time available for remediation-5-

Bioremediation
Exploiting ability of microorganisms to degrade/detoxify contaminants

Bioaugmentation
Introducing microbes in the contaminated media

Biostimulation (Our strength)
Stimulating native microbes

Aerobic degradation
Stimulating microbes that use oxygen for respiration

Facultative anaerobic microbes
Stimulating microbes that live in presence or absence of oxygen(EcoClean-M mediated Cr(VI) remediation)

Biostimulation
Stimulating microbes that live only when oxygen is absent(EcoClean mediated chlorinated solvent remediation)-6-

EcoClean Based Bioremediation is for……
Pumping and treating the water foryears without much success

Want to clean the site
in moderately shorttime

Want to save $Large size of contamination
plume

Deep andwidespreadcontamination

Moderately highconcentration

Running/active facility

Facilities has large
machines-7-

EcoClean the Wholesome Food for Dehalogenating Microbial Population

HydrogenAmino Acids

Energy source for themicroorganisms

Essential building
blocks of microbial cell

EcoClean™
Vitamins
Essential for
Dehalogenase
enzyme production

Patented ProductMinerals
Co-factors for microbial
enzymesA group of effective microbial consortium capable of respiring chlorinated compounds
is stimulated resulting in faster and complete degradation of contaminants.
-8-

EcoClean-E the Slow Hydrogen Releasing Biostimulant

HydrogenNatural Polymer

Energy source for themicroorganisms

Can stay longer in thesubsurface

EcoClean-E™
Microemulsions

Patented Product

Easily propagates in low
K soil

Bio-surfactant effectDesorption of sorbed
mass

Effective in low K soil, to degrade DNAPL and for coastal area
groundwater with high salt concentration.
-9-Target Contaminants Halogenated/Chlorinated Aliphatic Hydrocarbons (CAHs):

Perchloroethylene (PCE), Trichloroethylene (TCE),
Trichloroethane (TCA), Dichloromethane (DCM) which werebeing used as solvents and de-greasers in the industries like
automobile, hardware, electronic equipments, etc.
 And their daughter products like Dichloroethylene (DCE),

Dichloroethane (DCA), Vinyl chloride (VC) in Chemical andplastic raw material manufacturers.

- 10 -

What is Special in EcoClean Based Bioremediation① Short time: Complete remediation within 3～12 months② Remediation of high concentration VOCs contamination(our experience: 380mg/ℓ)③ High priority of safety issue: EcoClean is completely biodegradable
into carbon dioxide and water④ Low cost technology: 1/3 to 1/5 of conventional methods⑤ Easy to apply on the site and under the running facilities/factories- 11 -

EcoClean Gravity Injection

Mixing Tank

Injection Unit

Injection Well

Contamination

Monitoring Well
- 12 -

Injection with Direct Push Technology

Sturm Ruger,NH, USA

Wyman-Gordon
Grafton, Massachusetts, USAGeoProbe 6610-DT track rig
2.3 GPM capacity pump, with pressures up to ~1,000 PSI- 13 -

Double Packer Injection

 Injection points are designed based on
distribution of contamination
 Injection pressure, dilution, etc. depend onhydrogeology
 Precautions taken to avoid short-circuit- 14 -

Various Chlorinated Solvents and Their Decomposition Pathways
CO2
Carbon dioxide

CT
CarbonTetrachloride

PCE
Tetrachloroethylene

1,1,2-TCA
1,1,2-Trichloroethane

CF
Chloroform
DCM
Dichloromethane

1,2-DCA
1,2-Dichloroethane

TCE
Trichloroethylenetrans-1,2-DCE
trans-1,2Dichloroethylene

1,1,1-TCA
1,1,1-Trichloroethane

1,1-DCE
1,1-Dichloroethylene

1,1-DCA
1,1-Dichloroethane

VCVinyl Chloride

CMChloromethane

CO2Carbon dioxide

EtheneMethane
CO2Carbon dioxide

cis-1,2-DCE
cis-1,2-Dichloroethylene

Acetic Acid

CA
Chloroethane
Ethylene
GlycolEthanol

EthaneMicrobial degradation
Anaerobic microbial degradation

Abiotic Hydrolysis
Abiotic dechlorination

Chlorinated solvents are degraded by microorganism with EcoClean. The resulted end products of thewhole process would be non-toxic ethylene, inorganic chlorides, carbon dioxide and water.
- 15 -

Chlorinated Aliphatic Hydrocarbons Contaminated Site, Near Nagoya
Site Characteristics:
Former Metal Finishing Facility

Golf Driving Range

Planned Residential Area

Plume Size: 6,781m2x12m
Pollutants: TCE, cis-1,2-DCE
Hydrogeology: Medium to coarse Sand with some Silty Sand
Hydraulic Conductivity(K): 10-2 to 10-3cm/sec.
High Natural Organic Matter
Initial ORP : <-100 mv

Remedial Approach:
Failed ISCO followed by Enhanced Reductive Dehalogenation
Biostimulant Load: 7,000kgs biostimulant was diluted 100 times in water and injected inexisting wells (around 25 wells)
Specially Monitored and Regulated by Nagoya City
- 16 -

Post-injection Changes in ORP

Before EDC injection

After injection

After 1 week

After 20 days
- 17 -TCE Degradation

Before Injection

30 d post injection

60 d post injection

75d post injection

cis-1,2-DCE Degradation

Before Injection

30 d post injection

60 d post injection

75d post injection

Post remedial monitoring for 2 years (as per Japanese regulations) showed no sign of contamination rebound- 18 -

Permeable Reactive Bio-barriersSpecially designed forbiobarrier

Biostimulant Injection

EcoClean-B™

Contaminants？

Subsurface
Propagation ofBiostimulants

Activemicrobial
zone foranaerobic
degradation

Naturalpolymer

Patented Product
Natural polymer that can stayin subsurface for longer time

Merits of EcoClean Biobarrier over other Conventional Methods

Comparison of Pump & Treat！
 Running cost (electricity; carbon filters; stripper maintenanceetc.) is considerable
 Accelerated groundwater flow due to extraction may result in
spread of the contamination from the source

Comparison with Zero-valent Iron barrier！
 Cost: Around 1/3 of ZVI barrier.
 Large augur is used to mix ZVI into the soil that
consumes large working space and limits operation inactive facilities
 ZVI oxidizes resulting rusty groundwater down
gradient to ZVI barrier.

EcoClean biobarrier is low cost, environmental friendly and needs no maintenance.
- 19 -

EcoClean-M for Bioremediation of Hexavalent Chromium EcoClean-M is a bioremediation agent for soil andgroundwater contaminated with hexavalent chromium (VI).
 It is a mixture of well-balanced nutrients and energy source
for microbes capable of respiring Cr(VI).
 In this process the water soluble, highly toxic Cr(VI) isreduced to non-soluble and stable chromium (III).

EcoClean-M™
Patented Product

Advantages of EcoClean‐M based Bioremediation
 In-situ technology
 Easy to apply even in running facilities
 less than 1/3 the cost of conventional technologies
 Short remediation time (1-3 months)
 Environmentally green; the product is biodegradable to carbon dioxide
and water- 20 -

Site A: Near Tokyo, Automobile Parts Manufacturer
Site Characteristics:
Area: 10,000 m2; thickness of overburden aquifer: 5 m from GL

Concentration (mg/L)

Hydrogeology: Sandy Loam; Gravel; GW level: 2-3 m from the GL

Days after EDC-M injection

Post remedial monitoring for 5 years showed no sign of contamination rebound- 21 -

Green Clean for High Performance Aerobic Remediation
 Green Clean biostimulants are optimal nutritional source for aerobic
microorganisms capable of biodegradation of petroleum hydrocarbons(BTEX, light oils, etc.) cyanide compounds, and chlorinatedhydrocarbons.
 Green Clean is dissolved in water containing high concentration ofoxygen and injected to subsurface.

Green Clean™
Patented Product Made of food materials and food additives, and safe to use.

Green Clean Bioremediation Applicable to Gasoline Stands, Refineries
Petroleum Hydrocarbons Contamination (BTEX, gasoline, diesel, kerosene, light oil, etc.)
dicyclopentadiene, PAHs, etc.
 Chemical manufacturers & users
Ethylene dichloride, Dichloromethane, Vinyl Chloride, etc.
 Gas Manufacture, Electroplating Industries
Cyanides and cyanide metal complexes
Polyaromatic hydrocarbons (PAHs ,naphthalene, benzopyrene, etc.)- 22 -

In-situ Application of Green Clean- 23 -

Green Clean-BP for Remediation of Soil Contaminated with Petroleum Hydrocarbons• BIOTHERMOPILE is useful for treating aerobically degradable contaminants, on-site or off-site.
• Green Clean-BP is efficient to degrade various petroleum hydrocarbons of high concentrations in short times.
• Soil contaminated with petroleum hydrocarbons is made into a pile & Green Clean-BP is mixed to stimulate
natural microorganisms that degrade contaminants.

• Low cost, easy to apply and there is no environmental burden.
Soil Vapor
Monitoring Probes
Contaminated SoilNutrients andMoisture Addition

Air Injection

Ex-situ Application of BIOTHERMOPILE- 24 -Custom-made Nutrients for the Contaminants of Concern
We also manufacture custom-made bioremediation nutrients for a wide rangeof contaminants. Please contact EcoCycle Corporation for further information.
An Example of Contamination Concern

Custom-madeNutrients

Chlorobenzenes (dichlorobenzene, etc.), hexachlorobutadiene
Chlorophenols (pentachlorophenol's, etc.), BCEE, Toxaphene
Trinitrotoluene, Trinitrobenzene, perchlorate, chlorinated agriculturalchemicals & pesticides.

EcoClean-X

Polyaromatic hydrocarbons (PAHs, naphthalene, benzopyrene, etc.)
Dioxanes, MTBE, methyl ethyl ketone, dicyclopentadiene, etc.
Agricultural Chemicals (Simazine, Thiobencarb, Thiram, etc.)

Green Clean-X

Heavy metals of lead, arsenic, cadmium, selenium, copper, cobalt, zinc, &nickel※ Water soluble contaminants are biologically stabilized

EcoClean-MX*Please contact EcoCycle if the contaminants and/or groups are multiple.
Combination of nutrients are applied and/or new nutrients are designed & manufactured.
- 25 -Summary
 Bioremediation: Environmental friendly green technology
 Bioremediation: in-situ application; various contaminants aretargets.
 Bioremediation: High concentration of contaminants can bedegraded; shorter time; cheaper to many existing technologies.
 Bioremediation: Good for running facilities, almost no noise,
simple equipment are needed.
 Very good inter-disciplinary knowledge is needed to succeed.

- 26 -