Ralph Elliott (Butch) President, Enviro Water Quality Restoration And Douglas Dent

1. “Use of Bioremediation in the Treatment of Natural and Man-made Bodies of Water to Improve Water Quality and Reduce Organic Sediments” Ralph Elliott (Butch) President, Enviro Water Quality Restoration And Douglas Dent Senior Vice President Product Development Manager Ecological Laboratories, Inc.

2. Technologies Commonly Used to Remove Pollutants and Sediments from Natural and Man-made Bodies of Water • Physical Chemical Treatment • Oxidizers • Flocculants • Carbon • Zeolites • Mechanical Treatment • Dredging

3. Technologies Commonly Used to Remove Pollutants and Sediments from Natural and Man-made Bodies of Water • Physical Chemical Treatment • Oxidizers • Flocculants • Carbon • Zeolites • Mechanical Treatment • Dredging

4. Technologies Commonly Used to Remove Pollutants and Sediments from Natural and Man-made Bodies of Water • Physical Chemical Treatment • Oxidizers – Oxidize organic and certain inorganic compounds • Hydrogen Peroxide • Potassium Permanganate • Solid Peroxide Compounds e.g. Sodium Carbonate Peroxyhydrate

5. Technologies Commonly Used to Remove Pollutants and Sediments from Natural and Man-made Bodies of Water • Physical Chemical Treatment • Oxidizers • Flocculants • Carbon • Zeolites • Mechanical Treatment • Dredging

6. Technologies Commonly Used to Remove Pollutants and Sediments from Natural and Man-made Bodies of Water • Physical Chemical Treatment • Flocculants – Aggregate and settle out suspended solids • Polymers • Alum • Clays

7. Technologies Commonly Used to Remove Pollutants and Sediments from Natural and Man-made Bodies of Water • Physical Chemical Treatment • Oxidizers • Flocculants • Carbon • Zeolites • Mechanical Treatment • Dredging

8. Technologies Commonly Used to Remove Pollutants and Sediments from Natural and Man-made Bodies of Water • Physical Chemical Treatment • Carbon – Adsorbs organic pollutants, certain metals, etc. • Powdered Activated Carbon (PAC) • Granular Activated Carbon (GAC)

9. Technologies Commonly Used to Remove Pollutants and Sediments from Natural and Man-made Bodies of Water • Physical Chemical Treatment • Oxidizers • Flocculants • Carbon • Zeolites • Mechanical Treatment • Dredging

10. Technologies Commonly Used to Remove Pollutants and Sediments from Natural and Man-made Bodies of Water • Physical Chemical Treatment • Zeolites – Naturally occurring minerals that have an affinity for certain charged species like • Ammonium • Calcium • Magnesium

11. Technologies Commonly Used to Remove Pollutants and Sediments from Natural and Man-made Bodies of Water • Physical Chemical Treatment • Oxidizers • Flocculants • Carbon • Zeolites • Mechanical Treatment • Dredging

12. Technologies Commonly Used to Remove Pollutants and Sediments from Natural and Man-made Bodies of Water • Mechanical Treatment • Dredging – Physical removal of solids that have built up on the bottom of lakes, streams, rivers, from dead vegetation, droppings from water fowl, etc. • Generally done in a channel and does not include bank to bank removal. So in silt conditions the channel fills back rapidly.

13. Technologies Commonly Used to Remove Pollutants and Sediments from Natural and Man-made Bodies of Water • Physical Chemical Treatment • Advantages • Effective in some cases • Disadvantages • Done on a stoichiometric (balance) basis making it expensive to treat large bodies of water • Can be harmful to aquatic life • Can contribute to build up of sludge

14. Technologies Commonly Used to Remove Pollutants and Sediments from Natural and Man-made Bodies of Water • Mechanical Treatment • Dredging • Advantages • Fast • Disadvantages • Costly • Disposal of spoils • Disruptive to aquatic habitat • Not bank to bank • Does nothing for water quality

15. Dredging Environmental Issues Costly Causes Turbidity Destroys Flora and Fauna in Dredge Areas Disrupts Habitats Requires Spoils Disposal Area Impedes Navigation Noisy Large Fossil Fuel Use Negative Impact on Roads and Traffic Long Permitting Process Old Technology Bio remediation Environmentally Benign Cost Effective Minimal Turbidity Minimal Impact on Live Organisms in Dredge Areas Minimal Habitat Disruption No Spoil/Reduced Spoil Area No Impact on Navigation Very Little Noise Low Fossil Fuel Use Little to No Impact on Roads & Traffic Immediate Project Startup Technologically Advanced Dredging vs Bio remediation for Bottom Solids Reduction – Pros and Cons

16. Dredging Pre-treatment Survey Dredge and Fuel Tug and Fuel Pipe and Fusing Machine Dozer and Fuel Trucking and Fuel Weir Environmental Monitoring Endangered Species Monitoring Turbidity Testing 9-15 people plus Truck Drivers Upland Disposal Area Post Treatment Survey Bioremediation Pre-Treatment Survey Mobilization including Launching Boat with Spray Equipment Bio augmentation Product Spray Equipment Small Boat and Fuel 2-4 people Water Quality Testing Post Treatment Survey Dredging vs Bioremediation for Bottom Solids Reduction – Associated Costs

17. Bioremediation as the Natural Alternative for Treatment of Natural and Man-made Bodies of Water Bioremediation • Definition - any process that uses microorganisms, fungi, green plants or their enzymes to return the natural environment altered by contaminants to its original condition. • ELI technology differs significantly in fermentation process, culture consortium and oxidation reduction pathways and processes • Wider range of organic oxidation reduction • Increased pathways, aerobic, facultative and anoxic, denitrification • Capable of denitrification = Nitrate to nitrogen gas ,

18. Biological Oxidation PathwaysMICROBE-LIFT/PBL • Aerobic respiration = ATP 38 • Anaerobic respiration = ATP 2 • Anoxic respiration = ATP 34 • Anaerobic - vs - Anoxic = 17 times faster • Anoxic respiration takes place within the organic muck layer, results in denitrification with nitrate converted to nitrogen gas that bubbles off to the atmosphere

19. Bioremediation as the Natural Alternative for Treatment of Natural and Man-made Bodies of Water • Bioremediation - Based on Natural Processes • Biogeochemical Cycles • Carbon Cycle • Nitrogen Cycle • Use of conventional microbial products may result in temporary improvements • With end biological oxidation reduction byproduct being nitrate, contributing to eutrophication

20. Biological Oxidation Reduction Introduction To MICROBE-LIFT Technology • Truly a Unique Culture Consortium • Microorganisms Differ • ELI Culture Bank (novel bio-technology) • Fermentation Process Differs (enhances product value) • Novel Culture Consortium (grown as a consortium) • Biological pathways advantages, Aerobic, Facultative, Anaerobic , Anoxic, capabilities enhanced by the novel fermentation process, and select microorganisms Consortium value

21. Bioremediation as the Natural Alternative for Treatment of Natural and Man-made Bodies of Water • Bioremediation • Based on Natural Processes • Carbon Cycle

22. Life Cycles –Carbon Cycle Aerobic Bacteria + Oxygen Carbon Dioxide (CO2 ) Aerobic Bacteria + Oxygen Methane (CH4) Photosynthetic Plants Higher Life Anaerobic Bacteria

23. Bioremediation as the Natural Alternative for Treatment of Natural and Man-made Bodies of Water • Bioremediation • Based on Natural Processes • Nitrogen Cycle • Modern microbiology can speed oxidation reduction processes • Assist in the reduction of nitrogen compounds

24. Nitrogen Cycle Nitrate retained in aquatic ecosystems = algae, plant growth Nitrogen (N2) Denitrifying bacteria Nitrogen Fixing Bacteria Nitrate (NO3) Nitrobacter sp. Inorganic Nitrogen (NO3) Ammonia (NH3) Nitrite (NO2) Nitrosomonas sp.

25. Bioremediation as the Natural Alternative for Treatment of Natural and Man-made Bodies of Water • Bio remediation • Can Be Used to Reduce or Eliminate • Speeds the biological oxidation reduction process • Breakdown and reduce slow to degrade matter • Soluble organics that make up BOD, COD, etc. • Soluble inorganic compounds like nitrates • Suspended organic solids • Settled organic solids and organic sludge • Microbial activity primarily within littoral and benthic zones, anaerobic and anoxic pathways vital

26. Augmentation Technology • Consortium contains; phototrophs, autotrophs, heterotrophs, Chemotrophs • Vegetative consortium active on application • Functions all aquatic zones • Functions in aerobic, facultative, anaerobic and anoxic environments • Utilizes Carbon dioxide as a carbon source • Capable of anoxic respiration, denitrification

27. Accelerating Bio remediation Through Bio augmentation • Bio augmentation – The purposeful inoculation of an ecosystem with specialized microbes or a consortium in order to assist the natural populations, accelerating and enhancing the biological processes within the ecosystem. The process must exceed organic loading factors, loading vs removal • Targeting slow and difficult to degrade constituents that accumulate as organic muck • Requires denitrification pathway processes

28. Technology Review • Kinetic rate of organic constituents a factor in muck accumulation • Anoxic respiration speeds rate of bottom solids removal, ATP/38 -ATP/2 - ATP/34 • Anoxic reduction = 17 times faster • Eliminates nitrate, reduces green water • Vegetative and photosynthetic culture consortium reduces lysis and nutrients • Speeds biological oxidation slow and difficult to degrade constituents

29. Single-Celled-Microorganisms

30. Starting a biological process • Form base film • Attach due to charge • Change polymer production • Develop bio film matrix • Inhabit bio film • Generate new cells • Reduce organic waste

31. Four Stages Bacterial Growth Curve Why some on-site systems fail

32. Water restoration processes • Augmentation overcomes flowing water • Applied Microorganisms attach to surface areas within the aquatic environment • Microbes form bio films on all surface areas • Grow and cell divide • Initial feed rate overcomes wash out rate • Functions on and within muck layer • Utilize various pathways and processes • Requires specific oxidation reduction pathways to assure success

33. Biofilms are quite complex • Consist of multiple species of microorganisms. Combined with gel matrices surrounding the cells. The bio film matrices consist of polysaccharides (EPS), the primary structural component of bio films. EPS’s are critical to the formation of soil structure and the EPS exhibits toxicity protection to bio film biology

34. Attachment to soil particles via polymer productionform active bio films

35. Microbial attachment = polymers Microbes Microbes adhere to soil particles due to charge relationships, hydrophobic & hydrophilic forces, & extracellular polymers. Microbes form strong attachments through the production of polysaccharide matrix and cellular structures called fimbrae, polysaccharide adds to soil structure and nutrient retention.

36. Anoxic respiration takes place within the anaerobic zone (denitrification)

37. Restoration Performance • Enhances water quality (organics) • Reduces odorous biological reactions, controls hydrogen sulfide • Reduces fish kills • Achieves denitrification in anaerobic zone via anoxic respiration, reduces green water events, and aquatic weed growth • Increases bottom solids oxidation reduction process by 17 times (faster) • Capable of 6 inches to 12 inches reduction/year (non aeration)

38. Bio augmentation – Methods of Application

39. Bio augmentation – Methods of Application

40. Bio augmentation –Method of Application

41. Bioremediation of Organic Solids and Sediments • Where Does It Go? • Organics are converted to carbon dioxide and water just like wood in a fireplace except it is biological oxidation vs thermal oxidation via combustion. The non-biodegradable fraction is equivalent to the ash.

42. Determining Bio-treatability of Solids and Sludge • Acquire Representative Samples of Sludge • Perform Volatile Solids Analysis (VSS) • Indicates organic fraction which gives a good indication of the biodegradable fraction

43. Case Studies – Retention PondPenang, Malaysia

44. Penang Retention PondSources of Pollution

45. Bioremediation Project - Retention Pond Penang, Malaysia • Treatment Objectives • Reduce BOD/COD of water before discharge to ocean • Reduce odors affecting nearby condos • Reduce sludge in bottom of retention pond (added onto list of objectives after initial pond evaluation identified 24” of sludge in bottom of pond)

46. Bioremediation Project – Retention Pond, Penang, Malaysia • Treatment Program • Inoculation with MICROBE-LIFT/IND • Day 1 – 10 ppm • Day 8, 15, 22, 29 – 8 ppm • Maintenance – 2 ppm per week * Sold in the U.S. as MICROBE-LIFT/IND

47. Bioremediation Project – Retention Pond, Penang, Malaysia

48. Ground Level Water Level Sludge Level on 29 Aug 2005 Sludge Level on 17 Oct 2005 Approx. 300mm (11.3”) Approx. 600mm (22.6”) Appr 150 mm Sludge Level on 7 Nov 2005 Approx. 100mm Bottom datum of pond Sludge Level on 24 Feb 2006 Approx. 300mm Bioremediation Project -Retention Pond, Penang Malaysia

49. Bioremediation Project –Retention Pond, Penang, Malaysia • Program Summary • Odors Eliminated in One Month • Sludge Reduced from 24” to < 6” in Six Months • BOD/COD Reduced to Below Discharge Limits >90% of the time

50. Case Studies – Xiba RiverKunming, China