1. Bioscience, Inc. Allentown, PA www.bioscienceinc.com

2. The Biological Process of Wastewater Treatment Jay Hill Product Manager

3. Coarse Physical TreatmentCoarse Physical Treatment Settling of Suspended Solids/FloatablesSettling of Suspended Solids/Floatables Biological Treatment of Dissolved/ Fine SolidsBiological Treatment of Dissolved/ Fine Solids Recovery of BiomassRecovery of Biomass Removal of InorganicsRemoval of Inorganics DisinfectionDisinfection Excess Biomass RemovalExcess Biomass Removal Typical Wastewater Treatment Steps

4. Primary Treatment Bar ScreenBar Screen ClarifierClarifier –Primary sludge Sand/gritSand/grit Coarse organic matterCoarse organic matter –Floatables FOGFOG DAF or API SeparatorDAF or API Separator –FOG, petroleum HC, light solids

5. Activated Sludge Systems Untreated Discharge Aeration Tank Clarifier Recycled Sludge Biomass (Secondary Sludge) Clean Water O2O2 CO 2

6. Secondary Clarification ClarifierClarifier –Secondary sludge Fine organic matterFine organic matter –Floatables FOGFOG

7. Solids Processing Sludge ThickeningSludge Thickening Aerobic or Anaerobic DigestionAerobic or Anaerobic Digestion CentrifugeCentrifuge Belt PressBelt Press Incineration, Land Application, Fertilizer, LandfillIncineration, Land Application, Fertilizer, Landfill

8. Aerobic Microbial Respiration Respiration C,H + O 2 CO 2 + H 2 O + protein * * bacteria, N, P, pH, temperature

9. Composition of Wastewater Inorganics Inorganics Ammonia Ammonia Nitrate Nitrate Phosphate Phosphate Carbonate Carbonate Minerals Minerals Calcium Calcium Magnesium Magnesium Iron Iron Etc. Etc. Organics Organics Biodegradable (BOD) Biodegradable (BOD) Carbohydrates Carbohydrates Proteins (TKN) Proteins (TKN) FOG FOG Non-Biodegradable (COD-BOD) Non-Biodegradable (COD-BOD) Large particles Large particles Complex polymers (plastics, lignin) Complex polymers (plastics, lignin) Surfactants (some) Surfactants (some) Pesticides (some) Pesticides (some) Pharmaceuticals (some) Pharmaceuticals (some)

10. Temperature Temperature pH pH Water activity Water activity Energy source Energy source Nutrients Nutrients Carbon Carbon Nitrogen Nitrogen Phosphorus Phosphorus Minerals Minerals Vitamins/growth factors Vitamins/growth factors Requirements for Growth of Microbes

11. Thermophiles Thermophiles 40°C to >100°C 40°C to >100°C Mesophiles Mesophiles 10°C to ~45°C 10°C to ~45°C Psychrophiles Psychrophiles <5°C to ~35°C <5°C to ~35°C Temperature

12. Acidophiles Acidophiles pH 0-6 pH 0-6 Alkaliphiles Alkaliphiles pH 8-13 pH 8-13 Most bacteria prefer pH 6-8 Most bacteria prefer pH 6-8 Most fungi prefer pH 4-7 Most fungi prefer pH 4-7 pH

13. Salt content of water Salt content of water Fresh water <1% NaCl (most bacteria) Fresh water <1% NaCl (most bacteria) Brackish water ~1-3% NaCl (limits some species) Brackish water ~1-3% NaCl (limits some species) Seawater ~3.5% NaCl (salt tolerant only) Seawater ~3.5% NaCl (salt tolerant only) Saline water up to 30+% (saturated NaCl)- (only few species) Saline water up to 30+% (saturated NaCl)- (only few species) Soils (moisture content and salts) Soils (moisture content and salts) 50-100% FMC (most bacteria) 50-100% FMC (most bacteria) Fungi tolerate lower moisture content Fungi tolerate lower moisture content Water Activity

14. Oxygen (aerobes) C 6 H 12 O 6 + 6 O 2  6 CO 2 +6 H 2 O Oxygen (aerobes) C 6 H 12 O 6 + 6 O 2  6 CO 2 +6 H 2 O Autotrophs Autotrophs NH 4 + + 2 O 2  NO 3 - + H 2 O + 2 H + (Nitrifiers) NH 4 + + 2 O 2  NO 3 - + H 2 O + 2 H + (Nitrifiers) H 2 S + 2 O 2  SO 4 - - + 2 H + (sulfur oxidizers) H 2 S + 2 O 2  SO 4 - - + 2 H + (sulfur oxidizers) H 2 S + 0.5 O 2  S 0 + H 2 O (sulfur oxidizers) H 2 S + 0.5 O 2  S 0 + H 2 O (sulfur oxidizers) Nitrate (facultative) C 6 H 12 O 6 + 6 H 2 O  6 CO 2 + 12 H 2 / 5 H 2 +2 NO 3 - + 2 H +  N 2 + 6 H 2 O (denitrifiers) Nitrate (facultative) C 6 H 12 O 6 + 6 H 2 O  6 CO 2 + 12 H 2 / 5 H 2 +2 NO 3 - + 2 H +  N 2 + 6 H 2 O (denitrifiers) Sulfate (anaerobes) C 2 H 4 O 2  CO 2 / SO 4 --  H 2 S (sulfate reducers) Sulfate (anaerobes) C 2 H 4 O 2  CO 2 / SO 4 --  H 2 S (sulfate reducers) Carbon dioxide (anaerobes) CO 2 + 4 H 2  CH 4 +2 H 2 O (methanogens) Carbon dioxide (anaerobes) CO 2 + 4 H 2  CH 4 +2 H 2 O (methanogens) Fermentation C 6 H 12 O 6  2 CO 2 +2 C 2 H 5 OH Fermentation C 6 H 12 O 6  2 CO 2 +2 C 2 H 5 OH Energy Sources

15. Nutrients Required for Growth Carbon Carbon Usually from food source or CO 2 Usually from food source or CO 2 Nitrogen Nitrogen Usually from ammonia, nitrate or simple organics (amino acids) Usually from ammonia, nitrate or simple organics (amino acids) Phosphorus Phosphorus Inorganic phosphate Inorganic phosphate Sulfur Sulfur Inorganic sulfate or simple organics Inorganic sulfate or simple organics Minerals (Ca, Mg, K, Na, Fe) Minerals (Ca, Mg, K, Na, Fe) Trace elements (Ni, Co, Cu, Mo, Zn) Trace elements (Ni, Co, Cu, Mo, Zn) Growth factors/vitamins Growth factors/vitamins

16. Operation Limits pH 6-9 pH 6-9 <4: Most bacteria dead or inactive <6: Bacteria activity drops, fungi may create settling problem <6.5: Nitrification very poor 7.5: optimum for hydrocarbons, fog, nitrification, sulfide >9: Bacteria activity drops Nutrients BOD:N:P = 100:5:1 Effluent ammonium-N <2 mg/L may limit BOD removal or slow response to slug loading Effluent ammonium-N <0.5 mg/L probably deficient unless nitrifying (nitrate provides N) Effluent ortho-phosphate <1 mg/L may limit BOD removal or slow response to slug loading Effluent ortho-phosphate <0.2 mg/L probably deficient

17. Operation Limits Temperature Temperature <5°C Few bacteria are active <15°C Nitrification and most bacteria growth very slow 20-35°C Optimum for most bacteria 39-45°C Bacteria activity drops, death rate increases >45°C Only adapted or thermophilic processes occur Biomass MLSS normally 1500-6000 mg/L <1500 poor settling, dispersed >6000 oxygen limited?; may overflow clarifier weir MLSS/MLVSS 80-90% <80% low viable percentage, possible accumulation of inert <70% may occur in aerobically digested sludge >90% light (poor settling) floc

18. Operation Limits Sludge Age/MCRT Sludge Age/MCRT <3 days Poor settling/COD removal/high sludge production <8 days May have poor nitrification >20 days May have filament problems or pin floc; good for exotic chemical degradation and sludge digestion SOUR Complete mix system 3-15 mg O2/g MLSS per hour <3 Inhibition or severe underload >15 Slug load/ possible overloading Staged aeration –1 st Stage 30-100 mg O2/g MLSS per hour <20 Inhibition <30 Insoluble waste ` >100 Overloading

19. Operation Limits Dissolved Oxygen Dissolved Oxygen 2-7 mg/L normal range <0.5 Anaerobic <1 General BOD removal slows <2 Nitrification slows >7 Slow growth (inhibition) or underloaded >9 Bacteria dead or inactive Sludge Blanket Normal range 3-7 ft below surface <3 feet(1 meter) poor settling or compaction; biomass may washout with flow increase >7 feet (2 meters) Rapid settling may leave dispersed solids in effluent

20. Typical Upsets Heavy Organic Load Increased Growth/Respiration Reduced Dissolved Oxygen More Sludge to Clarifier Reduced Growth/Respiration Inadequate Removal Deflocculation of the Biomass Poor Settling Worse Effluent Elevated BOD/COD High Effluent Suspended Solids

21. Common Wastewater Problems Poor Settling Poor Settling Effluent Violation (TSS or BOD) Effluent Violation (TSS or BOD) Filamentous Forms Filamentous Forms Poor Nitrification Poor Nitrification Toxicity Toxicity Odors Odors

22. Aerobic Microbial Respiration Respiration C,H + O 2 CO 2 + H 2 O + protein * * bacteria, N, P, pH, temperature

23. Chemical Characterization of VISC 25 ParameterResultComments pH of 10% solution10.5 Alkalinity37 meq/100 gTitrated to pH 7 w/HCl Ammonium-Nitrogen400 mg/L Nitrite-Nitrogen5 mg/LColor fades rapidly Nitrate-Nitrogen<5mg/LTurns yellow after cadmium addition Phosphate-Phosphorus30 mg/L Chemical Oxygen Demand71,000 mg/L

24. Wastewater Treatment Plant Performance Testing Wastewater Treatment Plant Performance Testing Process control tests or performance evaluation tests to determine overall treatment process efficiency, identify or investigate problems, or evaluate specific ability to treat target compounds. Process control tests generally must be quick turnaround tests usually performed on-site to allow process adjustment in response to problems. However, some longer time-frame tests may be set up to predict or determine the effect of process changes or identify trends in process efficiency. Some of the investigations performed by Bioscience have been designed to: 1. evaluate foam or settling problems 2. measure nitrification rates or nitrification potential 3. measure FOG degradation rates or potential 4. measure permissible loading rates for potentially toxic waste streams or septage 5. measure effectiveness of bioaugmentation 6. measure biomass kinetic constants for process design.

25. Wastewater Treatment Plant Performance Testing Wastewater Treatment Plant Performance Testing Available methods include: Standard Methods 5210D Biochemical Oxygen Demand Respirometric Method (Respirometric Oxygen Uptake) Standard Methods 2710B Oxygen-Consumption Rate (Specific Oxygen Uptake Rate; Dissolved Oxygen Probe Method) OECD 209 Activated Sludge, Respiration Inhibition Test ASTM D5120 Standard test Method for Inhibition of Respiration in the Activated Sludge Process Short-Term BOD Test (EZ-BOD instrument test for influent or effluent BOD-5 estimation) Suspended Solids (Photometric Method) CONTRAL Biodegradation Kinetics Microscopic Evaluation of Biomass (Higher Forms and Filaments)

26. Wastewater Treatment Problems (AS) ProblemCauseCure FOG in collection VariousDNT-RF/GEL Odor/H 2 S Anaerobic condition DNT-RF/ANL FOG in aeration basin Slow digestion DNT-RF/SXM/NPN/TM High temperature/low activity Hot process water HT No or partial nitrification Toxicity/low temp/low SRT/nutrients XNC/XNL/TM Low COD or specific compound removal Low temp /various/industry specific HX, XR, XP, etc. Excessive filaments various XF, SXM, nutrients Excessive sludge FOG/cellulose/etc accumulation in sludge SR Poor sludge digestion Nutrient imbalance/FOG AD, SXM, TM

27. Wastewater Treatment Problems (Lagoon) ProblemCauseCure Odor/H 2 S Odor/NH 3 Anaerobic condition High NH 3 /High pH ANL/TN and/or aeration ECL FOG in aeration basin Slow digestion DNT-RF/SXM/NPN/TM High temperature/low activity Hot process water HT No or partial nitrification Toxicity/low temp/low SRT/nutrients XNC/XNL/TM Low COD or specific compound removal Low temp /various/industry specific LF, HX, XR, XP, etc. Excessive sludge FOG/cellulose/etc accumulation in sludge SR Animal waste consistency High solids DL Algae High nutrients (N,P) AL/ALN Poor denitrification Low facultative population DEN

28. Bioaugmentation

29. How Does Bioaugmentation Work? Numbers – By adding cultures regularly the minor cultures (but important cultures) gain a survival advantage (against the dominant cultures). Natural Genetic Interchange – Recent work indicates the possibility of transfer to the biomass of desirable and needed characteristics (but not permanently), particularly capabilities controlled by the plasmids in the cells and demanded by the conditions in the system.

30. Activated Sludge- Culture Selector The biomass is comprised of thousands of cultures of bacteria, fungi, protozoans, etc. The system “selects” cultures with both major/dominant populations and minor counts. Both populations are important in obtaining good effluent quality. The combination of cultures in the biomass continuously changes and adapts to changes in ambient conditions.

31. Major Versus Minor Cultures Major Cultures Grow rapidly settle well control the general nature of the biomass Minor Cultures Produce important results Are more difficult to maintain in the biomass

32. The Transfer of Plasmids a b c d Scientific American, January 1998, p. 68

33. Bioaugmentation Benefits Benefits for Wastewater Treatment: Reduce Effluent Peaks (NPDES outages) Reduce Effluent Peaks (NPDES outages) Reduce Effects of Toxic Compounds Reduce Effects of Toxic Compounds Improve Settling Thru Filament Control Improve Settling Thru Filament Control Enhance Process Stability Enhance Process Stability Reduce Sludge Production Reduce Sludge Production Minimize Downtime/Reduce Labor Minimize Downtime/Reduce Labor

34. Filamentous Populations Individual microbes do the work Microbes flocculate and form particles that settle But the filamentous forms inhibit settling

35. Nitrification 2NH 4 + + 3O 2 2NO 2 - + 4H + + 2H 2 O 2NO 2 - + O 2 2NO 3 -

36. NH3 Treatment System Removal of insolubles Removal of BOD Removal of NOD Clarifier Recycle of Sludge Discharge of Sludge