1. Bioavailable Phosphorus in Advanced Phosphorus Removal Facility Effluent Phase II Study Bo Li, Michael T. Brett Department of Civil and Environmental Engineering, University of Washington, Seattle, Washington 98195, USA; libo@u.washington.edu

2. INTRODUCTION Effluent total phosphorus (TP) concentration 3mg/L 500µg/L 50µg/L 20µg/L 100mg/L 200mg/L 250mg/L!! What is left here??

3. Phosphorus Characterization – Common Operational Categories: Total Phosphorus (TP) Total Reactive Phosphorus (TRP) Soluble Reactive Phosphorus (SRP) etc…. However…. These are all based on chemical analysis… INTRODUCTION

4. Phosphate (PO 4 -3 ) …. Inorganic P Apatite AlPO 4 FePO 4 Recalcitrant P Organic P Polyphosphate Inositol hexakisphophate L-α-phosphatidyl choline phosphoenol pyruvate Definition of Bio-available phosphorus (BAP) Phosphorus can be utilized by algae INTRODUCTION

7. METHODS PHASE I STUDY – Spokane Region Spokane City Pilot Plant City of Coeur d’Alene Post Falls Liberty Lake Hayden Area Regional Sewer Board Inland Empire Paper Spokane River

8. Previous study on effluent TP vs. BAP% – Spokane City Pilot Plant (Three alum additions + filtration process) PHASE I RESULTS – Effluent TP vs. BAP% A very strong statistical correlation between the effluent TP and BAP% As more chemicals were added, the BAP% in the effluent declined. KEY NOTE KEY NOTE

9. P bioavailability in Spokane Regional WWTP effluents BAP% varied in effluents from different advanced nutrient removal facilities Less than 60% of P in the effluents are bioavailable. BAP% varied in effluents from different advanced nutrient removal facilities Less than 60% of P in the effluents are bioavailable. KEY NOTE KEY NOTE PHASE I RESULTS – Effluent TP vs. BAP%

10. 21 P species – inorganic P, organic P, humic substances 75 samples from 17 Plants Treatment Process CategoryPlantsChemical Addition EBPR without chemical addition 3 MBR Processes 2 √ Single stage tertiary 6 √ Dual stage tertiary 6 √ METHODS WERF national *EBPR: Enhanced Biological P removal *MBR: Membrane Biological Reactor

11. Bioavailability of P species: P SpeciesReactivityBioavailability Al-P, Fe-P Apatite DNA, RNA Humic Substances 90% ≈15% WERF national RESULTS – P species Not all P are created equal Can’t estimate Bioavailable P merely based on chemical analysis Not all P are created equal Can’t estimate Bioavailable P merely based on chemical analysis KEY NOTE KEY NOTE

12. Find the best predictor of BAP – TRP vs. BAP RESULTS WERF national RESULTS – Effluent TRP vs. BAP TRP is a conservative measure of BAP! KEY NOTE KEY NOTE *TRP: Total Reactive P

13. BAP% & TP results RESULTS WERF national RESULTS – Effluent TRP vs. BAP Intensive chemical P removal processes were used to achieve low TP concentrations. KEY NOTE KEY NOTE WERF national RESULTS – Effluent BAP% vs. TP

14. CONCLUSIONS PHASE II STUDY – Objective 1 CBODP – Phosphorus associated with Carbonaceous Biochemical Oxygen Demand (CBOD) Assumptions: – CBODP = TP – SRP – k CBODP = k TP-SRP Lake Spokane CE-QUAL-W2 model *SRP: Soluble Reactive P BAP = CBODP*(1-e -kt )

15. CONCLUSIONS PHASE II STUDY– Objective 1  CBODP Decay rate (k CBODP ) Corresponding SourceDecay rate, day -1 Point Sources Liberty Lake WWTP0.0456 Spokane WWTP0.0736 Coeur D‟Alene WWTP0.0792 Post Falls STP0.066 Inland Empire Paper0.0186 Kaiser Aluminum0.1275 Hayden POTW0.0838 Hayden POTW summer discharge (for scenarios)0.0838 AVE0.07 Non-Point Sources Organic matter from Washington Tributaries0.066 Lake Coeur D‟Alene CBOD0.13 AVE0.10 Lake Spokane CE-QUAL-W2 model

16. CONCLUSIONS PHASE II STUDY – Objective 1 Objective 1: Determine dissolved phosphorus mineralization rates Hypothesis: The mineralization rate for the recalcitrant dissolved P for Spokane region WWTP effluents is likely to be different with the original value assumed by current TMDL model. Task: The algal dissolved P uptake experimental design can determine conservative dissolved organic P mineralization rates for any Spokane region effluent type. These mineralization rates can be integrated into the current Long Lake TMDL model.

17. CONCLUSIONS PHASE II STUDY– Objective 1 Objective 1: Determine dissolved phosphorus mineralization rates Experimental Design: Sample P Bioassay Dissolved P analysis (Days 0, 1, 2, 4, 8, 14 and 21) Dissolved P mineralization rate Autoclaved Selenastrum capricornutum Initial concentration:200,000 cells/ml. Incubate for 21 days. Continuous Illumination Temperature: 24 ± 2ºC Shake at 110 rpm.

18. CONCLUSIONS PHASE II STUDY– Objective 1 Objective 1: Determine dissolved phosphorus mineralization rates Experimental Design: Sample P Bioassay Dissolved P analysis (Days 0, 1, 2, 4, 8, 14 and 21) Dissolved P mineralization rate Autoclaved BAP = BAP%*TDP*(1-e -kt ) BAP%, K *TDP: Total Dissolved P

19. CONCLUSIONS PHASE II STUDY– Objective 1 Objective 1: Determine dissolved phosphorus mineralization rates Experimental Design: Sample P Bioassay Dissolved P analysis (Days 0, 1, 2, 4, 8, 14 and 21) Dissolved P mineralization rate Autoclaved BAP% = 75%, k = 1.1 day -1 BAP% = 43%, k = 1.5 day -1

20. CONCLUSIONS PHASE II STUDY– Objective 2 Objective 2: Resolving issues on bioassay methods from the Phase I Spokane BAP study Hypothesis: Ecology and EPA concerned that some of the very low effluent %BAP results we reported were at least partially a result of nutrient co-limitation (e.g., by nitrogen) and/or effluent toxicity. Task: We will conduct experiments to determine whether the low %BAP we have previously determined for Spokane region dischargers is partially an artifact of nutrient colimitation and/or effluent toxicity.

21. CONCLUSIONS PHASE II STUDY– Objective 2 Objective 2: Resolving issues on bioassay methods from the Phase I Spokane BAP study Experimental Design: Sample Bioavailability of P in the effluents amended with all of other nutrients P Bioassay amended with all of other nutrients + P

22. CONCLUSIONS PHASE II STUDY– Objective 3 Objective 3: The role of Humics in dissolved nutrient recalcitrance Hypothesis: It is hypothesized that both recalcitrant dissolved organic nitrogen (rDON) and dissolved organic P (rDOP) will be associated with large molecular weight size fractions, (> 10 kilo Daltons; kDa). Task: We will use ultra-filtration to size fractionate the "dissolved" or colloidal portion of effluents with a high proportion rDON and rDOP to determine whether nutrient recalcitrance is associated with a high proportion of nutrients in the large molecular weight size range associated with colloidal humic substances (i.e., > 10 kDa).

23. CONCLUSIONS PHASE II STUDY– Objective 3 Objective 3: Comparison of the humic hypothesis for rDON and rDOP Experimental Design: Sample Ultrafiltration (3, 10, 30 and 100 kDa) P and N analysis DON and DOP concentration in each size range Filtrate

24. CONCLUSIONS PHASE II STUDY– Objective 3 Objective 3: Comparison of the humic hypothesis for rDON and rDOP Experimental Design: Sample Ultrafiltration (3, 10, 30 and 100 kDa) P and N analysis DON and DOP concentration in each size range Filtrate Problem: 3kDa membrane might filter out some fractions of PO 4

25. Water Environment Research Foundation (WERF) Nutrient Challenge Program Spokane River Stewardship Partners (SRSP) ACKNOWLEDGEMENTS

26. QUESTIONS?