Anaerobic Conditions for Optimal Biological Phosphorus Removal

1. Anaerobic Conditions for Optimal Biological Phosphorus Removal Sarah Kloss and Laura Mar CEE 453 Research

2. Objectives • to determine whether or not the duration of the anaerobic state would affect the efficiency of phosphorus removal • to find a range for the optimum anaerobic time • to compare our findings with the literature supported estimate of 1-1.5 hrs

3. What are PAOs? • Phosphorus Accumulating Organisms • They release phosphorus during the anaerobic stage • Bacteria take up short-chain fatty acids and transform them to PHB • At the same time, they release phosphorus through the hydrolysis of polyphosphate, which creates energy to make ATP • They take up phosphorus during the aerobic state • the bacteria are able to grow on the stored carbon products • take up excess phosphorus to incorporate in biomass and polyphosphate Source: University of Queensland 2001

4. Past Research in CEE 453 • Sedorovich et al., 2003 tested the effects of pH on the system. They found the optimal pH to be 7, which is supported by the literature (Bond, 1998). • Burns, Mitszewski, and Bovee, 2004 found that there was indeed increased phosphorus removal with the addition of an anaerobic state to the sequencing batch reactor

5. Our Experimental Setup • Sequencing Batch Reactor • Physical setup did not deviate from the lecture notes

6. The States of Our Reactor

7. Parameters Controlling Exit Conditions

8. Timeline of Our Experiment • First Seven Days – Set the anaerobic time to 1.5 hrs and allowed our plant to assimilate • Day 8 – Began taking effluent samples twice daily from our reactor and Juan and Phil’s reactor • Day 15 – Changed anaerobic time to 0.75 hrs • Day 19 – Change anaerobic time to 3 hrs • Day 21 – Power Outage anaerobic time reset to 1.5 hrs • Day 26 – Change anaerobic times to 3 hrs again

9. Phosphorus Test(Colorimetric Wet Chemistry Technique) • Reagent was made from a mixture of four stock solutions • Highly reactive - stock solutions had to be recombined on a weekly basis • Created a standard absorbance curve with known concentrations • Measured 2 repetitions of each sample at 95% dilution • Combined the following in 1.5 mL cuvets • 50 µL of sample • 950 µL E Pure Water • 160 µL Reagent • Waited 10 minutes for reaction to occur • Recorded phosphate concentrations calculated by the Spectrophotometer program based on our absorbance curve

10. Data Analysis • Phosphate concentrations were converted to phosphorus concentrations • Phosphorus concentrations adjusted based on dilution • Phosphorus removal calculated based on assumed constant influent of 6.9 mg/L • For each effluent sample the average of the two repetitions was used • Averages were plotted as a function of time in order to look for trends in our data

11. Results • Removal efficiencies highly inconsistent even over intervals with the same anaerobic time • Our data describes phosphorus generation • Large variation in P concentration from same sample vial

12. Hypothesis: PAO Storage • During anaerobic conditions, PAO may have released stored phosphorus indicating P generation. • 40 minute anaerobic settling time – P released stored P that then was drained in effluent waste water. • Drain Cup – sludge from drain state, samples not collected immediately therefore potentiallyhours for PAO to release phosphorus into our samples prior to collection. • Refrigerator - assume no P release due to cold temperatures (dormant bacteria)

13. Hypothesis: P Contamination • Additional P may have been from outside sources • Found reagent to react with lab equipment (pipettes, beakers, storage containers, distilled water) • If there is P in distilled water it makes sense that most lab equipment was contaminated with P • Likely that sample bottles were also contaminated

14. Error in Lab Technique • We do not think error is from inaccurate pipette technique, this would not explain magnitude of our errors. • Reagent easily contaminated making absorbance reflect sample AND contaminated reagent • We had trouble getting our data to fit within standard absorbance curve even with 95 % dilution • Extrapolation less precise than interpolation • Pulling samples at different heights within the effluent sample vial • Did not to mix the vial because we didn’t want any of the settled solids • Concentrations may have varied over height of vial, especially if P was being absorbed from contaminated vial over time or if PAO were releasing P over time.

15. Inadequate Procedure • Studies have indicated that active PAO may take at least four months to develop after the introduction of an anaerobic stage in a lab scale setting (Kortstee et al., 1994). • Phosphorus removal calculations rely heavily on our assumed influent concentration. • Assumed that the influent phosphorus concentrations would be constant. • If influent concentration higher than expected it would account for apparent phosphorus production. • inadequate mixing of the 20x concentrated waste stock • Variable delivery of phosphorus • 100x stock solution was inadequately mixed • Variable concentrations in 20x bottles

16. System Stresses • Thanksgiving Break and City of Ithaca power outage (Nov. 24) • Disruption of system • Default settings restored interfering with our experiment • Ran out of stock solution • High bacterial death period (darker bacteria)

17. Suggestions for Future Projects • Long startup time with anaerobic phase for PAO assimilation • More assimilation time with each change • Rinse sample vials (and all lab equipment) with reagent • Rinse all equipment with E pure water • Mix stock waste frequently • Take influent samples daily • Take samples as soon as possible after draining • Centrifuge samples prior to testing them • Test more repetitions of the same sample for less error in results • Take highly detailed notes of all procedures and technical failures in the lab

18. Conclusions • With our data we are uncomfortable making conclusions about optimal anaerobic times • Experiment a learning experience • Able to provide better procedure for future experiments • Problems w/ Future Biological P Removal Projects • Relies heavily on establishing and maintaining the PAO population. • An effective PAO population can take up to four month to grow. • Semester (esp. with vacations) not enough time to run successful project

19. References • Burns, Peter, A. Mitszewski, and B. Bovee. (2004). “Investigation of Biological Phosphorus Removal in a Sequencing Batch Reactor.” CEE 453 Research Project, Spring 2004. • Lemos, Paulo, et al. (2003) Metabolic Pathway for Propionate Utilization by Phosphorus-Accumulating Organisms in Activated Sludge: 13C Labeling and In Vivo Nuclear Magnetic Resonance. Applied and Environmental Microbiology, January 2003, p. 241-251, Vol. 69, No. http://aem.asm.org/cgi/content/full/69/1/241 • Kortstee GJ, Appeldoorn KJ, Bonting CF, van Niel EW, and van Veen HW (1994). Biology of polyphosphate-accumulating bacteria involved in enhanced biological phosphorus removal. FEMS Microbiol Rev. Oct;15(2-3):137-53. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=7946465&dopt=Abstract • The University of Queensland, Australia.(2001). Discovery of Biological Phosphorus Removal Organisms. http://awmc.uq.edu.au/activities/BPR.html • Weber-Shirk, Monroe. (2004) Laboratory Research in Environmental Engineering, CEE 453, Fall 2004. Cornell University. Class notes, laboratory manual, and course resources.