Anaerobic Conditions for Anaerobic Conditions for Optimal Biological Optimal Biological

Phosphorus RemovalPhosphorus Removal

Sarah Kloss and Laura MarSarah Kloss and Laura MarCEE 453 ResearchCEE 453 Research

ObjectivesObjectives

to determine whether or not the duration of to determine whether or not the duration of the anaerobic state would affect the the anaerobic state would affect the efficiency of phosphorus removalefficiency of phosphorus removal

to find a range for the optimum anaerobic to find a range for the optimum anaerobic timetime

to compare our findings with the literature to compare our findings with the literature supported estimate of 1-1.5 hrs supported estimate of 1-1.5 hrs

What are PAOs?What are PAOs?

PPhosphorus hosphorus AAccumulating ccumulating OOrganismsrganisms

They release phosphorus during the anaerobic stage They release phosphorus during the anaerobic stage Bacteria take up short-chain fatty acids and transform them to Bacteria take up short-chain fatty acids and transform them to

PHB PHB At the same time, they release phosphorus through the At the same time, they release phosphorus through the

hydrolysis of polyphosphate, which creates energy to make ATPhydrolysis of polyphosphate, which creates energy to make ATP

They take up phosphorus during the aerobic stateThey take up phosphorus during the aerobic state the bacteria are able to grow on the stored carbon productsthe bacteria are able to grow on the stored carbon products take up excess phosphorus to incorporate in biomass and take up excess phosphorus to incorporate in biomass and

polyphosphate polyphosphate

Source: University of Queensland 2001

Past Research in CEE 453Past Research in CEE 453

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

Our Experimental SetupOur Experimental Setup

Refrigerator

Reactor

Pump

Sequencing Batch ReactorSequencing Batch Reactor

Physical setup did not deviate from the Physical setup did not deviate from the lecture noteslecture notes

The States of Our ReactorThe States of Our ReactorState Characteristics Main Exit Condition Secondary Exit

Condition

Fill with Waste Waste is fed by pump from the refrigerator to the reactor

Time in state > Maximum Waste Time

Fill with Water Tap Water is fed by pump from the refrigerator to the reactor, stirrer is on

Volume in tank > Maximum Reactor Volume

Time in state > Maximum Water Time

Anaerobic State Stirrer is on Time in state > Max Anaerobic Time

Aeration Stirrer is on, air flow valves are set toggle

Time in state > Max Aerobic Time

Settle Everything is off Time in state > Max Settle Time

Drain Effluent Valve is on Volume in tank < Minimum Tank Volume

Time in state > Max Drain Time

Parameters Controlling Exit Parameters Controlling Exit ConditionsConditions

Max Waste Time 31.6 seconds

Max Water Time 600 seconds

Max Tank Volume 4 L

Min Tank Volume 1.2 L

Anaerobic Time Varied as part of our experiment

Aerobic Time 6 hr

Settle Time 0.67 hr

Max Drain Time 2000 seconds

Flow Rate 384 mL/min

Timeline of Our ExperimentTimeline of Our Experiment

First Seven Days – Set the anaerobic time to 1.5 First Seven Days – Set the anaerobic time to 1.5 hrs and allowed our plant to assimilatehrs and allowed our plant to assimilateDay 8 – Began taking effluent samples twice Day 8 – Began taking effluent samples twice daily from our reactor and Juan and Phil’s daily from our reactor and Juan and Phil’s reactorreactorDay 15 – Changed anaerobic time to 0.75 hrsDay 15 – Changed anaerobic time to 0.75 hrsDay 19 – Change anaerobic time to 3 hrsDay 19 – Change anaerobic time to 3 hrsDay 21 – Power Outage anaerobic time reset to Day 21 – Power Outage anaerobic time reset to 1.5 hrs1.5 hrsDay 26 – Change anaerobic times to 3 hrs againDay 26 – Change anaerobic times to 3 hrs again

Phosphorus TestPhosphorus Test(Colorimetric Wet Chemistry Technique)(Colorimetric Wet Chemistry Technique)

Reagent was made from a mixture of four stock solutionsReagent was made from a mixture of four stock solutions Highly reactive - stock solutions had to be recombined on a Highly reactive - stock solutions had to be recombined on a

weekly basisweekly basis

Created a standard absorbance curve with Created a standard absorbance curve with known concentrationsknown concentrationsMeasured 2 repetitions of each sample at 95% dilutionMeasured 2 repetitions of each sample at 95% dilutionCombined the following in 1.5 mL cuvetsCombined the following in 1.5 mL cuvets

50 50 µL of sampleµL of sample 950 950 µL E Pure WaterµL E Pure Water 160 µL Reagent160 µL Reagent

Waited 10 minutes for reaction to occurWaited 10 minutes for reaction to occurRecorded phosphate concentrations calculated by the Recorded phosphate concentrations calculated by the Spectrophotometer program based on our absorbance Spectrophotometer program based on our absorbance curvecurve

Data Analysis Data Analysis

Phosphate concentrations were converted to Phosphate concentrations were converted to phosphorus concentrationsphosphorus concentrations

Phosphorus concentrations adjusted based on Phosphorus concentrations adjusted based on dilutiondilution

Phosphorus removal calculated based on Phosphorus removal calculated based on assumed constant influent of 6.9 mg/Lassumed constant influent of 6.9 mg/L For each effluent sample the average of the two For each effluent sample the average of the two

repetitions was usedrepetitions was used Averages were plotted as a function of time in order to Averages were plotted as a function of time in order to

look for trends in our datalook for trends in our data

ResultsResults

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

-400%

-300%

-200%

-100%

0%

100%

200%

0 5 10 15 20

Time Elapsed (days)

P R

em

ov

al E

ffic

ien

cy

1.5 hours

0.75 hours

3 hours

1.5 hours

3 hours

Hypothesis: PAO StorageHypothesis: PAO Storage

During anaerobic conditions, PAO may have During anaerobic conditions, PAO may have released stored phosphorus indicating P released stored phosphorus indicating P generation.generation. 40 minute anaerobic settling time – P released stored 40 minute anaerobic settling time – P released stored

P that then was drained in effluent waste water.P that then was drained in effluent waste water. Drain Cup – sludge from drain state, samples not Drain Cup – sludge from drain state, samples not

collected immediately therefore potentially hours for collected immediately therefore potentially hours for PAO to release phosphorus into our samples prior to PAO to release phosphorus into our samples prior to collection. collection.

Refrigerator - assume no P release due to cold Refrigerator - assume no P release due to cold temperatures (dormant bacteria)temperatures (dormant bacteria)

Hypothesis: P ContaminationHypothesis: P Contamination

Additional P may have been from outside Additional P may have been from outside sourcessourcesFound reagent to react with lab equipment Found reagent to react with lab equipment (pipettes, beakers, storage containers, (pipettes, beakers, storage containers, distilled water)distilled water) If there is P in distilled water it makes sense If there is P in distilled water it makes sense

that most lab equipment was contaminated that most lab equipment was contaminated with Pwith P

Likely that sample bottles were also Likely that sample bottles were also contaminatedcontaminated

Error in Lab TechniqueError in Lab Technique

We do not think error is from inaccurate pipette We do not think error is from inaccurate pipette technique, this would not explain magnitude of our technique, this would not explain magnitude of our errors. errors. Reagent easily contaminated making absorbance Reagent easily contaminated making absorbance reflect sample AND contaminated reagentreflect sample AND contaminated reagentWe had trouble getting our data to fit within standard We had trouble getting our data to fit within standard absorbance curve even with 95 % dilutionabsorbance curve even with 95 % dilution

Extrapolation less precise than interpolation Extrapolation less precise than interpolation

Pulling samples at different heights within the Pulling samples at different heights within the effluent sample vial effluent sample vial

Did not to mix the vial because we didn’t want any of the settled solidsDid 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 Concentrations may have varied over height of vial, especially if P was

being absorbed from contaminated vial over time or if PAO were being absorbed from contaminated vial over time or if PAO were releasing P over time.releasing P over time.

Inadequate ProcedureInadequate Procedure

Studies have indicated that active PAO may take Studies have indicated that active PAO may take at at least four monthsleast four months to develop after the introduction of an to develop after the introduction of an anaerobic stage in a lab scale setting (Kortstee et al., anaerobic stage in a lab scale setting (Kortstee et al., 1994).1994).Phosphorus removal calculations rely heavily on our Phosphorus removal calculations rely heavily on our assumed influent concentration. assumed influent concentration.

Assumed that the influent phosphorus concentrations would be Assumed that the influent phosphorus concentrations would be constant. constant.

If influent concentration higher than expected it would account If influent concentration higher than expected it would account for apparent phosphorus production. for apparent phosphorus production.

inadequate mixing of the 20x concentrated waste stockinadequate mixing of the 20x concentrated waste stock Variable delivery of phosphorus Variable delivery of phosphorus

100x stock solution was inadequately mixed100x stock solution was inadequately mixed Variable concentrations in 20x bottlesVariable concentrations in 20x bottles

System StressesSystem Stresses

Thanksgiving Break and City of Ithaca Thanksgiving Break and City of Ithaca power outage (Nov. 24)power outage (Nov. 24) Disruption of systemDisruption of system Default settings restored interfering with our Default settings restored interfering with our

experimentexperiment Ran out of stock solutionRan out of stock solution High bacterial death period (darker bacteria)High bacterial death period (darker bacteria)

Suggestions for Future ProjectsSuggestions for Future Projects

Long startup time with anaerobic phase for PAO assimilation Long startup time with anaerobic phase for PAO assimilation More assimilation time with each change More assimilation time with each change Rinse sample vials (and all lab equipment) with reagent Rinse sample vials (and all lab equipment) with reagent Rinse all equipment with E pure water Rinse all equipment with E pure water Mix stock waste frequently Mix stock waste frequently Take influent samples dailyTake influent samples dailyTake samples as soon as possible after drainingTake samples as soon as possible after drainingCentrifuge samples prior to testing themCentrifuge samples prior to testing themTest more repetitions of the same sample for less error in resultsTest more repetitions of the same sample for less error in resultsTake highly detailed notes of all procedures and technical failures in Take highly detailed notes of all procedures and technical failures in the lab the lab

ConclusionsConclusions

With our data we are uncomfortable making conclusions With our data we are uncomfortable making conclusions about optimal anaerobic timesabout optimal anaerobic timesExperiment a learning experienceExperiment a learning experienceAble to provide better procedure for future experimentsAble to provide better procedure for future experimentsProblems w/ Future Biological P Removal ProjectsProblems w/ Future Biological P Removal Projects

Relies heavily on establishing and maintaining the PAO Relies heavily on establishing and maintaining the PAO population. population.

An effective PAO population can take up to four month to grow. An effective PAO population can take up to four month to grow. Semester (esp. with vacations) not enough time to run Semester (esp. with vacations) not enough time to run

successful projectsuccessful project

ReferencesReferences

Burns, Peter, A. Mitszewski, and B. Bovee. (2004). “Investigation of Biological Burns, Peter, A. Mitszewski, and B. Bovee. (2004). “Investigation of Biological Phosphorus Removal in a Sequencing Batch Reactor.” CEE 453 Research Project, Phosphorus Removal in a Sequencing Batch Reactor.” CEE 453 Research Project, Spring 2004.Spring 2004.

Lemos, Paulo, et al. (2003) Metabolic Pathway for Propionate Utilization by Lemos, Paulo, et al. (2003) Metabolic Pathway for Propionate Utilization by Phosphorus-Accumulating Organisms in Activated Sludge: 13C Labeling and In Vivo Phosphorus-Accumulating Organisms in Activated Sludge: 13C Labeling and In Vivo Nuclear Magnetic Resonance. Applied and Environmental Microbiology, January Nuclear Magnetic Resonance. Applied and Environmental Microbiology, January 2003, p. 241-251, Vol. 69, No. http://aem.asm.org/cgi/content/full/69/1/2412003, 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). Kortstee GJ, Appeldoorn KJ, Bonting CF, van Niel EW, and van Veen HW (1994). Biology of polyphosphate-accumulating bacteria involved in enhanced biological Biology of polyphosphate-accumulating bacteria involved in enhanced biological phosphorus removal. FEMS Microbiol Rev. Oct;15(2-3):137-53. phosphorus removal. FEMS Microbiol Rev. Oct;15(2-3):137-53. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=7946465&dopt=Abstractcmd=Retrieve&db=PubMed&list_uids=7946465&dopt=Abstract

The University of Queensland, Australia.(2001). Discovery of Biological Phosphorus The University of Queensland, Australia.(2001). Discovery of Biological Phosphorus Removal Organisms. http://awmc.uq.edu.au/activities/BPR.htmlRemoval Organisms. http://awmc.uq.edu.au/activities/BPR.html

Weber-Shirk, Monroe. (2004) Weber-Shirk, Monroe. (2004) Laboratory Research in Environmental EngineeringLaboratory Research in Environmental Engineering, , CEE 453, Fall 2004. Cornell University. Class notes, laboratory manual, and course CEE 453, Fall 2004. Cornell University. Class notes, laboratory manual, and course resources.resources.