Anaerobic Conditions for
Optimal Biological
Phosphorus Removal
Sarah Kloss and Laura Mar
CEE 453 Research
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
What are PAOs?
Phosphorus Accumulating Organisms
Source: University of Queensland 2001
They release phosphorus during the anaerobic stage

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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

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the bacteria are able to grow on the stored carbon products
take up excess phosphorus to incorporate in biomass and
polyphosphate
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
Our Experimental Setup
Refrigerator
Reactor
Pump
Sequencing Batch Reactor
Physical setup did not deviate from the
lecture notes
The States of Our Reactor
State
Characteristics
Main 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
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
Secondary Exit
Condition
Time in state >
Maximum Water Time
Time in state > Max
Drain Time
Parameters Controlling Exit
Conditions
Max Waste Time
Max Water Time
Max Tank Volume
Min Tank Volume
Anaerobic Time
Aerobic Time
Settle Time
Max Drain Time
Flow Rate
31.6 seconds
600 seconds
4L
1.2 L
Varied as part of our
experiment
6 hr
0.67 hr
2000 seconds
384 mL/min
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
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

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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
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

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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
Results
200%
100%
P Removal Efficiency
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
1.5 hours
0%
0.75 hours
3 hours
-100%
1.5 hours
-200%
3 hours
-300%
-400%
0
5
10
15
Time Elapsed (days)
20
Hypothesis: PAO Storage
During anaerobic conditions, PAO may have
released stored phosphorus indicating P
generation.
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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 potentially hours for
PAO to release phosphorus into our samples prior to
collection.
Refrigerator - assume no P release due to cold
temperatures (dormant bacteria)
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
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

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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.
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.

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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
System Stresses
Thanksgiving Break and City of Ithaca
power outage (Nov. 24)

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Disruption of system
Default settings restored interfering with our
experiment
Ran out of stock solution
High bacterial death period (darker bacteria)
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
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

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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
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.