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Electronic Theses and Dissertations, 2004-2019
2006
The Effects Of Ph On Enhanced Biological Phosphorus Removal The Effects Of Ph On Enhanced Biological Phosphorus Removal
(ebpr) With Propionic Acid As The Dominant Volatile Fatty Acid (ebpr) With Propionic Acid As The Dominant Volatile Fatty Acid
(vfa) (vfa)
Seyed Malekjahani University of Central Florida
Part of the Environmental Engineering Commons
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STARS Citation STARS Citation Malekjahani, Seyed, "The Effects Of Ph On Enhanced Biological Phosphorus Removal (ebpr) With Propionic Acid As The Dominant Volatile Fatty Acid (vfa)" (2006). Electronic Theses and Dissertations, 2004-2019. 861. https://stars.library.ucf.edu/etd/861
THE EFFECTS OF pH ON ENHANCED BIOLOGICAL PHOSPHORUS REMOVAL (EBPR) WITH PROPIONIC ACID AS THE DOMINANT
VOLATILE FATTY ACID (VFA)
by
SEYED MOHAMADREZA MALEKJAHANI B.S. University of Sistan and Balouchestan, 1996
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science
in the Department of Civil and Environmental Engineering in the College of Engineering and Computer Science
at the University of Central Florida Orlando, Florida
Fall Term 2006
© 2006 Seyed Malekjahani
ii
ABSTRACT
pH control is a tool to improve some aspects of Enhanced Biological Phosphorus
Removal (EBPR) process. Filipe et al (2001a, 2001b, and 2001c) found strong evidence that the
stability of EBPR systems can be improved by increasing the pH of the anaerobic zone, thereby
creating conditions where phosphorus-accumulating organisms (PAOs) are able to take up
acetate faster than glycogen-accumulating organisms (GAOs). They explained this observation
by comparing the growth rate of phosphorus-accumulating organisms (PAOs) and glycogen-
accumulating organisms (GAOs) and found that pH has little effect on PAOs growth rate but
adversely affects GAOs growth rate when it increases (at pH values greater than 7.25, PAOs
would take acetate faster than GAOs would). They used synthetic wastewater rich in acetic acid.
In this study, we used real wastewater and the dominant volatile fatty acid available to
microorganisms was propionic acid in continuous EBPR system.
It was found that lower anaerobic zone pH (6.5 vs. 7.2) reduced the anaerobic P release
both on an MLVSS specific basis and also on a non-specific (absolute value for the process)
basis. In addition, the observed yield was significantly decreased. Aerobic P uptake was lower in
the low-pH system (on a non-specific basis) due to the lower observed yield, and thus lower
MLVSS concentration. Net P uptake was hard to interpret because of the effect of P release in
the secondary clarifier of Train 2 (high pH). However, on a specific basis it was clear that net P
uptake was either equal or better in the low-pH system regardless of how the secondary clarifier
iii
data was interpreted. Carbon transformations were not impacted in as consistent a fashion as
anaerobic P release was. On a specific basis, PHA content remained unchanged although the
PHV/PHB ratio was impacted with much lower PHV content in the low-pH system. Glycogen
content and the amount of labile glycogen (delta glycogen) were higher in the low-pH system, in
spite of the fact that MLVSS P content did not decrease. However, due to the impact of the low
observed yield at low pH, absolute values resulted in higher PHA content for the process reactors
as a whole, higher glycogen content, and unchanged labile glycogen. Low pH resulted in
increased biomass P content, however the lower observed yield offset this on a process basis so
that effluent P levels were nearly equal. So low pH improved P removal on a specific basis, but
not on a process basis. Since it is unknown if the low observed yield is repeatable, and due to the
impact of the secondary clarifier in the high pH system, it cannot be concluded that the effect of
low pH on net P removal would be similar in other EBPR systems.
iv
ACKNOWLEDGMENTS
I would like to thank God and my family members especially my wife, Mitra, whose
support and encouragement was essential in completing this study.
I also thank my advisor, Dr. Andrew A. Randall whose teaching, advising, and support
made this study possible.
My appreciation goes to my thesis committee members, Dr Debra R. Reinhart and Dr
John D. Dietz, to all UCF faculty and staff members with whom I had the opportunity to learn. I
thank my friends in UCF especially Terence McCue, who helped me throughout this study,
Andrea Rios, Nicole Berge, and Eyad Batarseh for their friendship, advice, and support.
v
TABLE OF CONTENTS
LIST OF FIGURES ..................................................................................................................... viii
LIST OF TABLES......................................................................................................................... ix
INTRODUCTION .........................................................................................................................15
Literature Review.......................................................................................................................18
Enhanced Biological Phosphorus Removal ...........................................................................18
The Effect of pH on EBPR ....................................................................................................23
The Effect of Temperature on EBPR.....................................................................................25
The Roll of VFAs in EBPR Efficiency..................................................................................26
RESULTS AND DISCUSSION....................................................................................................29
The Effects of pH on Enhanced Biological Phosphorus Removal (EBPR) with Propionate as
the Dominant Volatile Fatty Acid (VFA) ..................................................................................29
Introduction............................................................................................................................29
Methods and Materials...........................................................................................................31
Experimental Set up...........................................................................................................31
Sample Collection..............................................................................................................33
Analytical Methods............................................................................................................34
Results and Discussion ..........................................................................................................35
Operation Conditions of the CSTR Trains.........................................................................35
vi
Process Phosphorus Profiles ..............................................................................................37
Phosphorus Removal Performance ....................................................................................41
Solids Inventory and Observed Yield ................................................................................43
Normalization with TSS and VSS .....................................................................................44
Statistical Analysis.............................................................................................................47
PHA Biosynthesis Performance.........................................................................................53
Glycogen Formation Performance.....................................................................................56
PHA Yield and Modified PHA Yield ................................................................................59
Prel/VFA, Pup/PHA, and Pup/Prel Ratios ..............................................................................62
Nitrate, ORP, and DO Data................................................................................................65
Conclusions............................................................................................................................67
Phosphorus Removal .........................................................................................................67
Carbon Transformations (PHA and Glycogen) .................................................................68
Other Impacts.....................................................................................................................69
APPENDIX A METHODS AND MATERIALS..........................................................................70
Experimental Approach .............................................................................................................71
Analytical Methods....................................................................................................................73
APPENDIX B THESIS DATA .....................................................................................................76
APPENDIX C EXAMPLE CALCULATIONS...........................................................................111
REFERENCES ............................................................................................................................120
vii
LIST OF FIGURES
Figure 1: Anaerobic conditions in Comeau Model (Comeau et al, 1986) ................................... 21
Figure 2: Aerobic conditions of EBPR process in Comeau Model (Comeau et al, 1986)........... 21
Figure 3: Mino Model for EBPR process (Cruz, 2004)............................................................... 22
Figure 4: Simplied diagram of proposed EBPR metabolism (Pramanik et al, 1999). ................. 22
Figure 5: Specific acetate uptake rates under anaerobic conditions for PAOs and GAOs as
function of pH (Filipe et al, 2001c) ...................................................................................... 24
Figure 6: Comparison of the PHA available for growth by GAOs and PAOs: (A) Smolders et al
1994b, (B) Filipe et al 2001a ................................................................................................ 24
Figure 7: Flow chart of the continuous EBPR systems ............................................................... 33
Figure 8: SOP Profile of Influent and Aerobic Zones ................................................................. 39
Figure 9: SOP Profile of Anaerobic Zones and Influent.............................................................. 40
Figure 10: Sample Calculation for SOP .................................................................................... 112
Figure 11: Sample Calculation for TP ....................................................................................... 113
Figure 12: Sample Calculation for Glycogen ............................................................................ 115
viii
LIST OF TABLES
Table 1: Average pH in consecutive zones .................................................................................. 35
Table 2: SRT Values at Individual Dates..................................................................................... 36
Table 3: SOP Data for both Trains............................................................................................... 38
Table 4: TP Data for both Trains ................................................................................................. 38
Table 5: Phosphorus Mass Balance Results for Train 1 (mg-P/d); Anaerobic pH=6.5 ............... 42
Table 6: Phosphorus Mass Balance Results for Train 2 (mg-P/d); Anaerobic pH=7.2 ............... 42
Table 7: SRT, MLVSS, and Observed Yields ............................................................................. 43
Table 8: Phosphorus Mass Balance Results Normalized with Total System TSS for Train 1,
(mg/g*d)1 .............................................................................................................................. 45
Table 9: Phosphorus Mass Balance Results Normalized with Total System TSS for Train 2,
(mg/g*d)................................................................................................................................ 46
Table 10: Phosphorus Mass Balance Results Normalized with Total System VSS for Train 1,
(mg/g*d)................................................................................................................................ 46
Table 11: Phosphorus Mass Balance Results Normalized with Total System VSS for Train 2,
(mg/g*d)................................................................................................................................ 47
Table 12: RPD between P Mass Balance Averages Along with Statistical Analysis Results ..... 49
Table 13: RPD between P Mass Balance Averages Normalized with VSS Along with Statistical
Analysis Results.................................................................................................................... 50
Table 14: PHB, PHV, and PHA in both Trains ........................................................................... 54
Table 15: PHB, PHV, and PHA Normalized with TSS in both Trains ....................................... 55
ix
Table 16: PHB, PHV, and PHA Normalized with VSS in both Trains ....................................... 55
Table 17: RPD between System Parameters Along with Statistical Analysis Results ................ 56
Table 18: Glycogen Concentrations in Trains 1 and 2................................................................. 57
Table 19: Glycogen Formation/Consumption in both Trains ...................................................... 58
Table 20: Glycogen Formation/Consumption Normalized with TSS in both Trains .................. 58
Table 21: Glycogen Formation/Consumption Normalized with VSS in both Trains.................. 59
Table 22: VFA Values in Influent and Total VFA ...................................................................... 61
Table 23: PHA Yield (YPHA or PHA/VFA Ratio) in Trains 1 and 2 ......................................... 61
Table 24: PHA Yield Based on VFA and Glycogen (YPHA*) in Trains 1 and 2 ....................... 62
Table 25: Prel/VFA and Pup/PHA Ratios in Trains 1 and 2 .......................................................... 64
Table 26: Pup/Prel Ratio in Trains 1 and 2 .................................................................................. 64
Table 27: Statistical Analysis Results .......................................................................................... 65
Table 28: Nitrate, ORP, and DO Data ......................................................................................... 66
Table 29: SOP Data for both Trains............................................................................................. 77
Table 30: TP Data for both Trains ............................................................................................... 78
Table 31: TSS Data for both Trains ............................................................................................. 78
Table 32: VSS Data for both Trains............................................................................................. 79
Table 33: COD Data for both Trains ........................................................................................... 80
Table 34: VFA Data for the Influent............................................................................................ 81
Table 35: PHA Data for both Trains............................................................................................ 82
Table 36: Glycogen Data for both Trains .................................................................................... 82
Table 37: pH Data for both Trains ............................................................................................... 83
x
Table 38: Influent Flow Rate Data for both Trains...................................................................... 84
Table 39: Internal Recycle Flow Rates of both Trains................................................................. 85
Table 40: Observed Yield in Train 1............................................................................................ 85
Table 41: Observed Yield in Train 2............................................................................................ 86
Table 42: Individual PHA/VFA data (YPHA) for both Trains.................................................... 86
Table 43: Individual PHA/(VFA+Gly) Data (YPHA*) for both Trains...................................... 87
Table 44: Individual Prel/VFA Ratios for both Trains.................................................................. 87
Table 45: Individual Pup/PHA Data for both Trains..................................................................... 88
Table 46: Individual Pup/Prel Ratio for both Trains ...................................................................... 88
Table 47: Individual Phosphorus Mass Balance Data for Train 1, mg/d..................................... 89
Table 48: Individual Phosphorus Mass Balance Data for Train 2, mg/d..................................... 90
Table 49: Phosphorus Mass Balance Results Normalized with Total System TSS for Train 1,
(mg/g*d)1 .............................................................................................................................. 91
Table 50: Phosphorus Mass Balance Results Normalized with Total System TSS for Train 2,
(mg/g*d)................................................................................................................................ 91
Table 51: Phosphorus Mass Balance Results Normalized with Total System VSS for Train 1,
(mg/g*d)................................................................................................................................ 92
Table 52: Phosphorus Mass Balance Results Normalized with Total System VSS for Train 2,
(mg/g*d)................................................................................................................................ 92
Table 53: PHB, PHV, and PHA Normalized with TSS in both Trains ....................................... 93
Table 54: PHB, PHV, and PHA Normalized with VSS in both Trains ....................................... 93
Table 55: Glycogen Formation/Consumption Normalized with TSS in both Trains .................. 94
xi
Table 56: Glycogen Formation/Consumption Normalized with VSS in both Trains.................. 94
Table 57: Nitrate, ORP, and DO Data ......................................................................................... 95
Table 58: Paired t Test Results for Anaerobic P Releases........................................................... 95
Table 59: Paired t Test Results for Anoxic P Releases................................................................ 96
Table 60: Paired t Test Results for Total Biological P Releases ................................................. 96
Table 61: Paired t Test Results for Total System P Releases ...................................................... 97
Table 62: Paired t Test Results for Aerobic P Uptakes................................................................ 97
Table 63: Paired t Test Results for Clarifier P Uptake ................................................................ 98
Table 64: Paired t Test Results for Total Biological P Uptakes .................................................. 98
Table 65: Paired t Test Results for Total System P Uptake......................................................... 99
Table 66: Paired t Test Results for Net System P Removals ....................................................... 99
Table 67: Paired t Test Results for Net Biological P Removals ................................................ 100
Table 68: Summary of Statistical Analysis (Paired t Test) for Phosphorus Mass Balance ....... 100
Table 69: Paired t Test Results for Normalized Anaerobic P Releases with VSS..................... 101
Table 70: Paired t Test Results for Normalized Anoxic P Releases with VSS ......................... 101
Table 71: Paired t Test Results for Normalized Total Biological P Releases with VSS ........... 102
Table 72: Paired t Test Results for Normalized Total System P Releases with VSS................ 102
Table 73: Paired t Test Results for Normalized Aerobic P Uptakes with VSS ......................... 103
Table 74: Paired t Test Results for Normalized Clarifier P Uptake with VSS .......................... 103
Table 75: Paired t Test Results for Normalized Total Biological P Uptakes with VSS............ 104
Table 76: Paired t Test Results for Normalized Total System P Uptake with VSS .................. 104
Table 77: Paired t Test Results for Normalized Net System P Removals with VSS................. 105
xii
Table 78: Paired t Test Results for Normalized Net Biological P Removals with VSS............ 105
Table 79: Summary of Statistical Analysis (Paired t Test) for Normalized Phosphorus Mass
Balance with VSS ............................................................................................................... 106
Table 80: Paired t Test Results for Anaerobic PHA .................................................................. 106
Table 81: Paired t Test Results for Normalized Anaerobic PHA with VSS.............................. 107
Table 82: Paired t Test Results for Normalized Anoxic PHA with VSS................................... 107
Table 83: Paired t Test Results for Normalized Glycogen Formation with VSS ...................... 108
Table 84: Paired t Test Results for Prel/VFA.............................................................................. 108
Table 85: Paired t Test Results for Pup/PHA.............................................................................. 109
Table 86: Paired t Test Results for YPHA................................................................................. 109
Table 87: Paired t Test Results for YPHA*............................................................................... 109
Table 88: Paired t Test Results for Pup/Prel................................................................................. 110
Table 89: Sample Calculation for TSS/VSS.............................................................................. 114
Table 90: Sample Calculation for COD..................................................................................... 114
Table 91: Example Calculation for Observed Yield .................................................................. 115
Table 92: Sample Calculation for Paired t Test for Anaerobic P Release1................................ 116
xiii
LIST OF ACRONYMS/ABBREVIATIONS
COD Chemical Oxygen Demand CSTR Continuous-flow Stirred Tank Reactor DO Dissolved Oxygen EBPR Enhanced Biological Phosphorus Removal FID Flame Ionization Detector MCRT Mean Cell Residence Time
GAO Glycogen Accumulating Organisms
PAO Phosphorus Accumulating Organism PHA Polyhydroxyalkanoates PHB Poly-B-hydroxybutyrate PHV Poly-hydroxyvalerate Pi Phosphate Poly-p Polyphosphate
RPD Relative Percentage Difference
SBR Sequencing Batch Reactor TSS/VSS Total Suspended Solids/Volatile Suspended Solids VFA Volatile Fatty Acids WAS Waste Activated Sludge
xiv
INTRODUCTION
There are many governmental regulations, both federal and state, related to nutrient
removal (mostly nitrogen and phosphorus) from wastewater. These stringent laws do not allow
wastewater effluent to be discharged to water bodies or used for irrigation unless a certain level
of nutrient concentration is met. On the other hand, economical considerations call for lower
costs for wastewater treatment. Therefore, engineers are challenged to meet the governmental
laws and regulations in an economically attractive manner.
Nitrogen and phosphorus discharged to water bodies in excess will stimulate algae
growth, and hence, dissolved oxygen (DO) depletion. This process, which is named
eutrophication, causes undesired changes in aquatic population such as damage to the fish life.
Additionally, contaminated surface water may not be suitable or economical to be treated for
drinking water. As a result, advanced wastewater treatment plants are being used more and more.
The typical configuration of these plants is an anaerobic-anoxic-aerobic sequence. These
zones are the basic requirements for a Biological Nutrient Removal treatment plant. When total
nitrogen effluent standards are below 8 to 5 mg/L-N there are usually more than three zones. For
example, in the 5-stage Bardenpho process, there is one anaerobic zone followed by two
alternating anoxic/aerobic zones for a total of 5 zones. The resulting configuration is an
anaerobic-anoxic-aerobic-anoxic-aerobic sequence. Both nitrogen and phosphorus removal can
be achieved in this process. In the case of nitrogen removal, the aerobic zones (mostly the first
15
one) serves as a place for nitrification while anoxic zones are where denitrification occurs
resulting in removing nitrogen as N2 gas. In the case of phosphorus removal, the anaerobic zone
is the essential zone for VFA uptake by microorganisms responsible for P removal (Poly-P
bacteria) and the aerobic zone is where they uptake phosphorus. This process is called enhanced
biological phosphorus removal.
Enhanced Biological Phosphorus Removal (EBPR) is a biological process that can
produce an effluent with lower phosphorus content compared to the conventional activated
sludge process. Generally, the effluent total phosphorus (TP) concentration can be even lower
than 1 mg-P/l in a full-scale treatment plant. With EBPR, eutrophication can be avoided and
regulations for effluent phosphorus content can be achieved. It was a common practice in the
past to remove phosphorus by chemical precipitation using alum or iron salts. EBPR is more
economically efficient than chemical phosphorus removal because the cost of chemicals will be
eliminated and also less sludge will be produced since no chemical is used.
In the EBPR process, certain microorganisms named Phosphorus Accumulating
Organisms (PAOs) or Poly-P bacteria take up more phosphorus than they normally do, if they
are placed in a suitable environment. The only known suitable environment is an anaerobic-
aerobic (or anaerobic-anoxic-aerobic) sequence. When the sludge containing PAOs is wasted it
results in low phosphorus content in the wastewater effluent. Once in the anaerobic zone, PAOs
take up volatile fatty acids (VFAs) by employing glycogen and polyhydroxyalkanoates (PHAs)
and release phosphorus resulting in a high phosphorus concentration in the bulk liquid. When
they go to the aerobic zone, they will take up more phosphorus than they released in the
anaerobic zone so the overall phosphorus mass balance will show phosphorus uptake. The waste
16
activated sludge (WAS) has a significant role in phosphorus removal because the PAOs carrying
high amount of phosphorus in their cells are wasted in the WAS stream.
EBPR improves the overall treatment system in other ways too. Having an anaerobic
zone before the aerobic zone benefits the treatment process in two ways. First there is apparently
some reduction in aeration requirements due to biological activity in the anaerobic zone (about
10% of the aeration requirements). The reason for this is not known, and this so-called anaerobic
stabilization remains a controversial subject. Secondly, the oxygen transfer rate from the air
above the aerobic zone into the activated sludge is high. This is due to the fact that the mixed
liquor coming from the anaerobic zone does not contain dissolved oxygen. The mass transfer rate
of oxygen is directly proportional to the oxygen concentration gradient between the air (the
gaseous phase) and the aerobic zone (the liquid phase). Because the oxygen concentration
gradient is high between the activated sludge in the aerobic zone and the air above it, the oxygen
will dissolve in the aerobic zone very efficiently resulting in better oxygen mass transfer.
Objectives
This study was designed to compare phosphorus removal between two systems that were
operated at different pHs while the VFA content of the influent was mainly propionic acid. Filipe
et al (2001a, 2001b, 2001c) studied the effect of pH on phosphorus removal and found
interesting results. They found that pH control can be a powerful tool to improve EBPR
efficiency. However, they used a synthetic wastewater rich in acetic acid with sequencing batch
reactors (SBR). In this study, we investigated the effect of pH when real wastewater is used in a
continuous EBPR system and the VFA available to microorganisms is mainly propionic acid.
17
The objectives of this study are to observe the performance of two parallel continuous EBPR
systems; investigate the effect of pH on phosphorus removal; study the VFA-driven phosphorus
release; and study the PHA-driven phosphorus uptake.
Literature Review
Enhanced Biological Phosphorus Removal
Excess phosphorus in fresh water bodies will promote undesirable growth of algae and other
aquatic plants resulting in depression of dissolved oxygen (DO), a process named eutrophication.
Excess phosphorus can be removed by chemical processes but biological removal is much more
economical and produces sludge that is easier to dispose of. However, despite its widespread
acceptance, EBPR is associated with unpredictability (Pramanik, 1999). Enhanced biological
phosphorus removal (EBPR) occurs when phosphorus accumulating organisms (PAOs)
outcompete the non-phosphorus accumulating organisms (e.g. glycogen accumulating organisms
or GAOs). PAOs take up more phosphorus than they need for their life activities and then they
are wasted in waste activated sludge (WAS) reducing phosphorus content of the plant effluent.
The phosphorus content of these microorganisms can approach 35 percent on a
phosphorus-to-VSS (volatile suspended solids) basis (Wentzel et al., 1988). However, the
phosphorus content of activated sludge mixed liquor in real biological phosphorus removal
systems is about 6 to 8 percent (P/VSS) and as high as 14 percent as opposed to conventional
18
activated sludge P content of 2.3 percent (Daigger, 2004).
There are two basic models describing the EBPR process named the Comeau Model
(Comeau et al, 1986) and the Mino Model (Mino et al, 1987). These models explain that in order
for the EBPR process to occur, there has to be an anaerobic-aerobic sequence. In the anaerobic
zone, PAOs have an advantage against other heterotrophs by their ability of storing carbon
reserve materials such as polyhydroxyalkanoates (PHAs) in their cells by sequestering and
reducing volatile fatty acids (VFAs) to PHAs using polyphosphate as their energy source. Other
heterotrophs carry out fermentation and produce VFAs for PAOs.
During anaerobiosis, PAOs break down phosphorus bonds of polyphosphate (poly-P)
molecules (they are also called poly-P bacteria in some references) and consume glycogen so the
phosphorus concentration in the surrounding bulk liquid increases and intra-cellular glycogen
concentration decreases. Also, PHA concentration that consists of PHB (poly-B-
hydroxybutyrate), PHV (poly-hydroxyvalerate), and 3H2MV (methylated PHV) increases in the
cells.
When oxygen is available (in the aerobic zone), PAOs carry out aerobic respiration by
using the stored PHA and phosphorus that exist in the surrounding. They deplete all the PHA
they stored in their cells completely and as a result they take up more phosphorus than they
normally need to store PHA-derived energy in the form of polyphosphate. They also replenish
glycogen.
The only difference between the Comeau and the Mino models is their consideration of
where the reducing equivalents to convert VFA to PHA come from. The Mino Model proposes
that the reducing power necessary for anaerobic PHA synthesis comes from glycogen. However,
19
the Comeau Model proposes the reducing power comes from the TCA cycle. The reactions
taking place in each model are shown in Figures 1 to 3. Figure 4 shows a simplified diagram of
EBPR metabolism.
20
Figure 1: Anaerobic conditions in Comeau Model (Comeau et al, 1986)
Figure 2: Aerobic conditions of EBPR process in Comeau Model (Comeau et al, 1986)
21
Figure 3: Mino Model for EBPR process (Cruz, 2004)
Figure 4: Simplied diagram of proposed EBPR metabolism. Gray lines indicate aerobic metabolism and black lines indicate anaerobic metabolism. The asterisk indicates that NADH refers to all carriers of reducing equivalents: NADH, NADPH, FADH2 (Pramanik et al, 1999).
22
The Effect of pH on EBPR
Recent studies show that the performance of EBPR systems is affected by system pH. A
pH of around 7.5 has been found to be the overall working optimum pH for an EBPR system
with propionate as the sole carbon source by Pijuan et al. (2004). The pH range they studied was
6.5 to 8. They concluded and proposed a pH of 8 to be the best pH in the anaerobic zone. They
found that P uptake in the aerobic zone was highest at pH of 7.5 and 8.
A direct linear relationship between pH and phosphorus release to acetate uptake ratio
has been found in EBPR systems for pHs between 5.5 and 8.2 (Smolders et al., 1994a, Cokgor et
al., 2004). Smolders et al (1994a) observed a range of 0.25 to 0.75 mol-P/mol-C for P release per
acetate uptake ratio when pH increased from 5.5 to 8.2. Also, P release increased with increasing
pH in their research. The P release was 1.45 to 4.68 mmol-P/L when pH increased from 5.5 to
8.2.
The rate of acetate uptake is independent of pH in the case of PAOs and strongly
dependent on pH for GAOs, i.e., the rate of acetate uptake by GAOs is significantly decreased
when the pH of the medium is increased (Filipe et al., 2001a and 2001b). According to Filipe et
al, the specific rate of acetate uptake for GAOs decreases from 0.21 to 0.11 mmol-C/mmol-C.h
when pH increases from 6.5 to 8 (Filipe et al., 2001a and 2001b). This suggests pH as a
performance parameter that can be manipulated to favor PAOs in order to optimize phosphorus
removal. Figure 5 shows the specific acetate uptake rates under anaerobic conditions for PAOs
and GAOs as function of pH where the pH of 7.25 is the equivalence point for acetate uptake
rate of both populations.
23
Figure 5: Specific acetate uptake rates under anaerobic conditions for PAOs and GAOs as function of pH (Filipe et al, 2001c)
Figure 6: Comparison of the PHA available for growth by GAOs and PAOs after replenishment of the glycogen and polyphosphate: (A) Smolders et al 1994b, (B) Filipe et al 2001a
The amount of PHA available for growth by GAOs and PAOs after replenishment of the
glycogen and polyphosphate found by Filipe et al (2001a) is slightly different than that found by
Smolders et al (1994). Figure 6 shows the comparison. Figure 6a suggests that the amount of
24
PHA available in the aerobic phase for biomass growth are similar for GAOs and PAOs while
Figure 6b suggests that GAOs have a slightly greater energetic efficiency than PAOs over a
broader pH range (Filipe et al, 2001c).
Filipe et al (2001c) observed a fluctuation in the anaerobic phosphorus concentration in
part III of their research. They explained this to be the result of the changing patterns of pH.
When the PAO population became more dominant, more phosphorus was released in the
anaerobic zone. This caused the pH to decrease, and hence, GAOs had an advantage in terms of
their acetate uptake rate over PAOs. GAOs took up acetic acid but did not release phosphate
resulting in acidity removal and pH increase. At this point, PAOs displaced GAOs and pH
increased. They proposed the optimum anaerobic pH to be between 7.4 and 7.6 and aerobic pH
to be controlled above 7 in order for the EBPR system to become rich in PAO community and,
therefore, better performance in phosphorus removal is achieved (Filipe et al, 2001c). When pH
is high, PAOs have advantage over GAOs. Bond et al (1999) found that when the intracellular
pH of EBPR sludge was raised, substantial anaerobic phosphate release was caused without
volatile fatty acid (VFA) uptake.
The Effect of Temperature on EBPR
Temperature affects EBPR in different ways when different zones or configurations
are considered. Brdjanovic et al (1998) found that temperature had a strong effect on anaerobic
phase kinetics but had relatively weak effect on stoichiometry in the anaerobic phase. They
25
also found that temperature impact on aerobic P uptake rate was moderate in the long run
but it was strong on other metabolic process rates such as PHA consumption, OUR, and
growth. Decreasing temperature to 5 °C from 20 °C had no significant effect on the EBPR
process in a study by Helmer et al (1998). The same fact was found in a wastewater
treatment plant (Ydstebo et al, 2000). They found that enhanced biological phosphorus
removal was accomplished at low temperature of 5 °C with 0.6 mg/L total phosphorus in the
effluent.
Erdall et al (2003) propose that the partial or complete loss of EBPR functions at low
temperatures reported by some researchers (McClintock et al. 1991, Brdjanovic et al. 1997,
Beatons et al 1999) is probably related to unsuitable operational conditions such as low
SRT, low anaerobic detention time, etc. Generally, it is almost universally agreed that
EBPR can be accomplished at low temperatures and low temperature is even favorable for
PAOs.
The Roll of VFAs in EBPR Efficiency
Phosphorus-accumulating organisms have the ability to take up VFAs such as acetate
under anaerobic conditions for their survival. This ability gives them a competitive advantage
over other heterotrophs. VFAs are present in the influent and/or are produced in the anaerobic
zone. They get consumed very quickly (less than an hour) in batch reactors and are almost
impossible to measure in continuous reactors. PAOs store VFAs in the form of PHA because
26
VFAs cannot be oxidized under anaerobic conditions. The presence of VFAs is essential for a
successful EBPR process (Filipe et al, 2001 a).
The mass of VFAs, therefore, available to these microorganisms plays a significant
role in EBPR process efficiency. Some wastewaters contain a high concentration of VFAs,
mostly acetic acid. These VFAs are produced under anaerobic conditions which exist in
collection and sewer systems as wastewater flows to the treatment plant. This production can
occur if the condition is anaerobic and temperature, hydraulic residence time, sewage strength
and mixing conditions allow for fermentation (Barnard, 1992).
Anaerobic conditions are essential in VFA production from wastewater organic
polymers. When an electron acceptor such as oxygen or NOx is present, bacteria will hydrolyze
and oxidize influent organics instead of carrying out fermentation because they get more energy
for their growth through electron acceptor utilization. There only needs to be 0.1 mg/l dissolved
oxygen for the condition to be not completely anaerobic (Erdal et al, 2002).
In addition, if the wastewater is not septic enough, a prefermenter should be installed to
ensure the availability of VFAs because in-reactor fermentation is not reliable. Randall et al
(1994) found that in-reactor fermentation (in the anaerobic phase of an SBR) was far less
efficient in driving EBPR than pre-fermentation. In their study, the system with pre-
fermentation of glucose (acidogenesis) had much better phosphorus removal that the system
fed with starch-containing influent (no pre-fermentation). Prefermenter units are not a part
of the activated sludge treatment system and maintain true anaerobic conditions providing
a suitable environment for fermentation (Barnard, 1992). The VFAs produced are typically
acetic, propionic, and butyric acids. Other VFAs such as formic, and valeric acids are
27
produced in much lower amounts.
28
RESULTS AND DISCUSSION
The Effects of pH on Enhanced Biological Phosphorus Removal (EBPR) with Propionate as the Dominant Volatile Fatty Acid (VFA)
Introduction
Nutrient removal from wastewater is increasingly used as the adverse impacts of nitrogen
and phosphorus discharge to water bodies and/or lands are recognized. Municipal wasewaters are
typically rich in nitrogen and phosphorus and thus would cause severe eutrophication if
discharged to water bodies without treatment. With the advent of TMDLs (Total Maximum
Daily Load), nutrient removal is being considered as a higher priority by local and national
authorities. Although phosphorus can be removed by chemical processes, biological nutrient
removal (BNR) systems are typically much more economical for removal of nitrogen and
phosphorus.
Enhanced biological phosphorus removal has been studied by many researchers over the
past two decades (Comeau et al, 1986, Mino et al, 1987, Wentzel et al, 1988, 1989a, and 1989b,
Filipe et al, 2001a, 2001b, and 2001c, Baeza et al, 2004). Enhanced biological phosphorus
29
removal (EBPR) is a biological process that employs an anaerobic-aerobic sequence in order to
give certain types of microorganisms a competitive advantage over other microorganisms. These
microorganisms are called poly-P or PAO (polyphosphate-accumulating organisms). They
outgrow other microorganisms if they get circulated between anaerobic and aerobic conditions.
Under anaerobic conditions, they utilize volatile fatty acids (VFA) by storing them as
polyhydroxyalkanoates (PHA). They also release phosphorus during anaerobic conditions and
subsequently take up phosphorus when oxygen becomes available (aerobic condition). In this
way, PAOs store more phosphorus than they normally need and then they are wasted resulting in
very low phosphorus content in the effluent.
There is occasionally a different type of bacteria in EBPR systems that can decrease
phosphorus removal efficiency (Matsu, 1994). They are called glycogen-accumulating organisms
(GAOs) and are able to sequester VFAs under anaerobic conditions, and thus compete with
PAOs but do not contribute to phosphorus removal.
Filipe et al observed that pH has a significant impact on phosphorus removal (Filipe et al,
2001a, 2001b, and 2001c). They showed that acetate uptake by GAOs was significantly
decreased by increasing pH but the uptake rate for PAOs was independent of the pH for the
range studied (6.5 to 8). It means pH can be used as a powerful tool to control the growth of the
GAOs and, as a result, improving phosphorus removal by PAOs. However, they used a synthetic
wastewater rich in acetate and their system was sequencing batch reactor. This question is still
unanswered: if we use a real wastewater with propionate concentration higher than acetate and
employ continuous reactors, what results would we get as pH varies?
The purpose of this research is to study the effect of pH on continuous EBPR systems fed
30
with real wastewater rich in propionate, and to investigate VFA-driven P release and PHA-driven
P uptake at different pHs.
Methods and Materials
Experimental Set up
Two parallel continuous trains consisting of one anaerobic, one anoxic, and one aerobic
zone were employed. The flow chart of each train is shown in Figure 7. In this figure, AN, AX,
and AE denote anaerobic, anoxic, and aerobic reactors, respectively.
The HRTs (hydraulic retention time) were 1.4, 3.4, and 4.9 hours for anaerobic 1, anoxic
1, and aerobic 1 reactors, respectively. The HRTs of anaerobic 2, anoxic 2, and aerobic 2
reactors were 1.3, 3.2, and 4.6 hours, respectively. The reactor volumes were 1637, 3867, and
5582 ml for anaerobic, anoxic, and aerobic reactors, respectively. Each reactor was completely
stirred and separated from other reactors by an overflow-underflow baffle configuration. The
average SRTs (solids retention time) for Trains 1 and 2 were 8.5 and 8 days, and the MCRTs
(mean cell residence time) for Trains 1 and 2 were 8.7 and 8.1 days, respectively.
The influent flowed into the anaerobic zone at an average flow rate of 27.6 and 29.4 L/d
for Trains 1 and 2 and then entered an external clarifier after passing through the anoxic and the
aerobic reactors (Figure 7). The returned activated sludge (RAS) was pumped from the bottom of
31
the clarifier to the anoxic zone at a flow rate of approximately twice the influent flow rate.
Propionic acid was pumped to both anaerobic zones from a common reservoir by a common
pump with two separate pump heads. The flow rate of propionic acid was often checked to make
sure the correct amount of propionic acid was being delivered to each anaerobic zone. The flow
rate was measured with a stopwatch and a graduated cylinder.
There were two internal recycle lines (see Figure 1), which were nitrate recycle from the
aerobic to the anoxic reactors (NARCY) and anoxic recycle from the anoxic to the anaerobic
reactors (ARCY). This is a VIP or UCT configuration. In this configuration, the NARCY line is
used to bring back nitrate to the anoxic zone in order to help achieve complete denitrification.
The ARCY line is used to provide biomass to the anaerobic reactor. Please refer to Tables 38 and
39 for flow rates.
Waste activated sludge (WAS) was taken out from the end of the aerobic zone and
wasted at a flow rate of 1 L/d. The effluent coming out from clarifiers was retained in a tank for
future analyses. In order to make the influent VFA propionate-dominant, propionic acid was
added to the anaerobic zones at an amount of 1 mM-C (based on influent flow rate). The pH in
one of the anaerobic reactors (Train 1) was kept lower (acidic) than the pH in the other anaerobic
reactor by adding sulfuric acid. The pH of Train 2 was natural, unadjusted pH. The pH in the
anaerobic 1 reactor averaged 6.5 and that of anaerobic 2 was 7.2. The Trains were kept in the
Environmental Growth Chamber located on the 4th floor of the Engineering Building 2 at the
University of Central Florida in Orlando, Florida at a constant temperature of 20°C. The influent
feeding the reactors was real wastewater obtained from the Eastern Water Reclamation Facility
located in Orange County, Florida.
32
AN AX AE
Influent Tank Effluent
Tank Propionic Acid Reservoir
RAS
NARCYARCY WAS
Clarifier
Figure 7: Flow chart of the continuous EBPR systems
Sample Collection
Samples were taken several times a week to conduct total suspended solids (TSS),
volatile suspended solids (VSS), soluble orthophosphate (SOP), total phosphorus (TP), soluble
chemical oxygen demand (SCOD), total chemical oxygen demand (TCOD), volatile fatty acids
(VFA), polyhydroxyalkanoates (PHA), and glycogen tests. A portion of the samples was
immediately filtered by 0.45-µm membrane filter for use in aqueous phase tests such as SOP,
SCOD, and VFA. If the samples were not to be used immediately, they were kept in a constant-
temperature vault at 4 °C. The glycogen test was done immediately after sampling and PHA
samples were immediately centrifuged and frozen at -80 °C for future lyophilization.
33
Analytical Methods
Most tests were done immediately after sampling. If conducting some tests was not
possible immediately, the sample was kept refrigerated and the test was done within 24 hours.
TSS, VSS, SOP, TP, TCOD, and SCOD tests were done according to Standard Methods (Eaton
et al, 1995). PHAs were measured after methanolytic decomposition using a DB-1 column
(Supelco Inc., Bellefonte, PA) and a Schimadzu 14A gas Chromatograph (GC) with a flame
ionization detector and helium as the carrier gas. The injection port temperature was 230ºC, with
initial column temperature of 100ºC for 2 minutes followed by temperature ramping at 20ºC per
minute to 160ºC where it stayed for 2 minutes. The detector temperature was 230ºC. The sample
injection volume was 2 µL.
The glycogen content of the samples was measured using the Anthrone Test for
Carbohydrate (Murray, 1981). The VFAs were measured following the Supelco Bulletin 856B
(Supelco Inc., Bellefonte, PA); a Shimadzu gas Chromatograph equipped with flame ionization
detector (FID) was used for this analysis. The VFAs were separated by 3-mm internal diameter
glass column with 60/80 Carbopack C/0.3% Carbowax 20M/0.1% phosphoric acid packing
(Supelco Inc., Bellefonte, PA). The oven of the GC was programmed to begin the analysis at
105ºC, and to remain at that temperature for 2 minutes. The temperature then increased 5ºC per
minute until it reached 150ºC remaining at this temperature for 2 minutes. The sample injection
volume was 2 µL. The injection port and the detector were maintained at 200ºC. Helium was
used as a carrier gas at a flow rate of 30 ml/min.
34
Results and Discussion
Operation Conditions of the CSTR Trains
The two systems were operated continuously with slightly acidic (pH of 6.5) and slightly
basic (pH of 7.2) conditions for Train 1 and 2, respectively. The pH of the anaerobic zone of
Train 1 was controlled with sulfuric acid addition but Train 2 had natural, unadjusted pH. Table
1 shows pH in all zones of the systems.
Table 1: Average pH in consecutive zones
pH Systems
Influent Anaerobic Anoxic Aerobic Clarifier
Train 1 7.1 6.5 6.9 7.2 7.3
Train 2 7.1 7.2 7.4 7.5 7.5
∆pH 0 0.7 0.5 0.3 0.2
Table 2 shows SRT values at individual dates. As can be seen, there are outliers on 6/22
and 6/26. If average SRT is calculated based on all dates, they would be 8.2 d and 7.9 d in Train
1 and 2, respectively. However, calculating SRT by ignoring the outliers would result in an SRT
of 8.4 d for both systems. The effluent TSS was taken into consideration in the SRT calculations.
35
The effluent TSS was 9.65 mg/l and 9.8 mg/l in Train 1 and 2, respectively.
Table 2: SRT Values at Individual Dates
Date SRT 1 SRT 2
6/22 7.1 7.3
6/26 8.2 5.9
6/29 8.3 8.2
7/6 8.6 8.6
7/13 8.0 9.0
7/20 8.8 8.2
7/27 8.6 8.2
Avg. 1* 8.2 7.9
Avg. 2** 8.4 8.4 * Based on all data ** Based on data from 6/29 to 7/27
The target influent flow rate was 30 L/d for both trains. Actual flow rates were 27.6 and
29.2 L/d for Trains 1 and 2, respectively. Both trains received 1 mM-C propionic acid (based on
influent flow rate) added directly to the anaerobic zones.
The wastewater used in this study was from the Eastern Water Reclamation Facility
located in East Orange County, Florida. This wastewater typically contains significant amount of
VFAs of approximately 1.2 and 1.16 mM-C (mmol/l as C) of acetic acid and propionic acid,
respectively. The system was spiked with 1 mM-C Propionic Acid (CH3CH2COOH). The
anaerobic zones were spiked with Propionic Acid at the above-mentioned concentration (Figure
7). Therefore, the average acetic and propionic acid concentration including the propionic spike
was 1.2 and 2.16 mM-C of acetic acid and propionic acid, respectively. The influent COD was
36
397 mg/L while the influent TP after spiking with phosphorus was 10.8 mg-P/L. This resulted in
a COD/TP ratio of around 37, which is less than 40. This means the influent feeding the reactors
was COD limited.
Process Phosphorus Profiles
Tables 1 and 2 present SOP (Soluble Ortho Phosphorus) and TP (Total Phosphorus) and
TP data, respectively. Figure 8 shows influent SOP profiles along with aerobic SOP profiles.
Figure 9 shows influent SOP profiles along with anaerobic SOP profiles. It can be seen from
these figures that pseudo-steady state was achieved by approximately 6/15/04. For statistical
comparisons, data from 6/15/04 to 7/27/04 inclusive was used. It can be seen in Tables 3 and 4
that influent TP values were lower than SOP values in some points. That was because of an
analytical problem with TP since SOP values were consistent and checked by P balances and
found to be valid. It was expected that influent TP concentrations were at least 2 mg-P/l higher
than measured SOP concentrations based on historic data for the wastewater. As a consequence
analysis was conducted using SOP, meaning that P removals and biomass P contents are
underestimated. It should be noted that, in Table 4, CL represents samples taken from
supernatant of the clarifiers while EFF represents samples taken from effluent tank (see Figure
7).
37
Table 3: SOP Data for both Trains
SOP, mg/l as P Date
INF AN 1 AX 1 AER 1 CL 1 AN 2 AX 2 AER 2 CL 2
5/25/2004 7.54 10.22 12.44 8.93 - 12.07 10.04 6.43 - 5/30/2004 10.41 11.15 10.59 7.72 13.22 9.48 10.59 9.11 15.69 6/1/2004 7.63 7.35 3.74 1.52 8.96 6.98 3.37 1.24 3.09 6/5/2004 7.91 10.22 3.83 1.43 4.85 8.56 3.65 1.52 2.07 6/8/2004 7.54 11.89 3.65 1.15 1.52 11.7 4.11 1.24 1.7
6/15/2004 6.98 13.37 5.13 2.72 3.37 15.22 5.41 1.98 3.28 6/22/2004 10.31 13.28 6.24 4.02 4.3 19.3 7.17 4.3 6.19 6/29/2004 9.94 13.37 8.56 6.33 5.96 18.56 9.76 6.98 7.44 7/6/2004 9.2 10.04 4.02 1.43 0.87 15.87 7.54 4.11 5.41
7/13/2004 8 10.22 5.22 2.72 3.74 16.06 5.87 2.35 3.37 7/20/2004 10.96 15.69 9.76 6.7 6.33 16.43 9.76 8 11.3 7/27/2004 8.28 13.83 7.72 4.11 3.65 17.91 8.56 5.22 5.31
Avg. 8.7 11.7 6.7 4.1 5.2 14.0 7.2 4.4 5.9 Std Dev 1.36 2.28 3.02 2.72 3.48 4.13 2.64 2.78 4.26
Table 4: TP Data for both Trains
TP, mg/l as P Date
INF AER 1 CL 1 EFF 1 AER2 CL 2 EFF 2
5/25/2004 - 120 - 4 114.8 - 6.2 6/1/2004 9.60 118.50 10.70 8.16 115.30 3.62 3.20 6/8/2004 9.42 111.58 3.80 0.80 127.03 5.68 3.01 6/15/2004 9.42 80.68 5.60 0.53 125.30 5.68 2.80 6/22/2004 8.05 80.68 6.90 4.24 127.03 6.71 3.62 6/29/2004 9.08 80.68 7.50 3.21 121.88 13.93 4.24 7/6/2004 7.44 89.60 2.10 0.95 107.87 6.10 4.65 7/13/2004 5.38 102.30 5.70 8.16 107.87 3.21 2.18 7/20/2004 30.6 84.69 7.80 6.10 128.47 16.40 6.71 7/27/2004 8.29 128.47 5.45 4.35 125.90 6.71 5.48
Avg. 9.5 99.7 6.2 4.1 120.1 7.6 4.2 Std Dev 8.25 18.72 2.47 2.82 8.03 4.53 1.53
38
0
2
4
6
8
10
12
5/13 5/23 6/2 6/12 6/22 7/2 7/12 7/22 8/1
Date
SOP,
mg/
l
INFAER 1AER 2
Figure 8: SOP Profile of Influent and Aerobic Zones
39
0
5
10
15
20
25
5/13 5/23 6/2 6/12 6/22 7/2 7/12 7/22 8/1
Date
SO
P, m
g/l
INFAN 1AN 2
Figure 9: SOP Profile of Anaerobic Zones and Influent
By inspecting Table 3 we see that EBPR was achieved during the pseudo-steady state
period, i.e. anaerobic SOP values were high. Also, P content of MLVSS between 6/15 and 7/27
was 4.9% and 3.8% for Train 1 and 2, respectively. The P content is calculated for aerobic and
effluent MLVSS. This supports the fact that EBPR was achieved because normally the P content
of microorganisms is between 1.5% and 2.5%. In addition, since only influent SOP was used for
the calculations the results are low, and thus conservative estimates.
40
Phosphorus Removal Performance
The data presented an apparent contradiction with respect to net P removal. Both the
aerobic zone and effluent SOP values were lower for Train 1 than for Train 2 (Table 3).
However, Train 2 performance was slightly better than that of Train 1 based on phosphorus mass
balance results around the biological reactors (Tables 5 and 6) but not for a control boundary
around the entire system. Note that Net Biological P Removal refers to mass balance around the
biological zones only (aerobic, anoxic, and anaerobic) but Net System P Removal refers to mass
balance around the system including the biological zones and the clarifier. The reason for this
apparent contradiction was that there was a large P release in the Train 2 clarifier, combined with
a very high RAS recycle flow rate. This meant that if a mass balance was done excluding the
clarifier for the two systems, Train 2 showed significantly higher P loading (mostly coming into
the control volume via the RAS), and then a much higher P uptake even though the SOP
concentration in the aerobic zone was slightly higher than Train 1. In this case the P mass
balance around the biological reactors may not have any meaning in terms of describing P
removal that would translate into better process performance. The P in the RAS was released in
the clarifier, and then taken up again in the aerobic zone, in a futile cycle that is more descriptive
of secondary P release than EBPR. As a result, the data seems to imply that Train 1 had lower
SOP concentrations than Train 2, but it is unknown if Train 2s higher concentrations would have
been as elevated if there had not been significant secondary P release in the clarifier. The mass
balances are unable to resolve this question and thus the data is inconclusive with respect to
which Train would have the best net P removal for EBPR systems in general (i.e. both systems
41
with properly functioning clarifiers).As a result, the data is inconclusive with respect to which
system had the best net P removal.
Table 5: Phosphorus Mass Balance Results for Train 1 (mg-P/d); Anaerobic pH=6.5
Date AN
Release AX
Release Total Bio. Release
Total Sys. Release
AE Uptake
Clarifier Uptake
Total Bio. Uptake
Total Sys. Uptake
Net Sys.Removal
Net Bio. Removal
6/15/04 477 -180 477 534 517 -57 697 697 163 220
6/29/04 334 65 399 399 462 33 462 494 95 62
7/6/04 337 79 416 416 556 50 556 606 190 140
7/13/04 314 76 390 477 515 -87 515 515 38 125
7/27/04 395 285 679 679 763 36 763 800 120 84
Avg. 336 -6 408 511 537 -86 615 632 121 208
Std Dev 119 183 171 114 107 211 108 106 76 174
Table 6: Phosphorus Mass Balance Results for Train 2 (mg-P/d); Anaerobic pH=7.2
Date AN Release
AX Release
Total Bio. Release
Total Sys. Release
AE Uptake
Clarifier Uptake
Total Bio.Uptake
Total Sys. Uptake
Net Sys.Removal
Net Bio. Removal
6/15/04 615 -179 615 730 736 -115 915 915 185 300
6/29/04 625 -143 625 665 565 -40 708 708 43 82
7/6/04 588 -22 588 702 733 -114 755 755 54 168
7/13/04 662 -80 662 750 720 -88 801 801 51 139
7/27/04 622 -22 622 630 691 -7 712 712 83 90
Avg. 510 -66 517 593 634 -77 707 707 114 191
Std Dev 211 78 194 183 118 49 152 152 74 93
42
Solids Inventory and Observed Yield
One major difference between the two systems was the observed yield and MLVSS
inventories. Lowering the pH of the anaerobic zone resulted in a much lower observed yield
even though the SRT and COD removal in the two systems were almost identical. Table 7
shows the SRTs, MLVSS concentrations, and observed yields. The MLVSS concentrations are
the weighted averages of biological zones’ MLVSS values in each system from 6/22 to 7/27.
Please refer to Table 27 for individual MLVSS values. The MLVSS concentration of Train 1 in
Table 7 is 18.8% less than in Train 2. SRTs were 3.6% different considering data from 6/22 to
7/27 but they were equal ignoring outliers on 6/22 and 6/26 (8.4 d, see Table 2). It should be
noted that weighted average MLVSS ignoring two data points at 6/22 and 6/26 were 2079 and
2548 mg/l for Trains 1 and 2, respectively. The reason of having outliers is that the clarifier 2
malfunctioned on 6/22 and 6/26 and the TSS concentration of effluent was very high although
3.6% difference between SRTs can be negligible in a practical point of view. Observed yield in
the acidic system (Train 1) was lower than in the system with unadjusted pH (Train 2) by an
RPD of 29%.
Table 7: SRT, MLVSS, and Observed Yields
SRT (days) MLVSS (mg/L) Yobs (mgVSS/mgCOD)
Train 1 pH = 6.5 8.2 2050 0.28
Train 2 pH = 7.2 7.9 2526 0.38
43
Normalization with TSS and VSS
Because of the significant differences in the MLVSS concentrations in the two systems,
phosphorus and carbon transformations were analyzed while normalizing for the MLSS and
MLVSS concentrations to see how that affected conclusions concerning the systems. The results
are presented in Tables 8, 9, 10 and 11. Note that the normalization was done by dividing with
the total MLSS or MLVSS inventory of the entire reactor system to allow normalized
comparisons of not only each zone, but also of the clarifier release (where no MLSS or MLVSS
concentration is available). So the resulting values are not to be confused with process rates
(such as the anaerobic P release divided by the MLVSS inventory in the anaerobic zone). These
values are to facilitate a normalized process comparison which eliminates the difference in
MLSS/MLVSS concentration as a variable.
As can be seen in Tables 8 and 9, Net Biological P Removal normalized with the total
system TSS was the same in both Trains: 4.58 and 4.56 mg/g*d in Trains 1 and 2, respectively.
Also, Net Biological P Removal normalized with VSS was the same in both Trains too: 5.42
mg/g*d in both Trains (Tables 10 and 11). However the implications of this with respect to
conclusions that can be generalized to EBPR systems is suspect since the mass loading from the
clarifiers significantly affected the mass balances for Train 2.
Net System P Removal was higher in the acidic Train than in the basic Train based on
both normalized mass balance results. The difference was 3.88 mg/g*d and 4.57 mg/g*d for
normalized results with TSS and VSS, respectively. The RPD was 55.6% and 55.3% for
normalized results with TSS and VSS, respectively. This strongly implies that system P removal
44
was adversely affected by high pH if system P mass balance normalized with TSS or VSS is
used for comparison. However the impact of secondary P release in the clarifier increases the
likelihood that this observation is only true for these systems, and may not be true of EBPR
systems in general.
Table 8: Phosphorus Mass Balance Results Normalized with Total System TSS for Train 1,
(mg/g*d)1
Date AN Release
AX Release
Total Bio. Release
Total Sys.Release
AE Uptake
ClarifierUptake
Total Bio.Uptake
Total Sys. Uptake
Net Sys.Removal
Net Bio.Removal
6/15/04 17.29 -6.52 17.29 19.35 18.74 -2.06 25.26 25.26 5.91 7.97
6/29/04 12.09 2.37 14.47 10.91 16.72 1.18 16.72 17.91 7.00 2.26
7/6/04 12.22 2.86 15.08 10.42 20.16 1.80 20.16 21.96 11.54 5.08
7/13/04 11.38 2.75 14.14 14.53 18.67 -3.15 18.67 18.67 4.14 4.54
7/27/04 14.30 10.32 24.62 12.99 27.66 1.31 27.66 28.98 15.99 3.04
Avg. 13.46 2.36 17.12 13.64 20.39 -0.18 21.70 22.56 8.92 4.58
Std Dev. 2.40 5.97 4.37 3.59 4.24 2.26 4.60 4.62 4.81 2.21 1 Exact unit is (mg-P/d)/(g-TSS)
45
Table 9: Phosphorus Mass Balance Results Normalized with Total System TSS for Train 2,
(mg/g*d)
Date AN Release
AX Release
Total Bio. Release
Total Sys.Release
AE Uptake
ClarifierUptake
Total Bio.Uptake
Total Sys. Uptake
Net Sys.Removal
Net Bio.Removal
6/15/04 17.98 -5.24 17.98 16.12 21.52 -3.38 26.76 26.76 10.64 8.78
6/29/04 18.28 -4.18 18.28 15.26 16.51 -1.16 20.69 20.69 5.43 2.41
7/6/04 17.18 -0.65 17.18 19.88 21.45 -3.34 22.09 22.09 2.21 4.91
7/13/04 19.36 -2.35 19.36 19.58 21.07 -2.57 23.42 23.42 3.84 4.06
7/27/04 18.20 -0.64 18.20 17.77 20.19 -0.22 20.84 20.84 3.06 2.64
Avg. 18.20 -2.61 18.20 17.72 20.15 -2.13 22.76 22.76 5.04 4.56
Std Dev. 0.78 2.07 0.78 2.05 2.10 1.40 2.49 2.49 3.35 2.57
Table 10: Phosphorus Mass Balance Results Normalized with Total System VSS for Train 1,
(mg/g*d)
Date AN Release
AX Release
Total Bio. Release
Total Sys.Release
AE Uptake
ClarifierUptake
Total Bio.Uptake
Total Sys. Uptake
Net Sys.Removal
Net Bio.Removal
6/15/04 20.46 -7.71 20.46 22.90 22.18 -2.44 29.89 29.89 7.00 9.43
6/29/04 14.31 2.81 17.12 12.91 19.79 1.40 19.79 21.20 8.28 2.67
7/6/04 14.46 3.39 17.85 12.33 23.87 2.13 23.87 25.99 13.66 6.01
7/13/04 13.47 3.26 16.73 17.20 22.10 -3.73 22.10 22.10 4.90 5.37
7/27/04 16.93 12.21 29.14 15.37 32.74 1.55 32.74 34.30 18.92 3.60
Avg. 15.93 2.79 20.26 16.14 24.14 -0.22 25.68 26.70 10.55 5.42
Std Dev. 2.84 7.07 5.17 4.25 5.02 2.67 5.44 5.47 5.69 2.61
46
Table 11: Phosphorus Mass Balance Results Normalized with Total System VSS for Train 2,
(mg/g*d)
Date AN Release
AX Release
Total Bio. Release
Total Sys.Release
AE Uptake
ClarifierUptake
Total Bio.Uptake
Total Sys. Uptake
Net Sys.Removal
Net Bio.Removal
6/15/04 21.35 -6.22 21.35 19.14 25.56 -4.01 31.78 31.78 12.64 10.43
6/29/04 21.71 -4.97 21.71 18.12 19.61 -1.38 24.58 24.58 6.45 2.87
7/6/04 20.41 -0.77 20.41 23.61 25.47 -3.97 26.24 26.24 2.63 5.83
7/13/04 22.99 -2.79 22.99 23.25 25.02 -3.05 27.81 27.81 4.56 4.82
7/27/04 21.62 -0.76 21.62 21.11 23.99 -0.26 24.75 24.75 3.64 3.13
Avg. 21.62 -3.10 21.62 21.05 23.93 -2.53 27.03 27.03 5.98 5.42
Std Dev. 0.93 2.46 0.93 2.43 2.49 1.66 2.96 2.96 3.98 3.06
Statistical Analysis
Statistical analysis was done to see if the difference seen between P removal
performances based on phosphorus mass balance were significant. RPD values for the compared
Train average mass balance values are reported in Table 12 along with the confidence level at
which the values were different for a Paired t-test. Table 13 shows the same data for P mass
balance results normalized with VSS. The comparison is based on normalized data with VSS
because either normalized data with VSS or with TSS would result similar conclusions.
Furthermore, VSS is a better representative of the biomass than TSS. The RPD for anoxic P
release is not reported because Train 1 showed P release in the anoxic zone but Train 2 showed P
47
uptake.
It can be seen in Table 12 that the RPD between P release averages in the anaerobic zones
was 50.5% but RPD between aerobic P uptake averages was less than half of that, i.e. 20.2%.
The difference in Train 1 and 2 was shown to be statistically significant (Table 12) for both
anaerobic and aerobic P transformations (99.8 and 92% confidence levels) and thus our
observations do show a pH effect in both zones. This is consistent with prior literature for the
anaerobic zone, including that the P release should decrease as pH decreases (Smolders, et al.
1994 and 1995). However, the decrease in aerobic P uptake with decreasing pH was not
observed by Smolders, et al. (1994 and 1995).
It also superficially appears that pH affected anaerobic P release greatly but didn’t affect
aerobic P uptake to the same extent. However, it should be noted that pH was not controlled in
the aerobic zone and it rose to 7.2 resulting in a pH difference of 0.3 between the two aerobic
zones, which is less than half of the pH difference in the anaerobic zones (0.7). The reason for
the pH increase is the consumption of VFAs in the anaerobic zone which makes downstream
zones more basic. In addition, CO2 stripping in the aerobic zone elevates pH. If we normalize
the RPD data with pH, we see that 0.3 pH difference resulted in a 20.2% RPD difference
(67.3%/pH) in the aerobic zone, while a 0.7 pH difference resulted in a 50.5% RPD difference
(72.1%/pH) in the anaerobic zone. This suggests the possibility that the effect on P
transformations per unit pH was similar in both zones. However, it is also possible the results
may be coincidence.
When the same parameters normalized with VSS are considered, it is seen that pH
affected anaerobic P release in the same way: the lower the pH, the lower the anaerobic release.
48
The RPD value was 30.3% and the differences were significant (confidence level of 98%). This
supports the above conclusion about anaerobic P release. However, the above conclusion about
aerobic uptake doesn’t seem to be supported and is hard to be made because the RPD value is
only 0.9% and confidence level of 7% means the differences were not significant. In addition, P
release occurred in clarifier 2 more than it did in clarifier 1 and when returned biomass through
RAS line took up phosphorus, that uptake was counted in aerobic uptake but that uptake might
not be a sole result of EBPR. That means aerobic uptake in Train 2 might be calculated higher
than what it really was. In any case, it cannot be concluded from available normalized data that
pH affects aerobic P uptake because of the malfunctioning of the clarifiers.
Table 12: RPD between P Mass Balance Averages Along with Statistical Analysis Results
Zone AN Release
AX Release
Tot. Bio. Release
Tot. Sys. Release
AERUptake
Clar. Uptake
Tot. Bio. Uptake
Tot. Sys. Uptake
Net Sys.Removal
Net Bio. Removal
RPD 50.5 N/A1 27.4 32.4 20.2 -174.1 26.1 22.2 37.5 21.0
Comparison Based on
Avg 1<2 1>21 1<2 1<2 1<2 1>2 1<2 1<2 1>2 1<2
Conf. Level 99.8 96 94 96 92 94 96 93 75 91
Effect of Lowering pH - + - - - +2 - - +3 -
1 P release in AX 1 and P uptake in AX 2 2 Lowering pH resulted in a lower P release than the unadjusted pH 3 Unadjusted pH system (Train 2) had a high P release in the clarifier
49
Table 13: RPD between P Mass Balance Averages Normalized with VSS Along with Statistical
Analysis Results
Zone AN
Release/ VSS
AX Release/
VSS
Tot. Bio. Release/
VSS
Tot. Sys. Release/
VSS
AERUptake/
VSS
Clar.Uptake/
VSS
Tot. Bio. Uptake/
VSS
Tot. Sys. Uptake/
VSS
Net Sys.Removal/
VSS
Net Bio. Removal/
VSS
RPD 30.3 N/A1 6.5 26.4 0.9 -168.5 5.1 1.3 55.3 0.0
Comparison Based on
Avg 1<2 1>21 1<2 1<2 1>2 1>2 1<2 1<2 1>2 1=2
Conf. Level 98 93 40 89 7 90 39 10 71 1
Effect of Lowering pH - + none - none + none none none none
1 P release in AX 1 and P uptake in AX 2
As noted in Table 12, all of the comparisons made were statistically significant at a level
greater than 90% except for the Net System P Removal of which the confidence level was 75%.
Table 13, however, shows six parameters with statistically insignificant differences. Those are
Total Biological Release, Aerobic Uptake, Total Biological Uptake, Total System Uptake, Net
System Removal, and Net Biological Removal. In other words, there is no significant difference
between most parameters. The results shown in Table 13 imply that acidification of the system
resulted in lower P transformations and lower Net Biological P Removal. The phosphorus
uptake/release ratios for the systems were virtually identical at 1.28 for Train 1 and 1.25 for
Train 2 (see Table 22), indicating that the magnitude of P transformations was the key to the
higher observed Net P Removals at higher pH. But based on normalized data, Net Biological P
50
Removal was unaffected by pH in the pH range used in this study.
Table 12 shows that the RPD between anaerobic P releases was 50.5% and Tables 5 and 6
show that the difference was 251 mg-P/d: 371 mg/d in Train 1 and 622 mg/d in Train 2. This
translates to a reduction in anaerobic P release of 40.4% due to a 0.7 pH change which is very
significant.
The anoxic zone showed two different behaviors when exposed to different pHs. When
pH was acidic (Train 1), there was a net P release in the anoxic zone, but when pH was
unadjusted, a net P uptake occurred. It is thought that in anoxic zones both P release due to
RBCOD VFA uptake and P uptake due to denitrifying PHA degradation compete
simultaneously (Chuang et al., 1996). In Train 1, anoxic P release was apparently greater than
anoxic P uptake, perhaps because so much less P release had occurred in the anaerobic zone. In
Train 2, anoxic P uptake was greater than anoxic P release and this could be because the biomass
was polyphosphate limited after having such a large preceding anaerobic P release. This results
in the Total Biological P Release (third column in Tables 5 and 6) of Train 1 being greater than
the Anaerobic P Release for Train 1 (472 vs. 371 mg-P/d) while Train 2’s release remains
unchanged at 622 mg-P/d.
Because of this, when anaerobic and anoxic zones are considered together, the Total
Biological P Release shows lower RPD compared to Anaerobic P Releases (27.4% vs. 50.5%).
However, the difference between Total Biological P Releases remains very large at 150 mg-P/d.
Note that average values in Tables 5 and 6 are the averages of corresponding columns
independently, i.e. they don’t add up because in some cases individual terms contribute to total
release, and in other cases they contribute to total uptake.
51
Aerobic P uptake was affected by pH too based on non-normalized data. Aerobic P
uptake was 563 mg-P/d in Train 1 compared to 689 mg-P/d for Train 2 with an RPD value of
20.2%. This represents an 18.3% reduction in Aerobic P Uptake due to a decrease in pH of 0.3
units. As discussed before, the pH differences between the two anaerobic zones and between the
two aerobic zones were not equal. The difference in the aerobic P uptake per unit pH was 420
mg-P/d and that of the anaerobic P release was 359 mg-P/d.
It is possible that a slightly basic pH might improve the P uptake performance if our data
can be extrapolated. However, we looked at a limited range of data, and in addition since only 2
pH points were compared it was not possible to say if the pH effect is best modeled as a linear
relationship or if some other mathematical model would be more appropriate. When normalized
data are considered, pH didn’t affect aerobic uptake as discussed before. Average normalized
aerobic uptake was 24.14 and 23.93 mg/g*d in Trains 1 and 2, respectively, which are practically
equal.
As it was stated before, the anoxic zone of Train 2 acted as a P uptake zone. Also, at one
point (6/15/04), P uptake occurred in the anoxic zone of Train 1. Therefore, when calculating
Total Biological P Uptake, the anoxic zone uptake was taken into account along with the aerobic
P uptake. The Total Biological P Uptake was 599 mg-P/d in Train 1 and 778 mg-P/d in Train 2
(179 mg-P/d higher than Train 1) with an RPD value of 26.1%. Net Removals in Tables 5 and 6
were calculated by subtracting Total Biological P Release from Total Biological P Uptake for
each individual mass balance points and averaging the results.
As Tables 5 and 6 show, the Net Biological P Removal was 21% higher in Train 2 with
values of 126 and 156 mg-P/d for Trains 1 and 2, respectively. This means that net P removal
52
was decreased by 21% from 0.7 pH units in the anaerobic zone which would correspond to an
influent decrease of 0.7 pH units in a normal, non-pH controlled, full-scale system. Comparison
of normalized data reveals, however, that Net Biological P Removal wasn’t affected by pH at all.
The normalized average Net Biological P Removal was 5.42 mg/g*d in both systems.
PHA Biosynthesis Performance
PHA biosynthesis decreased with lowering pH when non-normalized results were
considered. Table 14 shows that total PHA in the anaerobic zone of Train 1 (AN 1) was lower
than that of Train 2 (AN 2) by 1.5 mmol-C/L or 21%. Anoxic zone PHAs differed little from
anaerobic zone values which indicated that PHA biosynthesis occurred under anaerobic
conditions with little activity in the anoxic zone. PHV contributed more than PHB in the
differences between Train 1 and 2. pH did not affect PHB biosynthesis significantly (3.36 vs.
3.69 mM-C in AN 1 and 2, respectively), while lowering pH decreased PHV biosynthesis
significantly (2.24 vs. 3.41 mM-C in AN 1 and 2, respectively). This phenomenon has been
reported in prior research (Filipe et al, 2001b). At low pH, PHV biosynthesis was 39% of PHA,
while at the unadjusted pH, PHV was 48% of the PHA.
PHA data normalized with TSS and VSS are shown in Tables 15 and 16. Normalized
PHA data show the same general trend as non-normalized PHA data: lower values in Train 1.
AN 1 PHA normalized with TSS was lower than that of AN 2 by only an RPD of 2.2% which
means they were practicaly equal. When PHA data normalized with VSS is considered, AN 1
53
was lower than AN 2 by an RPD of 2.6%. Again, they were practicaly equal. RPD values for
AX PHA normalized with TSS and VSS were 7.3% and 7.7%, respectively. It can be seen that
PHA biosynthesis isn’t proved to be much different between the two systems when comparison
is based on PHA data that are normalized with TSS or VSS. Statistical analysis supports this
conclusion. The statistical analysis results are presented in Table 17.
It is seen that P releases in anaerobic zones were directly proportional to PHA
concentration: Train 1 with lower anaerobic PHA content had lower anaerobic P release. Also,
the net P removal of Train 1 was lower than that of Train 2. Lowering pH resulted in less labile
polyphosphate (lower P release and uptake) and lower PHA biosynthesis.
Table 14: PHB, PHV, and PHA in both Trains
PHB, PHV, and PHA, mM-C
Date
PHB, AN 1
PHV, AN 1
PHA, AN 1
PHB, AX 1
PHV, AX 1
PHA, AX 1
PHB, AN 2
PHV, AN 2
PHA, AN 2
PHB, AX 2
PHV, AX 2
PHA, AX 2
6/22/2004 3.41 2.02 5.43 3.32 1.94 5.26 3.52 3.40 6.92 3.55 3.45 7.00
6/29/2004 3.10 2.25 5.35 2.95 2.17 5.12 3.78 3.50 7.28 3.83 3.55 7.38
7/6/2004 2.89 1.79 4.68 2.79 1.71 4.50 3.52 3.26 6.78 3.59 3.31 6.90
7/13/2004 3.82 3.08 6.90 3.68 3.00 6.68 3.68 3.24 6.92 3.70 3.29 6.99
7/20/2004 3.58 2.06 5.64 3.46 1.98 5.44 3.93 3.65 7.58 4.03 3.70 7.73
Avg. 3.36 2.24 5.60 3.24 2.16 5.40 3.69 3.41 7.10 3.74 3.46 7.20
Std Dev 0.37 0.50 0.81 0.37 0.50 0.80 0.18 0.17 0.33 0.20 0.17 0.35
54
Table 15: PHB, PHV, and PHA Normalized with TSS in both Trains
PHB, PHV, and PHA Normalized with TSS, (mmol/g)
Date
PHB, AN 1
PHV, AN 1
PHA, AN 1
PHB, AX 1
PHV, AX 1
PHA, AX 1
PHB, AN 2
PHV, AN 2
PHA, AN 2
PHB, AX 2
PHV, AX 2
PHA, AX 2
6/22/2004 1.37 0.81 2.18 1.33 0.78 2.11 1.14 1.10 2.24 1.15 1.12 2.27
6/29/2004 1.25 0.90 2.15 1.19 0.87 2.06 1.23 1.13 2.36 1.24 1.15 2.39
7/6/2004 1.16 0.72 1.88 1.12 0.69 1.81 1.14 1.06 2.20 1.16 1.07 2.24
7/13/2004 1.53 1.24 2.77 1.48 1.21 2.68 1.19 1.05 2.24 1.20 1.07 2.27
7/20/2004 1.44 0.83 2.27 1.39 0.80 2.19 1.27 1.18 2.46 1.31 1.20 2.51
Avg. 1.35 0.90 2.25 1.30 0.87 2.17 1.20 1.11 2.30 1.21 1.12 2.33
Std Dev 0.15 0.20 0.33 0.15 0.20 0.32 0.06 0.06 0.11 0.06 0.06 0.11
Table 16: PHB, PHV, and PHA Normalized with VSS in both Trains
PHB, PHV, and PHA Normalized with VSS, (mmol/g)
Date
PHB, AN 1
PHV, AN 1
PHA, AN 1
PHB, AX 1
PHV, AX 1
PHA, AX 1
PHB, AN 2
PHV, AN 2
PHA, AN 2
PHB, AX 2
PHV, AX 2
PHA, AX 2
6/22/2004 1.62 0.96 2.58 1.58 0.92 2.50 1.36 1.31 2.66 1.37 1.33 2.70
6/29/2004 1.47 1.07 2.54 1.40 1.03 2.43 1.46 1.35 2.80 1.47 1.37 2.84
7/6/2004 1.37 0.85 2.23 1.33 0.81 2.14 1.36 1.26 2.61 1.38 1.27 2.66
7/13/2004 1.82 1.46 3.28 1.75 1.43 3.18 1.42 1.25 2.66 1.42 1.27 2.69
7/20/2004 1.70 0.98 2.68 1.65 0.94 2.59 1.51 1.41 2.92 1.55 1.42 2.98
Avg. 1.60 1.07 2.66 1.54 1.03 2.57 1.42 1.31 2.73 1.44 1.33 2.77
Std Dev 0.18 0.24 0.39 0.17 0.24 0.38 0.07 0.07 0.13 0.08 0.07 0.13
55
Table 17: RPD between System Parameters Along with Statistical Analysis Results
Zone Prel Pup AN PHA AX PHA GLY Formation
Prel/ VSS
Pup/ VSS
AN PHA/ VSS
AX PHA/ VSS
GLY Formation/
VSS
RPD 27.39 26.07 23.57 28.57 2.42 6.48 5.14 2.60 7.69 18.61
Comparison Based on
Avg 1<2 1<2 1<2 1<2 1<2 1<2 1<2 1<2 1<2 1>2
Conf. Level 94 96 98 99 95 40 39 29 68 95
Effect of Lowering pH - - - - - none none none none +
Glycogen Formation Performance
The glycogen content of different zones in the two Trains is presented in Table 18. Train
2 had more glycogen in all zones than Train 1 had based on non-normalized values. Glycogen
content rose from 10.58, 10.52, and 13.2 mM-C in AN 1, AX 1, and AER 1, compared to 13.22,
13.27, and 15.89 mM-C in AN 2, AX 2, and AER 2. Glycogen formation/consumption (they are
equal at steady-state) was actually the same (Table 18). Glycogen formation/consumption in
Train 1 and Train 2 was 2.6 and 2.7 mM-C, respectively. Thus glycogen content of the biomass
increased with increasing pH (from 6.5 to 7.2) but glycogen formation/consumption (aerobic –
anaerobic glycogen) remained unaffected. It is possible (but speculative) that the increased
glycogen content reflected an increased GAO/PAO ratio in the biomass, but it is also possible
56
that the pH change just increased glycogen storage in the biomass rather than this specific
population shift. However, Filipe et al. (2001) concluded that elevated pH decreased the
GAO/PAO ratio, although they did not do any population studies (e.g. Fluorescent In-Situ
Hybridization or FISH, etc.).
Glycogen formation normalized with TSS and VSS are also calculated and shown in
Tables 19 and 20. As it can be seen, the acidic Train (Train 1) had apparently more glycogen
formation relative to both TSS and VSS. The RPD values for glycogen formation per TSS and
per VSS were 19% and 18.6%, respectively. This difference supports the possible conclusion
that GAO/PAO ratio was increased in the biomass in the acidic Train leading to the conclusion
that lowering pH will probably harm PAO population and won’t be a good practice for
phosphorus removal.
Table 18: Glycogen Concentrations in Trains 1 and 2
Glycogen, mM-C Date
AN 1 AX 1 AER 1 AN 2 AX 2 AER 2
6/26/2004 10.82 10.73 13.39 13.60 13.72 16.7
6/29/2004 10.63 10.50 13.23 13.83 13.88 15.89
7/13/2004 10.41 10.29 13.11 12.65 12.67 15.3
7/20/2004 10.12 10.62 12.82 12.84 12.8 15.52
7/27/2004 10.93 10.48 13.43 13.16 13.26 16.06
Avg. 10.58 10.52 13.20 13.22 13.27 15.89
Std Dev 0.33 0.17 0.25 0.50 0.54 0.54
57
Table 19: Glycogen Formation/Consumption in both Trains
Glycogen, mM-C Date
Train 1 Train 2
6/26/2004 2.57 3.10
6/29/2004 2.60 2.06
7/13/2004 2.70 2.65
7/20/2004 2.70 2.68
7/27/2004 2.50 2.90
Avg. 2.61 2.67
Std Dev 0.09 0.39
Table 20: Glycogen Formation/Consumption Normalized with TSS in both Trains
Gly Formation/TSS, mmol/g Date
Train 1 Train 2
6/26/2004 1.032 1.005
6/29/2004 1.045 0.668
7/13/2004 1.085 0.859
7/20/2004 1.085 0.869
7/27/2004 1.004 0.940
Avg. 1.050 0.868
Std Dev 0.035 0.127
58
Table 21: Glycogen Formation/Consumption Normalized with VSS in both Trains
Gly Formation/VSS, mmol/g Date
Train 1 Train 2
6/26/2004 1.222 1.194
6/29/2004 1.236 0.793
7/13/2004 1.284 1.021
7/20/2004 1.284 1.032
7/27/2004 1.189 1.117
Avg. 1.243 1.031
Std Dev 0.041 0.150
PHA Yield and Modified PHA Yield
Table 22 shows the VFA values in the influent. It shows all useful data but only data
after 6/15/04 were taken as steady-state and used in statistical analysis. As mentioned before, the
anaerobic zones were spiked with 1 mM-C Propionic acid. The last column shows total VFA
including the spike. Table 23 shows that PHA yield (YPHA=PHA/VFA) of Train 2 was 27.8%
higher than PHA yield of Train 1. In both systems, YPHA was greater than one implying that
for each mole of VFA carbon consumed, more than one mole carbon as PHA was produced. This
was observed more in Train 2 (with unadjusted pH) than in Train 1 (acidified system). YPHAs
greater than one show that in both systems the PHA biosynthesis could not be explained from
VFA uptake alone.
A modified PHA yield (YPHA*) was therefore calculated and shown in Table 23.
59
YPHA* included anaerobic glycogen consumption (equal to aerobic glycogen synthesis at steady
state) along with VFA to determine if PHA biosynthesis could be explained from both VFA and
glycogen. It should be noted that YPHA* is calculated assuming 100% of the glycogen would
end up as PHA which would only occur if the pyruvate produced from glycolysis proceeded
through the propionate-succinate pathway rather than the EMP pathway, where 1/3rd of the
carbon would leave as CO2 rather than proceeding to PHA.
The yields for Train 2 were greater than Train 1, and more importantly still greater than
1, even with 100% of the anaerobic glycogen consumption included which implies that there was
a significant utilization of so called “cryptic nutrients” (Louie et al., 2000), i.e. unknown carbon
sources, in Train 2. The lowering of pH in Train 1 reduced yields, perhaps by interfering with
the biotransformation of either cryptic nutrients, glycogen, or VFAs to PHA. It would be
necessary to do tracer studies to determine which was involved.
60
Table 22: VFA Values in Influent and Total VFA
VFA
Date Acetic
Acid, ppm Propionic Acid, ppm
Acetic Acid, mM-C
Propionic Acid, mM-C
Propionic Acid Spike,
mM-C Total VFA,
mM-C
5/25/2004 38.5 0.65 1.56 1 3.21
6/1/2004 37.5
19.4
42.5 1.25 1.72 1 3.97
6/8/2004 23.9 30.5 0.80 1.23 1 3.03
6/22/2004 41.0 24.2 1.37 0.98 1 3.35
29.6 20.2 0.99 0.82 1 2.81
7/6/2004 33.7 1.12 0.72 1 2.84
7/13/2004 67.2 26.1 2.24 1 4.30
Avg. 36.0 28.6 1.20 1.16 1
Std Dev 15.6 9.2 0.5 0.4 0
6/29/2004
17.9
1.06
3.36
0.60
Table 23: PHA Yield (YPHA or PHA/VFA Ratio) in Trains 1 and 2
Date PHA AN 1 mM-C
PHA AN 2 mM-C
VFA mM-C (PHA/VFA) 1 (PHA/VFA) 2
6/22/2004 5.43 6.92 3.35 1.62 2.07
6/29/2004 5.35 7.28 2.81 1.91 2.59
7/6/2004 4.68 2.85 1.64 2.38
7/13/2004 6.90 6.92 4.30 1.61 1.61
Avg. 5.59 6.97 3.32 1.69 2.16
Std Dev 0.94 0.21 0.69 0.14 0.43
6.78
61
Table 24: PHA Yield Based on VFA and Glycogen (YPHA*) in Trains 1 and 2
Date PHA AN 1 mM-C
PHA AN 2 mM-C
VFA mM-C
Glycogen Formation1
mM-C
Glycogen Formation 2
mM-C YPHA*1 YPHA*2
6/29/2004 5.35 7.28 2.81 2.6 2.06 0.99 1.50
7/13/2004 6.90 6.92 4.30 2.7 2.65 0.99 1.00
Avg. 6.13 7.10 3.55 2.65 2.36 0.99 1.25
Std Dev 1.09 0.25 1.05 0.07 0.42 0 0.35
Prel/VFA, Pup/PHA, and Pup/Prel Ratios
The two Trains showed a difference in Prel/VFA ratio of 0.68 mmol-P/mmol-C or 26.8%
as presented in Table 25. VFA-driven P release was lower in Train 1 (0.148 mmol-P/mmol-C)
than in Train 2 (0.216 mmol-P/mmol-C) and it suggests that the energy required to transport
VFAs increases with pH which agrees with Smolders, et al. (1994). However, higher P release
corresponded to higher P uptake in the aerobic zone, and hence, better overall (net) P removal
based on non-normalized P mass balance results.
Pup/PHA ratio of Train 2 was 20% higher than this ratio for Train 1 (Table 25) but this
difference was only marginally statistically significant shown in Table 27 (significant at
confidence level = 73%). This ratio represents PHA-driven P uptake. This suggests that the
higher aerobic P uptake and higher net P removal at high pH (based on non-normalized results)
was due to higher PHA storage rather than a change in PHA-driven P uptake efficiency.
The Pup/Prel ratio is calculated and displayed in Table 26. There was not much
62
difference between Pup/Prel ratios in the two Trains. This ratio for Train 1 was 1.28 versus 1.25
for Train 2. Also, the statistical analysis results shown in Table 27 reveal that the Null
Hypothesis of equal means could not be rejected confidently: the confidence level was only 66%.
It has been observed in past studies that for a given population this ratio will not change
in batch tests even though substrate is varied (Randall, 2002). However, in those cases the
magnitude of P release and uptake did not vary much. Here steady state exposure to a different
pH might be expected to result in a different population (Filipe et al., 2001) so the consistency of
the ratio suggests another hypothesis may be possible for interpreting P uptake/P release ratios
than the populations being the same. Also in this case the magnitude of P release and uptake did
vary. Thus magnitude of P release and uptake from pH change seems more likely to be due to a
population change if we accept the hypothesis of Filipe. Thus the consistency of the P uptake /P
release ratio in this study doesn’t imply the populations were necessarily similar, and the
glycogen data also arguably suggests this (Table 18).
63
Table 25: Prel/VFA and Pup/PHA Ratios in Trains 1 and 2
Date (Prel/VFA) 1 (Prel/VFA) 2 (Pup/PHA) 1 (Pup/PHA) 2
6/15/2004 0.1681 0.196 - -
6/29/2004 0.141 0.239 0.085 0.104
7/6/2004 0.161 0.239 0.131 0.129
7/13/2004 0.120 0.191 0.099 0.144
Avg. 0.148 0.216 0.105 0.126
Std Dev 0.022 0.026 0.024 0.020 1 The numbers are unitless. Actual unit is (mmol-P/d)/(mmol-C/d).
Table 26: Pup/Prel Ratio in Trains 1 and 2
Date Pup/Prel 1 Pup/Prel 2
6/15/2004 1.46 1.49
6/29/2004 1.16 1.13
7/6/2004 1.34 1.29
7/13/2004 1.32 1.21
7/27/2004 1.12 1.14
Avg. 1.28 1.25
Std Dev 0.14 0.15
A statistical analysis was completed to ensure the differences discussed above are
meaningful. The statistical analysis done on these values was the Paired t-test of equal means.
The results are shown in Table 27. As can be seen, the confidence level to reject equal means of
64
all parameters except Pup/PHA, Pup/Prel, and YPHA* was equal or higher than 90% and thus
these differences were statistically significant. A comparison based on average values has been
done and, as shown in Table 27, all parameters with a significant difference were greater in Train
2 than in Train 1.
Table 27: Statistical Analysis Results
Parameter AN PHA Prel/VFA Pup/PHA YPHA PHA/VFA
YPHA* PHA / (VFA+GLY) Pup/Prel
Conf. Level 98 98 73 96 51 66
Comparison Based on Avg 1<2 1<2 1=2 1<2 1=2 1=2
Nitrate, ORP, and DO Data
Very limited nitrate data have been obtained in different zones of the two systems to
make sure the level of nitrate in the Trains (especially in anaerobic zones) wasn’t harmful to
EBPR but the data appear to be invalid. Since conducting a complete nitrogen analysis was not
intended, no more samples for nitrate were taken. Please refer to Table 28 for nitrate, ORP
(Oxidation Reduction Potential), and DO (Dissolved Oxygen) data. Looking at the body of data
we have to interpret the conditions in the anaerobic zone using ORP, DO, nitrate, and anaerobic
P release. Three of the four of these parameters are consistent with strongly anaerobic conditions.
DO measurements were down below the effective range of the DO meter. ORPs were below -
65
150 mV consistently, and values would be 100 to 200 mV higher in the presence of nitrate.
Anaerobic P release (which is inhibited by the presence of nitrate) was robust throughout the
study. Because of this, it was concluded that the nitrate data was erroneous. The ORP data in
particular was completely inconsistent with the presence of nitrate. Quality control was done to
ensure the ORP and DO data were valid. Nitrate data were not used in the analyses.
Table 28: Nitrate, ORP, and DO Data
NO3, mg-N/l ORP, mV DO, mg/l Date
INF AER 1
AX 1
AN 1
CL 1 AER 1 AX 1 AN 1 AER 1 AN 1
5/25/2004 2.8 0.78 6/8/2004 6.4 2.6 -40 -130 -200 6/16/2004 50 -35 -180 6.5 0.21 6/22/2004 3 1.5 2.1 4.2 1.6 7/1/2004 2.7 0.2 7/13/2004 7/15/2004 10 -60 -160 2.5 0.2
INF AER 2
AX 2
AN 2
CL 2 AER 2 AX 2 AN 2 AER 2 AN 2
5/25/2004 5.1 1.7 6/8/2004 6.3 2.3 -10 -100 -180 6/16/2004 15 -45 -168 5.5 0.2 6/22/2004 3 1.7 2.7 6.3 2.6 7/1/2004 2.9 0.2 7/13/2004 0.31 5.7 2.9 0.71 2.4 7/15/2004 18 -10 -195 2.1 0.2
1 The lowest DO value the instrument could show was 0.2 mg/l.
66
Conclusions
Phosphorus Removal
The results show that lower anaerobic zone pH reduced the anaerobic P release both on
an MLVSS specific basis and also on a non-specific (absolute value for the process) basis. In
addition, the observed yield was significantly decreased. Aerobic P uptake was roughly the same
on a specific basis although non-specific aerobic P uptake was lower in the low-pH system due
to the lower observed yield, and thus lower MLVSS concentration.
Net P removal was hard to interpret because of the effect of P release in the secondary
clarifier of Train 2 (high pH). However, on a specific basis it was clear that net P removal was
either equal or better in the low-pH system regardless of how the secondary clarifier data was
interpreted. Unfortunately, it is not possible to make a conclusion regarding Net P Removal due
to the impact of the secondary clarifier P release.
Unfortunately, the most significant question (which process will have greater P removal)
was obscured since the P release in the clarifier of Train 2 changes the answer. If we include the
clarifiers, the low-pH system removed more P and thus had a very slightly (not significant) lower
effluent concentration. However on a specific basis the low-pH system had a higher P content,
albeit negated on a process level by the lower observed yield. Thus, we cannot conclude that the
67
low-pH system will have superior effluent P to a properly operating high-pH system since the
effluents were observed to be equal (for all practical purposes) even though the biomass P
content was higher. Further we do not know what the high-pH system biomass P content would
have been if there were not secondary P release in the clarifier. A final question is the reason for
the much lower observed yield in Train 1, and whether this was an artifact of this study or if it
can be generalized (which is doubtful). The firmest conclusions regard the behaviour of the
systems anaerobic zones. Anaerobic P release for propionate rich wastewaters behaves in a
similar manner to systems receiving only acetic acid, i.e. lower P release with lower pH.
Carbon Transformations (PHA and Glycogen)
Carbon transformations were not impacted in as consistent a fashion as anaerobic P
release was. PHA and glycogen analyses on specific and non-specific basis lead to different
conclusions. On a specific basis, PHA content remained unchanged although the PHV/PHB ratio
was impacted with much lower PHV content in the low-pH system. However, on non-specific
basis, PHA biosynthesis decreased with lowering pH. Also, on a specific basis, glycogen content
and the amount of labile glycogen (delta glycogen) were higher in the low-pH system, in spite of
the fact that MLVSS P content did not decrease. However, glycogen formation was higher in the
acidic train based on specific results.
68
Other Impacts
Most of the other impacts were direct results of the lower yield on the process
performance. PHA and aerobic P uptake did not change on a specific basis (unit MLVSS basis).
However, since the influent VFA content was the same going into both systems, it is significant
that the absolute value of PHA produced in the low-pH reactor was lower since this means a
lower PHA/VFA and PHA/(VFA+∆Glycogen) yield in the low-pH system.
In this study, steady state results were obtained and pH was allowed to vary as it would in
full-scale systems in downstream reactors from the anaerobic zone. It is possible that lowering
pH due to organic acids could be different than lowering pH due to inorganic acids, and also the
comparison of sequesterable organic acids (e.g. VFAs) versus other organic acids. This can be
an area for future research. In addition it would be interesting to do a similar study to this one
while characterizing the microbial populations.
69
APPENDIX A METHODS AND MATERIALS
70
Experimental Approach
In order to study the effects of pH on EBPR, two parallel trains with identical rectangular
shapes were operated as continuous completely-mixed reactors. Three reactors (anaerobic,
anoxic, and aerobic) per train were created by placing two plastic baffles in grooves in an
overflow-underflow configuration. The volumes were 1637, 3867, and 5582 ml for anaerobic,
anoxic, and aerobic reactors, respectively. The trains were connected to external clarifiers from
which returned activated sludge (RAS) was pumped to the anoxic reactors at a rate of
approximately twice the influent flow rate. The clarifiers had a bottom scraper rotating at 1 rpm
to prevent accumulation of biomass. The supernatants of the clarifiers were collected in effluent
tanks for analyses. The whole system was kept in the Environmental Growth Chamber located
on the 4th floor of the Engineering Building 2 at the University of Central Florida in Orlando,
Florida at a constant temperature of 20°C.
The trains were fed with real wastewater from the Eastern Water Reclamation Facility in
East Orange County, Florida. About 120 liters of wastewater was collected every other day and
stored in influent tank and was pumped to the anaerobic reactors at a flow rate of 27.6 and 29.2
L/d for trains 1 and 2, respectively.
The HRTs (hydraulic retention time) were 1.4, 3.4, and 4.9 hours for anaerobic 1, anoxic
1, and aerobic 1 reactors, respectively. The HRTs of anaerobic 2, anoxic 2, and aerobic 2
reactors were 1.3, 3.2, and 4.6 hours, respectively. The average SRTs (solids retention time) for
train 1 and 2 were 8.5 and 8 days, and the MCRTs (mean cell residence time) for train 1 and 2
were 8.7 and 8.1 days, respectively.
71
Waste activated sludge (WAS) was pulled out from end of the aerobic reactors at a flow
rate of 1 L/d. Propionic acid at a rate of 1 mmol-C/L (based on influent flow rate) was added to
the anaerobic reactors to make propionate concentration higher than acetic concentration. The
pH of anaerobic 1 reactor was controlled by adding sulfuric acid drop-wise and the pH of Train 2
was unadjusted. The average pHs of anaerobic 1 and 2 reactors were 6.5 and 7.2, respectively.
A University of Cape Town (UCT) configuration was chose to secure anaerobic
conditions in the anaerobic reactors as much as possible. In this configuration, there are two
internal recycles named ARCY and NARCY. The ARCY line pumps from the anoxic to the
anaerobic reactors in order to provide biomass to the anaerobic zone. The NARCY line serves as
nitrate recycle from the aerobic to anoxic reactors (Figure 1). The flow rate of ARCY was 41.8
and 39.8 L/d for train 1 and 2, respectively. The NARCY flow rate was 126 and 122.4 L/d for
train 1 and 2, respectively.
72
Analytical Methods
The analytical methods for the determination of parameters were from Standard Methods
for Examination of Water and Wastewater (Eaton et al, 1995). The PHA and VFA analyses were
run by gas chromatography, and the Glycogen analysis was done with the Anthrone Test for
Carbohydrate (Murray, 1981). Details follow:
Chemical Oxygen Demand (COD): Total and soluble chemical oxygen demand
concentrations were measured using closed reflux, titrimetric method (section 5220 C of
Standard Methods). The COD is the oxygen equivalent of the organic matter present in the
wastewater. The reagents used in this test were prepared in our laboratory.
Total and Soluble Phosphate (TP and PO4-P): Total phosphorous was measured based
on persulfate digestion method (section 4500-P B.5 of Standard Methods) followed by the
vanadomolybdophosphoric acid colorimetric method (section 4500-P C). This procedure
transforms all other forms of phosphorous to the soluble form and then estimates the overall
phosphorous concentration. Orthophosphate was measured using the vanadomolybdophosphoric
acid colorimetric method (section 4500-P C of Standard Methods).
Mixed Liquor Total and Volatile Suspended Solids (MLSS and MLVSS): Total and
volatile suspended solids were measured using section 2540 D. and 2540 E, respectively. The
total and volatile suspended solids were determined to estimate the amount of biomass and
suspended inorganic and organic solids present in the samples.
This amount was needed to calculate the SRT of the system and to estimate the specific
content of other parameters in our biomass.
73
PHAs: PHAs were measured using a DB-1 column (Supelco Inc., Bellefonte, PA) and a
Schimadzu 14A gas Chromatograph (GC) with a flame ionization detector and helium as the
carrier gas. The injection port temperature was 230ºC, with initial column temperature of 100ºC
for 2 minutes followed by temperature ramping at 20ºC per minute to 160ºC where it stayed for 2
minutes. The detector temperature was 230ºC. The samples of about 120-ml were first
lyophilized for about 2 days. About 150 mg dry sludge was put into 5.0-ml V-shaped Wheaton-V
vials. 2-ml benzoic acid in chloroform (50 mg Benzoic acid per 100 ml of chloroform) was
added into the vial as an internal standard and solvent, respectively. 2 ml of 20% H2SO4 in
methanol was added as the digestion/esterification reagent (methyl esters of the PHA are what
are actually extracted into the chloroform phase). The vials were capped tightly and incubated at
100ºC in an oven for 7 hours. Duplicates vials were used in case any vials leaked. Also, to reduce
the same risk, all sample caps were tightened again several times during the incubation period.
After being cooled down in tap water and centrifuged for 10 minutes, the chloroform phase was
removed using a syringe into GC vials for GC analysis.
Glycogen: The glycogen content of the samples was measured using the Anthrone Test
for Carbohydrate. First, prepare a series of glucose standards (duplicate concentrations of 50, 70
and 90 µg/ml of glucose, including blanks) and samples adjusting them all to a final volume of 1
ml with distillated water; 5-ml test tubes were used for this. The anthrone reagent was prepared
by mixing 200 mg of anthrone and 5 ml of Ethanol with 75% sulfuric acid to a final volume of
100 ml. Then we added 5.0 ml of the anthrone reagent to each tube and mixed them thoroughly,
and placed them immediately to an ice water bath for a couple of minutes. Next, we transferred
all the tubes to a boiling water bath for exactly 10 minutes. Finally we returned all the tubes to
74
the ice water bath to cool them down fast. Then we measured their absorbency at 625 nm.
Volatile Fatty Acids (VFA): The VFA were measured following the Supelco Bulletin
856B (Supelco Inc., Bellefonte, PA). A Shimadzu gas Chromatograph equipped with flame
ionization detector (FID) was used for the analysis. VFAs were separated by 3-mm internal
diameter glass column with 60/80 Carbopack C/0.3% Carbowax 20M/0.1% phosphoric acid
packing (Supelco Inc., Bellefonte, PA). The oven of the GC was programmed to begin the
analysis at 105ºC, and to remain at that temperature for 2 minutes. The temperature then
increased 5ºC per minute until it reached 150ºC. Then, it was maintained at 150ºC for 2 minutes
more. The sample injection volume was 2 µL. The injection port and the detector were
maintained at 200ºC. Helium was used as a carrier gas at a velocity of 30 ml/min. An auto
sampler injected the sample into the GC and integrated the results.
At the time of the sampling, the samples were filtered with 0.45-µm membrane filters,
and filtrate was collected in 1.5-ml GC vials. The vials were sealed with Teflon-lined septum and
screw caps and stored at 4 C. Calibration curves were established for acetic and propionic acids
by using pure reagents purchased from Fisher Scientific (New Jersey).
75
APPENDIX B THESIS DATA
76
Table 29: SOP Data for both Trains
SOP, mg/l as P Date
INF AN 1 AX 1 AER 1 CL 1 AN 2 AX 2 AER 2 CL 2
5/25/2004 7.54 10.22 12.44 8.93 - 12.07 10.04 6.43 -
5/30/2004 10.41 11.15 10.59 7.72 13.22 9.48 10.59 9.11 15.69
6/1/2004 7.63 7.35 3.74 1.52 8.96 6.98 3.37 1.24 3.09
6/5/2004 7.91 10.22 3.83 1.43 4.85 8.56 3.65 1.52 2.07
6/8/2004 7.54 11.89 3.65 1.15 1.52 11.7 4.11 1.24 1.7
6/15/2004 6.98 13.37 5.13 2.72 3.37 15.22 5.41 1.98 3.28
6/22/2004 10.31 13.28 6.24 4.02 4.3 19.3 7.17 4.3 6.19
6/29/2004 9.94 13.37 8.56 6.33 5.96 18.56 9.76 6.98 7.44
7/6/2004 9.2 10.04 4.02 1.43 0.87 15.87 7.54 4.11 5.41
7/13/2004 8 10.22 5.22 2.72 3.74 16.06 5.87 2.35 3.37
7/20/2004 10.96 15.69 9.76 6.7 6.33 16.43 9.76 8 11.3
7/27/2004 8.28 13.83 7.72 4.11 3.65 17.91 8.56 5.22 5.31
Avg. 8.7 11.7 6.7 4.1 5.2 14.0 7.2 4.4 5.9
Std Dev 1.36 2.28 3.02 2.72 3.48 4.13 2.64 2.78 4.26
77
Table 30: TP Data for both Trains
TP, mg/l as P Date
INF AER 1 CL 1 EFF 1 AER2 CL 2 EFF 2
5/25/2004 - 120 - 4 114.8 - 6.2 6/1/2004 9.60 118.50 10.70 8.16 115.30 3.62 3.20 6/8/2004 3.42 111.58 3.80 0.80 127.03 5.68 3.01 6/15/2004 3.42 80.68 5.60 0.53 125.30 5.68 2.80 6/22/2004 8.05 80.68 6.90 4.24 127.03 6.71 3.62 6/29/2004 9.08 80.68 7.50 3.21 121.88 13.93 4.24 7/6/2004 7.44 89.60 2.10 0.95 107.87 6.10 4.65 7/13/2004 5.38 102.30 5.70 8.16 107.87 3.21 2.18 7/20/2004 30.61 84.69 7.80 6.10 128.47 16.40 6.71 7/27/2004 8.29 128.47 5.45 4.35 125.90 6.71 5.48
Avg. 9.5 99.7 6.2 4.1 120.1 7.6 4.2 Std Dev 8.25 18.72 2.47 2.82 8.03 4.53 1.53
Table 31: TSS Data for both Trains
TSS, mg/l Date
INF AN 1 AX 1 AER 1 EFF 1 AN 2 AX 2 AER 2 EFF 2
5/25/2004 49 390 2540 1635 8.5 585 2510 2840 15 5/30/2004 77 394 940 902.5 103.3 507.5 1355 575 85 6/1/2004 58 950 2610 2065 4.1 1214 2314 2412 35 6/5/2004 75 1170 2740 2620 14 1060 2220 2360 5 6/8/2004 58 1210 2762 2839 25.2 1630 2995 3002 36 6/22/2004 70 1100 2800 3160 26.6 2060 3040 3380 26 6/26/2004 45 1100 2180 2340 6.5 3480 2360 3350 62 6/29/2004 65 1140 2780 2900 5.5 2100 3220 3280 12 7/6/2004 55 1240 2740 2820 3 1500 3160 3360 1.5 7/13/2004 47.5 1270 2100 2320 10.5 2000 3660 3580 1 7/20/2004 46 1580 2900 2900 2.25 2040 2760 3140 8.5 7/27/2004 55 1420 2760 2920 3.4 2160 3120 3520 9
Avg. 58.4 1080 2488 2452 17.7 1695 2726 2900 24.7 Std Dev 11.1 359.3 546.7 652.5 28.2 810.8 611.9 833.5 26.1
78
Table 32: VSS Data for both Trains
VSS, mg/l Date
INF AN 1 AX 1 AER 1 CL 1 EFF 1 AN 2 AX 2 AER 2 CL 2 EFF 2
5/25/2004 40.2 335 2110 1380 - 7 505 2140 2320 - 12.1
6/1/2004 47.8 815 2190 2065 - 3.4 1032 1640 2120 - 27
6/8/2004 47.2 1040 2720 2839 - 19.9 1402 2520 2560 - 31
6/22/2004 56.7 940 2300 2600 - 26 1760 2460 2720 - 20.5
6/29/2004 53.9 930 2320 2340 4.4 4.3 1760 2640 2600 248.8 9.4
7/6/2004 44 1030 2320 2300 3.2 2.4 1280 2560 2640 3.7 1.2
7/13/2004 38.9 1140 1840 2000 25.5 8.9 1800 3080 3020 6.2 0.9
7/20/2004 39.1 1400 2340 2320 2.8 1.8 2040 2760 3140 63.6 7.0
7/27/2004 45.1 1240 2300 2320 21.8 2.7 1820 2580 2940 38.6 7.7
Avg. 45.9 985.6 2271 2240 11.5 8.0 1489 2487 2673 72.2 13.0
Std Dev 6.3 300.6 232.1 409.7 11.2 7.6 485.4 403.6 328.2 101.8 10.8
79
Table 33: COD Data for both Trains
COD, mg/l Date
SINF TINF SAN 1 SAX 1 SAER 1 SCL 1 TEFF 1 SAN 2 SAX 2 SAER 2 SCL 2 TEFF 2
6/1/2004 207 462 169.13 69.80 93.96 80.54 124.2 126.17 155.70 61.74 75.17 119.9
6/8/2004 233 476 107.19 86.27 96.73 96.73 148.3 117.65 75.82 86.27 107.19 187.2
6/15/2004 214 397 47.06 62.75 88.89 26.14 43.1 57.52 36.60 125.49 26.14 16.8
6/22/2004 192 363 53.33 58.67 53.33 53.33 84.1 53.33 74.67 64.00 53.33 74.0
6/29/2004 176 331 33.12 12.74 48.41 7.64 15.3 33.12 2.55 12.74 7.64 18.7
7/6/2004 205 293 104.00 29.33 56.00 40.00 62.6 45.33 34.67 29.33 40.00 46.0
7/13/2004 185 328 97.44 46.15 30.77 25.64 45.3 56.41 66.67 41.03 164.10 265.6
7/20/2004 154 523 51.28 46.15 51.28 76.92 117.2 61.54 51.28 41.03 41.03 48.2
Avg. 195.8 396.6 82.8 51.5 64.9 50.9 80.0 68.9 62.2 57.7 64.3 97.1
Std Dev 24.48 82.31 45.23 23.22 24.69 31.40 46.40 33.96 45.03 35.56 50.48 88.77
80
Table 34: VFA Data for the Influent
VFA
Date HAc, ppm Propionic Acid,
ppm HAc, mM-C Propionic Acid, mM-C
5/25/2004 19.4 38.5 0.65 1.56
6/1/2004 37.5 42.5 1.25 1.72
6/8/2004 23.9 30.5 0.80 1.23
6/22/2004 41.0 24.2 1.37 0.98
6/29/2004 29.6 20.2 0.99 0.82
7/6/2004 33.7 17.9 1.12 0.72
7/13/2004 67.2 26.1 2.24 1.06
Avg. 36.0 28.6 1.2 1.2
Std Dev 15.65 9.18 0.52 0.37
81
Table 35: PHA Data for both Trains
PHB, PHV, and PHA, mM-C
Date
PHB, AN 1
PHV, AN 1
PHA, AN 1
PHB, AX 1
PHV, AX 1
PHA, AX 1
PHB, AN 2
PHV, AN 2
PHA, AN 2
PHB, AX 2
PHV, AX 2
PHA, AX 2
6/22/2004 3.41 2.02 5.43 3.32 1.94 5.26 3.52 3.40 6.92 3.55 3.45 7.00
6/29/2004 3.10 2.25 5.35 2.95 2.17 5.12 3.78 3.50 7.28 3.83 3.55 7.38
7/6/2004 2.89 1.79 4.68 2.79 1.71 4.50 3.52 3.26 6.78 3.59 3.31 6.90
7/13/2004 3.82 3.08 6.90 3.68 3.00 6.68 3.68 3.24 6.92 3.70 3.29 6.99
7/20/2004 3.58 2.06 5.64 3.46 1.98 5.44 3.93 3.65 7.58 4.03 3.70 7.73
Avg. 3.36 2.24 5.60 3.24 2.16 5.40 3.69 3.41 7.10 3.74 3.46 7.20
Std Dev 0.37 0.50 0.81 0.37 0.50 0.80 0.18 0.17 0.33 0.20 0.17 0.35
Table 36: Glycogen Data for both Trains
Glycogen, mM-C Date
AN 1 AX 1 AER 1 AN 2 AX 2 AER 2
6/26/2004 10.82 10.73 13.39 13.60 13.72 16.7
6/29/2004 10.63 10.50 13.23 13.83 13.88 15.89
7/13/2004 10.41 10.29 13.11 12.65 12.67 15.3
7/20/2004 10.12 10.62 12.82 12.84 12.8 15.52
7/27/2004 10.93 10.48 13.43 13.16 13.26 16.06
Avg. 10.6 10.5 13.2 13.2 13.3 15.9
Std Dev 0.33 0.17 0.25 0.50 0.54 0.54
82
Table 37: pH Data for both Trains
pH Date
AN 1 AX 1 AER 1 CL 1 AN 2 AX 2 AER 2 CL 2
6/15/2004 6.7 7 6/16/2004 6.3 7.1 6/17/2004 6.5 6.9 7 7 7.5 7.7 6/18/2004 6.9 7.2 7.3 7.3 7.7 7.8 6/22/2004 7.4 7.7 8 7.7 7.3 7.5 7.7 7.6 6/25/2004 7.2 7.8 8 8 7.1 7.5 7.7 7.5
7/1/2004 6.4 6.8 7.1 7.1 7.2 7.3 7.4 7.4 7/5/2004 6.2 6.7 7.1 7.1 7.1 7.5 7.5 7.5 7/6/2004 6.2 6.5 6.7 7.2 7.3 7.6 7/7/2004 6.3 7.2
7/12/2004 6.1 6.5 6.9 7.2 7.1 7.4 7.4 7.4 7/13/2004 6.5 6.8 7.2 7.2 7.4 7.4 7/15/2004 6.5 6.8 7.2 7.2 7.4 7.4 7/17/2004 6.6 6.8 7.2 7.3 7.4 7.4 7/19/2004 6.5 6.8 7.2 7.2 7.4 7.4 7/21/2004 6.4 6.8 7.2 7.3 7.4 7.4 7/22/2004 6.5 6.8 7.2 7.2 7.4 7.4 7/24/2004 6.3 6.8 7.2 7.1 7.4 7.4 7/26/2004 6.3 6.8 7.2 7.2 7.4 7.4 7/27/2004 6.3 6.8 7.2 7.2 7.4 7.4 7/28/2004 6.4 6.8 7.2 7.2 7.4 7.4 7/29/2004 6.3 6.8 7.2 7.3 7.4 7.4 7/31/2004 6.4 6.8 7.2 7.2 7.4 7.4
Avg 6.5 6.9 7.2 7.4 7.2 7.4 7.5 7.5 Std Dev 0.31 0.33 0.30 0.41 0.09 0.09 0.14 0.08
83
Table 38: Influent Flow Rate Data for both Trains
Flow Rate, L/d Date
INF 1 INF 2
6/8/2004 28.3 29.5 6/9/2004 29.8 34.8
6/15/2004 27.3 30.2 6/16/2004 27 30 6/17/2004 27.4 29.7 6/18/2004 26 30.6 6/19/2004 25.5 29.2 6/24/2004 27.5 28.9 6/25/2004 26.8 29.1 6/26/2004 26.5 29.5 6/29/2004 32.6 30.1 7/1/2004 29.3 31 7/2/2004 29.7 33 7/3/2004 29.4 30.4 7/5/2004 28.3 29.9 7/6/2004 29.2 27.8 7/7/2004 27.1 28.3 7/8/2004 28.3 29.4 7/9/2004 25.2 27.5
7/12/2004 26.2 28.7 7/13/2004 24.4 26 7/15/2004 28.2 29.7 7/16/2004 26.9 26.9 7/17/2004 28.2 28.2 7/19/2004 28 30.1 7/20/2004 25.5 27.5 7/21/2004 27.4 29.4 7/22/2004 29.5 30.5 7/24/2004 27.7 29.6 7/26/2004 25.1 27.1 7/27/2004 26.9 29.5 7/28/2004 26.6 27.7
Avg 27.6 29.4 Std Dev 1.69 1.70
84
Table 39: Internal Recycle Flow Rates of both Trains
Train 1 Flow Rate, L/d Train 2 Flow Rate, L/d Date
ARCY NARCY RAS INF ARCY NARCY RAS INF
6/1/2004 41.1 125.3 52.1 22.9 40.2 126.3 50.1 24
6/8/2004 39.5 124.1 49.3 28.3 40.3 119.2 49 29.5
6/15/2004 44.8 127.2 61.2 27.3 44.8 125.3 59.6 30.2
6/29/2004 40.3 120.5 56.7 32.6 38.6 118.4 57.1 30.1
7/6/2004 43.4 125.8 60.4 29.2 42.4 126.1 61.1 27.8
7/13/2004 39.2 121 61.8 24.4 37.7 118.1 61.2 26
7/20/2004 42.5 123.7 59.1 25.5 40.3 121.2 59.2 27.5
7/27/2004 40.2 132.8 52.9 26.9 36.2 124.6 54.2 29.5
Avg 41.4 125.1 56.7 27.1 40.1 122.4 56.4 28.1
Std Dev 2.00 3.88 4.72 3.02 2.69 3.55 4.83 2.20
Table 40: Observed Yield in Train 1
Date Inf Flow Rate l/d
Inf TCOD mg/l
Clarifier SCODmg/l
Delta CODmg/d
WAS+Eff VSS g/d
Yobs mg VSS/mg COD
6/15/2004 27.3 397 26.1 10124 3.27 0.32
6/29/2004 32.6 331 7.6 10541 2.48 0.23
7/6/2004 29.2 293 40.0 7387 2.37 0.32
7/13/2004 24.4 328 25.6 7377 2.21 0.30
7/27/2004 26.9 523 76.9 11999 2.39 0.20
Avg. 28.08 374.40 35.24 9486 2.54 0.27
Std Dev 3.05 91.18 25.97 2043 0.42 0.06
85
Table 41: Observed Yield in Train 2
Date Inf Flow Rate l/d
Inf TCOD mg/l
Clarifier SCODmg/l
Delta CODmg/d
WAS+Eff VSS g/d
Yobs mg VSS/mg COD
6/15/2004 30.2 397 26.14 11199 3.39 0.30
6/29/2004 30.1 331 7.64 9733 2.87 0.30
7/6/2004 27.8 293 40 7033 2.67 0.38
7/13/2004 26.0 328 164.1 4261 3.04 0.71
7/27/2004 29.5 523 41.03 14218 3.16 0.22
Avg. 28.72 374.40 55.78 9289 3.03 0.38
Std Dev 1.80 91.18 62.04 3825 0.27 0.19
Table 42: Individual PHA/VFA data (YPHA) for both Trains
Date PHA AN 1 mM-C
PHA AN 2 mM-C
VFA mM-C PHA/VFA 1 PHA/VFA 2
6/22/2004 5.43 6.92 3.35 1.62 2.07
6/29/2004 5.35 7.28 2.81 1.91 2.59
7/6/2004 4.68 6.78 2.85 1.64 2.38
7/13/2004 6.90 6.92 4.30 1.61 1.61
Avg. 5.59 6.97 3.32 1.69 2.16
Std Dev 0.94 0.21 0.69 0.14 0.43
86
Table 43: Individual PHA/(VFA+Gly) Data (YPHA*) for both Trains
Date PHA AN 1 mM-C
PHA AN 2 mM-C
VFA mM-C
Glycogen Formation 1
mM-C
Glycogen Formation 2
mM-C YPHA* 1 YPHA* 2
6/29/2004 5.35 7.28 2.81 2.6 2.06 0.99 1.50
7/13/2004 6.90 6.92 4.30 2.7 2.65 0.99 1.00
Avg. 6.13 7.10 3.55 2.65 2.36 0.99 1.25
Std Dev 1.09 0.25 1.05 0.07 0.42 0 0.35
Table 44: Individual Prel/VFA Ratios for both Trains
Date VFA, mM-C
INF 1, L/d
P release 1,mg-P/d
INF 2, L/d
P release 2,mg-P/d Prel/VFA 1 Prel/VFA 2
6/1/2004 3.97 22.9 96.85 24 133.53 0.034 0.045
6/8/2004 3.03 28.3 395.38 29.5 373.14 0.149 0.135
6/15/2004 3.35 27.3 476.99 30.2 614.65 0.168 0.196
6/29/2004 2.80 32.6 399.18 30.1 625.03 0.141 0.239
7/6/2004 2.85 29.2 416.22 27.8 587.55 0.161 0.239
7/13/2004 4.30 24.4 390.06 26 661.84 0.120 0.191
AVG 3.38 27.45 362.45 27.93 499.29 0.129 0.174
Std Dev 0.62 3.47 133.98 2.51 206.41 0.05 0.07
87
Table 45: Individual P /PHA Data for both Trains up
PHA AN 1, mM-C
PHA AN 2, mM-C
INF 1,L/d L/d
P 1, up P /PHA 1 up P /PHA 2
7.28 32.6 30.1
7/6/2004 4.68 755.44 0.131 0.129
INF 2, P 2, upDate upmg-P/d mg-P/d
6/29/2004 5.35 461.52 707.52 0.085 0.104
6.78 29.2 27.8 556.46
7/13/2004 6.90 6.92 24.4 26 515.28 800.70 0.099 0.144
AVG 5.64 6.99 28.73 27.97 511.09 754.55 0.105 0.126
Std Dev 1.14 0.26 4.12 2.06 47.60 46.59 0.024 0.020
Table 46: Individual Pup/Prel Ratio for both Trains
Date Pup 1, mg-P/d
Prel 1, mg-P/d
Pup 2, mg-P/d
Prel 2, mg-P/d up rel Pup/Prel 2
6/1/2004 667.98 96.85 425.61 268.76 6.90 1.58
6/8/2004 646.51 395.38 635.75 408.79 1.64 1.56
6/15/2004 696.96 476.99 914.84 730.09 1.46 1.25
6/29/2004 461.52 399.18 707.52 664.68 1.16 1.06
7/6/2004 556.46 416.22 755.44 701.82 1.34 1.08
7/13/2004 515.28 390.06 800.70 749.77 1.32 1.07
7/27/2004 763.38 679.38 712.48 629.70 1.12 1.13
Avg. 615.44 407.72 707.48 593.37 2.13 1.25
Std Dev 107.59 171.20 151.99 182.92 2.11 0.23
P /P 1
88
Table 47: Individual Phosphorus Mass Balance Data for Train 1, mg/d
Train 1
Date AN Release
AX Release
Total Bio. Release
Total Sys. Release
AE Uptake
Clarifier Uptake
Total Bio.Uptake
Total Sys. Uptake
Net Sys.Removal
Net Bio Removal
6/1/04 96.85 -224.84 96.85 647.41 443.15 -550.56 667.98 667.98 20.58 571.14
6/8/04 395.38 -143.41 395.38 423.72 503.10 -28.34 646.51 646.51 222.79 251.13
6/15/04 476.99 -179.84 476.99 533.86 517.12 -56.88 696.96 696.96 163.10 219.97
6/29/04 333.70 65.49 399.18 399.18 461.52 32.67 461.52 494.20 95.01 62.34
7/6/04 337.19 79.03 416.22 416.22 556.46 49.62 556.46 606.07 189.85 140.24
7/13/04 314.10 75.96 390.06 476.96 515.28 -86.90 515.28 515.28 38.32 125.22
7/27/04 394.65 284.73 679.38 679.38 763.38 36.25 763.38 799.62 120.25 84.00
Avg 335.55 -6.13 407.72 510.96 537.14 -86.31 615.44 632.37 121.41 207.72
Std Dev 118.69 182.84 171.20 113.83 106.57 211.11 107.59 105.76 75.79 174.12
89
Table 48: Individual Phosphorus Mass Balance Data for Train 2, mg/d
Train 2
Date AN Release
AX Release
Total Bio. Release
Total Sys. Release
AE Uptake
Clarifier Uptake
Total Bio.Uptake
Total Sys. Uptake
Net Sys. Removal
Net Bio Removal
6/1/04 82.24 51.29 133.53 268.76 425.61 -135.24 425.61 425.61 156.85 292.09
6/8/04 373.14 -69.59 373.14 408.79 566.16 -35.65 635.75 635.75 226.96 262.61
6/15/04 614.65 -179.02 614.65 730.09 735.81 -115.44 914.84 914.84 184.75 300.19
6/29/04 625.03 -142.94 625.03 664.68 564.59 -39.65 707.52 707.52 42.84 82.50
7/6/04 587.55 -22.10 587.55 701.82 733.34 -114.27 755.44 755.44 53.62 167.89
7/13/04 661.84 -80.39 661.84 749.77 720.31 -87.92 800.70 800.70 50.93 138.85
7/27/04 622.26 -21.98 622.26 629.70 690.50 -7.44 712.48 712.48 82.78 90.22
Avg 509.53 -66.39 516.86 593.37 633.76 -76.52 707.48 707.48 114.11 190.62
Std Dev 211.17 78.03 194.07 182.92 118.13 48.85 151.99 151.99 74.46 93.50
90
Table 49: Phosphorus Mass Balance Results Normalized with Total System TSS for Train 1,
(mg/g*d)1
Date AN Release
AX Release
Total Bio. Release
Total Sys.Release
AE Uptake
ClarifierUptake
Total Bio.Uptake
Total Sys. Uptake
Net Sys.Removal
Net Bio.Removal
6/15/04 17.29 -6.52 17.29 19.35 18.74 -2.06 25.26 25.26 5.91 7.97
6/29/04 12.09 2.37 14.47 10.91 16.72 1.18 16.72 17.91 7.00 2.26
7/6/04 12.22 2.86 15.08 10.42 20.16 1.80 20.16 21.96 11.54 5.08
7/13/04 11.38 2.75 14.14 14.53 18.67 -3.15 18.67 18.67 4.14 4.54
7/27/04 14.30 10.32 24.62 12.99 27.66 1.31 27.66 28.98 15.99 3.04
Avg. 13.46 2.36 17.12 13.64 20.39 -0.18 21.70 22.56 8.92 4.58
Std Dev. 2.40 5.97 4.37 3.59 4.24 2.26 4.60 4.62 4.81 2.21 1 Exact unit is (mg-P/d)/(g-TSS)
Table 50: Phosphorus Mass Balance Results Normalized with Total System TSS for Train 2,
(mg/g*d)
Date AN Release
AX Release
Total Bio. Release
Total Sys.Release
AE Uptake
ClarifierUptake
Total Bio.Uptake
Total Sys. Uptake
Net Sys.Removal
Net Bio.Removal
6/15/04 17.98 -5.24 17.98 16.12 21.52 -3.38 26.76 26.76 10.64 8.78
6/29/04 18.28 -4.18 18.28 15.26 16.51 -1.16 20.69 20.69 5.43 2.41
7/6/04 17.18 -0.65 17.18 19.88 21.45 -3.34 22.09 22.09 2.21 4.91
7/13/04 19.36 -2.35 19.36 19.58 21.07 -2.57 23.42 23.42 3.84 4.06
7/27/04 18.20 -0.64 18.20 17.77 20.19 -0.22 20.84 20.84 3.06 2.64
Avg. 18.20 -2.61 18.20 17.72 20.15 -2.13 22.76 22.76 5.04 4.56
Std Dev. 0.78 2.07 0.78 2.05 2.10 1.40 2.49 2.49 3.35 2.57
91
Table 51: Phosphorus Mass Balance Results Normalized with Total System VSS for Train 1,
(mg/g*d)
92
RemovalDate AN Release
AX Release
Total Bio. Release
Total Sys.Release
AE Uptake
ClarifierUptake
Total Bio.Uptake
Total Sys. Uptake
Net Sys.Removal
Net Bio.
6/15/04 20.46 -7.71 20.46 22.90 22.18 -2.44 29.89 29.89 7.00 9.43
6/29/04 14.31 2.81 17.12 12.91 19.79 1.40 19.79 21.20 8.28 2.67
7/6/04 14.46 3.39 17.85 12.33 23.87 2.13 23.87 25.99 13.66 6.01
7/13/04 13.47 3.26 16.73 17.20 22.10 -3.73 22.10 22.10 4.90 5.37
7/27/04 16.93 12.21 29.14 15.37 32.74 1.55 32.74 34.30 18.92 3.60
Avg. 15.93 2.79 20.26 16.14 24.14 -0.22 25.68 26.70 10.55 5.42
Std Dev. 2.84 7.07 5.17 4.25 5.02 2.67 5.44 5.47 5.69 2.61
Table 52: Phosphorus Mass Balance Results Normalized with Total System VSS for Train 2,
(mg/g*d)
Date AN Release
AX Release
Total Bio. Release
Total Sys.Release
AE Uptake
ClarifierUptake
Total Bio.Uptake
Total Sys. Uptake
Net Sys.Removal
Net Bio.Removal
6/15/04 21.35 -6.22 21.35 19.14 25.56 -4.01 31.78 31.78 12.64 10.43
6/29/04 21.71 -4.97 21.71 18.12 19.61 -1.38 24.58 24.58 6.45 2.87
7/6/04 20.41 -0.77 20.41 23.61 25.47 -3.97 26.24 26.24 2.63 5.83
7/13/04 22.99 -2.79 22.99 23.25 25.02 -3.05 27.81 27.81 4.56 4.82
7/27/04 21.62 -0.76 21.62 21.11 23.99 -0.26 24.75 24.75 3.64 3.13
Avg. 21.62 -3.10 21.62 21.05 23.93 -2.53 27.03 27.03 5.98 5.42
0.93 2.46 0.93 2.43 2.49 1.66 2.96 2.96 3.98 3.06 Std Dev.
Table 53: PHB, PHV, and PHA Normalized with TSS in both Trains
PHB, PHV, and PHA Normalized with TSS, (mmol/g)
Date
PHB, AN 1
PHV, AN 1
PHA, AN 1
PHB, AX 1
PHV, AX 1
PHA, AX 1
PHB, AN 2
PHV, AN 2
PHA, AN 2
PHB, AX 2
PHV, AX 2
PHA, AX 2
6/22/2004 1.37 0.81 2.18 1.33 0.78 2.11 1.14 1.10 2.24 1.15 1.12 2.27
6/29/2004 1.25 0.90 2.15 1.19 0.87 2.06 1.23 1.13 2.36 1.24 1.15 2.39
7/6/2004 1.16 0.72 1.88 1.12 0.69 1.81 1.14 1.06 2.20 1.16 1.07 2.24
7/13/2004 1.53 1.24 2.77 1.48 1.21 2.68 1.19 1.05 2.24 1.20 1.07 2.27
7/20/2004 1.44 0.83 2.27 1.39 0.80 2.19 1.27 1.18 2.46 1.31 1.20 2.51
Avg. 1.35 0.90 2.25 1.30 0.87 2.17 1.20 1.11 2.30 1.21 1.12 2.33
Std Dev 0.15 0.20 0.33 0.15 0.20 0.32 0.06 0.06 0.11 0.06 0.06 0.11
Table 54: PHB, PHV, and PHA Normalized with VSS in both Trains
PHB, PHV, and PHA Normalized with VSS, (mmol/g) Date
PHB, AN 1
PHV, AN 1
PHA, AN 1
PHB, AX 1
PHV, AX 1
PHA, AX 1
PHB, AN 2
PHV, AN 2
PHA, AN 2
PHB, AX 2
PHV, AX 2
PHA, AX 2
6/22/2004 1.62 0.96 2.58 1.58 0.92 2.50 1.36 1.31 2.66 1.37 1.33 2.70
6/29/2004 1.47 1.07 2.54 1.40 1.03 2.43 1.46 1.35 2.80 1.47 1.37 2.84
7/6/2004 1.37 0.85 2.23 1.33 0.81 2.14 1.36 1.26 2.61 1.38 1.27 2.66
7/13/2004 1.82 1.46 3.28 1.75 1.43 3.18 1.42 1.25 2.66 1.42 1.27 2.69
7/20/2004 1.70 0.98 2.68 1.65 0.94 2.59 1.51 1.41 2.92 1.55 1.42 2.98
Avg. 1.60 1.07 2.66 1.54 1.03 2.57 1.42 1.31 2.73 1.44 1.33 2.77
Std Dev 0.18 0.24 0.39 0.17 0.24 0.38 0.07 0.07 0.13 0.08 0.07 0.13
93
Table 55: Glycogen Formation/Consumption Normalized with TSS in both Trains
Gly Formation/TSS, mmol/g Date
Train 1 Train 2
6/26/2004 1.032 1.005
6/29/2004 1.045 0.668
7/13/2004 1.085 0.859
7/20/2004 1.085 0.869
7/27/2004 1.004 0.940
Avg. 1.050 0.868
Std Dev 0.035 0.127
Table 56: Glycogen Formation/Consumption Normalized with VSS in both Trains
Gly Formation/VSS, mmol/g Date
Train 1 Train 2
6/26/2004 1.222 1.194
6/29/2004 1.236 0.793
7/13/2004 1.284 1.021
7/20/2004 1.284 1.032
7/27/2004 1.189 1.117
Avg. 1.243 1.031
Std Dev 0.041 0.150
94
Table 57: Nitrate, ORP, and DO Data
NO3, mg-N/l ORP, mV DO, mg/l Date
INF AER 1
AX 1
AN 1
CL 1 AER 1 AX 1 AN 1 AER 1 AN 1
5/25/2004 2.8 0.78 6/8/2004 6.4 2.6 -40 -130 -200 6/16/2004 50 -35 -180 6.5 0.21 6/22/2004 3 1.5 2.1 4.2 1.6 7/1/2004 2.7 0.2 7/13/2004 7/15/2004 10 -60 -160 2.5 0.2
INF AER 2
AX 2
AN 2
CL 2 AER 1 AX 1 AN 1 AER 1 AN 1
5/25/2004 5.1 1.7 6/8/2004 6.3 2.3 -10 -100 -180 6/16/2004 15 -45 -168 5.5 0.2 6/22/2004 3 1.7 2.7 6.3 2.6 7/1/2004 2.9 0.2 7/13/2004 0.31 5.7 2.9 0.71 2.4 7/15/2004 18 -10 -195 2.1 0.2
Table 58: Paired t Test Results for Anaerobic P Releases
Date AN 1 Release
AN 2 Release
Calculated t df1 CL1
6/15/2004 476.99 614.65
6/29/2004 333.70 625.03
7/6/2004 337.19 587.55
7/13/2004 314.10 661.84
7/27/2004 394.65 622.26
AVG 335.55 509.53
-7.187 4 99.8%
1 df = Degree of Freedom, CL = Confidence Level
95
Table 59: Paired t Test Results for Anoxic P Releases
Date AX 1 Release
AX 2 Release
Calculated t df CL
6/15/2004 -179.84 -179.02
6/29/2004 65.49 -142.94
7/6/2004 79.03 -22.10
7/13/2004 75.96 -80.39
7/27/2004 284.73 -21.98
AVG -6.13 -66.39
2.997 4 96%
Table 60: Paired t Test Results for Total Biological P Releases
Date Total Bio. 1 Release
Total Bio. 2 Release
Calculated t df CL
6/15/2004 476.99 614.65
6/29/2004 399.18 625.03
7/6/2004 416.22 587.55
7/13/2004 390.06 661.84
7/27/2004 679.38 622.26
AVG 407.72 516.86
-2.648 4 94%
96
Table 61: Paired t Test Results for Total System P Releases
Date Total Sys. 1 Release
Total Sys. 2Release
Calculated t df CL
6/15/2004 533.86 730.09
6/29/2004 399.18 664.68
7/6/2004 416.22 701.82
7/13/2004 476.96 749.77
7/27/2004 679.38 629.70
AVG 510.96 593.37
-3.086 4 96%
Table 62: Paired t Test Results for Aerobic P Uptakes
Date AE 1 Uptake
AE 2 Uptake
Calculated t df CL
6/15/2004 517.12 735.81
6/29/2004 461.52 564.59
7/6/2004 556.46 733.34
7/13/2004 515.28 720.31
7/27/2004 763.38 690.50
AVG 537.14 633.76
-2.353 4 92%
97
98
Table 63: Paired t Test Results for Clarifier P Uptake
Date Clarifier 1 Uptake
Clarifier 2 Uptake
Calculated t df CL
6/15/2004 -56.88 -115.44
6/29/2004 32.67 -39.65
7/6/2004 49.62 -114.27
7/13/2004 -86.90 -87.92
7/27/2004 36.25 -7.44
AVG -86.31 -76.52
2.532 4 94%
Table 64: Paired t Test Results for Total Biological P Uptakes
Date Total Bio. 1 Uptake
Total Bio. 2 Uptake
Calculated t df CL
6/15/2004 696.96 914.84
6/29/2004 461.52 707.52
7/6/2004 556.46 755.44
7/13/2004 515.28 800.70
7/27/2004 763.38 712.48
AVG 615.44 707.48
-3.021 4 96%
Table 65: Paired t Test Results for Total System P Uptake
Date Total Sys. 1 Uptake
Total Sys 2 Uptake
Calculated t df CL
6/15/2004 696.96 914.84
6/29/2004 494.20 707.52
7/6/2004 606.07 755.44
7/13/2004 515.28 800.70
7/27/2004 799.62 712.48
AVG 632.37 707.48
-2.418 4 93%
Table 66: Paired t Test Results for Net System P Removals
Date Net Sys. 1 Removal
Net Sys. 2 Removal
Calculated t df CL
6/15/2004 163.10 184.75
6/29/2004 95.01 42.84
7/6/2004 189.85 53.62
7/13/2004 38.32 50.93
7/27/2004 120.25 82.78
AVG 121.41 114.11
1.356 4 75%
99
Table 67: Paired t Test Results for Net Biological P Removals
Date Net Bio. 1 Removal
Net Bio. 2 Removal
Calculated t df CL
6/15/2004 219.97 300.19
6/29/2004 62.34 82.50
7/6/2004 140.24 167.89
7/13/2004 125.22 138.85
7/27/2004 84.00 90.22
AVG 126.35 155.93
-2.249 4 91%
Table 68: Summary of Statistical Analysis (Paired t Test) for Phosphorus Mass Balance
Zone AN Release
AX Release
Tot. Bio. Release
Tot. Sys. Release
AERUptake
Clar. Uptake
Tot. Bio. Uptake
Tot. Sys. Uptake
Net Sys.Removal
Net Bio. Removal
Calc. t -7.187 2.997 -2.648 -3.086 -2.353 2.532 -3.021 -2.418 1.356 -2.249
Conf. Level 99.8 96 94 96 92 94 96 93 75 91
Comparison Based on Avg 1<2 1>21 1<2 1<2 1<2 1>2 1<2 1<2 1>2 1<2
1 P release in AX 1 and P uptake in AX 2
100
Table 69: Paired t Test Results for Normalized Anaerobic P Releases with VSS
101
Date AN 1 Release
AN 2 Release
Calculated t df CL
6/15/2004 20.46 21.35
6/29/2004 14.31 21.71
7/6/2004 14.46 20.41
7/13/2004 13.47 22.99
7/27/2004 16.93 21.62
AVG 15.93 21.62
-3.942 4 98%
Table 70: Paired t Test Results for Normalized Anoxic P Releases with VSS
Date AX 1 Release
AX 2 Release
Calculated t df CL
6/15/2004 -7.71 -6.22
6/29/2004 2.81 -4.97
7/6/2004 3.39 -0.77
7/13/2004 3.26 -2.79
7/27/2004 12.21 -0.76
AVG 2.79 -3.10
2.497 4 93%
102
Table 71: Paired t Test Results for Normalized Total Biological P Releases with VSS
Date Total Bio. 1 Release
Total Bio. 2 Release
Calculated t df CL
6/15/2004 20.46 21.35
6/29/2004 17.12 21.71
7/6/2004 17.85 20.41
7/13/2004 16.73 22.99
7/27/2004 29.14 21.62
AVG 20.26 21.62
-0.565 4 40%
Table 72: Paired t Test Results for Normalized Total System P Releases with VSS
Date Total Sys. 1 Release
Total Sys. 2Release
Calculated t df CL
6/15/2004 22.90 19.14
6/29/2004 12.91 18.12
7/6/2004 12.33 23.61
7/13/2004 17.20 23.25
7/27/2004 15.37 21.11
AVG 16.14 21.05
-2.022 4 89%
Table 73: Paired t Test Results for Normalized Aerobic P Uptakes with VSS
Date AE 1 Uptake
AE 2 Uptake
Calculated t df CL
6/15/2004 22.18 25.56
6/29/2004 19.79 19.61
7/6/2004 23.87 25.47
7/13/2004 22.10 25.02
7/27/2004 32.74 23.99
AVG 24.14 23.93
0.092 4 7%
Table 74: Paired t Test Results for Normalized Clarifier P Uptake with VSS
Date Clarifier 1 Uptake
Clarifier 2 Uptake
Calculated t df CL
6/15/2004 -2.44 -4.01
6/29/2004 1.40 -1.38
7/6/2004 2.13 -3.97
7/13/2004 -3.73 -3.05
7/27/2004 1.55 -0.26
AVG -0.22 -2.53
2.104 4 90%
103
Table 75: Paired t Test Results for Normalized Total Biological P Uptakes with VSS
104
Uptake Date Total Bio. 1 Total Bio. 2 Uptake
Calculated t df CL
6/15/2004 29.89 31.78
6/29/2004 19.79 24.58
7/6/2004 23.87 26.24
7/13/2004 22.10 27.81
7/27/2004 32.74 24.75
AVG 25.68 27.03
-0.554 4 39%
Table 76: Paired t Test Results for Normalized Total System P Uptake with VSS
Date Total Sys. 1 Uptake
Total Sys 2 Uptake
Calculated t df CL
31.78
6/29/2004 21.20 24.58
7/6/2004 25.99 26.24
7/13/2004 22.10 27.81
7/27/2004 34.30 24.75
AVG 26.7 27.03
-0.128 4 10%
6/15/2004 29.89
Table 77: Paired t Test Results for Normalized Net System P Removals with VSS
105
Removal Date Net Sys.
1
Net Sys. Removal
2 Calculated
t df CL
6/15/2004 7.00 12.64
6/29/2004 8.28 6.45
7/6/2004 13.66 2.63
7/13/2004 4.90 4.56
7/27/2004 18.92 3.64
AVG 10.55 5.98
1.208 4 71%
Table 78: Paired t Test Results for Normalized Net Biological P Removals with VSS
Date Net Bio. Removal
1
Net Bio. Removal
2 Calculated
t df CL
6/15/2004 9.43 10.43
6/29/2004 2.67 2.87
7/6/2004 6.01 5.83
7/13/2004 5.37 4.82
7/27/2004 3.60 3.13
AVG 5.42 5.42
0.0089 4 1%
Table 79: Summary of Statistical Analysis (Paired t Test) for Normalized Phosphorus Mass
Balance with VSS
Zone AN Release
AX Release
Tot. Bio. Release
Tot. Sys. Release
AER Uptake
Clar. Uptake
Tot. Bio. Uptake
Tot. Sys. Uptake
Net Sys. Removal
Net Bio. Removal
Calc. t -3.942 2.497 -0.565 -2.022 -0.092 2.104 -0.554 -0.128 1.208 -0.009
Conf. Level 98 93 40 7 90 39 10 71 1
Comparison Based on Avg 1<2 1>21 1<2 1<2 1>2 1>2 1<2 1<2 1>2 1=2
89
1 P release in AX 1 and P uptake in AX 2
Table 8 : Paired t Test Results for Anaerobic PHA 0
Date AN PHA 1 AN PHA 2 Calculated t df CL
6/22/2004 5.43 6.92
6/29/2004 5.35 7.28
7/6/2004 4.68 6.78
7/13/2004 6.9 6.92
7/20/2004 5.64 7.58
AVG 5.6 7.1
2.776 4 98%
106
Table 81: Paired t Test Results for Normalized Anaerobic PHA with VSS
Date AN PHA 1 AN PHA 2 Calculated t df CL
6/22/2004 2.58 2.66
6/29/2004 2.54 2.80
7/6/2004 2.23 2.61
7/13/2004 3.28 2.66
7/20/2004 2.68 2.92
AVG 2.66 2.73
-0.393 4 29%
Table 82: Paired t Test Results for Normalized Anoxic PHA with VSS
Date AX PHA 1 AX PHA 2 Calculated t df CL
6/22/2004 2.50 2.70
6/29/2004 2.43 2.84
7/6/2004 2.14 2.66
7/13/2004 3.18 2.69
7/20/2004 2.59 2.98
AVG 2.57 2.77
-1.139 4 68%
107
Table 83: Paired t Test Results for Normalized Glycogen Formation with VSS
Date Glycogen 1 Glycogen 2 Calculated t df CL
6/26/2004 1.222 1.194
6/29/2004 1.236 0.793
7/13/2004 1.284 1.021
7/20/2004 1.284 1.032
7/27/2004 1.189 1.117
AVG 1.243 1.031
2.840 4 95%
Table 84: Paired t Test Results for Prel/VFA
Date Prel/VFA 1 Prel/VFA 2 Calculated t df CL
6/15/2004 0.168 0.196
6/29/2004 0.141 0.239
7/6/2004 0.161 0.239
7/13/2004 0.120 0.191
AVG 0.129 0.174
3.182 3 98%
108
Table 85: Paired t Test Results for Pup/PHA
Date Pup/PHA 1 Pup/PHA 2 Calculated t df CL
6/29/2004 0.085 0.104
7/6/2004 0.131 0.129
7/13/2004 0.099 0.144
AVG 0.105 0.126
4.302 2 73%
Table 86: Paired t Test Results for YPHA
Date YPHA 1 YPHA 2 Calculated t df CL
6/15/2004 1.62 2.07
6/29/2004 1.91 2.59
7/6/2004 1.64 2.38
7/13/2004 1.61 1.61
AVG 1.68 2.10
3.182 3 96%
Table 87: Paired t Test Results for YPHA*
Date YPHA* 1 YPHA* 2 Calculated t df CL
6/29/2004 0.99 1.50
7/6/2004 0.99 1.00
AVG 0.99 1.25
12.706 1 51%
109
Table 88: Paired t Test Results for Pup/Prel
110
PDate up/Prel 1 Pup/Prel 2 Calculated t df CL
6/15/2004 1.46 1.49
6/29/2004 1.16 1.13
1.29
7/13/2004 1.32 1.21
7/27/2004 1.12 1.14
AVG 1.28 1.25
2.776 4 66% 7/6/2004 1.34
APPENDIX C EXAMPLE CALCULATIONS
111
SOP (7/6/04)
Sample Absorbance @ 420 nm SOP, mg/l-P
INF 0.099 9.20 AN 1 0.108 10.04 AX 1 0.043 4.02 AER 1 0.015 1.43 CL 1 0.009 0.87 AN 2 0.171 15.87 AX 2 0.081 7.54 AER 2 0.044 4.11 CL 2 0.058 5.41
Standards,
mg/l-P Absorbance @ 420 nm
0 0 0.5 0.005 1 0.011 2 0.020 5 0.054 10 0.108 25 0.268 50 0.532
SOP Standard Curve
y = 0.0107x + 0.0002R2 = 1
0
0.1
0.2
0.3
0.4
0.5
0.6
0 10 20 30 40 50 60Concentration, mg/l-P
Abso
rban
ce@
420
nm
Figure 10: Sample Calculation for SOP
112
TP Test (7/20/2004)
Sample Sample volume, ml
Dilution Factor
Absorbance @ 420 nm TP, mg/l
INF 10 3 0.060 30.61 AER 1 2 13 0.033 84.69 CL 1 25 1 0.030 5.89 EFF 1 25 1 0.031 6.10 AER 2 2 13 0.050 128.47 CL 2 25 1 0.081 16.40 EFF 2 25 1 0.034 6.71
Std,
mg/l-P Absorbance @ 420 nm
0 0 2 0.012 5 0.025 10 0.05 25 0.125 50 0.243
TP Std Curve
y = 0.0049x + 0.0014R2 = 0.9998
0
0.05
0.1
0.15
0.2
0.25
0.3
0 10 20 30 40 50 6
Concentration, mg/l-P
Abso
rban
ce @
420
nm
0
Figure 11: Sample Calculation for TP
113
Table 89: Sample Calculation for TSS/VSS
TSS/VSS Test (6/22/2004)
Sample Initial mass, g
Final mass
(105 C), g
Sample mass,
mg
Final mass
(550 C), g
Volume, ml
TSS, mg/l
VSS, mg/l VSS/TSS
INF 18.7562 18.7583 2.1 18.7565 30 70 60 0.857 AN 1 19.4358 19.4468 11.0 19.4374 10 1100 940 0.855 AX 1 19.7847 19.7987 14.0 19.7872 5 2800 2300 0.821
AER 1 18.0026 18.0184 15.8 18.0054 5 3160 2600 0.823 EFF 1 18.8864 18.8892 2.8 18.8865 105.2 27 26 0.964 AN 2 19.6930 19.7136 20.6 19.6960 10 2060 1760 0.854 AX 2 19.8416 19.8568 15.2 19.8445 5 3040 2460 0.809
AER 2 18.2299 18.2468 16.9 18.2332 5 3380 2720 0.805 EFF 2 18.6199 18.6225 2.6 18.6205 100 26 20.5 0.788
Table 90: Sample Calculation for COD
114
CODSCL 1 = (FAS vol. of blank – FAS vol. of SCL 1)*FAS
molarity*Dilution Factor*4000 = (14.7-
14.5)*.0127*1*4000 = 8 mg/l
COD (6/29/04)
Samples Avg FAS
vol, ml DilutionFactor
COD, mg/l
SCL 1 14.5 1 8 SAER 1 13.7 1 48 SAX 1 14.4 1 13 SAN 1 14.0 1 33 TINF 11.4 2 331 SINF 11.2 1 176 SCL 2 14.5 1 8 SAER 2 14.4 1 13 SAX 2 14.6 1 3 SAN 2 14.0 1 33 Blank 14.7 1 0 FAS Molarity 0.0127
115
1Table 9 : Example Calculation for Observed Yield
Date Inf 1 Flow Rate l/d
Inf 1 TCODmg/l
Clarifier 1 SCODmg/l
Delta CODmg/d
WAS+Eff 1 VSS g/d
Yobs 1 mg VSS/mg COD
6/15/2004 27.3 397 26.1 10124 3.27 0.32
Yobs = (WAS+Eff VSS) / [(Inf TCOD – Clarifier SCOD)*Inf Flow Rate] = 3.27*1000 / [(397-26.1)*27.3 =
0.32 mg VSS/mg COD
Sample Absorbance @625 nm
Glycogen, mg/L
Dilution Factor
Glycogen, mg/L
Glycogen, mM-C
AN 1 0.228 32.47 10 324.7 10.8 AX 1 0.227 32.20 10 322.0 10.7 AER 1 0.281 40.18 10 401.8 13.4 AN 2 0.285 40.81 10 408.1 13.6 AX 2 0.288 41.17 10 411.7 13.7 AER 2 0.348 50.12 10 501.2 16.7 Std, mg/L Glucose
Absorbance @625 nm
0 0 10 0.079 20 0.159 40 0.257 80 0.557
100 0.696
Glycogen Std Curve
y = 0.0069x + 0.0047R2 = 0.9978
00.10.20.30.40.50.60.70.8
0 20 40 60 80 100 120
Std, mg/L glucose
Abs
orba
ce @
625
nm
Figure 12: Sample Calculation for Glycogen
Table 92: Sample Calculation for Paired t Test for Anaerobic P Release1
∑=
=n
iiDA
1
2
n
DB
n
ii
2
1)(∑
==
)1(2 −−
=nn
BASd
2dd SS =
∑=
=n
iiXX
111
∑=
=n
iiXX
122
dSXXt 21 −=
339238.6 314858.9 1219.0 34.91 371.32 622.26 -7.19
1 X1 = AN1 P Release, X2 = AN2 P Release, n = 5, Di=X1i – X2i
116
Sample Calculations for Phosphorus Mass Balance
Phosphorus mass balance was done around each zone to obtain phosphorus mass balance
results. When calculating P release, phosphorus that was coming in the chamber was subtracted
from the phosphorus that was going out of the chamber. When calculating P uptake, phosphorus
that was going out of the chamber was subtracted from the phosphorus that was coming in the
chamber. The propionic acid flow rate was negligible and didn’t contain phosphorus so it wasn’t
included in the mass balance. Figure 7 is copied below to make seeing the flow rates and
terminology easier. Example calculations for Train 1 in 6/1/2004 (Table 31) are as follows:
Anaerobic P Release = (Inf + ARCY)(AN SOP) – (Inf)(Inf TP) – (ARCY)(AX SOP) = (22.9 +
41.1 L/d)(7.35 mg-P/L) – (22.9 L/d)(9.6 mg-P/L) – (41.1 L/d)(3.74 mg-P/L) = 96.85 mg-P/d
Anoxic P Release = (Inf +ARCY +NARCY + RAS)(AX SOP) – (Inf + ARCY)(AN SOP) –
(NARCY)(AER SOP) – (RAS)(CL SOP) = (22.9 + 41.1 +125.3 +52.1 L/d)(3.74 mg-P/L) – (22.9
+ 41.1 L/d)(7.35 mg-P/d) – (125.3 L/d)(1.52 mg-P/L) – (52.1 L/d)(8.96 mg-P/L) = -224.85 mg-
P/d
Aerobic P Uptake = (Inf + NARCY +RAS)(AX SOP) – (Inf + NARCY +RAS+WAS)(AER
SOP) = (22.9 +125.3 +52.1 L/d)(3.74 mg-P/L) – (22.9 + 125.3 + 52.1 + 1 L/d)(1.52 mg-P/L) =
443.15 mg-P/d
117
Clarifier P Uptake = (Inf + RAS – WAS)(AER SOP – CL SOP) = (22.9 + 52.1 – 1 L/d)(1.52–
8.96 mg-P/L) = -550.56 mg-P/d
AN AX AE
Influent Tank Effluent
Tank Propionic Acid Reservoir
RAS
NARCYARCY WAS
Clarifier
Total Biological P release = Anaerobic P Release = 96.85 mg-P/d (There was P uptake in the
anoxic zone in this day so Anoxic P Release is not included here)
Total System P Release = Anaerobic P Release – Clarifier P Uptake = 96.85 - (-550.56) =
647.41 mg-P/d (The Clarifier P Uptake was negative in this day so it is actually P release)
Total Biological P Uptake = Aerobic P Uptake – Anoxic P Release = 443.15 – (-224.84) =
667.98 mg-P/d
118
Total System P Uptake = Aerobic P Uptake – Anoxic P Release = 443.15 – (-224.84) = 667.98
mg-P/d (The clarifier had P release not uptake)
Net System P Removal = Total System P Uptake – Total System P Release = 667.98 – 647.41 =
20.58 mg-P/d
Net Biological P Removal = Total Biological P Uptake – Total Biological P Release = 667.98 –
96.85 = 571.14 mg-P/d
119
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