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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

REFERENCES

Arun, V., T. Mino, T. Matsu, “Biological Mechanism of Acetate Uptake Mediated by

Carbohydrate Consumption in Excess Phosphorus Removal Systems” Water Research,

Vol. 22, No. 5, pp. 565-570, 1988

Bond, P. L., J. Keller, L. L. Blackall, “Anaerobic Phosphate Release from Activated Sludge with

Enhanced Biological Phosphorus Removal: A Possible Mechanism of Intracellular pH

Control”, Biotechnology and Bioengineering, Vol. 63, pp. 507-515, 1999

Brdjanovic, D., M. C. M.van Loodsdrecht, C. M. Hooijmans, G. J. Alaerts, and J. J. Heijnen,

”Temperature effects on biological phosphorus removal” J. Env. Eng., Vol. 123, No. 2, pp.

144–154, 1997

Cech, J. S., and Hartman, P., “Glucose Induced Breakdown of Enhanced Biological Phosphorus

Removal”, Env. Tech., Vol. 11, p. 651, 1990

Chuang, S. H., C. F. Ouyang, Y. B. Wang, “Kinetic Competition Between Phosphorus Release

and Denitrification on Sludge Under Anoxic Condition”, Water Research, Vol. 30, No. 12,

pp. 2961-2968, 1996

Cokgor, E. U., N. O. Yagci, C. W. Randall, N. Artan, and D. Orhon, “Effects of pH and

Substrate on the Competition Between Glycogen and Phosphorus Accumulating

Organisms”, J of Env Sci and Health, Vol. A39, No. 7, pp. 1695-1704, 2004

120

Comeau, Y., K. J. Hall, R. E. W. Hancock, and W. K. Oldham, “Biological Model for

Enhanced Biological Phosphorus Removal”, Water Research, Vol. 20, No. 12, pp. 1511-

1521, 1986

Cruz, R., “The Effects of a Reduced Cycle Duration in Enhanced Biological Phosphorus

Removal Sequencing Batch Reactors”, Thesis Defended 2004, UCF, Orlando, FL

Daigger, G. T., Purdue University online article, http://bridge.ecn.purdue.edu/~alleman/w3-

class/456/article/daigger-nutrients/daigger.html

Erdal U. G., Z. K. Erdal, C. W. Randall, “A thermal adaptation of bacteria to cold temperatures

in an enhanced biological phosphorus removal system”, Water Science and Technology,

Vol. 47, No. 11, pp. 123–128, 2003

121

Erdal, U. G., C. W. Randall, “The effects of operational conditions on EBPR performance”, PhD

dissertation, Virginia Polytechnic Institute and State University, 2002

Eaton, A. D., L. S. Clesceri, A. E. Greenberg, “Standard Methods for the Examination of Water

and Wastewater”, 19th Edition, American Public Health Association, Washington D.C.

1995

Filipe, C. D. M., G. T. Daigger, C. P. L. Grady, “Stoichiometry and Kinetics of Acetate Uptake

under Anaerobic Conditions by an Enriched Culture of Phosphorus-Accumulating

Organisms at Different pHs”, Biotechnology and Bioengineering, Vol. 76, pp. 32-46, 2001a

Filipe, C. D. M., G. T. Daigger, C. P. L. Grady, “A Metabolic Model for Acetate Uptake under

Anaerobic Conditions by Glycogen Accumulating Organisms: Stoichiometry, Kinetics, and

the Effect of pH”, Biotechnology and Bioengineering, Vol. 76, pp. 17-31, 2001b

Filipe, C. D. M., G. T. Daigger, C. P. L. Grady, “pH as a Key Factor in the Competition Between

Glycogen-Accumulating Organisms and Phosphorus-Accumulating Organisms”, Water

Environ. Res., Vol. 73, p. 223, 2001c

Helmer, C., S. Kunst, “Low Temperature Effects on Phosphorus release and Uptake by

Microorganisms in EBPR Plants”, Water Science and Technology, Vol. 37, No. 4-5, pp.

531-539, 1998

Louie, T.M., T.J. Mah, W. Oldham, and W.D. Ramey, “Use of Metabolic Inhibitors and Gas

Chromatography/Mass Spectrophotometry to Study Poly-β-Hydroxyalkanoates

Metabolism Involving Cryptic Nutrients in Enhanced Biological Phosphorus Removal

Systems”, Water Research, Vol. 34, No. 5, pp. 1507-1514, 2000

McClintock, S., C. W. Randall, and V. Pattarkine, “The effects of temperature and mean cell

residence time on enhanced biological phosphorus removal”, Environmental Engineering,

Proceedings of the 1991 Specialty Conference on Environmental Engineering, ASCE, 319–

324, 1991

Matsuo, Y., “Effect of the Anaerobic SRT on Enhanced Biological Phosphorus Removal”, Water

Science and Technology, Vol. 28, No. 1, p. 127, 1994

Mino, T., V. Arun, Y. Tsuzuki, T. Matsuo, “Effects of Phosphorus Accumulation on Acetate

Metabolism in the Biological Phosphate Removal Process”, Advances in Water Pollution

Control: Biological Phosphate Removal from Wastewater, R. Ramadore , Pergamon Press,

Oxford, 1987

122

Murray, R. G. E., “Manual of Methods for General Bacteriology”, American Society for

Microbiology, Washington, D.C., 1981

Pijuan, M., A. M. Saunders, A. Guisasola, C. Casas, J. A. Baeza, L. L. Blackall “Enhanced

Biological Phosphorus Removal in a Sequencing Batch Reactor Using Propionate as the

Sole Carbon Source”. Biotechnology and Bioengineering, Vol. 85, No. 1, pp. 56-67, 2004

Pramanik, J. , P. L. Trelstad, A. J. Schuler, D. Jenkins and J. D. Keasling, “Development and

validation of a flux-based stoichiometric model for enhanced biological phosphorus

removal metabolism”, Water Research, Vol 33, No. 2, pg 462, 1999

Randall, A. A., L. D. Benefield, and W. E. Hill, “Effect of Fermentation Products on Enhanced

Biological Phosphorus Removal, Polyphosphate Storage, and Microbial Population

Dynamics”, Water Science and Technology, Vol. 30, No. 6, pt 6. pp 213-219, 1994

Randall A. A., Y. Chen, Y. H. Liu and T. McCue, “Polyhydroxyalkanoate form and

polyphosphate regulation: keys to biological phosphorus and glycogen transformations?”,

Water Science and Technology, Vol. 47, No. 11, pp. 227–233, 2003

Satoh, H., W. D. Ramey, F. A. Koch, W. K. Oldham, T. Mino, T. Matsu, “Anaerobic Substrate

Uptake by Enhanced Biological Phosphorus Removal Activated Sludge Treating Real

Sewage”, Water Science and Technology, Vol. 34, No. 1-2, pp. 9-16, 1996

Smolders, G. J. F., J. van der Meij, M. C. M. van Loosdrecht, “Model of the Anaerobic

Metabolism of the Biological Phosphorus Removal Process: Stoichiometry and pH

Influence”, Biotechnology and Bioengineering, Vol. 43, No. 6, pp. 461-470, 1994a

123

124

Wentzel, M. C., P. L. Dold, G. A. Ekama, Gv. R. Marais, “Enhanced polyphosphate organism

cultures in activated sludge systems. Part III: Kinetic model”, Water S.A., Vol. 15, No. 2,

pp. 89-102, 1989b

Smolders, G. J. F., J. van der Meij, M. C. M. van Loosdrecht, “Stoichiometric model of the

aerobic metabolism of the biological phosphorus removal process”, Biotechnology and

Bioengineering, Vol. 44, No. 7, pp. 837-848, 1994b

Smolders, G. J. F., J. van der Meij, M. C. M. van Loosdrecht, “pH: Key Factor in biological

Phosphorus Removal Process”, Water Science and Technology, Vol. 29, No. 7, pp. 71-74,

1994

Wentzel, M. C., R. E. Loewenthal, G. A. Ekama, Gv. R. Marais, “Enhanced Polyphosphate

Organism Cultures in Activated Sludge Systems-Part 1: Enhanced Culture Development”,

Water S.A., Vol 14, No. 2, pp. 81-92, 1988

Wentzel, M. C., G. A. Ekama, R. E. Loewenthal, P. L. Dold, Gv. R. Marais, “Enhanced

Polyphosphate Organism Cultures in Activated Sludge Systems. Part II. Experimental

Behaviour”, Water S.A., Vol. 15, No. 2, pp. 71-88, 1989a

Ydstebo, L., T. Bilstad, J. Barnard, “Experience with biological nutrient removal at low

temperatures”, Water Environment Research, Vol. 72, No. 4, pp. 444-454, 2000