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Institute of Environmental Studies - The University of Tokyo

Institute of Environmental Studies - The University of Tokyo

Enhanced Biological Phosphate Removal (EBPR)


A Promising Technology for Phosphate Removal from Wastewater with Mysterious Microbiology
MINO Takashi, Professor - Institute of Environmental Studies - The University of Tokyo -

Outlines of Presentation
1. Basics of Phosphate Removal in Activated Sludge Process 2. The EBPR Process 3. The EBPR Metabolism 4. Microbiological Puzzle 5. Final Remarks

Institute of Environmental Studies - The University of Tokyo

Institute of Environmental Studies - The University of Tokyo

Phosphorus Removal In Activated Sludge Process

1. Basics of Phosphate Removal in Activated Sludge Process

Influent P (P in)

Activated Sludge Process

Effluent P (P out)

P in Waste Sludge (P waste) P removed = P in - P out = P waste = CYPx


C Removed Carbon (Organic Matters) Y Yield (Biomass produced/Carbon removed) PxPhosphorus Content of Sludge

Institute of Environmental Studies - The University of Tokyo

Institute of Environmental Studies - The University of Tokyo

Phosphorus Removal In Activated Sludge Process


To increase P removal, C, Y or Px should be increased.

Phosphorus Removal In Activated Sludge Process


C? => Carbon removal is already high enough
Y? =>
and C is difficult to increase further. Practically, Y is not feasible to control.

Px?
P removed = P in - P out = P waste = CYPx
C Removed Carbon (Organic Matters) Y Yield (Biomass produced/Carbon removed) PxPhosphorus Content of Sludge

=> To be increased for P removal.

P removed = P in - P out = P waste = CYPx


C Removed Carbon (Organic Matters) Y Yield (Biomass produced/Carbon removed) PxPhosphorus Content of Sludge

Institute of Environmental Studies - The University of Tokyo

Institute of Environmental Studies - The University of Tokyo

Chemical Phosphate Removal

Are there any practical ways to increase Px?


----------- Yes! Either chemically, or biologically

Addition of Chemical Coagulant

Enhanced Biological Phosphate Removal (EBPR)


Introduction of Anaerobic Stage

=> Px increases.

Institute of Environmental Studies - The University of Tokyo

Institute of Environmental Studies - The University of Tokyo

Development of Enhanced Biological Phosphate Removal (EBPR) Process

2. The EBPR Process

James Barnard (1975) - A New Concept Proposed


- Introduction of an absolute anaerobic stage at the influent end of aeration tank facilitates the production of activated sludge with high phosphate removal capability. - The anaerobic stage may function as a biological selector to facilitate the proliferation of phosphate accumulating organisms with a high P content.

Institute of Environmental Studies - The University of Tokyo

Institute of Environmental Studies - The University of Tokyo


Inf DP Ae DP : Anaerobic Zone Sed Eff
for P removal

The Anaerobic Aerobic Activated Sludge Process for Enhanced Biological Phosphate Removal (EBPR)
A modification of the conventional activated sludge process Introduction of an anaerobic stage where no electron acceptors (O2, NOx-) are available Circulation of activated sludge through alternating anaerobic and aerobic stages, allowing the sludge to contact the wastewater (carbon sources) only in the anaerobic stage Activated sludge with high a phosphorus content formed

Ae : Aerobic Zone DN : Anoxic Zone for


Denitrification

RS A. Anaerobic Aerobic Process () MLR

RA : Reaeration Sed : Sedimentation RA Sed

Inf

DP

DN

Ae

DN

Eff

Various EBPR Processes

B. Phoredox Process DP Inf MLR DN MLR Ae

RS

Sed

Eff

C. UCT Process Ae Inf Sed Eff

RS

Inf : Influent Eff : Effluent MLR : Mixed Liquor Recycle

RS(1) RS(2) SR D. Phostrip Process DPS CP

RS : Return Sludge SR : Supernatant Recycle

DPS : Anaerobic P Stripper CP : Chemical Precipitation

Institute of Environmental Studies - The University of Tokyo

Institute of Environmental Studies - The University of Tokyo

The anaerobic aerobic process is an established process from technological points of views.

3. The EBPR Metabolism


Design criteria developed Significant full-scale experiences in South Africa, Europe, US, Japan, etc. Performance acceptable Easy retrofit from conventional activated sludge process Green technology compared to chemical P removal Some drawbacks (P release in thickeners, less stable)

Institute of Environmental Studies - The University of Tokyo

Institute of Environmental Studies - The University of Tokyo

Why does the anaerobic zone favor phosphate accumulating organisms (PAOs)?

Structure of Polyphosphate
O O O Mg O O K

-P-O-P-O-P-O-

Because hydrolysis of polyphosphate stored in the cell can supply energy for the uptake of carbon sources under the anaerobic conditions where no electron acceptors are available.

High Energy Orthophosphate Group

Hydrolysis of polyphosphate can supply energy.

Institute of Environmental Studies - The University of Tokyo <Anaerobic> TOC in the Wastewater <Aerobic> Poly-hydroxyalkanoates (PHA) in the sludge

Institute of Environmental Studies - The University of Tokyo

EBPR Metabolism by Poly-P Accumulating Organisms (PAOs)


<Anaerobic> Carbon Sources O2 <Aerobic>

PO4-P
Energy

PHA Cell Structure


Energy

CO2

Typical Profiles of Key Components In the Anaerobic and Aerobic Stages of EBPR Process

Poly-P

PO4-P

Institute of Environmental Studies - The University of Tokyo

Institute of Environmental Studies - The University of Tokyo

Structure of PHA
O -O - CH - CH - C R1
3-hydroxybutyrate (3HB) Free Acid In PHA Precursors
OH CH3-CH-CH 2-COOH

How is the reducing power for the synthesis of PHA obtained? PHA = Reduced compound

R2

Acetate (Substrate) Acetyl-CoA Acetoacetyl-CoA

PHA
Hydroxybutyryl-CoA

3-hydroxyvalerate (3HV)
-

3-hydroxy-2-methylbutyrate 3-hydroxy-2-methylvalerate (3H2MB) (3H2MV)


OH CH3 CH3-CH-CH-COOH OH CH3 CH3-CH 2-CH-CH-COOH CH3 CH2 CH3 (-O-CH-CH-CO-)

OH CH3-CH 2-CH-CH 2-COOH CH3 CH2 (-O-CH-CH 2-CO-)

CH3 (-O-CH-CH 2-CO-)

CH3CH3 (-O-CH-CH-CO-)

2 acetyl-CoA

1 acetyl-CoA 1 propionyl-CoA

1 acetyl-CoA 1 propionyl-CoA

2 propionyl--CoA

NADP NADPH (reducing reagent)

Institute of Environmental Studies - The University of Tokyo <Anaerobic> TOC in the Wastewater <Aerobic> Polyhydroxyalkanoates (PHA) in the sludge

Institute of Environmental Studies - The University of Tokyo

EBPR Metabolism by Poly-P Accumulating Organisms (PAOs)


<Anaerobic> Carbon Sources Glycogen O2 CO2 PHA
Energy

<Aerobic>

Glycogen in the sludge PO4-P

CO2 Cell Structure


Energy

Typical Profiles of Key Components In the Anaerobic and Aerobic Stages of EBPR Process

Poly-P

PO4-P

Institute of Environmental Studies - The University of Tokyo

Institute of Environmental Studies - The University of Tokyo

A few Questions about the EBPRMetabolism


How is glycogen completely oxidized to CO2 under anaerobic conditions? Via the TCA cycle? Why are acetate and short chain fatty acids (SCFA) suitable substrates for EBPR? How are carbon sources other than SCFA metabolized? How can PAOs maintain the oxidation-reduction balance in the cell when reduced compounds (PHA) is produced from various carbon sources?

The Extended Mino Model


Glycogen External Substrates
CO2 Phosphenolpyruvate

[H]
(Reducing Power Produced)

Glycolytic pathway
Pyruvate

External Substrates

SuccinatePropionate Pathway [H]

0xaloacetate Succinate

[H]
(Reducing Power Produced) CO2

(Reducing Power Succinyl-CoA cunsumed) CO2

Acetyl-CoA Formation External Substrates

Propionyl-CoA PHA Accumulation

Acetyl-CoA [H]

3HA Unit (PHA)

(Reducing Power Consumed)

Institute of Environmental Studies - The University of Tokyo

Institute of Environmental Studies - The University of Tokyo

Stoichiometry for Anaerobic Carbon Metabolism by PAOs


Theoretical or Observed Glycogen (or CarboHydrate) consumed 1 1.2 1 1.1 Substrate Taken up PHA Produce d % of Acetyl-CoA or Propionyl-CoA needed for PHA synthesis Acetyl-CoA 6 6 6 6 4 3.9 4 4.0 100 95 25 25 PropionylCoA 0 5 75 75

Mysterious Biochemistry (1)


Presence of glycogen accumulating organisms (GAOs) and their metabolisms. GAOs are possible competitors for PAOs. GAOs can be a cause of poor stability and deterioration of EBPR.

Acetic Acid

Theoretical Observed

Propionic Acid

Theoretical Observed

Institute of Environmental Studies - The University of Tokyo

Institute of Environmental Studies - The University of Tokyo

Reported Presence of GAOs


Reference Matsuo et al , (1982) Fukase et al , (1984) Carbon Sources Diluted night soil Ac, Pep, YEx Causative Operation Seed sludge from night soil treatment plant Long SRT and HRT (54 days) Addition of Glucose Unclear Limited Phosphorus feeding Note When seeded by EBPR sludge, good P removal achieved. PHB accumulated anaerobically. PHB accumulated and glycogen consumed anaerobically. P removal recovered by extending anaerobic retention time. PHB accumulated and glycogen consumed anaerobically. PHB accumulated and glycogen consumed anaerobically.

Possible Metaabolism of GAOs


<Energy Generating Metabolosm>
CO2 2NAD 2ATP NAD NADH

Acetyl -CoA

NADH H+

Cech/Hartman Ac, Glu (1990/1993) Matsuo et al , (1994) Liu et al (1994) Satoh et al (1994) Pep, Ac, Prop Ac, Pep

Glucose
2NADH 2ADP + + 2H+ 2Pi

2 Pyruvate
2NADH 2H+

H+ 2NAD ADP+Pi NAD

3-HA Unit (PHA)

ATP

Propionyl -CoA

Ac, Prop, Unclear Pep, YEx

<Overall Reaction> 5(-C6H10O5-) + 12CH3COOH Q 11(-3HA-) + 7CO2

Institute of Environmental Studies - The University of Tokyo

Institute of Environmental Studies - The University of Tokyo

Mysterious Biochemistry (2)


Storage compounds other than PHA and glycogen suspected.
Glucose => Glycogen Glutamic Acid => Poly-glutamate Poly-lactate ?

Mysterious Biochemistry (2)


Diverse metabolisms other than EBPR may be possible for anaerobic energy generation in the anaerobic-aerobic process. These metabolisms can be competitive to the EBPR metabolism.

Institute of Environmental Studies - The University of Tokyo

Institute of Environmental Studies - The University of Tokyo

Who are responsible for EBPR? Who are PAOs?

4. Microbiological Puzzle

<Difficulty in isolation of PAOs>


* Microlunatus phosphovorus (Nakamura et al.) - Anaerobic P release and C uptake observed - No anaerobic acetate uptake accompanied by PHA storage * Loss of key characteristics of the EBPR metabolism after isolation (anaerobic PHA storage lost)

Institute of Environmental Studies - The University of Tokyo

Institute of Environmental Studies - The University of Tokyo

Who are PAOs?


Acinetobacter spp. ?
1975 (Fuhs & Chen) - 1980s - Isolation and quantification based on culture-dependent techniques (strong culturing biases expected). - Acinetobacter spp. dees not show the typical EBPR metabolism (especially, anaerobic PHA storage). - Culture-independent techniques have verified that Acinetobacter is a minor member of EBPR communities.

The biases
0 The whole community 100%

Acinetobacter

Others

=> No Acinetobactor as primary PAOs

Cultivable Non-cultivable

Institute of Environmental Studies - The University of Tokyo

Institute of Environmental Studies - The University of Tokyo

Molecular Approaches
Microbial Community Samples Extraction of DNA
35%

Molecular Identification
A.c. C.a. E.c. R.e. i

seed 3 M

17 31 45 days 13 22 38 51 c

a b d e

Purification and Amplification of <== PCR or Cloning Target Gene/DNA fragment Sequencing Phylogenetic Analysis /Probe or primer design
55%

h k j

EBPR Microbial Community Changes as seen on DGGE Patterns

Institute of Environmental Studies - The University of Tokyo

Institute of Environmental Studies - The University of Tokyo


(A) (B)

Rhodocyclus Related PAO Group


E.coli Clone GCP112 AF204248 274 378 254 460 405
A)

Propionibacter pelophilus AF016690 Dechlorimonas agitatus AF047462 Dechlorisoma suilla AF170348 Rhodocyclus tenuis D16208 995 Rhodocyclus tenuis D16210 RHC439
A)

(C)

(D)

460

434

Rhodocyclus tenuis D16209 band h Clone SBRB34 AF204247 PAO462 RHX456

196

Rhodocyclus sp. R6 AJ224937 B) 979 Clone SBRA245A AF204245 A) 688 clone SBRA220 AF204244 A) 0.1 A) Crocetti et al (2000)

Microscopic Images of Possible PAOs


(A)Phase contract, (B)DAPI for poly-P, (C)Phase Contrast (D)FISH Image with PAO Probe

B) Hesselmann et al (1999)

Institute of Environmental Studies - The University of Tokyo

Institute of Environmental Studies - The University of Tokyo

DAPI
DAPI for Total cell & Phosphorus

Merged image of DAPI & FISH


DAPI for Total cell Phosphorus + PAO462

The rods have two polyphosphate granules inside the cell.

It was clearly and directly shown that the bacteria represented by the band h really accumulated polyphospahte.

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