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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
Influent P (P in)
Effluent P (P out)
Px?
P removed = P in - P out = P waste = C Y Px
C Removed Carbon (Organic Matters) Y Yield (Biomass produced/Carbon removed) PxPhosphorus Content of Sludge
=> Px increases.
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
Inf
DP
DN
Ae
DN
Eff
RS
Sed
Eff
RS
The anaerobic aerobic process is an established process from technological points of views.
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.
Institute of Environmental Studies - The University of Tokyo <Anaerobic> TOC in the Wastewater <Aerobic> Poly-hydroxyalkanoates (PHA) in the sludge
PO4-P
Energy
CO2
Typical Profiles of Key Components In the Anaerobic and Aerobic Stages of EBPR Process
Poly-P
PO4-P
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
PHA
Hydroxybutyryl-CoA
3-hydroxyvalerate (3HV)
-
CH3CH3 (-O-CH-CH-CO-)
2 acetyl-CoA
1 acetyl-CoA 1 propionyl-CoA
1 acetyl-CoA 1 propionyl-CoA
2 propionyl--CoA
Institute of Environmental Studies - The University of Tokyo <Anaerobic> TOC in the Wastewater <Aerobic> Polyhydroxyalkanoates (PHA) in the sludge
<Aerobic>
Typical Profiles of Key Components In the Anaerobic and Aerobic Stages of EBPR Process
Poly-P
PO4-P
[H]
(Reducing Power Produced)
Glycolytic pathway
Pyruvate
External Substrates
0xaloacetate Succinate
[H]
(Reducing Power Produced) CO2
Acetyl-CoA [H]
Acetic Acid
Theoretical Observed
Propionic Acid
Theoretical Observed
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+
ATP
Propionyl -CoA
4. Microbiological Puzzle
The biases
0 The whole community 100%
Acinetobacter
Others
Cultivable Non-cultivable
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
Propionibacter pelophilus AF016690 Dechlorimonas agitatus AF047462 Dechlorisoma suilla AF170348 Rhodocyclus tenuis D16208 995 Rhodocyclus tenuis D16210 RHC439
A)
(C)
(D)
460
434
196
Rhodocyclus sp. R6 AJ224937 B) 979 Clone SBRA245A AF204245 A) 688 clone SBRA220 AF204244 A) 0.1 A) Crocetti et al (2000)
B) Hesselmann et al (1999)
DAPI
DAPI for Total cell & Phosphorus
It was clearly and directly shown that the bacteria represented by the band h really accumulated polyphospahte.
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