20150320 MCE - Storage Polymers
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Transcript of 20150320 MCE - Storage Polymers
1
Exploiting the role of
intracellular storage polymers
in
Microbial Community Engineering
Robbert Kleerebezem, Katja Johnson, Yang Jiang,
Helena Moralejo Garate, Leonie Marang, Jelmer Tamis,
Emma Korkakaki, Gerard Muyzer, Mark C. M. Van Loosdrecht
Delft University of Technology
Department of Biotechnology
Julianalaan 67, 2628BC Delft, The Netherlands
Outline
Ecology of storage polymers
Process 1: phosphate removal from sewage
Process 2: feast famine regime for PHA production from
wastewater
2
3
Why microorganisms make storage compounds?
Microbial fat!
Balancing growth
Induced by nutrient limitation
Opportunistic hoarding (hamsteren)!
4
Microbial storage compounds
Compound Stored for Examples
Glycogen/Polyglucose C, energy animals, yeast, prokaryotes
Starch C, energy plants
Polyphosphate P, energy e.g. PAOs
Polyhydroxyalkanoates C, energy prokaryotes
Triacylglycerols C, energy eukaryotes, few prokaryotes
Wax esters C, energy few prokaryotes, jojoba seed
Cyanophycin C, N, energy some cyanobacteria
Polyglutamic acid C, N, energy few bacilli
Sulphur e-, energy sulphur bacteria
3
PO4-REMOVAL FROM SEWAGE
Role of storage polymers in nutrient removal from sewage
5
6
Why wastewater treatment?
< 1970 Hygiene – Sanitation
1970-1990 Nature protection – Organic carbon/ammonium
>1990 Ecosystem protection – Nutrients/Micropollutants/Metals
4
Activated sludge based sewage treatment
Primary:
Removing Grit and Coarse and Suspended Material, Buffertank, pH adjustment
Secondary:
Biological removal of COD (organic compounds) and Nutrients and Pathogens
Tertiary:
Polishing, Disinfection
Microbial Community Engineering for combined
C, N, and P removal from sewage
Organic carbon: Aerobic and anoxic (with NO3-1) heterotrophic
degradation,
Nitrogen: Nitrification and denitrification,
Phosphorous: Phosphate accumulating bacteria
Activated
sludge
process
CH2O,
NH4+1
HPO4-2
Clean Water
Biomass with
HPO4-2
CO2
N2
Treatment objective:
5
9
Phosphate Accumulating Organisms (PAOs)
Alternating anaerobic and aerobic conditions,
Polyphosphate for Energy storage,
Two organic carbon storage polymers: Glycogen and PHA
Selective pressure:
acetate supply in the anaerobic phase!
Competition with Glycogen accumulating organisms
2
4n HPO ATP
Metabolism PAOs and GAOs
10
NADH
PHA GlycogenAcetate
ATP
GAOs
PAOs
Anaerobic:
Phosphate Poly-PPAOs
6
Metabolism PAOs and GAOs
11
Phosphate Poly-PPAOs
PHA Glycogen
ATP
GAOs
PAOs
Aerobic:
Biomass
12
PAO/GAO biochemistry (anaerobic)
ATP
acetate
EMP (3ATP)ED (2ATP)
ATP ADP
NADH NAD+
ATP
ADP
NADH
NAD+
NAD+
NADH
ATP
ADP
AMP
PHA
NADH
NAD+
CO2
hydroxyacyl CoA
Acetate
acetyl-CoA (A)
Glycogenpyruvate
propionyl CoA (P)
3-hydroxybutyrate (A+A)3-hydroxyvalerate (A+P)
3-hydroxy-2-methylbutyrate (A+P)3-hydroxy-2-methylvalerate (P+P)
ATP
ADP
PoliPn
PoliPn-1
NADH
TCA
CO2
NAD+
ATP
ADPH2PO4
- H2PO4-
M+
H+
M+
H+
PAO
OH-
OH-
7
Stoichiometry
Polyphosphate Accumulating Organisms (PAO)
1C-mol acetate + 0.5C-mol glycogen 1.33 C-mol PHA
Glycogen Accumulating Organisms (GAO)
1C-mol acetate + 0.9-1.2 C-mol glycogen 1.68-1.91 C-mol PHA
PAO-GAO competition determined by:
Temperature,
Carbon source (acetate vs. propionate)
…
Sequencing Batch Reactor
14
Start phase
10 min.
Filling phase
3 min.
Anaerobic
phase 122 min.
Aerobic phase
135 min.
Settling phase
80 min.
Effluent phase
10 min.
Cycle: 6 h
HRT: 0.5 d
SRT: 6 d
8
Anaerobic Aaerobic
Liquid
Solid (Biomass)
Conversions
Who competes with PAOs and GAOs?
16
1. What is the main selection criterion for PAOs/GAOs?
Anaerobic organic carbon (acetate) uptake!
2. Who is more capable of anaerobic organic carbon (acetate) uptake?
Aceticlastic methanogens!
1 1
2 3 2 4 2C H O H CH CO
3. How to avoid methanogenesis?
Aerobic period (toxicity), solid retention time (growth rate)
9
In activated sludge process
Bioreactor
CH2O,
NH4+1
HPO4-2
Clean Water
Clarifier
Sludge discharge
Questions:
• Why is there a clarifier?
• How to design the bioreactor to implement P-removal?
Biological P-removal process
Bioreactor
CH2O,
NH4+1
HPO4-2
Clean Water (with N)
Clarifier
Sludge discharge
Anaerobic
Air
Aerobic
10
Biological N-removal process
Bioreactor
CH2O,
NH4+1
HPO4-2
Clean Water (with P)
Clarifier
Sludge discharge
Anoxic
denitri-
fication
Air
Aerobic
nitrification
NO3-1
CH2O oxidation
Nitrate reduction
Nitrogen gas production (CH2O oxidation)
Ammonium oxidation
Oxygen reduction
Nitrate production
Combining N- and P-removal?
Bioreactor
CH2O,
NH4+1
HPO4-2
Clean Water
Clarifier
Sludge discharge
Anaerobic
Air
Anoxic
Tip: P-uptake can also be established with NO3-1/NO2
-1 as electron acceptor
Aerobic
P-release,
Glycogen fermentation,
Acetate uptake,
PHA production.
P-uptake,
Glycogen production,
PHA-degradation,
Nitrate reduction,
Nitrogen gas production.
Ammonium oxidation,
Oxygen reduction,
Nitrate production.
11
Wastewater Innovation: Granular Sludge
Advantages: 75% less space, 30% less energy, less construction materials 25 % less investment costs
Principle: Sequencing Batch Process, Short settling phase
granules, Anaerobic feeding/Aerobic
reaction period
2. Dynamics in Space and Time
NH4+ NH4
+
NO2-
NO3-
O2
NO3-
N2
N2
PO4-3 PO4
-3
PP
PO4-3
PP
PA
OA
OB
NO
B
DP
AO
+ O2
COD
PO4-3 PO4
-3
PP
PO4-3
PP
PA
O
DP
AO
-O2
Aerobic granular sludge: combined N, P, and C-removal through space and time dependent conversions
12
Summary
23
Biological P-removal depends on storage of
polyphosphate and two organic carbon polymers: glycogen
and PHA
P-removing bacteria compete with glycogen accumulating
organisms
Combined C, P, and N-removal from sewage is a nice
example of microbial community engineering
Selective environments can be created in space (different
tanks or in a biofilm) or in time (e.g. on/off aeration)
PHA PRODUCTION FROM
WASTEWATER
Feast-Famine regime for enrichment of PHA-producing bacteria
13
Polyhy-b-droxyalkanoate (PHA)
Poly(3-hydroxybutyrate)
C
C C CO
Ox
Common and widespread storage material in microorganisms (>90 genera of archaea and eubacteria)
Storage in granules (amorphous)
Often under limiting or dynamic conditions
Up to 80% of cell dry weight
Properties similar to petrochemical plastics
Properties depend on monomers and chain length: thermoplastic - elastomeric
Biologically degradable
Made from renewable resources
Monomers potential product
14
Introducing the enemy:
Industrial Biotechnology
Process development in Industrial Biotechnology
Work horse Genome analysis
Genetic
engineering
Product
15
Mirel ® process
E. coli Genome known
Add PHB-producing
enzymes
PHB
The competition: Mirel ® bioplastic process
Metabolix and ADM partnership
GMO-based industrial PHB production
Substrate = corn based glucose
50,000 ton/year commercial plant
Startup 2nd half 2008
16
Microbial Community Engineering
Microbial community Selective pressure
ProductDominant work horse
Substrate 40-50% process costs
Choi and Lee, 1997
17
PHA production strategy
Cultivation: selection of PHA producing mixed culture
Biomass
Fatty acids (with NH4)
1
Fatty acids (without NH4)
PHASequencing
batch reactorFed batch reactor
Accumulation: maximizing the cellular PHA content2
Ecological role of bioplastics
Intracellular Insoluble
Polymeric compounds:
• Glycogen
• Polyphosphate
• Polyhydroxyalkanoates
Microbial
Fat=
Time
Feed
supply
Pulse feeding of substrate favors growth of bioplastic
producing microorganisms: feast famine regime
18
Selective pressure for PHA-accumulating bacteria
Sequencing Batch ProcessAlternating absence and presence of substrate
resulting in a feast famine regime
Start
phase 10
min.
Filling phase 3
min. Reaction phase
700 min.
Effluent
phase 7 min.Operational cycle: 12 h
HRT/SRT: 24 h
Mechanism selection
Acetate Acetyl-CoA
CO2
Biomass
Substrate limited growth
O2
ATP
ATP
19
Mechanism selection
Acetate Acetyl-CoA
CO2
PHB
Biomass
1. Feast phase
Mechanism selection
Acetate Acetyl-CoA
CO2
PHB
Biomass
1. Feast phase
ATP
O2
ATP
ATP
20
Mechanism selection
Acetate Acetyl-CoA
CO2
PHB
Biomass
1. Feast phase
ATP
O2
ATP
ATP
Mechanism selection
Acetate Acetyl-CoA
CO2
PHB
Biomass
1. Feast phase
ATP
O2
ATP
ATP
21
Mechanism selection
Acetate Acetyl-CoA
CO2
PHB
Biomass
1. Feast phase
ATP
O2
ATP
ATP
Mechanism selection
Acetate Acetyl-CoA
CO2
PHB
Biomass
1. Feast phase
ATP
O2
ATP
ATP
22
Mechanism selection
Acetyl-CoA
CO2
PHB
Biomass
2. Famine phase
ATP
O2
ATP
0
10
20
30
40
50
60
0 100 200 300 400 500 600 700
Time [min]
Am
ou
nt
[(C
)mm
ol]
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Fra
ctio
n P
HB
[C
mo
l/C
mo
l]
23
Mechanism accumulation
Acetate Acetyl-CoA
CO2
PHB
Biomass
1. Accumulation phase
ATP
O2
ATP
ATP
Mechanism accumulation
Acetate Acetyl-CoA
CO2
PHB
Biomass
1. Accumulation phase
ATP
O2
ATP
ATPNH4
+
24
Acetate Acetyl-CoA
CO2
PHB
O2
ATP
ATP
Mechanism accumulation
1. Accumulation phase
0
20
40
60
80
100
0 2 4 6 8 10 12
Time [h]
PH
B [
%]
Feb 2007
July 2007
April 2008Evolution based
system improvement
25
Optimization of enrichment
0
5
10
15
20
25
30
35
40
0 360 720 1080 1440 1800 2160Time
PH
A c
on
ten
t
3 cycles
9 cycles
Minimize the number of cycles per SRT,
Or maximize the Feed to Biomass ratio…
Propionate-acetate mixtures utilization
0
20
40
60
80
100
120
0 50 75 100
Fraction of acetate (%)
rela
tive
fra
ctio
n (
%)
PHB
PHV
26
Mechanism
PHB PHV BiomassBiomassATP
HPrHAc
PrCoAAcCoANADH2
R1 R2
R3R8
R4 R5R6R7R9
R11R10
PHB PHV BiomassBiomassATP
HPrHAc
PrCoAAcCoANADH2
R1 R2
R3R8
R4 R5R6R7R9
R11R10
Modeling
0
10
20
30
40
50
0 120 240 360 480 600 720
Time [min]
Pr,
X [
Cm
mo
l];
CO
2,
O2 [
mm
ol]
;NH
4+ [
mm
ol]
0.0
0.1
0.2
0.3
0.4
0.5
0.6
PH
B,
PH
V [
Cm
mo
l/C
mm
ol]
Simplified metabolic network,
Example: cultivation on propionate
27
Microscopy-I
Microscopy II
28
Dry weight percentage PHB = 85-90% (previous maximum 65%)
world record for wild type strain/mixed culture
Accumulation phase ~ 7 h
Novel strain: Plasticicumulans acidivorans
Genome sequenced
Electron efficiency ~ 70%
Acetate and other VFAs
Main selection criteria:
No cycles/SRT
Temperature
Status
Comparison with Metabolix E. coli process
Origin Metabolix TU Delft
Biomass GMO E. coli Open-mixed culture
Substrate Glucose, propionate VFA rich wastewater
Operation Disinfection,
High tech process
No disinfection,
Simple process control
Process Batch (semi)Continuous
Product yield/Time 85-90% / ~ 40 h 85-90% / < 10 h
Product
characteristics
Well defined Dependent on substrate
29
Unique strain
or
general strategy?
Substrate that cannot be degraded by P. acidovorans
Substrate that cannot be degraded by P. acidovorans: Lactate
Enrichment conditions:
HRT = SRT = 24 h
Cycle Length = 12 h
Aerobic
pH = 7
Temperature = 30°C
Inoculum: activated sludge
30
Operational cycle: 70
0
20
40
60
80
100
120
0 120 240 360 480 600 720
Time [min]
DO
[%
]; P
HB
[w
t %
]; L
ac
[C
mm
ol/
L]
0
20
40
60
80
100
120
0 120 240 360 480 600 720
Time [min]
DO
[%
]; P
HB
[w
t %
]; L
ac
[C
mm
ol/
L]
Operational cycle: 220
31
Time evolution of the length of the feast phase
0
60
120
180
240
300
0 50 100 150 200 250
Cycle number
Le
ng
th f
ea
st
ph
as
e [
min
]
Microbial community structure
32
33
PHB production on lactate
Acetate – Lactate mixture
34
Acetate versus glycerol grown community
Fatty acid grown Glycerol grown
but the end product remains the same…
Glycerol: PHB/Polyglucose production
35
Example experiment
0
10
20
30
40
50
60
70
0 6 12 18 24 30 36 42 48
Time [h]
Gly
cero
l [m
M]
0
10
20
30
40
50
60
70
80
po
lyg
luco
se,
PH
B [
%]
glycerol
PG
PHB
Amaricoccus dominated community
36
Implementation: Poly-glucose production from glycerol
e.g. corn Evap.
1% Glycerol
Pre-treatment
EOH
YeastFerment.
Glucose10% EOH
1% Glycerol
Poly glucose
CultivationAccumu-
lationwater
72
Modeling PHA production
Feast Famine regime based selection of
a PHA producing microbial community
37
73
Stoichiometry
Acetate uptake and activation:
1 1 1HAc ATP AcCoA
PHB production: 1 0.25 1AcCoA NADH PHB
PHB degradation: 1 0.25 1 0.25PHB ATP AcCoA NADH
Catabolism: 21 2 1AcCoA NADH CO
Oxidative phosphorylation:21 0.5NADH O ATP
3
1.8 0.5 0.2 2
1.267 0.2 2.16
1 0.434 0.267
AcCoA NH ATP
CH O N NADH CO
Anabolism:
C-mol based
74
Stoichiometry
Acetate uptake and activation:
1 1 1HAc ATP AcCoA
PHB production: 1 0.25 1AcCoA NADH PHB
PHB degradation: 1 0.25 1 0.25PHB ATP AcCoA NADH
Catabolism: 21 2 1AcCoA NADH CO
Oxidative phosphorylation:21 0.5NADH O ATP
3
1.8 0.5 0.2 2
1.267 0.2 2.16
1 0.434 0.267
AcCoA NH ATP
CH O N NADH CO
Anabolism:
Feast Phase: 5 reactions
38
75
Stoichiometry
Acetate uptake and activation:
1 1 1HAc ATP AcCoA
PHB production: 1 0.25 1AcCoA NADH PHB
PHB degradation: 1 0.25 1 0.25PHB ATP AcCoA NADH
Catabolism: 21 2 1AcCoA NADH CO
Oxidative phosphorylation:21 0.5NADH O ATP
3
1.8 0.5 0.2 2
1.267 0.2 2.16
1 0.434 0.267
AcCoA NH ATP
CH O N NADH CO
Anabolism:
Famine Phase: 4 reactions
76
Feast phase stoichiometry
Conserved moieties: ATP NADH AcCoA
Feast phase: 5 reactions, 3 conserved moieties 2 degrees of freedom 3 reaction stoichiometries: Catabolism, Growth, PHB production
Feast phase
Growth 2
/
0.1 3.16
2 1
feast
CO XY
2/
3.21
2 1
feast
O XY
/
2 1
2.1 2.16
feast
X AcY
3/
0.2feast
NH XY
PHB production 2
/
0.25 1
2 1
feast
CO PHBY
2/
1.125
2 1
feast
O PHBY
/
2 1
2.25
feast
PHB AcY
Catabolism 2
/1
feast
CO AcY
2/
1feast
O AcY
/2 1
feast
ATP AcY
1
39
77
Feast phase kinetics
/ / 2 / 2
feast feast feast feast feastAc Ac X X ATP C ATP PHB C PHBq Y q Y m Y q
limiting rate
limiting rate
estimate
calculate
Empty cells
/ / 2 / 2
feast feast feast feast feastAc Ac X X ATP C ATP PHB C PHBq Y q Y m Y q
calculate
limiting rate
estimate
limiting rate
Full cells
78
Feast phase kinetics
Acetate uptake
- with PHB inhibition
max
,1
( )( )
( )
Ac
Ac Ac
Ac Ac
c tq t q
K c t
,2
/ /
1 1( ) ( )
feast feast
Ac PHB Acfeast feast
X Ac PHB Ac
q t t q mY Y
If ,1 ,2
feast feast
PHB PHBq q
If ,1 ,2
feast feast
PHB PHBq q
Growth 3
3 3
max
( ) ( )( )
( ) ( )
NHfeast Ac
Ac AcNH NH
c t c tt
K c t K c t
Maintenance /
ATP
Ac feast
ATP Ac
mm
Y
PHB production
- with PHB inhibition
feast
,1 Ac PHB/Ac
/
1( ) q ( ) ( ) Y
feast feast
PHB Acfeast
X Ac
q t t t mY
max
,2 PHB max
( ) ( )( ) q 1
( ) ( )
feast Ac PHB
PHB
Ac Ac PHB
c t f tq t
K c t f t
If ,1 ,2
feast feast
PHB PHBq q
If ,1 ,2
feast feast
PHB PHBq q
CO2 evolution 2 2 2 2
/ / /( ) ( ) ( )
feast feast feast feast feast feast
CO i i CO X PHB i CO PHB Ac CO Acq t t Y q t Y m Y
O2 uptake 2 2 2 2
/ / /( ) ( ) ( )
feast feast feast feast feast feast
O i i O X PHB i O PHB Ac O Acq t t Y q t Y m Y
NH3 uptake 3 3
/( ) ( )
feast feast feast
NH i i NH Xq t t Y
1
40
79
Famine phase stoichiometry
Conserved moieties: ATP NADH AcCoA
Famine phase: 4 reactions, 3 conserved moieties 1 degree of freedom 2 reaction stoichiometries: Catabolism, Growth, PHB production
Famine phase
Growth 2
/
2.41 0.15
2.25 0.25
fam
CO XY
2/
2.693
2.25 0.25
fam
O XY
/
2.25 0.25
2.1 2.16
fam
X PHBY
3/
0.2fam
NH XY
Catabolism 2
/1
fam
CO PHBY
2/
1.125fam
O PHBY
/0.25 2.25
fam
ATP PHBY
1
80
Shrinking particle model
22
33
4
4 3
surface rk f f PHB
volume r
41
81
Famine phase kinetics
PHB degradation ,1
2 3( ) ( )
fam
PHB PHBq t k f t
Maintenance /
ATP
PHB fam
ATP PHB
mm
Y
Growth /
( ) ( ( ) )fam fam fam
X PHB PHB PHBt Y q t m
CO2 evolution 2 2 2
/ /( ) ( )
fam fam fam fam
CO CO X PHB CO PHBq t t Y m Y
O2 uptake 2 2 2
/ /( ) ( )
fam fam fam fam
O i i O X PHB O PHBq t t Y m Y
NH3 uptake 3 3
/( ) ( )
fam fam fam
NH i i NH Xq t t Y
1
82
Model parameters
Parameter / initial conditions Value Constant or estimated
Half-saturation constant for acetate 0.2Ac
CmmolK
l Constant
Half-saturation constant for ammonia 3
0.0001NH
mmolK
l Constant
Efficiency of oxid. phosphorylation
2
mmol ATP
mmol NADH Constant
Maintenance ATP requirement ATP
m Estimated
Max. acetate uptake rate max
Acq Estimated
Max. growth rate feast max
Estimated
Max. PHB production rate max
2PHB
Cmmolq
Cmmol h
Constant
Exponent of PHB inhibition term 1.24 Constant
Max. fraction of PHB max
PHBf Estimated in fed-batch, constant in SBR
experiments (value of fed-batch experiment)
Rate constant PHB degradation k Estimated
Initial concentration of acetate ( 0)Ac
c t Estimated
Initial concentration of PHB ( 0)PHB
c t Steady state assumption
Initial concentration of biomass ( 0)X
c t Steady state assumption
Initial concentration of ammonia 3( 0)
NHc t Estimated
Initial carbon dioxide evolution ( 0) 0 cumCE t mmol Constant
Initial oxygen uptake ( 0) 0 cumOU t mmol Constant
1
42
83
Initialisations
Parameter / initial conditions Value Constant or estimated
Half-saturation constant for acetate 0.2Ac
CmmolK
l Constant
Half-saturation constant for ammonia 3
0.0001NH
mmolK
l Constant
Efficiency of oxid. phosphorylation
2
mmol ATP
mmol NADH Constant
Maintenance ATP requirement ATP
m Estimated
Max. acetate uptake rate max
Acq Estimated
Max. growth rate feast max
Estimated
Max. PHB production rate max
2PHB
Cmmolq
Cmmol h
Constant
Exponent of PHB inhibition term 1.24 Constant
Max. fraction of PHB max
PHBf Estimated in fed-batch, constant in SBR
experiments (value of fed-batch experiment)
Rate constant PHB degradation k Estimated
Initial concentration of acetate ( 0)Ac
c t Estimated
Initial concentration of PHB ( 0)PHB
c t Steady state assumption
Initial concentration of biomass ( 0)X
c t Steady state assumption
Initial concentration of ammonia 3( 0)
NHc t Estimated
Initial carbon dioxide evolution ( 0) 0 cumCE t mmol Constant
Initial oxygen uptake ( 0) 0 cumOU t mmol Constant
1
84
Feast phase
Parameter / initial conditions Value Constant or estimated
Half-saturation constant for acetate 0.2Ac
CmmolK
l Constant
Half-saturation constant for ammonia 3
0.0001NH
mmolK
l Constant
Efficiency of oxid. phosphorylation
2
mmol ATP
mmol NADH Constant
Maintenance ATP requirement ATP
m Estimated
Max. acetate uptake rate max
Acq Estimated
Max. growth rate feast max
Estimated
Max. PHB production rate max
2PHB
Cmmolq
Cmmol h
Constant
Exponent of PHB inhibition term 1.24 Constant
Max. fraction of PHB max
PHBf Estimated in fed-batch, constant in SBR
experiments (value of fed-batch experiment)
Rate constant PHB degradation k Estimated
Initial concentration of acetate ( 0)Ac
c t Estimated
Initial concentration of PHB ( 0)PHB
c t Steady state assumption
Initial concentration of biomass ( 0)X
c t Steady state assumption
Initial concentration of ammonia 3( 0)
NHc t Estimated
Initial carbon dioxide evolution ( 0) 0 cumCE t mmol Constant
Initial oxygen uptake ( 0) 0 cumOU t mmol Constant
1
43
85
Famine phase
Parameter / initial conditions Value Constant or estimated
Half-saturation constant for acetate 0.2Ac
CmmolK
l Constant
Half-saturation constant for ammonia 3
0.0001NH
mmolK
l Constant
Efficiency of oxid. phosphorylation
2
mmol ATP
mmol NADH Constant
Maintenance ATP requirement ATP
m Estimated
Max. acetate uptake rate max
Acq Estimated
Max. growth rate feast max
Estimated
Max. PHB production rate max
2PHB
Cmmolq
Cmmol h
Constant
Exponent of PHB inhibition term 1.24 Constant
Max. fraction of PHB max
PHBf Estimated in fed-batch, constant in SBR
experiments (value of fed-batch experiment)
Rate constant PHB degradation k Estimated
Initial concentration of acetate ( 0)Ac
c t Estimated
Initial concentration of PHB ( 0)PHB
c t Steady state assumption
Initial concentration of biomass ( 0)X
c t Steady state assumption
Initial concentration of ammonia 3( 0)
NHc t Estimated
Initial carbon dioxide evolution ( 0) 0 cumCE t mmol Constant
Initial oxygen uptake ( 0) 0 cumOU t mmol Constant
1
86
SBR cultivation
12 h cycle, 24 h SRT
44
87
Fed batch accumulation
88
SBR cultivation
4 h cycle, 24 h SRT
45
89
Fed batch accumulation
90
Are we ready?
No!Don’t forget data processing!
Measured data need to be processed to evaluate model.Steps to be taken: Delay in gas measurements Correction for sampling Calculate mass balance values (cumulative moles) Inorganic carbon partitioning Check mass and electron balances
46
91
Delay in off-gas measurement
measured O2 transfer rate
“true” O2 transfer rate
92
Inorganic carbon
1 1
3HCO H
2 1
3CO H
2CO
2( )CO g
2 2,L CO COk a Kh
2 3H COKa
3HCOKa
L
G
Acetate
measurements
pH-control
estimated aqueous species
total inorganic carbon production
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93
C-balance
0
10
20
30
40
50
60
0 2 4 6 8 10 12
Time [h]
Ac,
PH
B,
bio
ma
ss,
CO
2 [
C-m
mo
l]
A Total C
biomass
CO2
PHB
94
e-balance
0
50
100
150
200
250
0 2 4 6 8 10 12
Time [h]
Ac,
PH
B,
bio
ma
ss,
O2
[e
-mm
ol]
Total e
biomass
-O2
PHB
48
THE REAL WORLD
Tests with industrial wastewater
PHA as product?
High value
biodegradable plastic
hydroxybutyrate monomer
Low value
paper additive,
low grade plastics (bermpaal)
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Current work
TU/e
Pilot plant
Cultivation: selection of PHA producing enrichment
Biomass
2
PHASequencing batch
reactorFed batch reactor
Accumulation: maximizing the cellular PHA content3
CSTR
Acidification: Production of VFA1
Waste water
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Design• TUD-Paques
• V ~ 300 L
• First test location: Mars(Veghel, The Netherlands)
• Startup: December 2012
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In summary…
Microbial Community Engineering (MCE) is a feasible
alternative for Genetic Engineering for production of PHA
MCE does not require aseptic conditions (E-consumption) and
is waste based (No competition with food production)
Different substrates result in different communities, but the
same functionality
Non-fermented substrates may enable polyglucose production
Industrial implementation is a challenge requiring a
multidisciplinary approach
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103
Agroindustry
Sugar beet
Sugar
Sugar extraction
Fermentation
Fermentation
Food
Biofuel
Biochemical
104
Wastewater sludge
Wastewater treatment
Purification
Conversion with methanol
Direct chemical conversion
Plastic
Biofuel
Biochemical
Sludge + PHA PHA
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