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

[email protected]

Outline

Ecology of storage polymers

Process 1: phosphate removal from sewage

Process 2: feast famine regime for PHA production from

wastewater

mailto:[email protected]

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


Acetate Acetyl-CoA

CO2

PHB

Biomass


ATP

O2

ATP

ATPNH4

+

24

Acetate Acetyl-CoA

CO2

PHB

O2

ATP

ATP



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

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


1 1 1HAc ATP AcCoA





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


1 1 1HAc ATP AcCoA





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


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



CmmolK

l Constant


0.0001NH

mmolK

l Constant


2

mmol ATP

mmol NADH Constant


m Estimated


Acq Estimated


Estimated


2PHB

Cmmolq

Cmmol h

Constant







c t Estimated






NHc t Estimated



1

84

Feast phase



CmmolK

l Constant


0.0001NH

mmolK

l Constant


2

mmol ATP

mmol NADH Constant


m Estimated


Acq Estimated


Estimated


2PHB

Cmmolq

Cmmol h

Constant







c t Estimated






NHc t Estimated



1

43

85

Famine phase



CmmolK

l Constant


0.0001NH

mmolK

l Constant


2

mmol ATP

mmol NADH Constant


m Estimated


Acq Estimated


Estimated


2PHB

Cmmolq

Cmmol h

Constant







c t Estimated






NHc t Estimated



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

47

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)

49

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

50

Design• TUD-Paques

• V ~ 300 L

• First test location: Mars(Veghel, The Netherlands)

• Startup: December 2012

51

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

52

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

20150320 mce - storage polymers

Documents

energy storage

cmol acetate

cmol glycogen

paospolyhydroxyalkanoates

combined c

examplesglycogenpolyglucose

prokaryotesstarch c

energy eukaryotes