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Metabolic modeling of mixed substrate uptake forpolyhydroxyalkanoate (PHA) production

Yang Jiang, Marit Hebly, Robbert Kleerebezem, Gerard Muyzer, Mark C.M. van Loosdrecht*

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands

a r t i c l e i n f o

Article history:

Received 25 May 2010

Received in revised form

23 September 2010

Accepted 12 October 2010

Available online 20 October 2010

Keywords:

Mixed culture

Mixed substrate

Metabolic model

Copolymers

Plasticicumulans acidivorans

* Corresponding author. Tel.: þ31 15 278 109E-mail addresses: Y.Jiang@tudelft.nl (Y. Ji

tudelft.nl (G. Muyzer), M.C.M.vanLoosdrecht0043-1354/$ e see front matter ª 2010 Elsevdoi:10.1016/j.watres.2010.10.009

a b s t r a c t

Polyhydroxyalkanoate (PHA) production by mixed microbial communities can be estab-

lished in a two-stage process, consisting of a microbial enrichment step and a PHA accu-

mulation step. In this study, a mathematical model was constructed for evaluating the

influence of the carbon substrate composition on both steps of the PHA production process.

Experiments were conducted with acetate, propionate, and acetate propionate mixtures.

Microbial community analysis demonstrated that despite the changes in substrate

composition the dominant microorganism was Plasticicumulans acidivorans in all experi-

ments. A metabolic network model was established to investigate the processes observed.

The model based analysis indicated that adaptation of the acetate and propionate uptake

rate as a function of acetate and propionate concentrations in the substrate during culti-

vation occurred. The monomer composition of the PHA produced was found to be directly

related to the composition of the substrate. Propionate induced mainly polyhydroxy-

valerate (PHV) production whereas only polyhydroxybutyrate (PHB) was produced on

acetate. Accumulation experiments with acetate-propionate mixtures yielded PHB/PHV

mixtures in ratios directly related to the acetate and propionate uptake rate. The model

developed can be used as a useful tool to predict the PHA composition as a function of the

substrate composition for acetateepropionate mixtures.

ª 2010 Elsevier Ltd. All rights reserved.

1. Introduction axenic culture and waste organic carbon as raw material.

Polyhydroxyalkanoates (PHAs) are biopolymers produced

by many different bacteria as an intracellular carbon and

energy reservematerial (Steinbuchel andValentin, 1995). They

attract considerable attention as alternative for petroleum-

based plastics because they are biodegradable and made

from renewable resources (Braunegg et al., 1998). Industrial

production of PHAs generally uses pure cultures of natural or

genetically modified PHA producing bacteria. The price of

PHAs is much higher compared to petro-chemical based

plastic (Choi and Lee, 1999). Potentially, a reduction of the

costs of PHA production can be established by using a non-

1; fax: þ31 15 278 2355.ang), M.Heblij@tudelft.nl (@tudelft.nl (M.C.M. van Lier Ltd. All rights reserved

Agriculture is responsible for 95% global water usage, imp-

lying we should recycle organic materials from agricultural

sources as much as possible. The products from fermented

agro-residues can be used as the substrate of PHA synthesis

(Kleerebezem and van Loosdrecht, 2007; Reis et al., 2003).

For waste based production of PHA, a two-step process has

been proposed, consisting of (1) enrichment of a PHA

producingmixed culture with a feast and famine regime in an

open sequencing batch reactor (SBR) and (2) PHA production in

a fed-batch reactor under growth limited condition. This

process when fed with acetate has been reported to accumu-

late up to 89 wt% cellular PHB content (Johnson et al., 2009a),

M. Hebly), R.Kleerebezem@tudelft.nl (R. Kleerebezem), G.Muijzer@oosdrecht)..

Nomenclature

Ci concentration of the inhibitor [Cmol/L]

CPHA Concentration of PHA [Cmol/L]

CPHASRT Concentration of PHA calculated from SRT

[Cmol/L]

Cs concentration of substrate [Cmol/L]

CX concentration of active biomass [Cmol/L]

CXSRT concentration of active biomass calculated from

SRT [Cmol/L]

EM error between model and measurements

ESS error of model deviating from steady state

fPHA;X fraction of PHA on active biomass [Cmol/Cmol]

fPHB fraction of PHB over total PHA [Cmol/Cmol]

fPHB,X fraction of PHB on active biomass [Cmol/Cmol]

fPHV,X fraction of PHV on active biomass [Cmol/Cmol]

fPr fraction of propionate uptake to total carbon

uptake

k rate constant of PHA degradation

[(Cmol/Cmol)1/3/h]

KNH3 half-saturation constant for ammonia [mol/L]

KS half-saturation constant for substrate [Cmol/L]

Ki half-saturation constant for inhibitor [Cmol/L]

mS biomass specific substrate requirement for

maintenance [Cmol/Cmol/h]

mATP biomass specific ATP requirement for

maintenance [mol/Cmol/h]

mPHA biomass specific PHA requirement for

maintenance [Cmol/Cmol/h]

MwX molecular weight of active biomass per one mole

of carbon (incl. ash)¼ 25.1 g/Cmol

MwPHA molecular weight of PHA per one mole of carbon

(depends on the PHA composition) [g/Cmol]

PHA% PHA content of the biomass

qS biomass specific substrate uptake rate [Cmol/

Cmol/h]

qPHAfamine biomass specific PHA degradation rate in the

famine phase [Cmol/Cmol/h]

qSfeast biomass specific substrate uptake rate in the feast

phase [Cmol/Cmol/h]

qsmax maximum biomass specific substrate uptake rate

[Cmol/Cmol/h]

SSrelEi sum of squared relative errors between

measurements and model for compound i

t Model time [min]

Yfaminei;j stoichiometric yield of compound i on compound j

in the famine phase [Cmol/Cmol]

Yfeasti;j stoichiometric yield of compound i on compound j

in the feast phase [Cmol/Cmol]

Yi;j yield of compound i on compound j [Cmol/Cmol]

a exponent of PHA inhibition term

d efficiency of oxidative phosphorylation

[mol ATP/mol NADH2]

mfamine biomass specific growth rate in the famine phase

[1/h]

mfeast biomass specific growth rate in the feast phase

[1/h]

mmax maximum biomass specific growth rate in the

feast phase [1/h]

Abbreviations

DO dissolved oxygen

FISH fluorescence in situ hybridization

GAOs glycogen accumulating organisms

HB hydroxybutyrate

HRT hydraulic retention time

HV hydroxyvalerate

NaAc sodium acetate

NaPr sodium propionate

PAOs phosphate accumulating organisms

PHA polyhydroxyalkanoate

PHB polyhydroxybutyrate

PHH polyhydroxyhexanoate

PHMV polyhydroxymethylvalerate

PHV polyhydroxyvalerate

SBR sequencing batch reactor

SRT solid retention time

TSS total suspended solid

X biomass

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 3 0 9e1 3 2 11310

which is similar to the values reported for geneticallymodified

Escherichia coli strains (Slater et al., 1988). A general metabolic

model for both enrichment experiments in SBR and accumu-

lation experiment in fed-batch with acetate as substrate has

been constructed (Johnson et al., 2009b). This model has

shown excellent correlation with experimental data.

For full scale applications, however, it is aimed to use fer-

mented organicwastewater. Althoughacetate is usually among

the main fermentation products, significant concentrations of

propionate, butyrate, lactate and ethanol have been reported as

well in pre-fermented substrates (Bengtsson et al., 2008;

Temudo et al., 2007). It is therefore of interest to study the

impact of other fatty acids and mixtures thereof on the PHA

productionprocess.Asa result of usingpropionate as substrate,

a copolymer is normally synthesized consisting of 3-hydrox-

ybutyrate (HB) and 3-hydroxyvalerate (HV) monomers (Lemos

et al., 2006). The polymer properties of the homopolymer HB,

as generated from acetate as sole substrate, are stiff and brittle.

The copolymer, poly(hydroxybutyrate-hydroxyvalerate)

(PHBV), has properties more similar to polypropylene and is

thereforemore interesting. Until now, relatively fewmodels for

propionate and/or acetate usage are available. Dias et al. (2008)

describes a model for copolymers production on mixtures of

acetate and propionate, only focusing on the feast phase.

Oehmen et al. (2005, 2006, 2007) developed anaerobic and

aerobic models to simulate the behavior of polyphosphate-

accumulating organisms (PAOs) and glycogen-accumulating

organisms (GAOs) with propionate as substrate.

This paper aims to extend the existingmetabolic flux based

models for PHB production to describe the production of

copolymers from propionate as sole substrate, or mixtures of

acetate and propionate. The kinetic and stoichiometric prop-

erties of a microbial community using different substrates are

evaluated. Extensive analysis of the data allows predicting the

behavior of the process and the composition of the products

by this model.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 3 0 9e1 3 2 1 1311

2. Materials and methods

2.1. Enrichment culture in SBR

2L working volume double-jacket glass bioreactors (Applikon,

TheNetherlands) were used to enrich PHAproducing cultures.

Four SBR runs were conducted with four different acetate/

propionate ratios (100/0, 75/25, 50/50 and 0/100, Cmole based).

The reactors were operated as SBRs under non-axenic condi-

tions. Each SBR cycle consisted of four phases: (1) a 7 min start

phase, (2) a 10 min influent phase used to supply fresh

medium, (3) a 683 min reaction phase, and (4) a 20 min effluent

phase used to remove effluent from the reactor. The solid

retention time (SRT) and the hydraulic retention time (HRT)

were both maintained at one day. The end of an operational

cycle was immediately followed by the start phase of the next

cycle. Each enrichment culture was inoculated with biomass

from a SBR operated in the same conditions with acetate as

sole substrate formore than three years (Johnson et al., 2009a).

A steady state was considered to be obtained when stable

values were obtained for the length of the feast phase, total

suspended solids (TSS) concentration and the ammonium

concentration at the end of the cycle for at least five subse-

quent SBR cycles. The biomass for fed-batch accumulation

experiments was harvested from SBR after steady state was

achieved. The general reactor setup and biocontroller systems

were described by Johnson et al. (2009a).

2.2. PHA production in fed-batch reactor

In order to evaluate themaximumPHAproduction, a fed-batch

protocol was applied. 1L of biomass from the enrichment SBRs

was mixed with 1 L of carbon substrate and ammonium free

medium and introduced in the reactor (same composition as

for SBR, but no carbon source and NH4Cl). To avoid substrate

inhibition, a fed-batch approach was used to supply substrate

to the system. The initial substrate concentration was set to

30 mM (acetate, propionate, or a mixture of acetate and

propionate with desired ratio). Additional substrate was

supplied from a 1.5 M substrate stock solution (acetic acid,

propionic acid, or acetic/propionic acidmixture) in a fed-batch

mode using the pH-controller. Growth was restricted to the

first fewhours in these experiments as no nitrogen sourcewas

supplied and only a small amount of nitrogen source remained

from the previous SBR cycle. The progress of the experiments

was monitored via online (DO, pH, acid and base dosage, off-

gas CO2 and O2) and offline (acetate, propionate, TSS, PHA,

ammonium) measurements. A detailed description of the

analytical procedures for onlineandofflinemeasurements can

be found elsewhere (Johnson et al., 2009b).

2.3. Medium and cultivation methods

The medium for the SBR was dosed to the reactor from three

separate bottles containing respectively carbon substrate,

nutrients and dilution water. The medium for propionate-fed

SBR enrichment contained: 131 mM NaPr as carbon substrate;

67.5 mM NH4Cl, 24.9 mM KH2PO4, 5.6 mM MgSO4, 7.2 mM KCl,

15 ml/L trace elements solution according to (Vishniac and

Santer, 1957) in the nutrient source. In the other experi-

ments on acetate or mixtures of acetate and propionate, the

total amount of organic carbon-moles in the carbon source

was kept constant and the nutrient source was the same as

the propionate experiment. Allylthiourea (100 mg/L) was

added through a 0.2 mmfilter to the nutrients after autoclaving

to prevent nitrification. In the influent phase of each batch

cycle, 100 ml of carbon source, 100 ml of nutrient source and

800 ml of dilution water were mixed and pumped into the

reactor. All reactors were operated at 30 �C; the pH in the

reactor was controlled to 7� 0.05 by addition of 1 M HCl and

1 M NaOH.

2.4. Fluorescence in situ hybridization (FISH)

FISH was performed to investigate the microbial community

structure in all cultures. The oligonucleotide probes and

procedures used were the same as described in Johnson et al.

(2009a).

2.5. Calculations

TSS was assumed to be composed of active biomass (X ) and

PHA.ThemeasuredPHAcontentof thesludgewasexpressedas:

PHA% ¼ PHBþ PHVTSS

100% ðg=gÞ (1)

The active biomass concentration was calculated by sub-

tracting the amount of PHA from TSS.

Xðg=LÞ ¼ TSS

�1� PHA%

100

�(2)

And the fraction of PHA over active biomass fPHA;X was

expressed as:

fPHB;X ¼ PHB%100� PHA%

MwX

MwPHBðCmol=CmolÞ (3a)

fPHV;X ¼ PHV%100� PHA%

MwX

MwPHVðCmol=CmolÞ (3b)

fPHA;X ¼ fPHB;X þ fPHV;X ðCmol=CmolÞ (3c)

The molar weight of the active biomass includes the ash

content.

3. Metabolic model

3.1. Metabolism and basic reactions

Dias et al. (2008) proposed a metabolic model for PHB and PHV

production from a mixture of acetate and propionate. This

model focused on biopolymer production and therefore

described only the conversions during the feast phase. We

based our model on the Dias-model but several extensions

and modifications were made:

1. The metabolic reactions during the famine phase were

added to the model structure,

2. The biomass synthesis from acetyl-CoA and propionyl-CoA

were individually specified.

PHB PHV BiomassBiomassATP

HPrHAc

PrCoAAcCoANADH2

R1 R2

R3R8

R4 R5R6 R7R9

R11R10

Fig. 1 e A schematic representation of the PHAmetabolism.

Solid lines indicate the reactions involved in the basic

anabolism.Thedot lines represent the reaction for substrate

uptake during the feast phase. The dashed lines state the

PHA degradation reaction during the famine phase.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 3 0 9e1 3 2 11312

3. Maintenance ATP requirement (mATP) was used as estima-

tion parameter in the kinetics whereas the P/O ratio was

fixed.

4. The TCA cycle was assumed inactive when propionate was

the sole carbon source in the medium.

A schematic overview of the metabolic reactions involved

in thePHAproductionprocess is shown inFig. 1 anda complete

overview of the reaction stoichiometries are presented in

Table 1. All reactions are expressed on a carbon-mole basis.

Both acetate and propionate are assumed to be taken up by

the cells by active transport, requiring one mole of ATP per

mole of carbon source (Gottschalk, 1986). They are subse-

quently converted into acetyl-CoA and propionyl-CoA by

consuming another mole of ATP per mole of substrate. The

net reactions for acetate or propionate are denoted as R1 and

R2 respectively (Dias et al., 2008). It was observed that the

biomass consumed acetate and propionate simultaneously in

this work. To calculate the stoichiometric yields on mixed

substrate, the fraction of propionate uptake rate in the total

substrate uptake rate is defined as fPr:

Table 1 e Reactions considered in the metabolic model on a caphosphorylation.

Reaction

1 HAc uptake

2 HPr uptake

3 Conversion from PrCoA to AcCoA

4 PHB production

5 PHV production

6 Growth on AcCoA

7 Grwoth on PrCoA

8 Catabolism

9 Oxidative phosphorylation

10 PHB consumption

11 PHV consumption

fPr ¼ R2

R1 þ R2(4)

It has been reported that a fraction of the propionyl-CoA

formed can be converted into acetyl-CoA via five different

metabolic pathways (Lemos et al., 2006). The net material and

electron balances are identical in these pathways. Only the

electron carriers used and consequently the amount of energy

generated differs between these pathways. We used the same

conversion pathway from propionyl-CoA to acetyl-CoA as

described by Dias et al. (2008). The net reaction for this

conversion is shown as R3 (Table 1).

Except PHB and PHV, PHMV (polyhydroxymethylvalerate)

can theoretically be produced when using propionate as

carbon source. The monomer of PHMV can be formed by

coupling two propionyl-CoA units. However, no PHMV

formation was observed in this study. PHB and PHV were

therefore the only polymers taken into account for the current

model. The net reactions for polymer formation are shown in

R4 and R5 (Dias et al., 2008; Johnson et al., 2009b). As generally

observed in this study, HB and HV monomeric units were

polymerized simultaneously. The fraction of PHB in the total

PHA fPHB is defined by the following equation:

fPHB ¼ R4

R4 þ R5(5)

Biomass formation was assumed to occur from both acetyl-

CoA and propionyl-CoA. The energy requirements for biomass

production from acetyl-CoA and propionyl-CoA were esti-

mated as 2.16 (van Aalst van Leeuwen et al., 1997) and 1.38 mol

ATP per C-mol of active biomass (Dias et al., 2008). The reac-

tions are shown in R6 and R7 in Table 1. The active bio-

mass formula used in the stoichiometric calculation was

CH1.8O0.5N0.2 with a molecular weight of 25.1 g/Cmol,

including 2% ash (Beun et al., 2002).

It is not identifiable whether active biomass is formed from

either acetyl-CoA or propionyl-CoA. The experimental data

from Dias et al. (2008) were obtained under ammonia limiting

condition where cell growth is negligible. Here we assumed

that acetyl-CoA and propionyl-CoA use for active biomass

synthesis were proportional to the flux through acetyl-CoA

and propionyl-CoA. In other words, fPr and fPHB determine the

ratio between the growth occurring on acetyl-CoA and pro-

pionyl-CoAduring the feast and the faminephase respectively.

rbon-mole base. d is the efficiency of oxidative

Stoichiometry

1HAcþ 1ATP/ 1AcCoA

1HPrþ 2/3ATP/ 1PrCoA

1.5PrCoA/ 1 AcCoAþ 0.5CO2þ 1.5NADH2

1AcCoAþ 0.25NADH2/ 1PHB

0.4AcCoAþ 0.6PrCoAþ 0.2NADH2/ 1PHV

1.267AcCoAþ 0.2NH3þ 2.16ATP/ 1Xþ 0.267CO2þ 0.434NADH2

1.06PrCoAþ 0.2NH3þ 1.38ATP/ 1Xþ 0.06CO2þ 0.373NADH2

1AcCoA/ 1CO2þ2NADH2

1NADH2þ 0.5O2/ dATP

1PHBþ 0.25ATP/ 1AcCoAþ 0.25 NADH2

1PHVþ 0.2ATP/ 0.4AcCoAþ 0.6PrCoAþ 0.2NADH2

Table 2e Stoichiometric yields derived from themetabolic reactions and balances for the conservedmoieties, expressed asa function of the efficiency of oxidative phosphorylation d.

Feast phase

GrowthYfeastO2 ;X

¼ 13:9fPr þ 2:3f2Pr � 57:836dþ 6fPr þ 6dfPr � 18

; YfeastCO2 ;X

¼ 3d� 33:4fPr � 10dfPr þ 94:860dþ 10fPr þ 10dfPr � 30

;

YfeastX;S ¼ �6dþ fPr þ dfPr � 3

6:3d� 2:3fPr þ 6:5;

YfeastN;X ¼ �0:2

PHA productionYfeastO2 ;PHA ¼ 48fPr þ 9fPHB � 3fPrfPHB � 144

240dþ 40fPr þ 40fPrd� 120; Yfeast

CO2 ;PHA ¼ �20fPr � 24dþ 20dfPr þ 9dfPHB � 60120dþ 20fPr þ 20dfPr � 60

;

YfeastPHA;S ¼ �120dþ 20fPr þ 20dfPr � 60

144d� 9dfPHB;

MaintenanceYfeastO2 ;S

¼ fPr6

þ 1;YfeastCO2 ;S

¼ �1;

YfeastATP;S ¼ 1� fPr

3� dfPr

3� 2d;

Famine phase

GrowthYfamineO2 ;X

¼ 106fPHB � 7f2PHB þ 2246fPHB � 288dþ 18dfPHB þ 24

; YfamineCO2 ;X

¼ �250fPHB � 90d þ 45dfPHB þ 47415fPHB � 720d þ 45dfPHB þ 60

;

YfamineX;PHA ¼ 1:5fPHB � 72dþ 4:5dfPHB þ 6

63dþ 23:4fPHB � dfPHB þ 41:4;

YfamineN;X ¼ �0:2

Maintenance YfamineO2 ;PHA ¼ 1:2� 0:075fPHB; Yfamine

CO2 ;PHA ¼ �1;

YfamineATP;PHA ¼ 0:05fPHB � 2:4dþ 0:15fPHBdþ 0:2

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 3 0 9e1 3 2 1 1313

R6

R7¼ 1� fPr

fPr(6)

R6

R7¼ fPHB

1� fPHB(7)

Acetyl-CoA is converted to CO2 through the tricarboxylic acid

cycle (TCA, R8). The net reaction was described by van Aalst

van Leeuwen et al. (1997). The amount of ATP generated by

oxidizing one mole of NADH2 is expressed by the P/O ratio (d),

which represents the efficiency of oxidative phosphorylation.

This reaction is expressed in R9.

Degradation of PHB and PHV to acetyl-CoA and propionyl-

CoA require one mole of ATP per mole of building block. The

net reactions are shown in R10 and R11. Like the assumption

made during PHA production, PHB and PHV are assumed to be

simultaneously degraded to produce acetyl-CoA and pro-

pionyl-CoA.

R10

R11¼ fPHB

1� fPHB(8)

3.2. Determination of overall stoichiometry

The stoichiometric yield equations on mixed substrate were

calculated from the metabolic reaction stoichiometries and

mass and charge balances using the symbolic solver from

software MathCAD (Table 2). The conserved moieties, NADH2,

ATP, acetyl-CoA and propionyl-CoA and the elements C, O, H,

N were used to balance all the reactions.

3.2.1. Feast phaseIn the feast phase nine, lumpedmetabolic reactions (R1e9, Table

1) are active. Due to the four conserved moieties and three

constraints (Eqs. (4)e(6)); the degree of freedom is reduced from

nine rates to two rates. If two reaction rates are defined, the

remaining reaction rates can be calculated throughHerbert-Pirt

typeof relationshipduring the feast phase (Eq. (9)). This relation

couples substrate uptake to growth, PHA production and

maintenance through defined yields.

qfeastS ¼ mfeast

YfeastX;S

þ qfeastPHA

YfeastPHA;S

þmS (9)

3.2.2. Famine phaseIn the famine phase, only seven lumped metabolic reactions

(R3 and R6e11, Table 1) are active. As described for the feast

phase, the four conserved moieties and two constrains (Eqs.

(7) and (8)) decrease the degree of freedom from seven rates to

one rate. The HerbertePirt relation only contains PHA

consumption, growth and maintenance during the famine

phase (Eq. (10)).

qfaminePHA ¼ mfamine

YfamineX;PHA

þmS (10)

All the stoichiometric maximum yields during both the feast

phase and the famine phase are listed in Table 2.

3.3. Kinetic model

3.3.1. Feast phaseTwo limiting cases canoccur (Johnsonet al., 2009b). At lowPHA

contents the substrate uptake rate proceeds at a maximum

rate, limiting growth and PHA production. In this case, the

substrate uptake can be expressed by regular saturation

kinetics (eq. (3), Table 3) and the PHA synthesis rate (eq. (1),

Table 3) can be regarded as a resultant from substrate uptake,

minus substrate required for growth and maintenance

purposes. When PHA reaches a certain concentration in the

cell, the PHA synthesis rate and the growth rate are limiting

substrate uptake. In this case, the PHA synthesis rate can be

described using the product inhibition kinetics as previous

established by Johnson et al. (2009b) (eq. (2), Table 3). Herewith

the substrate uptake rate can be calculated as the sum from

Table 3 e Model kinetics.

Feast phase

PHA productionqfeastPHA;1ðtÞ ¼

qSðtÞ � mfeastðtÞ 1

YfeastX;S

�mS

!Yfeast

PHA;S if qfeastPHA;1 � qfeast

PHA;2 (1)

With PHA inhibitionqfeastPHA;2ðtÞ ¼ qmax

PHA

CSðtÞKS þ CSðtÞ

"1�

fPHA;XðtÞfmaxPHA;X

!a#if qfeast

PHA;1 � qfeastPHA;2 (2)

Substrate uptakeqS;1ðtÞ ¼ qmax

S

CSðtÞKS þ CSðtÞ if qfeast

PHA;1 � qfeastPHA;2 (3)

With PHA inhibitionqS;2ðtÞ ¼ mfeastðtÞ 1

YfeastX;S

þ qfeastPHA

1

YfeastPHA;S

þmS if qfeastPHA;1 � qfeast

PHA;2 (4)

GrowthmfeastðtÞ ¼ mmax CNH3

ðtÞKNH3

þ CNH3ðtÞ

CSðtÞKS þ CSðtÞ (5)

MaintenancemS ¼ mATP

YfeastATP;S

(6)

CO2 evolution qfeastCO2

ðtÞ ¼ mfeastðtÞYfeastCO2 ;X

þ qfeastPHA ðtÞYfeast

CO2 ;PHA þmSYfeastCO2 ;S

(7)

O2 uptake qfeastO2

ðtÞ ¼ mfeastðtÞYfeastO2 ;X

þ qfeastPHA ðtÞYfeast

O2 ;PHA þmSYfeastO2 ;S

(8)

NH3 uptake qfeastNH3

ðtÞ ¼ mfeastðtÞYfeastNH3 ;X

(9)

Famine phase

GrowthmfamineðtÞ ¼ Yfamine

X;PHA

�qfaminePHA ðtÞ �mPHA

�(10)

PHA degradation qfaminePHA ðtÞ ¼ kfPHA;XðtÞ2=3 (11)

MaintenancemPHA ¼ mATP

YfamineATP;PHA

(12)

CO2 evolution qfamineCO2

ðtÞ ¼ mfamineðtÞYfamineCO2 ;X

þmSYfamineCO2 ;PHA (13)

O2 uptake qfamineO2

ðtÞ ¼ mfamineðtÞYfamineO2 ;X

þmSYfamineO2 ;PHA (14)

NH3 uptake qfamineNH3

ðtÞ ¼ mfamineYfamineNH3 ;X

(15)

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 3 0 9e1 3 2 11314

substrate utilization for PHA production, growth, and main-

tenance purposes according to the HerbertePirt equation (eq.

(4), Table 3). All other rates are the same in both cases. The

growth rate can be described by Monod-type relation with

limitation by the substrate and ammonium (eq. (5), Table 3).

The substrate based maintenance rate is related to the ATP

based maintenance rate through the ATP yield on substrate

(eq. (6), Table 3). All other conversion rates can be obtained by

rearrangement of the HerbertePirt relation with the corre-

sponding yield factors. The stoichiometric relations for carbon

dioxide, oxygen and ammonium are included by eqs. (7)e(9) in

Table 3.

3.3.2. Famine phaseThe HerbertePirt relation describes growth as function of PHA

conversion andmaintenance use (eq. (10), Table 3). It has been

generally assumed that in the case of growth on a storage

polymer, the PHA conversion is the rate limiting step (Beun

et al., 2002; van Loosdrecht and Heijnen, 2002). The PHA

degradation rate during the famine phase was suggested to

depend on the PHA surface area and was therefore defined as

a two-third order function of the cellular PHA concentration

(Murnleitner et al., 1997) (eq. (11), Table 3). The consumption

of PHA for maintenance purposes was derived from the ATP

maintenance rate through the ATP yield on PHA (eq. (12),

Table 3). The conversions of carbon dioxide, oxygen and

ammonium are stoichiometrically derived from the Herbert-

Pirt relation (eqs. (13)e(15), Table 3).

3.4. Model calibration

For each sampling time point (ti), the modeled data for each

compound were compared with the measured data. The

relative errors were calculated, squared and summed up as

shown for the example of propionate in Eq. (11).

SSrelEPr ¼XNi¼1

�nmeasurePr ðtiÞ � nmodel

Pr ðtiÞnmeasurePr ðtiÞ

�2(11)

All the measurements were treated equally without bias on

the accuracy. The squared relative errors for different

measurements were summed to the total error between

measurements and model (Eq. (12)).

EM ¼X

SSrelEi (12)

with i¼Ac, Pr, NH3, PHB, PHV, CO2, O2.

In steadystate theamountof solid (activebiomassandPHA)

that is produced during one operational cycle is equal to the

amount of the solids removed at the end of the cycle. The SRT

definition can be used to calculate the concentration change

for active biomass and PHA in one cycle (Eqs. (13) and (14)):

CSRTX ðtendÞ ¼ CXð0Þ SRT

SRT� tcycle(13)

CSRTPHAðtendÞ ¼ CPHAð0Þ SRT

SRT� tcycle(14)

Table 4 e The experimental data sets collected foranalysis in this study.

Acetate/ Cycle Fed-batch

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 3 0 9e1 3 2 1 1315

The steady-state error was defined as Eq. (15).

ESS ¼hCSRTX ðtendÞ�Cmodel

X ðtendÞi2þhCSRTPHAðtendÞ�Cmodel

PHA ðtendÞi2

(15)

Propionate ratioa measurement measurement

SBR-I 100/0 þ þSBR-II 75/25 � þSBR-III 50/50 � þSBR-IV 0/100 þ þ

þ: The experiment preformed.

�: The experiment not performed.

a Cmole based.

For the cultivation experiments therewith the total error is

defined as the sum of EM and ESS. By minimizing this total

error using the solver tool in Microsoft Excel, all the kinetic

rates and concentrations of all relevant compounds were

calculated. Similar to the method used by Johnson et al.

(2009b), some parameters like half-saturation constant for

acetate, propionate and ammonia, P/O ratio, initial CO2

evolution andO2 uptakewere kept constant to compute all the

rates and concentrations. Some other relevant parameters

like, maintenance ATP requirement (mATP), maximum

substrate uptake rate (qmaxS ), the fraction of propionate uptake

rate in the total substrate uptake rate (fPr), maximum growth

rate (mmax), maximum PHA production rate (qmaxPHA), the fraction

of PHB in the total PHA (fPHB), exponent of PHA inhibition term

(a) andmaximum fraction of PHA (fmaxPHA ) were estimated by the

calibration procedure.

4. Results

4.1. Microbial diversity evaluation

The Plasticicumulans acidivorans dominated microbial

community was found to be very well capable of degrading

short chain fatty acids different from the acetate which it was

originally grown on. Even though the uptake rate was initially

much lower when the substrate was changed to propionate, it

took less than five SRTs to reach a steady-state. All the

experiments described here were conducted when the reac-

tors had been operated stably for three weeks. The microbial

community composition (Fig. 2) was proven to be stable and

independent of the substrate composition by FISH analysis. In

all operated SBRs, P. acidivorans was found as the only domi-

nant bacterial species (over 80% of the total culture). The

influence from changes in microbial community structure

could therefore be excluded and all the differences in

Fig. 2 e The fluorescence microscopic photographs of the mixe

ratio (left: 75/25, middle:/50/50, right: 0/100, Cmol based). The g

(EUB338 mix, red) and the specific probe for P. acidivorans was

indicates that both probes hybridized with the same bacteria (F

legend, the reader is referred to the web version of this article.)

metabolic activity were the result of variation in themetabolic

activity of a highly comparable microbial community.

4.2. Measurement and model evaluations

As described in the materials and methods section, we oper-

ated four SBRs using four acetate/propionate ratios as

substrate: 100/0, 75/25, 50/50 and 0/100 (Cmole based). The

measured data obtained from six distinct experiments were

analyzed in this study, including two data sets from cycle

measurements as obtained from the SBR used for enrich-

ments with only acetate or sole propionate and the other four

data sets from PHA accumulation experiments in fed-batch

reactors fed with all the tested acetate/propionate rations

(Table 4).

As an indication of the accuracy of the measurements, the

carbon balances and the electron balances of the data were

calculated (Table 5). The relative errors were small (less than

10%), indicating the measurements were adequate to be used

as the basis for the modeling.

Theconcentrationprofiles for thedifferent experiments are

given in Fig. 3. The experiments with acetate as sole substrate

gave comparable results compared to the work described by

Johnson et al. (2009b). Fig. 3 therefore displays only the

performance of the experiments conducted with sole propio-

nate or acetate/propionate mixtures. In the graph of the cycle

d culture in SBR fed with different acetate and propionate

eneral 16S rRNA probe for Eubacteria was labeled with Cy3

labeled with fluorescein (UCB823, green). The yellow color

or interpretation of the references to color in this figure

Table 5 e Observed variables and estimated parameters from both cycle measurements and accumulation experiments.

Unit Ac-SBR Ac-Batch 75%Acþ25%Pr-Batch

50%Acþ50%Pr-Batch

Pr-Batch Pr-SBR

Measured

data

C-balance [%] 2.8 (�4.8) 0.5 (�1.8) 0.57 (�4.2) �3.8 (�2.7) �1.5 (�0.8) 4.6 (�5.0)

e-balance [%] 0.4 (�4.4) �0.4 (�6.7) �0.1 (�5.0) �10.8 (�7.0) �1.0 (�0.7) 4.9 (�4.8)

PHAmax a [wt %] 53.0 87.7 85.2 80.2 60.0 33.1

Model

parameters

qSmax [Cmmol/Cmmol/h] �3.20 �2.00 �1.66 �1.45 �0.65 �0.94

fPr [Cmol/Cmol] 0.00 0.00 0.23 0.38 1.00 1.00

qPHAmax [Cmmol/Cmmol/h] 1.95 1.25 1.01 0.93 0.35 0.43

fPHB [Cmol/Cmol] 1.00 1.00 0.77 0.53 0.12 0.08

mmax [Cmmol/Cmmol/h] 0.05 0.09 0.08 0.06 0.12 0.27

k [(Cmol/Cmol)1/3/h] �0.25 N/A N/A N/A N/A �0.16

mATP [mmol/Cmmol/h] 0.013 0.000 0.000 0.000 0.227 0.057

a [e] 2.0 1.7 3.3 8.6 1.0 1.6

fPHA,Xmax [Cmol/Cmol] N/A 8.3 6.9 4.9 1.8 N/A

Model based

variables

YX,S [Cmol/Cmol] �0.02 �0.04 �0.05 �0.04 �0.19 �0.32

YPHA,S [Cmol/Cmol] �0.61 �0.61 �0.61 �0.64 �0.43 �0.42

qProCoA,AcCoA [Cmmol/Cmmol/h] 0.00 0.00 0.12 0.17 0.24 0.26

Ac: acetate.

Pr: propionate.

S: substrate (carbon source).

SBR: sequencing batch reactor.

X: active biomass.

N/A: not available.

a In SBR, PHAmax indicates the PHA content at the end of feast phase.

Fig. 3 e Modeling results. Figure shows the results obtained cycle measurement fed with sole propionate (A) and

accumulation experiments fed by a mixture of acetate and propionate with different ratio (B: 0/100, C: 50/50, D 75/25, Cmol

based). Full symbols represent the experimental data and full lines indicate the modeling results. (*) acetate, (-)propionate,

(3) ammonia, (:) active biomass, (A) PHB, (C) PHV, (e) oxygen and (D) carbon dioxide.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 3 0 9e1 3 2 11316

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 3 0 9e1 3 2 1 1317

measurement on sole propionate (Fig. 3, A), all measured

concentrations are shown in the graph, except for CO2 and

O2 concentrations that are presented as cumulative data

calculated from off-gas concentrations. In the graphs for

the accumulation experiments (Fig. 3, BeD), the substrate

concentration shown is calculated by assuming that all

substrate was supplied to the system at the beginning of the

experiment. Fig. 4 shows the coefficient of determination (R2)

between the experimental data and modeled data in all

experiments, excluding experiments performed on sole

acetate. The values of R2were all very close to 1, indicating that

the model could adequately describe the dynamic behavior of

the different cultures during feast and famine periods of the

cultivation experiments as well as PHA accumulation in the

accumulation experiments in absence of ammonium.

4.3. Observed variables and estimated parameters

Somemeasured experimental data, estimatedparameters and

model-based variables are shown in Table 5. The maximum

PHAcontentwasderiveddirectly fromthemeasurements. The

0

100

200

300

400

500

0 100 200 300 400 500Measured Acetate [Cmmol]

Mod

eled

Ace

tate

[Cm

mol

]

Mod

eled

Pro

pion

ate

[Cm

mol

]

0

50

100

150

200

250

0 50 100 150 200 250

Mod

eled

PH

B [C

mm

ol]

Mod

eled

PH

V [C

mm

ol]

R2=0.969

R2=0.996

Measured PHB [Cmmol]

Fig. 4 e Modeling results. Figures show the comparison betwee

(solid lines) from both cycle measurement and accumulation ex

propionate) and two major products were analyzed. (-) The ex

measurement, (:) 100% propionate fed-batch experiment, (A) 5

(C) 75% acetate and 25% propionate fed-batch experiment.

PHA content at the end of the feast phase during the cycle

measurements and at the end of the accumulation experi-

ments are listed in the top part of Table 5. In both the cycle

experiment and the accumulation experiment, the maximum

PHAcontentwasabout 20wt% lowerwhenpropionatewas the

sole substrate.

Stoichiometric and kinetic parameter values were esti-

mated by calibration of themodel with the experimental data.

Key model parameters are listed in the middle part of Table 5.

Some parameters are only functional in the famine phase and

were therefore only estimated for the cycle measurements

(e.g. the k-value describing the PHA degradation rate). Other

parameters could only be estimated from the accumulation

experiments, like fmaxPHA;X. It was noticed that the qmax

S -values

obtained from cycle measurements were higher than those

estimated from the accumulation experiments with the same

substrate. This is potentially due to substrate inhibition

related to the higher initial substrate concentration in the

accumulation experiments. Some clear trends can be

observed: at the higher fractions of acetate in the substrate,

higher values were found for qmaxS , qmax

PHA and fPHB but a lower

2

0

100

200

300

400

500

0 100 200 300 400 500Measured Propionate [Cmmol]

0

20

40

60

80

100

0 20 40 60 80 100

R =0.991

R2=0.938

Measured PHV [Cmmol]

n the experimental data (full symbols) and modeled data

periments. Totally, two carbon-sources (acetate and

perimental data are from propionate SBR cycle

0% acetate and 50% propionate fed-batch experiment and

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 3 0 9e1 3 2 11318

value for fPr. The values found for mATP were low and had

amarginal impact on the reaction stoichiometry. The biomass

specific acetate uptake rate and propionate uptake rate can be

calculated from the overall biomass specific substrate uptake

rate (qmaxS ) and fPr. The maximum acetate uptake rate was

obtained from the experiment with acetate as sole substrate,

whereas the maximum propionate uptake rate was obtained

with propionate as sole substrate. In the mixed substrate

experiments, both acetate and propionate uptake rates were

lower than theirmaximumvalue. By increasing the fraction of

propionate in the substrate, the acetate uptake rate decreased

almost linearly. In contrast, the propionate uptake rate only

showed a significant decreasewhen the acetate fraction in the

substrate was 75% (Fig. 5).

The yield of active biomass on substrate (YX;S) and the yield

of PHA on substrate (YPHA;S) were calculated from the model

parameters to define the carbon fractions used for storage and

for growth on both substrates. The higher YX;S-values and

lower YPHA;S-values obtained from propionate fed experi-

ments indicated that more substrate was used for active

biomass synthesis and less substrate was used for PHA

production. One reaction, oxidizing propionyl-CoA to acetyl-

CoA (R3, Fig. 1), only occurs when propionate is present in the

substrate. It can be noticed this reaction rate calculated by the

model increasedwith an increase of the propionate fraction in

the substrate.

5. Discussion

5.1. Influence of the substrate composition on the PHAcontent

The PHA production capacity of the highly similar mixed

culture was strongly influenced by the composition of the

substrates. The maximum PHA contents obtained from fed-

batch experiments were consistently higher than 80 wt%

when acetate was present in the substrate. The maximum

PHA content from the fed-batch experiment with propionate

as the sole substrate was only about 60 wt %. Despite this

0.00

0.05

0.10

0.15

0.20

0.25

0 20 40 60 80 100

Fraction of Acetate [Cmmol %]

q [m

mol

/Cm

mol

/h]

0.0

0.2

0.4

0.6

0.8

1.0

1.2

q [m

mol

/Cm

mol

/h]

Fig. 5 e Biomass specific substrate uptake rates as

a function of the fraction acetate in the feed. The influence

of substrate composition on the acetate uptake rate (A)

and propionate uptake rate (:). The solid and dashed line

indicates the trend of acetate and propionate uptake rate

change.

lower value, it still is the highest PHA-content with propionate

as sole substrate reported till now (Dias et al., 2006; Lemos

et al., 2008). The high accumulation capacity seems strongly

related to the dominance of P. acidivorans in the microbial

community. A P. acidivorans dominated microbial community

was previously shown to have a superior PHB storing capacity

on acetate (Johnson et al., 2009a).

The reason for the lower maximum PHA content when

propionate was used as sole substrate remains unclear. A

explanation is that both PHA synthesis and degradation occur

simultaneously (Ren et al., 2009). At an increasing PHA

content, the PHA synthesis rate gets limited by product inhi-

bition, whereas the PHA degradation rate increases. When the

PHA production rate equals the PHA degradation rate, the

maximum PHA content is reached. Our results demonstrated

that the propionate uptake rate is significantly lower than the

acetate uptake rate. This suggests that PHA synthesis equals

PHA degradation at a lower PHA content when propionate is

used as substrate.

In the propionate fed SBR, a lower PHA content at the end

of the feast phasewas observed as well (Table 5). This is due to

the lower substrate uptake rate and higher growth rate

observed with propionate. The values of YX;S also indicated

that more substrate (about 30%, Cmmol basis) was directed

towards active biomass when the culture was fed with

propionate only. In contrast, YPHA;S values were higher when

acetate was the substrate (Table 5). Therefore, with respect to

rates andmaximal storage capacity, acetate is a more suitable

substrate for PHA production than propionate.

5.2. Influence of the substrate composition on polymercomposition

The composition of the final polymers strongly depended on

the composition of the substrate. The homopolymer PHB was

usually observed when biomass was fed with acetate as sole

substrate as described before by others (Dionisi et al., 2004;

Lemos et al., 2006; Dias et al., 2008). When propionate was

used as sole substrate, the composition of copolymer in this

study varied slightly from previous studies. Both Lemos et al.

(2006) and Dias et al. (2008) reported amixedmicrobial culture

enriched on propionate. The polymers composition these

authors described was approximately 20 Cmol % HB and 80

Cmol % HV, compared to 11 Cmol % HB and 89 Cmol % HV

observed in our work. Dionisi et al. (2004) enriched a mixed

microbial culture on a mixture of acetate, propionate and

lactate. When this culture was fed with propionate only, pure

PHV was obtained.

No PHMV was observed in this study. This can probably be

attributed to the specific properties P. acidivorans that is

dominating the microbial community described here. The

PHA synthase of P. acidivorans belongs to class III (based on

genome sequencing, data not shown). Class III PHA synthase

preferably produces short chain length PHA, although some

papers have reported that it also had a slight affinity to

synthesize 3HA middle chain length PHA (e.g. Poly-

hydroxyhexanoate, PHH) (Yuan et al., 2001). It seems like that

the PHA-synthase in P. acidivorans prefers to bind one mole-

cule of acetyl-CoA with one molecule of acyl-CoA (e.g. acetyl-

CoA, propionyl-CoA, butyryl-CoA). The synthase seems to lack

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 3 0 9e1 3 2 1 1319

the capacity to bind two propionyl-CoAmolecules as required

for PHMV production. Whether this is a general characteristic

of class III PHA-synthase genes remains to be elucidated.

Microorganisms related to PAOs or GAOs were reported to

produce PHMV from propionate (Oehmen et al., 2005, 2006,

2007). These microbial enrichments were selected under

alternating anaerobic and aerobic condition. PHAs are gener-

ated in the anaerobic period from fatty acids and aerobically

stored glycogen. As opposed to our P. acidivorans dominated

culture; two propionyl-CoA units can be combined to produce

PHMV. Lemos et al. (2006) and Dias et al. (2008) reported PHMV

production also with a mixed microbial culture enriched on

propionate under aerobic condition. However, the dominant

microorganism in their enrichments was reported as Amar-

icoccus sp. (Lemos et al., 2008), which is closely related to GAOs.

Therefore, the PHMV producing capacity is strongly depen-

dent on bacterial species.

5.3. Influence of the substrate composition on thesubstrate uptake rates

The specific total substrate uptake rate decreased at an

increasing fraction of propionate in the feed, and this decrease

was mainly the result of an apparent decrease in the acetate

uptake rate. Considering that the microbial community

structures in all enrichment were highly similar, the micro-

organisms adapted their substrate uptake strategy to the new

acetate/propionate ratio in the medium. It is an interesting

observation, because the PHA composition is also closely

related to the acetate and propionate uptake rate. Most of the

PHB produced is derived from the consumed acetate while

a small part (about 10%) originates from the consumed

propionate. However, the mechanism by which microorgan-

isms adapt their relative substrate uptake rate to the substrate

composition remains unclear. Over-expression of specific

transporters may play a role but other bottlenecks in the

biochemical pathways cannot be excluded.

The influence of the substrate composition on the biomass

specific substrate uptake rate has been reported before

(Dionisi et al., 2004; Lemos et al., 2006; Dias et al., 2008). All

these authors investigated the response of biomass to the

external substrate after a short-term substrate shift, while our

experiments were conducted after prolonged cultivation on

the modified substrate. A direct comparison of our results

with these previous results is therefore difficult.

5.4. Rate-limiting step during PHA accumulation

Reaction R3 (Fig. 1), converting propionyl-CoA to acetyl-CoA,

has been suggested as rate-limiting step when using propio-

nate as sole substrate (Dionisi et al., 2004; Lemos et al., 2006;

Dias et al., 2008). The low reaction rate of R3 can limit the

propionate uptake rate. In presence of acetate, acetyl-CoA can

be produced directly from acetate uptake (R1), decreasing the

need for converting propionyl-CoA to acetyl-CoA (R3). Conse-

quently, the propionate uptake rate can be enhanced by

acetate uptake if R3 is rate-limiting. This phenomenon has

been observed by short-term substrate shift experiments

conducted by Dionisi et al. (2004), Lemos et al. (2006) and Dias

et al. (2008).

In contrast, the long-term substrate shift experiments

from this study suggest that in our case the biomass specific

propionate uptake rate may be the rate-limiting. As described

above, the need of R3 is less when acetate is present. There-

fore, it can be expected that the PHV synthesis rate will be

augmented by increasing the fraction of acetate in the

substrate. In the mixed substrate experiment (50/50, Cmmol

based), PHVwasmore efficiently synthesized compared to the

sole propionate condition. However, when the proportion of

acetate was further increased (75/25, Cmmol based), the PHV

synthesis rate was unexpectedly decreased. This suggests

that not R3 but the specific substrate uptake rates were

determining the flux through the metabolic network, with

a preference for acetate uptake over propionate.

5.5. Maximum P/O ratio

The P/O ratio is a crucial parameter in themodel and is used to

provide insight in the efficiency of oxidative phosphorylation.

Like in many metabolic modeling studies (Beun et al., 2000;

Johnson et al., 2009b), we also used a fixed P/O ratio in our

study. Usually it is not possible to identify the P/O ratio

independently from the ATP need formaintenance: a high P/O

ratio and a high maintenance ATP consumption can give

a similar result as a low P/O ratio and a low maintenance ATP

consumption (Johnson et al., 2009b; Lopez-Vazquez et al.,

2009). When the metabolism is rather simple, there however

exists a possibility to independently estimate the P/O ratio. For

instance, Smolders et al. (1994) estimated the P/O ratio directly

from macroscopic conversions from PAOs. The value of P/O

ratio calculated in their study (d¼ 1.85).

In our case for the conversion of propionate to PHA with

propionate as the sole substrate, also only a limited amount of

biochemical reactions are involved. Stoichiometric analysis

demonstrated that the TCA cycle is reversed if choosing a P/O

ratio value exceeds two, suggesting there is a maximum P/O

ratio value when biomass was fed with propionate only.

Adequate reducing power can potentially be generated by

converting propionyl-CoA to acetyl-CoA, reducing the require-

ment of TCA cycle to generate reducing power. Theoretically,

TCA cycle has no need to be active when a maximal P/O ratio

can be established. To investigate at which P/O ratio the TCA

cycle is not needed for energy production, we assumed that the

flux through the TCA cycle was negligible in accumulation

experiments with propionate as sole substrate. In absence of

a flux through the TCA cycle, the maximum P/O ratio can esti-

mated from the fraction of PHB in the polymer (fPHB, Eq. (16))

d ¼ 4fPHB þ 1617fPHB þ 8

(16)

This equation suggests that if only PHV is produced (fPHB ¼ 0),

the maximum P/O ratio amounts 2. The average value of fPHB

identified from experiments with propionate as sole subs-

trate was approximately 10% (Table 5), corresponding to a

maximum P/O ratio around 1.7. This P/O ratio is similar to the

one calculated by Smolders et al. (1994). Considering the stable

microbial community, an identical P/O ratio of 1.7 mmol ATP/

mmol NADH2 was assigned to all experiment in this study.

However, it should be noted that the solid evidence for the

absence of TCA cycle in sole propionate experiment under

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 3 0 9e1 3 2 11320

aerobic condition is still missing. Only Pijuan et al. (2008)

found the contribution of NADH2 from TCA cycle was negli-

gible when feeding PAOs with only propionate in anaerobic

condition. A lower P/O ratio is required when there is a flux

through the TCA cycle.

6. Conclusions

In this study a metabolic model has been developed that

adequately describes the dynamics of a microbial community

producing PHA dominated by P. acidivorans. The model

includes regulation for simultaneous acetate and propionate

uptake, and the production of both PHV and PHB as a function

of the acetate-propionate substratemixture composition. Both

cultivation experiments in SBRs and fed-batch experiments

were modeled. The stoichiometric and kinetic parameters

obtained from the experiments with different substrate

mixtures can be used for predicting the polymer composition

and rate of production.

Acknowledgements

We thank Gert van der Steen for the analytical work. The

investigation was supported by the Netherlands Organisation

for Scientific Research (NWO) in the NWO-ACTS research

programme B_BASIC and by the Foundation for Technical

Sciences (STW).

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