Bioresource Technology 148 (2013) 3038

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Impact of sustaining a controlled residual growth on

polyhydroxybutyrate yield and production kinetics
in Cupriavidus necator
Estelle Grousseau a,b,c,d,, Elise Blanchet a,b,c,d, Stphane Dlris d, Maria G.E. Albuquerque d,
Etienne Paul a,b,c, Jean-Louis Uribelarrea a,b,c
a

Universit de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France
INRA, UMR792 Ingnierie des Systmes Biologiques et des Procds, F-31400 Toulouse, France
c
CNRS, UMR5504, F-31400 Toulouse, France
d
VEOLIA Environnement, Centre de Recherche sur lEau, Chemin de la Digue, BP 76, F-78603 Maisons-Laftte Cedex, France
b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 A complementary modeling and

experimental approach for PHB

production was used.
 The anabolic requirements control
NADPH regeneration pathways and
availability.
 Sustaining a residual growth does not
alter PHB production yield.
 Sustaining a residual growth
improves PHB specic production
rate.

a r t i c l e

i n f o

Article history:
Received 20 June 2013
Received in revised form 31 July 2013
Accepted 2 August 2013
Available online 26 August 2013
Keywords:
Cupriavidus necator
Polyhydroxybutyrate (PHB)
Volatiles fatty acids (VFA)
Fed-Batch fermentation
Metabolic Flux Analysis (MFA)

a b s t r a c t
In this study a complementary modeling and experimental approach was used to explore how growth
controls the NADPH generation and availability, and the resulting impact on PHB (polyhydroxybutyrate)
yields and kinetics. The results show that the anabolic demand allowed the NADPH production through
the Entner-Doudoroff (ED) pathway, leading to a high maximal theoretical PHB production yield of
0.89 Cmole Cmole1; whereas without biomass production, NADPH regeneration is only possible via
the isocitrate dehydrogenase leading to a theoretical yield of 0.67 Cmole Cmole1. Furthermore, the maximum specic rate of NADPH produced at maximal growth rate (to full biomass requirement) was found
to be the maximum set in every conditions, which by consequence determines the maximal PHB production rate.
These results imply that sustaining a controlled residual growth improves the PHB specic production
rate without altering production yield.
2013 Elsevier Ltd. All rights reserved.

Corresponding author. Address: Laboratoire dIngnierie des Systmes Biologiques et des Procds INSA, UMR INSA/CNRS 5504 UMR INSA/INRA 792, 135
Avenue de Rangueil, 31077 Toulouse Cedex 4, France. Tel.: +33 (0)5 61 55 94 70.
E-mail addresses: estelle.grousseau@insa-toulouse.fr (E. Grousseau), blanchet.
elise@gmail.com (E. Blanchet), stephane.deleris@veoliaeau.fr (S. Dlris), Maria.
ALBUQUERQUE@veolia.com (M.G.E. Albuquerque), etienne.paul@insa-toulouse.
fr (E. Paul), uribelarrea@insa-toulouse.fr (J.-L. Uribelarrea).
0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2013.08.120

1. Introduction
There exists increasing industrial interest in the microbial
production of polyhydroxyalkanoates (PHA). PHA are biobased
and biodegradable polymeric materials that can be an interesting
substitute to petroleum derived plastics. PHA production naturally

E. Grousseau et al. / Bioresource Technology 148 (2013) 3038

31

Nomenclature
ai
DCW
kED
kTCA
nCi
qi
qNADPH
qNADPH
QPHB
RQ
rj
rs

molar
accumulation
of
the
ith
metabolite
(mole Cmole1 h1)
dry cell weight (g L1)
proportional factor of NADPH generation by ED to
NADPH requirement for anabolism
maximum NADPH ux generated by the tricarboxylic
acid cycle (TCA) (mmole Cmole1 h1)
number of carbon of the metabolite i (Cmole mole1)
specic i production rate (Cmole Cmole1 h1)
Xrmax maximum specic rate of NADPH generated
(reached at lmax) (mmole Cmole1 h1)
required for anabolic demand

Xr NADPH
(mmole Cmole1 h1)
overall volumetric PHB productivity (g L1 h1)
respiratory quotient (Cmole mole1)
specic molar ux of the reaction j (mole Cmole1 h1)
substrate consumption rate (Cmole L1 h1)

occurs under the unfavorable growth conditions of nutrient limitation such as oxygen, nitrogen, phosphorus, with adequate availability of carbon (Koller et al., 2010). About 90 monomers of
hydroxyalkanoates have been reported in the literature (Steinbuchel and Valentin, 1995). Among them 3-hydroxybutyrate (3HB)
is the most common naturally occurring from various carbon
sources.
Hundreds of bacterial genera are able to synthesize and store
PHA (Koller et al., 2010). Among them Cupriavidus necator (formerly known as Ralstonia eutropha) has been widely used because
of its industrial interest (Verlinden et al., 2007). This strain is a
gram-negative obligate aerobic bacterium living autotrophically
or heterotrophically using a variety of carbon sources.
Despite signicant decreases in recent years, PHA production
costs are still high compared to current traditional plastics. High
substrate conversion yield, high productivity, and high PHA content in microbial cells are important industrial criteria to decrease
production costs (Choi and Lee, 1999). The use of low cost, renewable carbon sources is also of major interest for an economic and
sustainable production of PHA (Koller et al., 2010). For example,
the by-products from biodiesel industry (Garcia et al., 2013; Vrana
Spoljaric et al., 2013) or Volatile Fatty Acids (VFA) are both suitable
carbon sources. VFA such as valeric, butyric, propionic, lactic, or
acetic acids, can be obtained from acidogenic fermentation of different kinds of waste streams such as industrial, agricultural or
municipal wastewater (Du et al., 2004; Li and Yu, 2011; Yu,
2001; Marang et al., 2013). PHA biosynthesis has been extensively
investigated on carbohydrates as single substrate and very high
titers, productivities and yields have been achieved (Ryu et al.,
1997). In contrast, limited knowledge is available on VFA substrates utilization for PHA production. In particular, butyric acid
has not been extensively studied (Du et al., 2004; Yu and Si,
2004; Zinn et al., 2003) despite great conversion yield (Shi et al.,
1997; Marang et al., 2013). In order to determine the carbon ux
distribution during PHA biosynthesis from various carbon sources,
stoichiometric and Metabolic Flux Analysis (MFA) approaches have
been proposed (Shi et al., 1997; Yamane, 1993; Yu, 2001). Although
all of these studies pointed out the critical role of NADPH in PHA
synthesis, the inuence of NADPH generation pathway on PHA production has not been intensively investigated. Moreover few
authors have evaluated the effects of sustaining growth during
PHA-production on organic acids (Kobayashi et al., 2000; Shimizu
et al., 1993; Yu and Si, 2004).

S
Xr
YATP,Xr
YP,Xr
YS,i
Y tS;i
Y lim
S;i
Y theo
S;i
%PHA

l
ei;j

substrate
residual biomass
energetic biomass yield (gXr MoleATP1)
overall residual biomass production on phosphorus
yield (Cmole Mole1)
overall i production on substrate yield (Cmole Cmole1)
instantaneous i production on substrate yield
(Cmole Cmole1)
limit i production on substrate yield (Cmole Cmole1)
theoretical
i
production
on
substrate
yield
(Cmole Cmole1)
intracellular PHA content (g g1)
growth rate (h1)
stoichiometric coefcient for the metabolite i in the
reaction j

In this study, well controlled phosphorus limited fed-batch cultures were carried out with butyric acid as substrate under noninhibitory feeding conditions. A stoichiometric model and a kinetic
model of the organism were constructed. The experiments and
modeling were integrated to explore how the microbial growth
sustained by a controlled phosphorus feeding inuences the
NADPH generation pathway and availability, and to explain the
resulting impacts on polyhydroxybutyrate (PHB) yields and
kinetics.
2. Methods
2.1. Micro-organism
The strain C. necator DSM 545 (or H1 G+3) was obtained from
DSMZ (Germany). The strain was stored at 80 C in liquid NB
medium (Nutrient Broth Merck: Meat Peptone 5 g L1, Meat extract 3 g L1) with 20% glycerol (v/v). Strain DSM 545 is a spontaneous mutant of the strain DSM 529 (H1) and is able to
catabolize glucose unlike the wild type C. necator DSM 428 (H16).
2.2. Seed cultures
One glycerol stock was streaked on a NB medium Petri dish (NB
with addition of Agar 15 g L1). The plate was incubated for
2448 h at 30 C. One colony was used to inoculate the rst seed
culture grown for 12 h in tube with 10 mL of Liquid NB medium
(30 C, 200 RPM). The second seed culture was grown for
1215 h with 150 mL of mineral-salt medium in a 1 L Erlenmeyer
ask (30 C, 100 RPM). The mineral salt medium composition is described in the literature by Aragao et al., 1996 except for the carbon
source (Glucose added to a nal concentration of 4 g L1) and for
the phosphorus source (2 mL/L of a solution of 224 g L1 of Na2HPO412 H2O and 37.5 g L1 of KH2PO4). This latter culture was
used to inoculate the bioreactor.
2.3. Fed-Batch culture conditions and bioreactor system
The Fed-Batch culture was performed in a 2 L (working volume)
fermentor (Braun Biotech, Melsungen Germany) with an initial
medium volume of 1.2 L, equipped with pH, dissolved oxygen
(DO), temperature, pressure and Anti-foam controllers. The on-line

32

E. Grousseau et al. / Bioresource Technology 148 (2013) 3038

monitoring and control systems of the reactor were handled by the

software BIOPAT MFCS/win. Both CO2 and O2 in inlet and outlet
gazes were analyzed using 1313 fermentation monitor INNOVA
Airtech Instrument, and the 1309 multipoint sampler (LUMASENSE
TECHNOLOGIES A/S, Ballerup, Denmark). The DO level in the reactor was controlled above 20% of air saturation by varying stirring
speed and/or inlet air ow rate. An overpressure of 75 mBar was
applied in the bioreactor headspace. The pH was maintained at
7.0 by addition of a 14% (v/v) NH3 solution. Temperature was maintained at 30 C. The culture medium was the mineral salt medium
described by Aragao et al., 1996, except for the initial amount of
phosphorus (0,654 mmole L1, 8 mL L1 of a solution of 25 g L1
of Na2HPO412 H2O and 1.6 g L1 of KH2PO4) and the carbon source
(butyric acid at a concentration of 190,6 g L1). Two bottles of carbon sources were prepared. In one of them, phosphorus was added
to support growth during the rst PHB production phase
(9.3 mL L1 of a solution of 224 g L1 of Na2HPO412 H2O and
37.5 g L1 of KH2PO4). The added masses of substrate and NH3
were monitored in real time course by weight.
2.4. Analytical procedures
Culture supernatant was obtained by centrifuging (MiniSpin
Eppendorf, USA) fermentation broth samples in Eppendorf tubes
at 13400 rpm for 3 min and used directly for volatile acids determination. The harvested biomass was used for dry cell weight
and PHB measurements.
The residual volatile fatty acids were quantied by gas chromatography in a gas chromatograph (GC) equipped with a ame
ionization detector (FID) by injection of the supernatant with an
internal standard (4:1; propan-2-ol, 10 g L1 in sulphuric acid
1 N) into a fused silica column (0,53 mm  50 m Poraplot
Q CHROMPACK, Varian Inc., Hansen Way, Palo Alto, USA-). The
residual glucose from the seed culture in the supernatant was measured by a 2700 Select Biochemistry Analyzer (YELLOW SPRINGS
INSTRUMENT Inc., Yellow Springs, Ohio, USA). The residual phosphorus was determined by colorimetric assay (Allen, 1940).
Total biomass growth was monitored by measuring the optical
density at 620 nm (OD620nm) using a visible spectrophotometer
(BIOCHROM LIBRA S4) with a 2 mm absorption cell (HELLMA).
OD620nm was calibrated against dry cell weight (DCW) measurement. The cells were harvested by centrifugation as previously described and then dried in an oven (HERAEUS, France) at 60 C
under vacuum (200 mmHg) in presence of silica gel for at least
48 h. The residual biomass (Xr) concentration was calculated by
difference between DCW and PHB in g L1.
The amount of PHB was determined by gas chromatography
after methanol esterication (Brandl et al., 1988) of the dried biomass used for DCW measurement. The exact amount of biomass
used was weighted. The system was the CPG VARIAN 430 (Varian
Inc., HansenWay, Palo Alto, USA) with nitrogen as carrier gas
(25 mL min1) and ame ionization detection (270 C). The column
was a CP-Wax 58 (FFAP) CB column (25  0.23  0.70). The injection was carried out at 250 C. The temperature program of the
oven was: 115 C during the initial 5 min then increasing to nal
temperature of 230 C at a rate of 20 C min1 and an isotherm of
10 min.
2.5. Calculation
2.5.1. Rates and yields calculation
The specic substrate (butyric acid) uptake rate (qs), specic
PHB production rate (qPHB) and growth rate (l) were calculated
from their measured data by means of the respective mass balance
equation, taking into account both the volumetric feed medium
rate and the withdrawal volume from the sampling.

All Yields are expressed as carbon ratios except for the energetic
yield. Instant Yield was the ratio of specic i production rate and
specic substrate consumption rate: Y tS;i qi =qS . Overall Yield
was the ratio of produced quantities of i and consumed quantities
of substrate during a time interval (t2t1): YS,i = |(it2  it1)/
(St2  St1)|.
2.5.2. Modeling metabolic descriptor
The metabolic descriptor was composed of two equation
systems:
(1) The rst is an anabolic network (53 reactions: Supplementary online material S2) leading to the synthesis of different
macromolecular components of biomass from 28 intracellular intermediate metabolites. This network has been used to
calculate the global molar stoichiometry (reaction r0, Supplementary online material S1) for the production of 1 g of
biomass, assuming the biomass formula (C1H1.77O0.44N0.25,
4% ash, MMr = 25.35 g mole1) and the mass fraction of macromolecular components (Proteins 74.00%, RNA 3.58%, DNA
1.85%, Phospholipids 6.49%, Polysaccharides 4.34%, Peptydoglycane 2.64%, LPS 4.98%, Polyamine 0.11%, water 7.01%,
ashes 3.92%).
(2) The second is a catabolic network (Supplementary online
material S1) which describes carbon assimilation including
both PHB synthesis and the central metabolic pathways such
as glyoxylate shunt (GXS), tricarboxylic acid cycle (TCA), gluconeogenesis (45 reactions, 46 intermediate metabolites:
Supplementary online material S3 and S4).
2.5.3. Modeling metabolic routes
Metabolic routes used in this study were obtained from a bibliographic study and available databases such as KEGGS. It is currently accepted that the metabolic routes from basic precursors
to small molecules are common to a wide variety of organisms.
Where specic data were not available for C. necator, composition
data of Escherichia coli were used instead.
Catabolism of butyric acid: Butyric acid in un-ionized form penetrates into the cells and is activated by the sequential action of
butyrate kinase and phosphate butyryltransferase (r1, Supplementary online material S1). Butyryl-CoA is converted to acetoacetylCoA via b-oxidation (Shi et al., 1997) (r2 to r4). It is then either
decomposed to acetyl-CoA by a b-ketothiolase to produce biomass,
energy and co-factors via the central metabolism or it is incorporated into PHB (r5 to r6).
Central metabolic pathways: The acetyl-CoA is then directed towards either the tricarboxylic acid cycle (TCA, r8 to r15) or towards
the glyoxylate shunt (GXS, r29 to r30). There are two paths for bioconversion of C4 to C3 (Bruland et al., 2010) to feed gluconeogenesis: (i) the malic enzyme (EM, r17) which decarboxylates malate to
pyruvate, (ii) the PEP (Phospho-Enol-Pyruvate) carboxykinase
(PCK, r16) which catalyzes the decarboxylation of oxaloacetate into
P-enolpyruvate. The glycolysis pathway and the PentosePhosphate Pathway (PPP) are incomplete in C. necator because
the key enzymes fructose-1,6-bisphophatase (FBP) and gluconate
6-phosphate dehydrogenase (GND) are lacking. (Pohlmann et al.,
2006). However, the Entner-Doudoroff pathway (ED, r27 and r28)
overcomes this limitation.
Biosynthesis of PHB from acetoacetyl-CoA: acetoacetyl-CoA is reduced to R-hydroxybutyryl-CoA by a NADPH acetoacetyl-CoA
reductase (r5). The last necessary enzyme in PHB production is
the PHA synthase (r6) which polymerises monomers (Braunegg
et al., 1998).
The NADPH consumed by the PHB synthesis reaction (r5) must
be generated by some other biochemical reactions in order to
ensure continuous PHB synthesis. As indicated before, the

E. Grousseau et al. / Bioresource Technology 148 (2013) 3038

Pentose-Phosphate Pathway cannot be considered. Four candidates

are conceivable: (i) glucose-6-phosphate dehydrogenase (G6PD,
r26) of Entner-Doudoroff Pathway (ED), (ii) isocitrate dehydrogenase of the TCA cycle (r10) (iii) malic enzyme (EM, r17) (iv)
transhydrogenases. As a rst assumption, although putative transhydrogenase gene regions are apparent from the genome sequence (Cramm, 2009) and transhydrogenase subunit have been
identied (Raberg et al., 2011), transhydrogenase activity has not
been considered because of its low activity (Lee et al., 1995). (This
assumption is discussed in the Results and Discussion section).
2.5.4. Modeling mathematical solution
The complete descriptor was a set of linear equations. The system was solved on the basis of the mass balance. The accumulation
P
of the ith metabolite is ai j45
j0 ei;j :r j (where ei,j is the stoichiometric coefcient for the metabolite i in the reaction j, and rj is the
molar ux, and i is in the range of 045. See Supplementary online
material S4). ai was set to zero for intracellular metabolites based
on the pseudo-steady-state assumption (Stephanopoulos et al.,
1998) and equal to specic ux qi for exchanged metabolites
(growth, consumption, production).
The model was used in two ways: (1) to simulate the theoretical
yield that could be reached by the strain as explained below, (2) to
calculate the intracellular ux based on experimental results (minP
exp 2
imization of i45
where qexp
was the experimental
i0 nCi :ai  qi
i
specic rate of production or consumption of the metabolites
i and nCi the number of carbon of the metabolite i).
The theoretical yield of PHB production is calculated using the
descriptor previously described by maximizing the ratio
Y theo
S;PHB r 6 =r 1 with a null biomass production (r0 = 0) and with a selected NADPH generation reaction (ED, TCA or EM as listed
previously).
To calculate the theoretical biomass production yield, the rst
step is to calculate the energetic growth yield (YATP,Xr) which corresponds to the quantity of biomass produced in gram per mole of ATP
consumed. The biomass composition chosen leads to an anabolic
demand equivalent of a maximum YATP,Xr of 19,21 gXr moleATP1, if
no ATP spilling is considered (r45 = 0) using YATP,Xr = a0.MXr/
(e45,0.r0 + e45,45.r45) (where a0 is the biomass production molar ux,
MXr is the biomass molar mass (25.35 g mole1), r0 is the molar ux
associated with the biomass production reaction, e45,0 is the stoichiometric coefcient of the 45th metabolite (ATP) for the reaction r0,
and e45,45 is the stoichiometric coefcient of the 45th metabolite
(ATP) for the ATP spilling reaction r45. See S2 and S3). By solving
the reactions network with experimental data of biomass

33

production from glucose (unpublished data from G. M. F Aragao)

(additional reaction: 1 Glucose + 1 ATP ? 1 Glucose-P), an energetic
growth yield of 14 gXr moleATP1 was calculated (equivalent to an
ATP spilling (e45,45.r45) of 0.019 mol for one gram of biomass). Then
the theoretical biomass production yield from butyric acid is calculated by maximizing the ratio Y theo
S;PHB r 0 =4r 1 with YATP,Xr set to
14 gXr moleATP1. To note, the maximal theoretical value of YATP,Xr,
calculated from the energetic of the anabolic pathways, is generally
more than twice the experimental yield (Neijssel and Demattos,
1994). This difference can be explained by futile cycle, protein and
nucleic acid turn-overs, and by useful maintenance (ionic transports, cellular homeostasis. . .). However, in our case, the maximal
theoretical yield (19.21 gXr moleATP1) is only 1.4 times the experimental yield (14 gXr moleATP1).
Another calculated parameter is the ATP spilled compared to
carbon consumed: spilled ATP = r45/4r1. This ratio cannot be null
since there are some ATP requiring phenomena that are not described by the model, as explained above.
Limit experimental yield (Y lim
S;i ) is the yield that could be reached if
no by-products have been synthesized. During the PHB production
theo
phase: Y lim
S;PHB jPHBt2  PHBt1 =St2  St1  Xrt2  Xrt1 =Y S;Xr j.
3. Results and discussion
3.1. Fed-Batch culture of C. necator grown on butyric acid as sole
carbon source
VFA are inhibitory: for example, total inhibition of growth from
6 g L1 of acetic acid and reduction of growth rate of C. necator to
80% of the maximum from 1 g L1 of acetic acid (Wang and Yu,
2000) were observed. In order to avoid this phenomenon, the cultivation was carried out in fed-batch mode under carbon-limiting
conditions (0.0 residual acid concentration or lower than
0.25 g L1) with carbon being fed based on an exponential or linear
prole, adjusted to the biological demand (see Supplementary online material S5 for more details).
For the fed-batch culture carried out, four phases were identied (Fig. 1): (A) was the growth phase with an initial supply of
phosphorus estimated to produce about 0.05 Cmole biomass (or
1.3 g; YP,Xr = 67 9 Cmole mole1 calculated during exponential
growth, using an average of seven cultures with butyric, propionic
or acetic acid as substrate, data not shown). After 10 h of cultivation, the initial phosphorus was completely exhausted. (B) phase
corresponded to a transient period where PHB synthesis started;
(C) was a PHB production phase during which a controlled

Fig. 1. Time course of Fed-Batch culture of C. necator with butyric acid as carbon source.

34

E. Grousseau et al. / Bioresource Technology 148 (2013) 3038

Table 1
Comparison of PHA production with C. necator from butyric acid in Fed-Batch mode.
Reference

This work

Yu (2001)

Du et al. (2004)

Carbon source

Butyric acid

Butyric acid

Limiting element
Process time (h)
DCW (g L1)
PHB (g L1)
PHV (g L1)
%PHA (g g1)
QPHB (g L1 h1)
YS,PHA (Cmole Cmole1)

P
67.4
46.7
38.4
0.0
82%
0.57
0.62

N
46.0
20.0
9.4
0.0
47%
0.20
nr

Acetic, propionic,
butyric and
lactic acid
nr
79.0
22.7
16.0
0.5
73%
0.21
nr

DCW Dry Cell Weight, PHB, PHV polyhydroxybutyrate, and polyhydroxyvalerate

concentrations reached at the end of the process, %PHA intracellular PHA content,
QPHB overall volumetric PHB productivity, YS,PHA overall PHA on substrate yield, and
nr not reported.

Table 2
Experimental and limit overall yields, Respiratory quotient (RQ), during each stage of
the Fed-Batch culture (strain C. necator).
Fed-Batch culture phase

YS,Xr (Cmole Cmole1)

YS,PHB (Cmole Cmole1)
RQ (Cmole mole1)

0.59
0.04
0.64

0.15
0.62
0.58
0.80

0.07
0.67
0.61
0.75

1
Y lim
S;PHB (Cmole Cmole )

phosphorus feeding strategy was applied, maintaining a constant

Phosphorus to Carbon ratio (P/C in mMole Cmole1 set at 0.97) targeted to support residual growth. During this period, residual biomass increased by 4.5 times due to the continuous supply of
phosphorus (3.2 mmole total). At the end of this phase, the accumulation of PHB within biomass reached a plateau of 75% (g g1).
(D) was the nal PHB production phase without any phosphorus
supply. As a consequence biomass production was almost stopped
while PHB percentage reached a maximum at 82% (Table 1).
These results were the highest reported (Table 1), compared to
literature data obtained using the same strain and with the same
substrate: the total biomass produced was 46.7 g L1 versus
22.7 g L1 by Du et al., 2004 with 82% of PHB versus 73% by Du
et al., 2004 and the overall productivity was 0.57 g L1 h1 versus
0.2 by Du et al. (2004) and Yu (2001). The overall PHB on substrate
yield was 0.62 Cmole Cmole1.
During the growth phase (A), the experimental yield of biomass
production was 0.59 Cmole Cmole1 (Table 2) which corresponds
to 8892% of the theoretical growth yield calculated from the
1
descriptor. Y theo
S;Xr is in the range of 0.640.67 Cmole Cmole
depending on the NADPH generation pathway and associated with
a Respiratory Quotient (RQ) between 0.61 and 0.63 Cmole Mole1
(Table 3). The Y theo
S;Xr calculated by Shi et al., 1997 was about
0.6 Cmole Cmole1. The difference between this yield and our
calculation was mainly due to the consideration of a higher ATP

spilling by Shi et al., 1997. In our model a yield of 0.6 was reached
with a YATP,Xr of about 10.5 gXr moleATP1 instead of 14.
During phase C, the overall experimental PHB production yield
was 0.62 Cmole Cmole1 (Table 2) occurring simultaneously with a
biomass production. The decrease in biomass production in subsequent phase (D), did not signicantly increase the overall experimental PHB production yield (0.67 Cmole Cmole1, Table 2).
3.2. Sustaining a low residual growth did not alter the PHB production
yield
It is generally accepted in the literature that PHB yield increases
with biomass yield decrease (Shi et al., 1997). In fact, nutrient starvation is often used as a trigger for batch or fed-batch PHA production processes. As depicted in Fig. 2-A, while instantaneous
experimental PHB production yield (Y tS;PHB ) from butyric acid are
clearly increasing when the growth rate decreased from 0.33 h1
down to 0.06 h1, however, below 0.06 h1, this relationship
changes. Our current results shown that sustaining a low residual
growth (l equal or lower than 0.06 h1) via a controlled phosphorus feeding, did not affect signicantly the instantaneous experimental PHB production yield (Y tS;PHB ) from butyric acid. To
understand what happened for this growth rate range, limit PHB
production yields (Y lim
S;PHB ) from butyric acid have been calculated
and are depicted versus growth rate on the Fig. 2-B. This Figure shows that with the decrease in growth rate from 0.06 h1 to
0 h1, the limit PHB yield decreased from about 0.83 to
0.71 Cmole Cmole1. In order to understand the carbon allocation
during PHB production phases (C and D), the theoretical PHB production depending on the NADPH sources (ED, TCA, or EM described in Supplementary online material S1) have been
simulated using the metabolic descriptor. This simulation shows
that the theoretical PHB production yield was greatly dependent
on NADPH generation pathway (Table 3):
(1) If NADPH is generated by the isocitrate dehydrogenase
NADPH linked to the TCA or by the EM, the theoretical yield
of PHB production is 0.67 Cmole Cmole1, and the RQ is
0.67 Cmole Mole1. This is in accordance with Yamane,
1993 and Shi et al., 1997, who respectively calculated 0.66
and 0.68 using similar hypotheses. In our simulation, ATP
is generated in association with PHB production and is not
limiting: the ratio of ATP spilled to Carbon consumed is
between 1330 and 1500 mmole Cmole1 (Table 3) which is
4 to 5 time higher than during growth. As seen in Table 3,
results were the same if NADPH was generated by TCA or
EM. Therefore to simplify the system, EM pathway was
removed.
(2) If NADPH is generated by a cyclic carbon circulation in the
ED pathway, the theoretical PHB production yield is
0.89 Cmole Cmole1 and the RQ is 0.45 Cmole Mole1. In this
case, ATP generation is limiting (0 mmole of spilled ATP per
Cmole, Table 3). Energy spilling reaction, protein turn-over,
maintenance (ionic transports, homeostasis. . .) must be

Table 3
Theoretical Yields of i production on substrate (butyric acid) calculated with stoichiometric modeling ( parameters set).
i

Growth

PHB production

Total substrate decarboxylation

Xr

PHB

PHB

PHB

CO2

Y theo
(Cmole Cmole1)
S;i

EM/TCA/ED
0.640.67

EM or TCA
0.67

ED
0.89

ED
0.82

EM/TCA/ED
1.00

RQ (Cmole Mole1)
Spilled ATP (mmole.Cmole1)
YATP,Xr (g moleATP1)

0.630.61
320330
14

0.67
13301500

0.45
0

0.55
300

0.80
4390

NADPH generation pathway

E. Grousseau et al. / Bioresource Technology 148 (2013) 3038

35

Such high yield of PHB production on butyric acid with C. necator has been reported once in the literature and a similar statement
has been used by Shi et al., 1997: these authors suggested the
cyclic use of pentoses-phosphate pathway (which leads to
1
Y theo
). However, from the literature data,
S;PHB = 0.82 Cmole Cmole
the gluconate 6-phosphate dehydrogenase (GND) is lacking in
C. necator (Pohlmann et al., 2006), and therefore, the pentosesphosphate pathway cannot be used. Alternatively such high yields
may be achieved by considering the activity of a transhydrogenase.
But this hypothesis seemed less plausible because there is no
objective reason to correlate this enzyme activity to anabolism contrary to the Entner-Doudoroff pathway. High yields with butyric acid
have also been reported in microbial enrichment cultures instead of
axenic cultures of C. necator (Marang et al., 2013). These results
emphasized the need of optimization of waste-streams digestion to
promote butyrate yield over other acids in resulting mixture.
To note, there is a debate in literature on isocitrate dehydrogenase regarding the existence of NAD+-dependent activity: two
genes (icd1 and icd2) coding for isocitrate dehydrogenase were
identied (Wang et al., 2003), but no NAD+-dependent isocitrate
dehydrogenase activity was detected in cells extracts. Nevertheless
an older study cited by Wang et al. (2003) revealed an isocitrate
dehydrogenase with NAD+-dependent activity. Two isocitrate
dehydrogenases with respective afnity to NADP+ and NAD+ were
separated and partially puried. Sequence analysis identied an
additional gene coding for an isocitrate dehydrogenase icd3 (locus
H16_B1016) in C. necator (Pohlmann et al., 2006) although not
experimentally studied. This third gene could encode for a NAD+
dependent enzyme. No studies have been reported on regulation
of isocitrate dehydrogenase isoenzymes with respect to NADH or
NADPH generation. Considering all these informations, both
NAD+- and NADP+-dependent activities were considered in the
stoichiometric model.

(Y tS;PHB )

Fig. 2. (A) Instantaneous experimental PHB production yield

and (B) Limit
PHB production yield (Y lim
S;PHB ), versus growth rate (l) during Fed-Batch culture of C.
necator on butyric acid.

considered. If about 300 mmole of dissipated ATP per Cmole

is considered (a level similar to the one reached during
growth, Table 3), PHB production yield decreases to
0.82 Cmole Cmole1 with an RQ of 0.55 Cmole Mole1.
Despite this reduction, this value remains high relative to
that obtained through other NADPH generation pathways.
Considering the results of these simulations, the only possibility
for the strain to produce PHB with a limit PHB production yield
around 0.83 Cmole Cmole1 (Fig. 2-B) is to generate NADPH by circulation of carbon in the Entner-Doudoroff pathway. It seems that
this pathway is linked to anabolic requirements of the strain since
limit PHB production yield decreases with decreasing growth rate
from 0.83 Cmole Cmole1 to 0.71 near the theoretical PHB production yield calculated considering the TCA for the NADPH source
(Fig. 2-B). This assumption seems logical regarding the metabolism
of C. necator on VFA, where there is no need to use the gluconeogenesis pathway for substrate assimilation, except for providing
biomass precursors (Supplementary online material S1). NADPH
allocation was further investigated by calculating the ux distribution using the descriptor at different time points of the fed-batch
culture. Measured instantaneous yields are reported in the Table 4
and are in very good agreement with simulated instantaneous
yields (values in brackets in the Table 4). The gradual decrease of
NADPH generation by ED for decreasing cell growth rates was
conrmed.

3.3. Support of a low residual growth improved PHB specic

production rate
The experimental maximal growth rate (lmax) observed during
phase A of the fed-batch culture is 0.34 h1 0.01 using butyric
acid as substrate. When using other VFA as substrate, similar values were measured: 0.32 h1 with acetic acid and 0.33 h1 with
propionic acid (data not shown). The bibliographic data for growth
rate of C. necator on those acids vary between 0.26 and 0.30 h1
(Ampe et al., 1996; Kim et al., 1992; Wang and Yu, 2000).
The maximal specic PHB production rate obtained, of
0.26 Cmole Cmole1 h1, was reached in association with a growth
rate of 0.05 h1 (Fig. 3). For growth rates above 0.05 h1, an inverse
coupling was observed: a decrease of specic PHB production with
increase of growth rate. For growth rates under 0.05 h1, a partial
coupling between the PHB synthesis rate and the cell growth rate
was observed. Such a relationship has already been observed
(Shimizu et al., 1999) but no explanation was proposed to this phenomenon, since it was not the goal of that paper. In this study, is
proposed a simple mechanism based on NADPH ux and availability which describes the maximal kinetic relationship between
specic PHB production rate and growth rate (reachable under
non-inhibitory and carbon non-limiting conditions):
(1) the maximum specic rate of NADPH generated is dened
to full biomass requirement at a maximal growth rate
lmax of 0.33 h1 (qNADPH#Xr max ) and is equal to 101.1
mmole Cmole1 h1 (See Table 4 and Supplementary material online S6 A).
(2) a decrease in growth rate (controlled by applying a limiting
phosphorus ux), allows dedication of a part of the maximal
NADPH generation capacity to PHB production, explaining

36

E. Grousseau et al. / Bioresource Technology 148 (2013) 3038

Table 4
Experimental and simulated data (in brackets) of the Fed-Batch of C. necator with butyric acid as carbon source.
t = 30,4 h

t = 32,2 h

t = 37,7 h

t = 43 h

t = 48,6 h

t = 57,3 h

t = 58,8 h

t = 65,3 h

t = 67,5 h

l (h1)

0,33a

0,056

0,048

0,045

0,038

0,034

0,014

0,013

0,012

0,007

0,004

YATP,X (gXr moleATP1)

(14)

(9,54)

(3,94)

(3,57)

(3,21)

(4,9)

(1,63)

(1,49)

(1,49)

(0,95)

(0,49)

Spilled ATP (mmole Cmole1)

(316)

(206)

(575)

(635)

(855)

(653)

(1137)

(938)

(888)

(1009)

(1275)

YS,X (Cmole Cmole1)

0,65
(0,65)

0,14
(0,15)

0,12
(0,11)

0,12
(0,11)

0,13
(0,13)

0,17
(0,17)

0,08
(0,08)

0,06
(0,06)

0,06
(0,06)

0,04
(0,04)

0,025
(0,026)

qPHB (Cmole Cmole1 h1)

0,26
(0,23)

0,26
(0,29)

0,25
(0,27)

0,18
(0,18)

0,12
(0,12)

0,11
(0,11)

0,14
(0,15)

0,14
(0,15)

0,14
(0,12)

0,12
(0,11)

YS,PHB (Cmole Cmole1)

0,64
(0,64)

0,67
(0,67)

0,66
(0,66)

0,60
(0,60)

0,58
(0,58)

0,62
(0,62)

0,68
(0,68)

0,70
(0,70)

0,70
(0,70)

0,68
(0,67)

QR (Cmole Mole1)

0,63
(0,63)

0,56
(0,57)

0,56
(0,58)

0,57
(0,58)

0,62
(0,62)

0,64
(0,60)

0,68
(0,64)

0,60
(0,62)

0,63
(0,61)

0,66
(0,62)

0,65
(0,65)

NADPH produced through ED

(mmole Cmole1 h1)

(45.5)

(72.0)

(44.9)

(40.2)

(20.1)

(18.1)

(6.7)

(14.0)

(15.1)

(10.1)

(4.0)

NADPH produced through TCA

(mmole Cmole1 h1)

(55.6)b

(3.1)

(15.9)

(41.6)

(35.5)

(21.4)

(24.8)

(26.8)

(25.2)

(22.5)

(23.5)

10

Total NADPH
(mmole Cmole1 h1)

(101.1)

(75.1)

(86.2)

(81.8)

(55.6)

(39.5)

(31.5)

(40.9)

(40.4)

(32.6)

(27.5)

11

qNADPH?Xr (mmole Cmole1 h1)

(101.1)c

12d

NADPH through ED qNADPH?Xr

(mole mole1)

13

Butyric Acid consumed

(mmole Cmole1 h1)

lmax.
kTCA.
qNADPH#Xr max .
Average of row 12: kED = 3 1.

t = 25,6
30,4 h

b
c

Growth

(128.2)

(17.2)

(14.7)

(13.8)

(11.6)

(10.4)

(4.3)

(4.0)

(3.7)

(2.2)

(1.2)

(4.2)

(3.1)

(2.9)

(1.7)

(1.7)

(1.6)

(3.5)

(4.1)

(4.7)

(3.3)

(91.1)

(106.7)

(102.4)

(73.2)

(50.1)

(43.8)

(54.2)

(53)

(43.8)

(39.2)

the inverse linear relation between qPHB and l. With a

molar conversion of NADPH to PHB (r5, Supplementary
online material S1), the corresponding equation is
qPHB qNADPH#Xr max 1  l=lmax nCPHB =1000 (Eq. 1) with
nC PHB = 4.
(3) The NADPH ux generated through ED is linked to the
NADPH required for the biomass synthesis (qNADPHXr). A
proportional factor between these two uxes was designated kED and set to 3 1 (according to data in Table 4,

Fig. 3. Relationship between specic PHB production rate (qPHB) and growth rate
(l) considering simulated and experimental data during Fed-Batch culture of
C. necator on butyric acid.

row 12). The NADPH ux generated by the TCA cannot be

higher than a maximum value designated kTCA. kTCA is
initially equal to the maximal ux of NADPH produced
through TCA calculated during growth at lmax
(55.6 mmole Cmole1 h1, see Table 4). The corresponding
equation
is
qPHB qNADPH#Xr max l=lmax kED  1 kTCA
nCPHB =1000 (Eq. 2).
The maximal kinetic model was drawn by calculating the minimum of equations 1 and 2, represented on Fig. 3. The maximal kinetic model included the experimental data (Fig. 3). This
agreement showed that simple kinetic constraints on NADPH generation are able to describe the kinetic behavior of the strain. The
theoretical optimum would be a specic production rate about
0.35 Cmole Cmole1 h1 in association with a growth rate of about
0.05 h1 (See Fig. 3 and Supplementary online material S6 D).
This optimum is in accordance with the specic production rates
of 0.340.35 Cmole Cmole1 h1 measured during other fed-batch
cultures carried out with volatiles fatty acids (data not shown).
The maximal kinetic model is valid for C. necator cultivated on
different VFA substrates such as butyric acid, propionic acid,
and acetic acid since the growth rate and consequently the
maximal specic rate of NADPH produced are identical
(101.1 mmole Cmole1 h1). This hypothesis has been validated
with data obtained in acetic acid fed-batch culture (data not
shown) and with data obtained in valeric and butyric acid fedbatch culture by Shimizu et al. (1999).
For a null growth rate, considering Eq. (2), the specic PHB production rate depends only on kTCA (qPHB = (kTCA)(nC PHB/1000)) and
is equal to 0.22 Cmole Cmole1 h1 (Fig. 3 and Supplementary online material S6 B). However, the experimental specic PHB production rate was lower than this value. The maximal kinetic model

E. Grousseau et al. / Bioresource Technology 148 (2013) 3038

for very low growth rate may be further rened by considering the
reduction of catalytic capacity with phosphorus limitation and to
the operation of physical constraints with PHB accumulation as
has been shown in previous studies (Heinzle and Lafferty, 1980).
To take these phenomena into account kTCA and kED could decrease
with the growth rate instead of being held constant.
This model shows that maximal specic PHB production rate is
dened by the maximum specic rate of NADPH produced, which
depends on anabolism of the strain. It therefore follows that the
higher the maximal growth rate of the strain, the higher is the specic PHB production rate. Since PHA production is closely related
to growth of the strain, the study of growth capacity should not
be neglected in order to optimize the PHA production process.
Moreover, the choice of a PHB production microorganism can be
principally driven by this criterion.
Among existing models on PHB production (Gahlawat and Srivastava, 2013; Shi et al., 1997; Vrana Spoljaric et al., 2013; Yamane,
1993; Yu and Si, 2004), this work used a stoichiometric model
combined with a simple kinetic model. This strategy proved to
be a useful tool to understand biological phenomenon and to guide
in designing operating strategies to further improve PHB
production.
4. Conclusion
In this work, the complementary modeling and experimental
approach validates that sustaining a residual growth do not alter
PHB production yield and improves PHB specic production rate
during cultivation of C. necator strain with volatile fatty acids as
sole substrate. Further, that a specic growth rate of about
0.05 h1 is the optimum set point. Under these conditions, high
specic productivity of 0.35 Cmole Cmole1 h1, high PHB production yield of 0.67 Cmole Cmole1 and by consequence high PHB
content (about 85%) could be simultaneously reached. Moreover
this paper highlights the importance of the knowledge of the strain
anabolism to optimize PHB production.
Acknowledgements
This work was supported by Veolia Environment Research and
Innovation, by grants from the French National Research Agency
(ANR-08-ECOT-O17-001) and the French National Research and
Technology Association (ANRT, Cifre). We thank Dr. Nathalie
Gorret, Dr. Stphane Guillouet, and Dr. Dores Cirne for the critical
review of this manuscript; Dr. Anne-Sophie Lepeuple and
Dr. Pierre-Alain Hoffmann for their support throughout the course
of this study. And we also kindly thank Mr. John W. Quimby for his
help in correcting the English version of the manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2013.08.
120.
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