European J, Appl. Mierobiol. Biotechnol.
11, 8-16 (1980)
A Kinetic Model for Growth and Synthesis of Poly-/3-Hydroxybutyric
Acid (PHB) in Alcaligenes eutrophus H 16
E. Heinzle* and R. M. Lafferty
Institut ftir Biotechnologie, Mikrobiologie und Abfalltechnologie, T.U. Graz, Schl6gelgasse 9, A-8010 Graz, Austria
Summary. A structured model is presented giving a mathe-
matical description of batch cultures ofAlcaligenes eutro-
phus strain H 16 under chemolithoantotrophic growth
conditions. A mass-spectrometer with a membrane inlet
system was used to measure and control the concentra-
tions of the dissolved gaseous substrates H2, 02 and CO 2.
Growth and storage of PHB (Poly-/~-hydroxybutyric acid)
are described as a function of the limiting substrate S (N H +4),
the residual biomass R, and the product P (PHB), Model
parameters were evaluated by regression analysis followed
by linearization. The differential equation model was
solved numerically and compared with experimental data.
1. Introduction
PHB (poly-/~-hydroxybutyric acid), a carbon and energy
reserve material synthesized by many microorganisms, is
accumulated under certain unbalanced growth conditions
(Dawes et al. 1973; Repaske and Mayer 1976). In some
microorganisms this polyester can be accumulated in
amounts up-to more than 80% of the total dry mass
(Dawes et al. 1973).
Alcaligenes eutrophus strain H 16 can grow and store
PHB on fully synthetic medium containing only several
ions and the gaseous substrates H~, 02, and CO2. It is
possible to provide the organism with separate energy and
carbon sources. This organism is thus a very suitable model
organism for kinetic studies.
Simple models are necessary in order to have a solid
basis for the design of fermentation processes, for eco-
nomic calculations, and for the control of fermentation
processes. Simplifications of the complex biological sys-
* Present address: Techn. Chem. Labor, ETH-Ztirich, Universi-
t~itsstr. 6, CH-8006 ZUrich, Switzerland
0171-1741/80/0011/0008/$01,80
Applied
Microbiologyand
Biotechnology
9 by Springer~Verlag 9 1980
tern are necessary prerequisites and are at the same time
a major goal of modelling. In this work a semi-empirical
model based on a simple mechanistic description is given.
The influence of gas transfer is to be eliminated by main-
taining the concentrations of the dissolved gases constant.
This can be achieved by using a continuous measuring
system for gas concentrations and varying individual gas
flow rates to fulfill the required experimental conditions.
2. Materials and Methods
2.1. Microorganism and Medium
Alcaligenes eutrophus strain H 16 (DSM 428) was cultivated in a
mineral medium (Schlegel et al. 1961) containing 2.3 g/1 of
(NH4)2SO4 and was provided with a sterile gas mixture of 02,
H2, and CO2. The strain H 16 was maintained on nutrient agar
slants by monthly subculture. Inocula were prepared in a 1-1 pre-
culture of mineral medium in a glass fermentor at 30 ~ Gases
for the inoculum preeulture were supplied in a ratio of H 2 : 02 : CO2
= 6: 2:1 and a total flow rate of 1.5 vvm.
2.2. Culture Conditions
The bacteria were grown batchwise using a Biostat-V-fermentor
(Braun-Melsungen, Melsungen, F.R.G.) with 8 1 liquid volume,
equipped with three conventional turbine impellers and 4 conven-
tional baffles. Temperature was kept at 30 ~ (• pH was auto-
maticaUy maintained at 6.9 (-+0.1) by adding 2 N NaOH. Impeller
speed was kept at 400 rpm. The concentrations of the dissolved
gases were manually controlled by changing the individual gas
flow rates.
2. 3. Analytical Procedures
Cell dry weight (X), PHB (P), Protein (Prot), and NH~ (S) were
determined as described by Sonnleitner et al. (1979). Dissolved
oxygen was measured with a polarographic electrode IL-Model
530. Dissolved H2, 02, and CO2 were monitored with a mass
 E. Heinzle and R. M. Lafferty: Kinetic Model for Growth and Synthesis of Poly-/3-Hydroxybutyrie 9

X, P, Prof [gl[] 5; [g/i]

20

15) +~'§
~ z x ~ z~

I0~ *\S
+%
\
\ /
/
~ x

\<,~ •
J

~ •
I , . I I 0
10 20 30 ~,0 50 60
f [h]
Fig. 1. Growth and accumulation of PHB in Alcaligenes eutrophus strain H 16 under chemolithoautotrophic growth conditions. Con-
centradons of dissolved gases as in Table 1. S -- concentration of the limiting substrate NH~ (g/1 (NH4)2SO4); +, X - cell dry weight;
z~, p _ PHB; x, Prot - protein; |

spectrometer (Varian MAT, Model GD 150) equipped with a teflon Table 1. Concentrations of dissolved gases (c) during fermentation
membrane inlet system (Heinzle and Lafferty 1978). of Alcaligenes eutrophus strain H 16

2.4. Model Evaluation and Simulation Procedures Gas c [mg/1] P* 1%1

Measured data were fitted by the least square method using simple H2 0.3-0.7 23-55
functions with up to 4 parameters (polynomial functions and func- 02 2.2-3.0 6.5-9.8
tions containing logarithmic and exponential parts) as has been CO2 70-120 6.3-10.7
described by Sonnleitner et al. (1979) and by Heinzle (1978). A
piecewise fitting was used for calculation of reaction rates unless
otherwise stated, The system of first order differential equations p* - Equilibrium partial pressure in the gas phase corresponding
to e
was solved by Runge-Kutta integration on a Univac 1100 com-
puter equipped with a digital plotter (Heinzle 1978).

3. Results and Discussion
Batch fermentation results with constant concentrations
of the dissolved gases (Table 1) are shown in Fig. 1. Two
phases can be clearly recognized: the growth phase and
the storage phase (Sonnleitner et al. 1979). During the
growth phase there is sufficient NH~ to permit protein
synthesis. When the limiting substrate NH~ (S) is exhaust-
ed, the protein synthesis ceases and the production rate of
PHB is increased. During the storage phase only PHB is
produced and no further protein.
3.1. Modelling
The most important reactions and regulations in case of
constant concentrations of the dissolved gaseous substrates
are shown schematically in Fig. 2. The whole cell dry mass
(X) consists of two main parts, namely PHIl (P) and resid-
ual biomass (R), where R is calculated as the difference
between the total cell dry weight and the concentration
ofPHB (R = X - P). R can be considered as the catalyti-
cally active biomass including proteins and nucleic acids.
This is manifested by Figs. 3 and 4, which show that R is
proportional to tile protein concentration (Prot).
10 E. Heinzle and R. M. Lafferty: Kinetic Model for Growth and Synthesis of Poly-C~-I-lydroxybutyric

x ......... ~:~ . . . . \~-) Prof [g/L]

3t
I , i O
I

i 2
t 1

S = , ]
l

1
Fig. 2. Schematic diagram of growth (synthesis of residual bio-
mass R) and synthesis of the intracellular product PHB (P) in
A. eutrophus H 16 under chemolithoautotrophic conditions with
constant concentrations of the dissoNed gaseous substrates H2,
02, and CO 2 (SG). X is the whole dry biomass (X = R + P). 0
Dashed lines characterize regulatory mechanisms and full lines 0 1 2 3 z~
R [g/I]
mass flows
Fig. 4. Correlation between the synthesis of residual biomass (R)
and the synthesis of protein (Prot). The average content of protein
[g/t] in the residual biomass under these conditions is 70%
9

[h-ll

3 0 0 0 0 0

-+ .+///x-.x--x--X-x
,-'~~--~-'~--•215Prol- .2 o /

2 -+S+.+ o o
o

/x +

0 0-- 5' 10
' 15 20
' 2*5 30' .0.0 .5 1.0 1.5 210
f [h] S [gill

Fig. 3. Utilization of the limiting substrate S (NH~ as (NH4)2SO4); Fig. 5. Specific rate of synthesis of residual biomass/~(# = rR/R)
+ and synthesis of protein (Prot); x and residual biomass (R); | as a function of the concentration of the limiting substrate S
(NH~ as (NH4)2SO4). o, experimental data (data points from
regression with pieces of functions linear in parameters)

Since the gaseous substrates were maintained within
narrow ranges of concentrations (Table 1) they are assumed
to have no influence or at the most a constant influence
on the kinetics o f the process. The limiting substrate
NH~ (S) is essential to produce R and limits its synthesis
at low concentrations: The synthesis of R is described as
follows,
this is based on the postulate that there exist two different
mechanisms for the assimilation of NH~ in procaryotes
(Kavanagh et al. 1975;Mtiller et al. 1977). This formula-
tion is not a mechanistic one, since in reality the enzyme
system using energy to assimilate NH~ is repressed by
high concentrations o f NH~. The following equation is
thus proposed,

s (S/Ks,2) n
dR _ rR =/2" R (1)
/2 =/~1 +/2~ = t2m, ~ 9 Ks,t + S + ~m,2 1 + (S/Ks,2) n (2)
dt

where r R is the rate o f synthesis o f R and/a is the specific
rate of synthesis of R. F o r the description of g as a func-
tion o f S the experimental results cannot be fitted by a
simple saturation function. There is some sigmoidal behav-
iour as seen in Fig. 5. The mathematical description of
The sum of/2m,1 and ~m,2, and/2m,2 were determined by
a semilogarithmic plot o f the concentration o f R versus
time to be 0.21 and 0.13 h -1 , respectively. By difference
/22m,1 is equal to 0.08 h -1 . Ks,1 and Ks,2 can be directly
estimated from Fig. 5 to be approximately 0.1 and 1.0 g/1
 E. Heinzle and R. M. Lafferty: Kinetic Model for Growth and Synthesis of Poly-~-Hydmxybutyric 11

rS [g/It.hi Figure 6 shows that Eq. (3) is adequate to describe the

O,A relationship between the rate of synthesis of residual bio-
Y R I 5 _ = ~ mass ( r a ) and the rate of consumption of the limiting sub-
strate (r s).

dS 1
. . . . . rR (3)
0,2 0 ~ / ~ 2 ~ - rs YR/S

A mean differential evaluation (mean value of all YR/S

= -- dR/dS) yields a value o f Y R / s = 1.48 +- 0.14 whereas
the integral evaluation (YR/s = - (R~o - R0)/(S~o - So) )
gives YR/s = 1.59. The subscript o indicates the beginning
I [ t I I of fermentation and oo the time after all S is consumed.
0 0,2 0,L 0,6 For further calculations and simulations the mean differ-
rR [g/It-hi
ential value of YR/s was used.
Fig. 6. Correlation between the rate of utilization of the limiting The rate of synthesis of P(rp) is assumed to be the sum
substrate (rS) and the rate of synthesis of residual biomass (rR). of a growth associated term (rv,1) and a biomass associated
YR/S yield coefficient. Data points from regression with pieces
term (rp,2) as is formulated in Eq. (4),
-

of a function linear in parameters

dP
rp [g/t.h] dT = rp = rp, 1 + rp, 2 (4)

.3 o12
The correlation between the rate of production of P(rp)
and the rate of synthesis of residual biomass R ( r a ) in the
growth phase is shown in Fig. 7. As there are only limited
experimental data a linear correlation [Eq. (5)1 was tried
as a first approach,
o11
.2
rp,1 = YP/R" ra (5)

o10 This has been done using only the data points with the
numbers 1 to 8. Starting with point 9, rp is obviously in-
o9
creasing independent of r R . This fact will be described by
~ ~~Yp/R = Pp/PR rv,2. From this time onwards, where rp, 2 gives a consider-
able contribution to the synthesis of PHB, rR as well as
P/R rp, 1, the latter of which is proportional to rR, gradually
approach a zero value. The non-growth associated term
of the synthesis of P(rp,2) is assumed to be a function of
t I I I I (
the limiting substrate S, of the residual biomass R, and of
.0 .I .2 .3 ./+ .5 pR [y',,Jr~a.k~ the product P. At high contents of PHB in the cells the
rate of synthesis of P is decreased, which can be formally
Fig. 7. Correlation between the rate of synthesis of PHB (rp) and described as an inhibition.
the rate of synthesis of residual biomass (rR) during the growth
In the case of a high PHB content, a good linear rela-
phase (point 1 to 8) and the beginning phase of storage of PHB
(point 9 and further). YP/R, yield coefficient for product tbrma- tionship could be found between the specific rate of pro-
tion during the growth phase. Data points from regression with duction o f P ( v p = rp/P) and the quotient X/P as shown in
pieces of a function linear in parameters Fig. 8 which leads to Eq. (6),

as (NH4)2SO4. The coefficient n for the sigmoidal correla-
tion is calculated by taking the mean of all values for n;
n = in (/~2/(#m,2 - g2))/ln (S/Ks,2) which yields n = 5.
In Fig. 5 experimental results and values calculated from
the model [Eqs. (1) and (2)] are compared and show suf-
ficient agreement.
rp = d P + kX (6)
d is the intersection with the ordinate and k is the slope
of the straight line in Fig. 8. The value of d was found to
be - 0 . 2 2 5 ( - 0 . 2 2 4 ) h -1 and that o f k to be 0.180 (0.176)
h -1 . Specifications outside the parentheses are the results
of a regression with a square root parabola (o); those
12 E. Heinzle and R. M. Lafferty: Kinetic Model for Growth and Synthesis of Poly-fl-Hydroxybutyric

[h-l]
• X

.B

.2

//
.1

I I I

• 2 3 /," 5
• [-]

Fig. 8. Synthesis of PHB in the late storage phase./zp - - specific rate of synthesis of PHB ( / ~ p = rp/P). X, cell dry weight; P, PHB. o, data
points from regression of the experimental data with one square root paxabota; x, data from regression with pieces of a function linear
in parameters

inside are the results of regressions with elements of a Using Eqs. (4) and (8), a dimensionless rate of non-growth
function containing a logarithmic and an exponential associated synthesis of PHB ( U p ) can be evaluated,
part (x). By substituting X by P + R Eq. (7) results
rp -- YP/R " rR Kr
- (9)
rp, 2 = -k~ 9P + k2 9R (7) rp (-k 1 . P + k 2 - R ) KI+S

In this case k 1 = d + k and k 2 = k and thus k 1 = 0.045 By taking the reciprocal value of Eq. (9), KI can be
(0.048) h -1 and k2 = 0.18 (0.176) h -1 . This description determined by plotting 1/r-P-eversus S,
is not mechanistic, since no turnover of PHB could be
found in the cells during synthesis of it (Gottschalk 1964;, = 1 + _.1 s (lO)
Hippe 1967). Such a turnover would be an energy wasting r~- Kr
metabolic pathway.
The last remaining aspect of the model to be described K] was determined to be between 0.036 and 0.047 g/l
is the initialization of the non-growth associated produc- (NHa)2 SO4 as shown in Fig. 9.
tion of P, and the inhibition of it by the limiting substrate Table 2 summarizes all the parameters evaluated which
NH~ (S). To describe this inhibition rp,2 in Eq. (7) is mul- are required to solve the final set of equations [Eqs. (1),
tiplied with a function that is used to describe allosteric (2), (3), (4), (5), and (8)]. Figure 10 shows the whole
substrate inhibition. This yields the following equation: model in the form of a block diagramm.

K~ - - ,
( - k I P + k2 R)
, 9
(8) 3. 2. Simulation o f the Model
re,2 - KI + S

K I is the inhibition constant representing the substrate Figure [ 1 shows the results of a numerical integration of
concentration at half maximum rate of production of P. the model with one set of parameters as given in the figure.
 E. Heinzle and R. M. Lafferty: Kinetic Model for Growth and Synthesis of Poly-/3-Hydroxybutyric 13

-1 [-] Table 2. Evaluated parameters for the model equations

I
15 Parameter Value Dimension Equation no.

YR/S 1.5 - (3)

K=. • t ~m,1
KSA
0.13
0.1
h -1
g/1
(2)
(2)
/~m,2 0.08 h -1 (2)
10 KS,2 1.0 g/1 (2)
n 5 - (2)
YP/R 0.105-0.16 - (5)
KI 0.036-0.047 g/1 (8)
k1 0.045-0.048 h -1 (8)
k2 0.18-0.176 h -1 (8)

Fig. 9. Evaluation of the inhibition constant K I for the non growth

associated synthesis of PHB corresponding to Eq. (10). x, evalua-
i i i ! ! i 1 tion with YP/R = 0.16; o, evaluation with YP/R = 0.105
0 .2 .L~ .6
s [g/l]

R
f~
I

dS I
~ - - I m ~ = rS S -' ~ ~ ~(s) rs I
(

dY

~ __~ qT-= %,z § ~p,2 F rp, 2 = R KI + S

,
(-kl P + k 2 R) 'rP~2
I
z (s/Ks,2) ~
~(s) ~ "m,1 -KS,l+ S + Urn,2 "1"+ (S/Ks,2)n Fig. 10. Block diagramm of the
model

Concentrations and reaction rates are plotted versus time. describing the batch growth and accumulation of PHB in
The time course of calculated concentrations is compared A. eutrophus under chemolithoautotrophic growth condi-
with experimental values.
tions with controlled concentrations of the dissolved
Further comparison of the model with the experiment gaseous substrates.
is shown in Fig. 12a and b. In Fig. 12a the rate o f synthe- A/though not considered in the present work, it is
sis o f P H B (rp) is plotted versus the content of PHB in the interesting to speculate how the model could be altered
dry cell mass (%-P) and in Fig. 12b the specific rate of to accomodate the effects of changing concentrations of
synthesis o f P H B (pp = rp/P) is plotted versus the quo- gaseous substrates. Additional liquid phase balances for
tient X/P. These comparisons demonstrate the good agree-
H2, 02, and CO 2 would be needed to account for the gas-
ment o f the data from simulations of the model with the
liquid transfer and biological uptake of these quantities.
experimental data. Thus the proposed model is useful in
Although the equations as presented would remain basi-
14 E. Heinzle and R, M. Lafferty: Kinetic Model for Growth and Synthesis of Poly-#-Hydroxybutyric

S [g/t] X,P,R [g/t] P/X l-]

3
O Q "

.+ /o /. x 6

"-\+s ,A /
"~x.,. / / X/1c .5
\. // z/

\ ).~2' - ' ? ~ -/.: ' R .2

. 1

~--------x= x-x'X " +~ J ' .0

0 10 20 30 /,,0
a t [hi

r [g/l-h]

.6

~x
.4

Fig. 11 a and b
Integration of the model equations with following set o f
parameter values: YR/S = 1,54, Vm 1 = 0. t3 h -1, K S 1 =
.2 0.1 g/l,/~m 2 = 0.08 h - 1 , KS 2 = 1.'0 g/l, n = 5, YP/R =
0.105, K I ~ 0.041 g/I, k 1 = ()'.045 h - l , k2 = 0.I8 h -1.
Initial conditions: R 0 = 0.22 g/l, SO = 2.3 g/t, PO = 0.02
g/i. b Shows the rate curves and a shows the corresponding
rp2 concentration curves, which are compared with experimen-
tal values. +, substrale; A, biomass; x, PttB; o, residual bio-
~-rP1, I
mass; o, PHB-content
.0 30 40
0 10 2O
b f [h]
 E. Heinzle and R. M, Lafferty: Kinetic Model for Growth and Synthesis of Poly-C~-Hydroxybutyric 15

rp [g//.h] Abbreviations

DSM Deutsche Sammlung yon Mikroorganismen, G6ttingen,

.6- o
R.R.G.
9 Q PHB Poly-C~-hydroxybutyric acid

Symbols

c 1 [g/ll Concentration
KI [g/ll Inhibition constant, g/1 (NH4)2SO 4

1
.2 n [-1 Hill coefficient
P [g/l] Product concentration (PHB)
Prot [g/l] Protein concentration
r [g/1 hl Reaction rate
R [g/l] Residual biomass (R = X - P)
0 .2 .4 .5 .~ 1.0 rp,red
[-] Reduced rate of synthesis of PHB
a P/X [-] S [g/1] Limiting substrate NH~ as (NH4)2SO 4
X [gill Cell cry mass concentration
Up [b-1) YR/S [g/gl Yield coefficient, g residual biomass / g substrate
6 YP/R [g/gl Yield coefficient, g PHB/g substrate
[h -11 Specific rate of synthesis of R(rR/R )
~Zm,1 [h -11 Maximum specific growth rate
urn,2 [h - l ] Maximum specific growth rate
Up [h-1 l Specific rate of synthesis of P(rp/P)
%'P [%1 PHB content of the cell dry mass

Subscripts

b X/P l I
Product
Residual biomass
Fig. 12a and b. Comparison of the synthesis of PHB in the experi- Substrate
ment and in the model, a Shows a plot of the rate of PHB-synthe- Cell dry mass
sis versus the PHB content and b shows a plot of the specific rate Beginning of fermentation
of PHB-synthesis versus the reciprocal content of PHB. e, regres- After consumption of all S in fermentation
sion of the experimental data with one square root paraboly; o,
regression with pieces of a function linear in parameters; X, cell
dry weight; P, PHB; rp, rate of synthesis of PHB; ~p, specific rate
of synthesis of PHB (pp = rp/P). Parameters and initial conditions
as in Fig. 11 References

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cally the same, m a n y of the parameters w o u l d have to be
expressed as f u n c t i o n s of the dissolved gas concentrations.
As with a n y multisubstrate growth model, the c o m p l e x i t y
o f the m o d e l would increase as the influence o f each sub-
strate is incorporated. An interesting situation could exist
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the cells (Repaske R and Repaske AC 1976). Since the
reaction o f dissolved C 0 2 with water to yield H2CO 3 is a
relatively slow reaction, this could become the limiting
step if the activity of the biomass is large enough.
Acknowledgements. Our thanks are due to the "Fonds zur F6r-
derung tier wissenschaftlichen Forschung" (project number 3296)
for having supported this work.
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Received November 23, 1979