European J. Appl. Microbiol.

Biotechno]. 7, 1--10 (1979)

European
Jo~,~,o,
Apphed
Microbiology and
Biotechnology
9

9 by Springer-Verlag 9 1979

Formal Kinetics of Poly-/~-HydroxybutyricAcid (PHB)

Production in Alcaligenes eutrophus H 16 and

Mycoplana rubra R 14 With Respectto the Dissolved

Oxygen Tension in Ammonium-Limited Batch Cultures
B. Sonnleitner, E. Heinzle, G. Braunegg, and R.M. Lafferty
Institut ffir Biochemische Technologie und Lebensmittelchemie der TU Graz,
Schl6gelgasse 9, A - 8010 Graz, Austria

Summary. Under chemolithoautotrophic growth conditions with the organism

Alcaligenes eutropbus HI6 the exponential growth phase is characterized by two

different growth rates, each associated with different specific rates of ammonium
consumption. On the basis of the analytical determination of Poly-13-hydroxybutyric acid (PHB), it can be conclusively shown that PHB is synthesized even during
the exponential growth phase at a specific rate proportional to the specific growth
rates of total biomass. After complete consumption of ammonium, the increase
of biomass is exclusively due to PHB synthesis, whereas protein and rest biomass
(cell dry weight minus PHB) remain constant. After an extended period of fermentation, the PHB content reaches a saturation value. The transient phase between the growth and the storage phase is very short in comparison to the duration of the whole fermentation. In the case of Alcaligenes eutropbus, strain H 16,
high concentrations of dissolved oxygen strongly influence growth as well as PHB
synthesis.

Introduction

Poly-~-hydroxybutyric acid (PHB) is a carbon and energy storage material accumulated

by a wide variety of microorganisms usually under certain conditions of nutrient limitation, such as nitrogen, phosphorous a n d / o r oxygen limitation (Dawes and Senior, 1973;
Emeruwa and Hawirko, 1973; Nuti and Lepidi, 1974; Repaske and Repaske, 1977). The
polyester of the D(-)13-hydroxybutyric acid could possibly find industrial applications
as a biodegradable substance with a number of physical properties related to conventional plastics now in use (Lafferty and Heinzle, 1977). In addition to many conventional
substrates, a mixture of H2, 02, and CO 2 can be utilized by Alcaligenes eutrophus to
synthesize PHB (Schlegel et al., 1961).
In the case ofAlcaligenes eutrophus H 16, PHB can accumulate in amounts up to 65%
of dry weight (Schlegel et al., 1961) when the culture is nitrogen limited, and up to 28%
(Schuster and Schlegel, t967; Morinaga et al., 1978) under conditions of oxygen limitation. A significant synthesis of PHB, however, during exponential growth of Alcaligenes
eutrophus has not yet been cited.
0171--1741/79/0007/0001/~ 02.00

B. Sonnleitner et al.

PHB synthesis has also been observed in methanol-utilizing bacteria, such as Mycoplana
(Braunegg, 1978). In Azotobacter beijerinckii oxygen limitation is more effective in stimulating PHB accumulation than is ammonium or nitrogen limitation (Dawes and Senior,
1973; Ward et al., 1977).
The aim of this work was to study the correlation of PHB synthesis with cell growth
and the influence of the dissolved oxygen tension on both processes.
Results presented are based on a series of experiments carried out under identical conditions.
2. Materials and Methods

2.1 Organisms and Media

Alcaligenes eutrophus, strain H 16 (Deutsche Sammlung yon Mikroorganismen Nr. 428,
G6ttingen, F.R.G.), was cultivated in a mineral medium (Schlegel et al., 1961) containing
2.8 g/1 (NH4)2SO 4 and gassed with a sterile mixture of H2, 02, and CO 2 in variable
ratios. This strain was also grown on the same mineral medium with 2% (w/v) lactic acid
as a carbon source. Mycoplana rubra, strain R 14, (Braunegg, 1978) was cultivated in
the same mineral medium with 0.5 to 1.5% (v/v) methanol as a carbon source supplemented with 50 rag/1 Fe-NH4-citrate.
2.2 Inocula
One liter of the chemolithotrophie inoculum of Alcaligenes eutrophus H 16 was prepared
using a 2 1 glass fermenter at 30~ (Quickfit, Wiesbaden, F.R.G.); gases were supplied in
a ratio of H2:O2:CO 2 = 6:2:1 and at a total flow rate of 1.5 vvm. The inocula for chemoorganotrophic fermentations were grown in shaking flasks using the same media as in the
fermentations.

2.3 Fermentation Conditions

A Braun-Biostat fermenter (Braun-Melsungen, Melsungen, F.R.G) with a working volume
of 8 1 and a Bioengineering fermenter type L 1523 (Bioengineering AG., Riki, Switzerland) of 18 1 total volume which were equipped with two conventional turbine-type impellers and baffles were employed. The temperature was maintained at 30~ (-+ 1 ~ and
pH was kept at a constant value of 6.9 by an automatic pH-measuring and titrating system
(type Meredos PH/R, Jungkeit, G6ttingen, F.R.G.) using 2 N NaOH. During chemolithoautotrophic fermentations, the impeller speed was 400 rpm; the partial pressures of the
gases were adjusted only by regulating sterile gas flow. The resulting concentrations of
the dissolved gases were: H2:0.3 to 0.7 rag/l, 02.2.2 to 2.8 mg/1,CO2:70 to 120 rag/1.
During the chemoorganotrophic fermentations the dissolved oxygen tension (d.o.t.) was
usually maintained between 2 and 3 rag/1 by varying impeller speed as well as the flow
rate of sterile air.

2.4 Analytical Methods

The d.o.t, was measured with a polarographic oxygen electrode (Instrumentation Laboratories, type IL-530, Ingold, Frankfurt a. M., F.R.G.) and/or with a mass spectrometer

Formal Kinetics (PHB)

(GD 150 Varian Mat, Bremen, F.R.G.) equipped with a membrane inlet system (teflon
membrane D 602, Radiometer, Copenhagen, Denmark). With the latter system the concentrations of dissolved H 2 and CO 2 were also determined (Heinzle and Lafferty, 1978).
PHB was determined using a gas chromatographic method (Braunegg et al., 1978) with
benzoic acid as an internal standard (gas chromatograph model HP 5840 A, Hewlett
Packard, USA). Parallel determinations were carried out after extraction of the polymer
from acetone-dried cells employing ethylene carbonate (Lafferty and Heinzle, 1977)
according to a spectrophotometric method of analysis (Slepecky and Law, 1969;Sonnleitner, 1976; Heinzle, 1978). Ammonium concentrations were determined using a modified Berthelot reaction (Klinisches Labor, Merck, F.R.G. 1974). Cell dry weight was
determined by the membrane filter method (0.2/ira, type SM 11307, Sartorius, G6ttingen, F.R.G.) after air drying for 24 h at room temperature and heating to 100~ for 48
h. Optical density was measured at 436 nm using a spectrophotometer (Zeiss, model
PMQ II, Oberkochen, F.R.G.) after dilution of the cell suspension with 0.9% NaCI solution to a proper density (0.1 <~ E < 0.6). Protein was determined according to Lowry
(modified by Ruhr, 1977) and/or using the Biuret method (modified by Oeding, 1972).
Lactic acid as well as methanol were determined gas chromatographically, methanol directly on an 8 ft. glass column of 0.1 in. diameter, packed with 10% Hallcomid M 18/OL
on chromosorb G AW 60/80. After freeze drying of the sample, lactic acid was treated
with 3% (v/v) H2SO 4 in methanol, then heated to 100~ for 1 h to obtain lactic acid
methyl ester. This was separated from other substances on a 8 ft. glass column packed
with 2% Reoplex 400 on chromosorb G AW 60/80 and detected quantitatively with a
FID (Hewlett Packard, USA). All chemicals used for analytical purposes were of
analytical grade; those used for nutient solutions of either analytical grade or of DAB 7
quality and purchased from E. Merck, Darmstadt, F.R.G.
2.5 Evaluation Methods
Measured data were fitted by simple functions; the least square method was used to find
the optimum fit. One method used for fitting was to calculate a single regression function for each phase of a fermentation; the other method was to use segments of parabolas with a narrow region of validity to fit the entire fermentation. The latter method
was carried out using a computer (Univac 1180) and a digital plotter. Both methods
gave practically the same results. The following functions were used:
Growth phase: lne i = k I + k2.t
Storage phase: ci = k 3 + k4.t1/2 + k4.t
stepwise fit: c i = k 5 + k6.t + k7.t2,
whereby c i = concentration, kj. = constant, t = time.
For further evaluations regression data were used.
2.6 Dynamic Measurements
Only those dynamic measurements carried out during chemoorganotrophic fermentations with lactic acid as the sole carbon source are described in this paper. In order to
measure the 02 consumption rate, the sterile air flow was stopped and at the same time
the impeller speed was reduced to 50 rpm. When the reading of the oxygen electrode

B. Sonnleitner et al.

was approximately 0.5 mg/1, the air flow rate and the impeller speed were re-adjusted to
their original values. The slope of the plot cO2,L (d.o.t.) versus time is assumed to equal
the actual c o m s u m p t i o n rate (r0) of the suspension, since the oxygen transfer under
these conditions is negligible. The response time of the 0 2 electrode was d e t e r m i n e d as
being a negligible factor.

3. Results and Discussion

Alcaligenes eutropbus H 16 grown in a m m o n i u m - l i m i t e d c h e m o l i t h o a u t o t r o p h i c batch
cultures is able to accumulate up to 78% of PHB in the late storage phase. Even during
the early exponential growth phase, PHB is synthesized and is definitely growth-associated (Figs. 1-3). It is obvious from Figure 1 that the c o n c e n t r a t i o n time courses of
cell dry weight (X), PHB and rest biomass (R, that is, cell dry weight minus PHB) have
the same slope in a semilogarithmic plot at a m m o n i u m - c o n c e n t r a t i o n s above 1 g/l,
calculated as NH4Cl, while at lower a m m o n i u m concentrations, i.e., under 1 g NH4C1/1
the slopes are different. This indicates that PHB synthesis is fully associated with cell
growth even when there is no substrate limitation. In an analogous m a n n e r to the
equation
px:_dlnX
dt

= d_XX. L
dt
X

where X = cell dry weight, formal kinetic parameters

sfas

15
o

10

Z,

10 12

lz, i-, 7,-,

lZ, 16 ~' 20

30

40

50

60

70

Fermentation time (h)

Fig. 1 a and b. Growth and PHB synthesis of A. eutropbus H 16 in ammonium-limited

chemolithoautotrophie fermentation. The growth phase is shown in a semilogarithmic plot (a)
- up to 16.5 h - and the storage phase in a linear plot (b) - after 17 h. Fermentation conditions:
temperature: 30~ pH: 6.9; Dissolved gas tensions were as follows: cO2,L : 2.5 ~- 0.3 mg/l;
CH2,L : 0.5 ~-0.2 mg/l; cco2, L : 100 "7 30 mg/l. The sterile gas flow was varied hetween 0.15 and
0.20 vvm; the impeller speed was kept constant at 400 rpm.
The graphical presentation compiles the results of a number of independent experiments each
done under identical conditions, o Substrate concentration "S": calculated as g/l NH4CI;
OCell dry weight "X"; 9 PHB "P"; 9 Rest biomass "R" = cell dry weight minus PHB

Formal Kinetics (PHB)

/~p . d In
. P H. B .
d t

d PHB

d t

PHB

for the product PHB and

dlnR
/~R

dR.

dt

-dt

},/IX

.5

ul

~.2

10

20

30

40

50

60

Fermentation time (h)

b
4

0~Z
176176

~, 2
o_
tO

0
0

10

20

30

40

50

60

Fermentation time (h)

Fig. 2 a and b. a Growth rate and PHB production rate o f A . eutropbus H 16 in chemolithoautotrophic fermentation. (~ r X = dX/dt; 9 rR= dR/dt; 9 rp = dP/dt, b Specific rates of growth and
PHB production. (~/~X = r x / X ; 9 #R = rR/R; 9 #P = rp/P. Fermentation conditions were as in
Figure 1

B. Sonnleitner et al.

can be defined and evaluated. In this case/Ip is the specific rate of product formation
and ~R is the corresponding value for rest-biomass formation. #p is equal to/~R during
the first part of the exponential growth phase. In the second part of this phase (i.e.,
after 11 h as show in Fig. 1), #X and/~R are significantly smaller (/aX = 0.13 h-1 and
~R = 0.11 h-l); /~p, however, remains constant during the whole exponential phase.
In the case of a chemolithoautotrophic culture,/.tX,ma x =/~P,max =/~R,max -- 0,23 h -1.
The production of PHB is, therefore, apparently an autocatalytic reaction during the
exponential growth phase (Fig. 2b).
However, during the storage phase when the nitrogen source is depleted, the increase
of cell dry weight is only due to the accumulation of PHB as can be seen from the time
course of change in rest biomass when linearly plotted as in Figure 1. Seventy percent
of the rest biomass consisted of protein. Rest biomass as well as the protein concentration remains constant throughout the entire storage phase. Furthermore, it can be seen
that PHB must be the only storage material synthesized by Alcaligenes eutropbus H 16
under the described conditions. Similar results were reported by Ward et al. (1977)
in the case of Azotobacter beijerinckii.
Within a very short period after the ammonium concentration has reached practically
undetectable low values, the rate of PHB synthesis exhibits a maximum and then
decreases asymptotically to zero as shown in Figure 2a. From the very beginning of the
storage phase the PHB production rate (rp = d PHB/d t) is equal to the rate of cell dry
weight increase (r X = d X/d t). The PHB content approaches a maximum value (Fig. 4).
Therefore, one can conclude that the total yield of PHB is indirectly a function of the
initial ammonium concentration in an ammonium-limited culture.
The exponential growth phase and the storage phase are separated by an extremely
short transition period which is almost comparable to the action of a switching
mechanism. This transition period coincides with the depletion of the nitrogen source
and only lasts for less than one generation time. The formal kinetic interpretation
suggests that a drastic change of metabolic regulation takes place. The rates of rest-biomass and protein production decrease to zero and remain at this level throughout the
remaining time of fermentation. The PHB production rate increases significantly during
the transition period. During the very short transition period, it is, however, difficult
to obtain sufficient experimental data. Although samples were taken from the fermenter
every 20 min, there are not enough conclusive data available to be able to fully describe
the transition period in detail.
The rate of PHB production (rp) is linearly correlated with the rate of cell dry weight
production (rx) and with the rate of rest-biomass production (r R) during both parts of
the growth phase. Following the transition period, rp is equivalent to the value of r X.
Only PHB is produced in significant amounts as a storage material (Fig. 3a and b).
The plot of PHB production rate (rp) versus the PHB content of the cells (% of cell
dry weight) (Fig. 4) shows a maximum content which cannot be exceeded and suggests
that there exists a minimum content of PHB during the exponential growth phase under
the described discontinuous culture conditions. It is of course logical that the maximum
content of PHB will depend on the particular organism and growth conditions. In the
authors' laboratory the highest maximum content of PHB in chemolithoautotrophically
grown Alcaligenes eutropbus H 16 was found to be 78%. To clarify whether or not the
synthesis of PHB is directly associated with growth only in the case of chemolithoauto-

Formal Kinetics (PHB)

o_

)
)

(1)

o
~U

.6

~s

/4

)
o
Q

@.3

o
EL

s3 .2
I
EL

D_

.1

.1

.6

.7

9
_

/
I

L.

.1

.2

.3

I _

.5

Rate of rest biomass production ( rR ) (g/l h)

Growth rate ( rx ) ( g / [ h)

Fig. 3 a a n d b. Correlation between PHB production rate (rp) and the rate o f synthesis of cell dry
weight (rx) a or rest biomass (r R) b during a chemolithoautotrophic fermentation of
A. e~tropbus H 16, rp = dP/dt; r X = dX/dt; r R = dR/dr. F e r m e n t a t i o n conditions were as in
Figure i

o\
o

o
o

J~

100

gs

6O

40

o
o

o)

0
0

Q_

0
0
0
0

T
0_ .2

of
.1

O
O
O
O

o
o

0
4

20

/40

60

PHi3 Content (%PHB)

i
80
5

%
t

20

I _ I
I
I
I
t
2
3
/4
5
Dissolved oxygen tension (Coz,L) (rag/t)

Fig. 4. PHB production rate (rp) versus PHB c o n t e n t (% of cell dry weight) o f A . eutropbus H 16
grown under chemolithoautotrophic conditions. F e r m e n t a t i o n conditions were as in Figure 1
Fig. 5. Effect of dissolved oxygen tension (cO2,L) on the specific respiration rate r o / R , which
was evaluated from d y n a m i c measurements. Experiments were carried o u t with A. eutropbus
H 16 under chemoorganotrophic conditions with lactic acid as sole carbon source. Fermentations
conditions: temperature : 3 0 ~ pH : 7.0; impeller speed: 400 rpm, during d y n a m i c measurements:
50 r p m

B. Sonnleitner et al.

!"1

I00

[]

[]

c
o

~- E'
~, ,_o[~ 50

I"1

10

20

30

Fermentation time (hours)

Fig. 6. Maximal specific respiration rate ro/R during growth of A. eutropbus H 16 under ammonium- limited chemoorganotrophic conditions with lactic acid as sole carbon source. The nitrogen
source was fully depleted after 16,5 h. Fermentation conditions were as in Figure 5

trophically grown cells of Alcaligenes eutropbus H 16, this strain and a different
organism were grown chemoorganotrophically for purposes of comparison and each
then subjected to analysis as described before. Lactic acid was the only carbon source
used for Alcaligenes eutropbus. The organism used for comparison was Mycoplana rubra
strain R 14 with methanol as the carbon source. The results of a series of growth experiments with Mycoplana rubra R 14 were qualitatively identical with the results obtained in the case of chemolithotrophically grown Alcaligenes eutrophus H 16.
The only difference between Mycoplana rubra and Alcaligenes eutrophus under
chemoorganotrophic growth conditions are the numerical values for the rates of biomass synthesis (Alcaligenes eutrophus H 16: #X,max = 0.42 H-1;Mycoplana rubra R 14:
/IX,ma x = 0.13 h-l).
Previous experiments with Alcaligenes eutrophus H 16 which were carried out under
ehemolithoautotrophic growth conditions at high dissolved oxygen tensions (from
4 mg/1 up to 7 mg/l) showed that significantly smaller, specific growth rates were
measured than with a d.o.t, of 2 to 3 mg/1. Whenever the d.o.t, at the beginning of a
fermentation exceeded 3 mg/1 and extended "lag-phase" was observed. A decrease of the
d.o.t, to 2.5 rag/1 during the fermentation was always correlated with an increase in
the specific growth rate. Dynamic measurements during growth of Alcaligenes eutrophus
H 16 under chemoorganotrophic conditions at different cell densities and during
different phases of the fermentation revealed a sigmoid decrease of the d.o.t, with a
maximum slope (r O) at a d.o.t, of approximately 2.5 mg/1 (Fig. 5). From the results
of several dynamic experiments carried out during a fermentation, maximum oxygen
consumption rates (to) were evaluated according to the described method and
normalized on the basis of the rest biomass.

Formal Kinetics (PHB)

Figure 6 shows the time course of the maximal specific respiration rate calculated on
the basis of rest biomass. This plot appears to be an unsteady function, however, it is
the graphical presentation which best reveals the abruptness of the transient phase.
Calculation of the maximal respiration rate on the basis of total biomass results in a
similar plot. This plot also shows the transient phase, but much less pronounced than
when only rest biomass is used as the basis of calculation. This is justifiable since
stored PHB has no connection with respiratory activity of cells.
As can he seen from Figure 6 two distinct phases exist. During the exponential
growth phase the maximal values of r 0 are nearly constant. After the depletion of the
nitrogen source the values of ro/R decrease abruptly to about 40% of the value found
in the growth phase and then decrease slightly during the further period of fermentation.
On the basis of all described results it can definitely be asserted that in the case of
both Alcaligenes eutrophus and Mycoplana rubra, where the growth conditions are
such that PHB synthesis can take place there are three definitely recognizable phases.
These are: a phase of exponential cell growth and directly associated PHB production,
a second transient phase where the rate of PHB production accelerates to a maximum
and the rate of rest biomass production approaches zero, and a third phase characterized
exclusively by PHB accumulation the rate of which approaches zero value as the percentage
of PHB content of the cells asymptotically approaches a maximum value.

Acknowledgments. Our thanks are due to the "Fonds zur F/Jrderung der wissenschaftlichen
Forschung" (project number 3296) for having supported this work.

Abbrevations
c o 2 ,L

concentration of oxygen in the liquid phase (dissolved oxygen tension: d.o.t)

CH2,L

concentration of hydrogen in the liquid phase

c c o 2,L concentration of carbon dioxide in the liquid phase

rR

limiting substrate, concentration of

total biomass, concentration of; total cell dry weight
product; PHB, concentration of
rest biomass: X - P, concentration of
= dX/dt growth rate
= dP/dt rate of PHB synthesis
= d R / d t rate of rest biomass production

ro

= dcO2,L/dt rate of oxygen consumption

/ax
/.tp

= dX/dt 9 1/X = r X 9 1/X specific growth rate

= dP/dt 9 1/P = rp 9 1/P specific rate of product formation
= d R / d t 9 1/R = r R 9 1/R specific rate of rest biomass formation
specific respiration rate

S
X
P
R
rX
rp

//R
ro/R

10

B. Sonnleitner et al.

References

Braunegg, G. (1978). Lecture at Annual Meeting of the "Osterr. Ges. f. Hygiene,

Mikrobiologie und Pr~iventivmedizin", May 1978, Graz
Braunegg, G., Sonnleitner, B., Lafferty, R.M. (1978). Eur. J. Appl. Microbiol. Biotechnol.
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Dawes, E.A., Senior, P.J. (1973). Adv. Microb. Physiol. 10,203-253
Emeruwa, A.C., Hawirko, R.Z. (1973). J. Bacteriok 116,989-993
Heinzle, E. (1978). Dissertation, Graz
Heinzle, E., Lafferty, R,M. (1978). 1st European Congress Biotechnology, Interlaken,
Switzerland
Lafferty, R.M., Heinzle, E. (1977). Chem. Rundsch. 30, (41), 14-16
Fa. Merck (1974). Klinisches Labor, 12th ed., Darmstadt, F.R.G.
Morinaga, Y., Yamanaka, S., Ishizaki, A., Hirose, Y. (1978) Agric. Biol. Chem.
42, (2), 439-444
Nuti, M.P., Lepidi, A.A. (1974). Proc. 4th Int. Symp. Yeasts, part 1. 123-124
Oeding, V. (1972). Dissertation, G6ttingen, F.R.G.
Repaske, R.,Repaske, A.C. (1976). Appk Environ. Microbioi. 32,585--591
Ruhr, EM. (1977). Dissertation, G6ttingen, F.R.G.
Schlegel, H.G., Gottschalk, G., Bartha, R. (1961). Nature (Lond.) 1919, 463--465
Schuster, E., Schlegel, H.G. (1967). Arch. Microbiol. 58,380-409
Slepecky, R.A., Law, J.H. (1969). Anal. Chem. 32, 1 6 9 7 - 1 6 9 9
Sonnleitner, B. (1976). Diplomarbeit, Graz
Ward, A.C., Rowley, B.I., Dawes, E.A. (1977). J. Gen. Microbiol. 102, 61-68
Received December 18, 1978