JOURNALOF FERMENTATIONAND BIOENGINEERING

Vol. 71, No. 4, 254-257. 1991

Production of Poly-/ -Hydroxybutyric Acid from Carbon

Dioxide by Alcaligenes eutrophus ATCC 17697 T
A Y A A K I I S H I Z A K I * AND K E N J I T A N A K A
Department of Food Science and Technology, Faculty of Agriculture, Kyushu University,
46-09 Hakozaki, Higashi-ku, Fukuoka 812, Japan
Received 8 October 1990/Accepted 5 January 1991

The characteristics of PHB production from carbon dioxide by autotrophic culture of Alcaligenes eutrophus
ATCC 17697 x using a recycled gas closed circuit culture system under the condition of oxygen limitation were
investigated. Cell concentration increased to more than 60 g/l after 60 h of cultivation, while the PHB concen-
tration reached 36 g/l. PHB accumulation in the oxygen-limited culture was superior than that in an am-
monium-deficient culture. The PHB produced was identified as a homopolymer of D-3-hydroxybutyrate by 1H
and 13C NMR analysis. The stoichiometry for PHB production from CO2 under the oxygen limitation condi-
tion was indicated to be as follows: 33H2 + 1202 + 4CO2 ~ C4H602 + 30H20. This stoichiometry shows that
the hydrogen consumption per one mole of CO2 for PHB production is larger than that for cell formation.

In a previous study (1), a recycled gas closed circuit culture
system was employed to obtain a high cell mass concentra-
tion of hydrogen-oxidizing bacteria with complete utiliza-
tion o f substrate gas and without danger of detonation.
Alcaligenes eutrophus A T C C 17697 T accumulates poly-
i3-hydroxybutyric acid (PHB) as a storage polymer under
culture conditions that limit the mineral nutrients (2, 5) or
oxygen (4, 3). P H B is attracting industrial attention as a
raw material for b i o d e g r a d a b l e plastic. At present, P H B is
p r o d u c e d by using an organic acid, such as pentanoic acid,
as substrate. In this paper, we describe the characteristics
o f fermentation for the p r o d u c t i o n o f P H B from COz by
batch culture of A. eutrophus using a recycled gas closed
circuit culture system under the oxygen limitation condi-
tion.
MATERIALS AND METHODS
Cultivation The microorganism, media, cultivation
system, pH, temperature, agitation speed and the gas
feeding rate used in this experiment were the same as
previously reported (1) unless otherwise described. P H B in
the cells was accumulated by limitation o f oxygen,
hydrogen and a m m o n i u m . In oxygen-limited culture, the
partial pressure of oxygen in the gas phase was maintained
under 0.15 arm during cultivation; the partial pressure of
dissolved oxygen then became zero when the cell concentra-
tion exceeded 15 g/l, and thus the oxygen limitation condi-
tion was formed.
Analytical methods The determinations o f substrate
gas composition, gas c o n s u m p t i o n in the fermentation,
partial pressure o f dissolved oxygen, cell concentration
and the elementary composition o f the product were car-
ried out by the methods described earlier (1). It was verified
that the calibration curve used for determination o f cell
concentration was correct in the P H B accumulation phase
by drying the cells in the broth at 105C and measuring its
constant weight.
Protein concentration was determined by the Biuret
m e t h o d (6). Nucleic acid concentration was determined by
* Corresponding author.
254
the extraction m e t h o d of Schneider using perchloric acid
(7). P h o s p h a t e concentration was determined by the
m e t h o d o f Allen (8).
P H B concentration was determined by the method o f
Sonnleitner et al. using gas c h r o m a t o g r a p h y (9). The cells
produced were centrifuged and dried by freeze-drying.
Two milliliters of methanol acidified with 3 ~ ( v / v ) H/SO 4
and 2 m l o f c h l o r o f o r m were added to the dried cells,
then the suspension was heated at 100C for 3.5 h for
depolymerization and methanolysis of PHB. After cool-
ing, 1 ml o f H20 was a d d e d and the suspension was shaken
well for 10 rain. After two phases were allowed to separate
by standing, 2 / d o f the organic phase was injected to a gas
c h r o m a t o g r a p h i c analyzer equipped with a flame ioniza-
tion detector (JGC-20K GAS C H R O M A T O G R A P H ,
JEOL. Co., Ltd., Tokyo). A glass column(3 m 4 m m i.d.)
was filled with 2 ~ Reoplex 400 on C h r o m o s o r b G A W 60
to 80 mesh and used under the following conditions: initial
temperature = 90 C; final temperature = 150 C; the rate o f
temperature increase was set at 8C/rain. The sodium salt
of D,L-3-hydroxybutyrate was used for the preparation of
a calibration curve.
The p r e p a r a t i o n o f P H B used for N M R analysis was
carried out according to the method o f Williamson and
Wilkinson (10). The cells produced under oxygen limita-
tion were centrifuged and treated with 6//00 sodium
hypochlorite solution at 37C for 2 h. After centrifuga-
tion, particles o f P H B were washed with distilled water,
acetone and ethanol. Extracted P H B was purified with
chloroform.
IH and ~3C nuclear magnetic resonance (NMR) analysis
using a J E O L G S X 400 spectrometer was carried out to
determine the chemical structure o f P H B produced from
CO2 in the oxygenqimited culture. The 400 M H z N M R
spectra o f ~H and ~3C were recorded from CDC13 solution
o f PHB. N M R analysis of P H B was entrusted to Mitsu-
bishi Petrochemical Engineering Co., Ltd., Tokyo.
RESULTS AND DISCUSSION
Cell growth and accumulation o f P H B in batch
culture Figure 1 shows typical time courses, indicating
Voz. 71, 1991 P H B F R O M CO2 255

cell mass concentration, P H B concentration and the 50

decrease of partial pressure of dissolved oxygen. The in-
itial composition of substrate gas mixture was set at H2 :
02 : C O 2 = 7 5 : 15 : 10. The microorganism grew at a con-
stant specific growth rate until the partia ! pressure o f
dissolved oxygen in the culture liquid decreased to about
zero, when the cell growth became linear. After the cell
concentration exceeded 39 g/l, the growth was gradually
suppressed and almost ceased at cell concentrations above
a p p r o x i m a t e l y 50 g/l. S u p p l e m e n t a t i o n o f mineral nu-
trients to the culture medium was not carried out during
the cultivation in this experiment. The accumulation of
P H B was mainly observed after the oxygen limitation con-
dition was formed. Partial pressure o f dissolved oxygen in-
dicated zero in the P H B accumulation phase. As the
fermentation proceeded, the percentage o f P H B in the
cells increased gradually, and reached to 53% at the end of
the fermentation. Protein f o r m a t i o n a l m o s t ceased in the
oxygen-limited condition.
In the fermentation test shown in Fig. 2, the initial gas
c o m p o s i t i o n was set at H 2 : 0 2 : C O 2 = 6 5 : 25 : 10. A s a
result, exponential cell growth continued until the cell con-
centration reached 15 g/l, but the percentage o f P H B ac-
cumulation was low. The partial pressure of dissolved ox-
ygen during linear growth was maintained at a b o u t 0.05,
atm, which is above the critical level for oxygen limitation.
Therefore, it m a y be suspected that limitation o f hydrogen
was created and this suppressed the cell growth. The p o o r
accumulation o f P H B in this experiment agreed with the
result o f hydrogen-limited culture reported by Schlegel
and Lafferty (21).
Figure 3 shows a batch culture profile when mineral
nutrients were supplied during the cultivation. A small
quantity (3 ml) o f concentrated solution (MgSO4.7H20;
20 g/l, FeSO 4 7H20; 2.0 g/l, CaSO4; 0.4 g//) was aseptically
a d d e d to the culture liquid with a working volume o f
100 ml. Since the c o n s u m p t i o n rate o f p h o s p h a t e ion in the
culture liquid under oxygen limitation was faster than that
under oxygen sufficiency, feeding o f KH2PO4 (50 g//) was
carried out periodically. The a m o u n t of p h o s p h a t e con-
sumption required for the f o r m a t i o n o f 1 g biomass in the
P H B accumulation phase (at 50 h of cultivation) was 1.26
times greater than that in the exponential growth phase in
A4c
O~
'Z 2c 0.20 ~
'4 0
i lc o.lo ~
0 10 20 30 40 50 60
Cultivation time ( h )
FIG. 2. Time course of oxygen-limited culture of A. eutrophus
under the initial gas composition of H2 : 02 : CO2= 65 : 25 : 10. Sym-
bols: concentrations of cell ( ) , P H B ( C ) , protein (A), and nucleic
acid (~), partial pressure of dissolved oxygen ( [] ).
which P H B was not accumulated. The phosphate concen-
tration was maintained above 0.05 g/l in the P H B ac-
cumulation phase. According to the results o f our experi-
ment, if the phosphate concentration was set above
0.35 g/l at the start o f cultivation~ and maintained above
0.02 g/l during cultivation under oxygen-sufficient condi-
tions, neither a decrease o f the cell growth rate nor o f P H B
accumulation was observed. Thus, in the cultivation
shown in Fig. 3, it was concluded that p h o s p h a t e limita-
tion was not formed. As a result, the cell concentration in-
creased to about 60 g/l after 60 h o f cultivation, and the
P H B concentration reached 3 6 g / l . The decrease in the
growth rate above 60 g/I cell concentration was due to an
unstable gas supply caused by f o a m filling the gas return
line o f the fermentor. However, in the case of the culture
using a 2 1 fermentor, foaming was not as great as that in
the culture using a 200 ml fermentor, so that the cell con-
centration o f the 2 l fermentor reached 85 g/l. The max-
i m u m concentrations of cells and PHB, and the percentage
6o _ t~

50_ ;
/ !
4c~_

_0.15 ~ ~o 30~
// /l 0.15 2
q-I A

"~ 2C- ", ~/ /d __.~0.10

u
g lO
\
', 0.05 .~ Z i
w 1C-
",
"D o e-~

\ 0
o ~, t I Jo 0
0 10 20 30 40 50 60 0 2010 30 40 50 60
Cultivation time ( h ) C u l t i v a t i o n time (h)
FIG. 1. Time course of oxygen-limited culture of A. eutrophus FIG. 3. Time course of oxygen-limited culture of A. eutrophus
without supplement of mineral nutrients under the initial gas composi- to which supplementation of KH2PO 4, MgSO4-VH20, FeSO4-7H20
tion of H2 : O~ : CO~ 75 : 15 : 10. Symbols: concentrations of cell and CaSO4 was carried out. Symbols: concentrations of cell ( e ) ,
( ) , P H B ( o ) , protein (~), and nucleic acid (ix), partial pressure of P H B ( ), protein (A) and nucleic acid (zx), partial pressure of dissolved
dissolved oxygen ( [] ). oxygen ( [] ).
256 ISHIZAKI A N D T A N A K A J. FERMENX, B]OENG.,

of PHB accumulation in the cells, obtained in our ex- 3O i

periments under the oxygen-limited condition were higher j ~

than those of previous reports (11-13). It was supposed 25 m

that this high productivity was due to the high gas transfer
capacity of the fermentor used. 2O m

/
Ammonium-deficient culture Many studies of PHB
accumulation by A. eutrophus or other microorganisms .,Q 15 m 0) e,

have been carried out under heterotrophic culturing condi- // to

tions, and several investigations on the effect of the C/N +~ lO /
/ ] .02to*
~
ratio on PHB accumulation have been reported (22-24). +j
\/
/

However, reports on PHB production under autotrophic
culturing conditions are few, and most of those which
have appeared were carried out under the condition of am-
monium deficiency (14, 18, 19). We investigated the pro-
duction of PHB from CO2 in ammonium-deficient culture
under conditions of oxygen sufficiency, and in oxygen-
limited culture under conditions of ammonium sufficiency,
and compared the two results. Figure 4 indicates a batch
culture profile of ammonium-deficient autotrophic culture
using the recycled gas closed circuit culture system. In this
experiment, the initial (NH4)2SO4 concentration in the
medium was 5 g/l, and instead of NH4OH, NaOH was
used for pH control In this fermentation, the cell growth
was gradually suppressed when the ammonium-deficient
condition was formed, resulting in the poor cell concentra-
tion of 27 g/l with PHB of 16 g/l at 80 h cultivation. The
PHB accumulation rate in the oxygen-limited culture was
about 1.5 g/lh, and the concentrations of cell and PHB
were higher than those in the ammonium-deficient culture
According to previous reports (18, 19), the PHB percent-
age in the cells of ammonium-deficient cultures was high,
but the cell and PHB concentrations were low. Our results
supported these findings.
Chemical structure of PHB In view of the physical
property of polyesters, a copolyester containing hydroxy-
alkanoates such as D-4-hydroxybutyrate, D-3-hydroxy-
valerate as constituent units is superior than the homo-
polymer of D-3-hydroxybutyrate (15-17) Hence, 1H and
~3C NMR analysis for PHB obtained from CO2 under
the oxygen-limited culture was carried out to determine its
0
/\ \ / ,& ~ A - ~
0.1
u
O 10 20 30 40 50 60 70 80 918 ~.~
Cultivation time ( h )
FIG. 4. Time course of ammonium-limited culture of A.
eutrophus. Symbols: concentrations of cell ( ) , P H B ( C ) , protein
(A), and (NH4)2SO 4 (A), partial pressure of dissolved oxygen ( [] ).
chemical structure. Figure 5 shows the ~H and ~3C NMR
spectra of the sample PHB. If the polyester contains D-3-
hydroxyvalerate, the IH spectrum has a chemical shift for
C H 3- of the ethyl group around 1 ppm. However, such a
shift was not detected on the NMR spectra of the sample
PHB. This result indicated that the polyester produced
by autotrophic cultivation of A. eutrophus under oxygen
limitation contained neither of o-3-hydroxyvalerate nor
D-4-hydroxybutyrate, and that it was a homopolymer of
D-3-hydroxybutyrate.
Stoichiometry for PHB production The stoi-
chiometry for PHB production from CO2 in the oxygen-
limited culture of A. eutrophus was investigated. The
consumption of substrate gas, cell concentration, PHB
concentration and elementary composition of products
were accurately measured by batch culture using the recy-
cled gas closed circuit culture system (1). The material
balance for the production of biomass, residual biomass
and PHB in the oxygen-limited culture is shown in Table
1. The values of gas consumption for PHB production

Oh 0 OJt~
A 8 8 ~,o B
O ~ OO r,Q 4

I I tl I _( cH3 0

O-CH- CH2-C
3 2 I
CHB
( r 9
\ O-CH-CH2- C
- a b 7n

b 3 2 4 i

..1 5, i. ,
i .... ~ P-~- Yg6iggf~digoigbiZo1N6i~bT~"di~g~g~~'6g~gS~~5'oi
6~J
--tO ro ~)1"~- CO Ot~ tXl~3(~ 0 (M 030 ~-

qO N~['~ CO "~ --0_

FIG. 5. The 400 M H z N M R spectra of ~H and J3C of P H B produced by A. eutrophus under the oxygen limitation condition. (A): ~H NMR
spectrum, (B): ~3C N M R spectrum.
VoL. 71, 1991
TABLE 1. Material balance for production of biomass, PHB and residual biomass in the oxygen limited culture
Product Amount of product
(g/dl) Hz
Biomass including PHB 3.05 0.833
Residual biomass 2.04 0.376
PHB 1.01 0.457
were estimated by subtracting the values of residual
biomass production from those of production of biomass
including PHB. The elementary composition and PHB
c o n t e n t in cells were d e t e r m i n e d f o r b i o m a s s i n c l u d i n g
P H B . By u s i n g t h e s e t w o d a t a , t h e f o r m e r v a l u e o f r e s i d u a l
b i o m a s s was c a l c u l a t e d . T h e s t o i e h i o m e t r y f o r P H B p r o -
d u c t i o n is t h u s i n t r o d u c e d as t h e f o l l o w i n g f o r m u l a .
33H2+ 1202+4CO2 ~ C4H6Oz+30H20
C o m p a r i n g t h e s t o i c h i o m e t r y o b t a i n e d in t h i s e x p e r i m e n t
w i t h t h a t in t h e p r e v i o u s o n e (1), it w a s f o u n d t h a t t h e
amount of hydrogen required for the fixation of 1 mol of
C O : w h i c h is c o n v e r t e d i n t o P H B was a b o u t 1.5 t i m e s as
large as t h e a m o u n t o f h y d r o g e n n e e d e d f o r b i o m a s s p r o -
d u c t i o n . T h i s s t o i c h i o m e t r y d o e s n o t a g r e e w i t h t h e re-
sults o b t a i n e d b y t h e a m m o n i u m - d e f i c i e n t c u l t u r e o f
H y d r o g e n o m o n a s H 16 w i t h t h e m a n o m e t r i c m e t h o d (20).
A s d e s c r i b e d in t h e p r e v i o u s s e c t i o n , t h e c o n s u m p t i o n o f
phosphate increased under oxygen limitation; this may be
d u e t o t h e d e c r e a s e o f e n e r g y u t i l i z a t i o n efficiency f o r t h e
f i x a t i o n o f CO2 u n d e r s u c h a c o n d i t i o n . W e are n o w s t u d y -
ing t h e r e l a t i o n s h i p s a m o n g t h e c o n s u m p t i o n o f p h o s -
phate, the accumulation of polyphosphate and ATP
f o r m a t i o n in a u t o t r o p h i c c u l t u r e o f t h i s m i c r o o r g a n i s m .
T h i s will b e r e p o r t e d o n in t h e n e a r f u t u r e .
ACKNOWLEDGMENT
We express our sincere appreciation to Mitsubishi Petrochemical
Engineering Co., Ltd., for NMR analysis of the chemical structure of
PHB.
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PHB FROM COs 257
Gas consumption (mol) Elementary composition
O2 CO2
0.247 0.132 C4.38Hv.0702.17N0,3s
0.114 0.045 C409H713Or s9N0.76
0.133 0.087 C4H602
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