JOURNAL OF BIOSCIENCE AND BIOENGWEERING

Vol. 88, No. 4, 404-409. 1999

Optimization of L-Lactic Acid Feeding for the Production of

Poly-D-3-Hydroxybutyric Acid by Alcaligenes eutrophus
in Fed-Batch Culture
TAKEHARU TSUGE, KENJI TANAKA, MITSUYA SHIMODA, AND AYAAKI ISHIZAKI*
Department of Food Scienceand Technology, Faculty of Agriculture, Kyushu University, 6-10-I Hakozaki,
Higashi-ku, Fukuoka 812-8581, Japan
Received 29 March 1999/Accepted 9 July 1999

We investigated optimization of the feeding of c-lactic acid for the production of poly-o&hydroxybutyric
acid [P(3HB)] by Alculigenes eutrophus in a fed-batch culture system. An acidic substrate solution was fed
automatically so as to maintain the pH of the culture liquid at 7.0. Feeding of a substrate solution containing
45% (w/v) L-lactic acid, 6.2% (w/v) sodium L-lactate, 5.8% (w/v) ammonia water and 1.8% (w/v) potassium
phosphate [at a molar ratio of carbon to nitrogen (C/N molar ratio) of 101, allowed the L-lactate concentration
in the culture liquid to be maintained at approximately 2 g/Z and the cell concentration reached 27.4 g/Z after
15 h of cultivation. To promote P(3HB) production, a two-stage fed-batch culture consisting of a culture for cell
growth and one for P(3HB) accumulation was carried out. When the substrate solution, whose C/N molar ratio
was 23, was fed during the P(3HB) accumulation phase, the cell concentration and the PQHB) content in the
cells reached 103 g/Z and 57.6% (w/w), respectively, in 51.5 h.
[Key words: Alcaligenes eutrophus, poly-D-3-hydroxybutyric acid, L-lactic acid, pH-stat substrate feeding,
high cell density cultivation]

Polyhydroxyalkanoate (PHA) is a polyester that is
used as a raw material for the production of biodegrada-
ble thermoplastics. Many microorganisms accumulate
PHA as an intracellular storage material under restricted
growth conditions (1). Alcaligenes eutrophus is one of
the best known PHA-producing bacterium and is also
used for the commercial production of PHA (2).
However, a major hurdle in the practical applications of
PHA is the high production cost, as compared to that of
petrochemical thermoplastics. One approach to reduce
the production cost of PHA is to use more inexpensive
carbon sources, for example, starch (3), plant oils (4),
whey (5) and molasses (6). Agricultural waste materials,
like lignocellulose, are considered to be desirable as a
carbon source for the production of PHA.
Although xylose is one of the main components of
lignocellulose, few commercial products can be obtained
from xylose at present. Pseudomonas cepacia (7, 8),
Pseudomonas pseudoflava (9) and recombinant Esch-
erichia cofi harboring PHA biosynthesis genes from
A. eutrophus (10) produced PHA from xylose but high
productivity of PHA was not obtained. In contrast, a
lactic acid bacterium Lactococcus lactis IO- 1, isolated in
our laboratory, produces L-lactic acid together with a
small amount of other organic acids from xylose at a
high rate (ll-13), A. eutrophus cannot assimilate xylose
(14) but can utilize L-lactate and acetate which results in
a competitive growth rate (15, 16). Therefore, we have
developed a culture method for the production of PHA
from xylose employing these two bacteria. Our method
consists of an initial culture stage for the conversion
of xylose to L-lactate by L. lactis IO-1 and a subsequent
culture stage for the production of a kind of PHA, poly-
D-3-hydroxybutyrate [P(3HB)], from L-lactate by A. eu-
trophus. Highly purified L-lactic acid is also transformed
* Corresponding author.
404
into polylactate, which is one of the chemically synthe-
sized biodegradable plastics. However, our fermentation
system enables the use of lactic acid solution, which con-
tains other organic acids such as acetic acid and formic
acid. Our previous studies (15, 16) demonstrated that the
growth of A. eutrophus was significantly inhibited when
the concentration of L-lactate (as sodium salt) in the cul-
ture system reached more than 5 g/l. When organic acids
are used as the carbon source in fermentation, their con-
centrations in the culture liquid must be kept at low
levels in order to achieve a high cell density. However, it
is very difficult to maintain the concentrations of organic
acids at low levels over long culture periods by the usual
control methods. Recently, we succeeded at maintaining
the acetic acid concentration in the culture liquid at
approximately 1 g/I using a pH-stat feeding method during
the heterotrophic growth of A. eutrophus, in the pre-
culture stage of the two-trophic phase culture for the
autotrophic production of P(3HB) from CO2 (17). This
feeding method enabled high cell concentrations to be
obtained using organic acids as the carbon source.
In this paper, we report optimization of the feeding
of L-lactic acid for the production of P(3HB) by A.
eutrophus in a two-stage fed-batch culture system. We
attempted to maintain the L-lactate concentration in the
culture liquid at a low level during the cell growth. We
subsequently examined the effect of the molar ratio of
carbon to nitrogen (C/N molar ratio) in the substrate
solution, on the P(3HB) yield.
MATERIALS AND METHODS
Microorganism, media and culture conditions A.
eutrophus ATCC 17697T was used in this study. The
inoculum (25 ml) was grown in a test-tube in a medium
consisting of 7.5 g/I sodium L-lactate (Showa-Kakou
Co. Ltd., Osaka), 5.Og/l polypeptone (Nihon Seiyaku
VOL. 88, 1999 P(3HB) PRODUCTION FROM LACTIC ACID 405

TABLE 1. Compositions of the substrate solutions used in this study

Composition (w/v %)
C/N molar ratioa
L-Lactic acid Sodium L-lactate NHIOH K2HP0db
Experiment 1 - 20.0 - -
Experiment 2 10 33.3 - 3.9
Experiment 3-1 10 50.0 - 5.8 1.8
3-2 10 45.0 6.2 5.8 1.8
3-3 10 40.0 12.4 5.8 1.8
Experiment 4 phase A 10 45.0 6.2 5.8 1.8
phase B 23 50.0 - 2.5 0.8
nhase C 46 50.0 - 1.3 0.4
phase D 181 50.0 - 0.3 0.1
a mol carbon/mol nitrogen.
b P/N molar ratio (mol phosphorus/mol nitrogen) was set at 6.10 X 1O-2.

Co. Ltd., Tokyo) and 5.Og/l yeast extract (Difco Labo- (15, 16), cell cultivation at a high density could not be
ratories, Detroit, MI, USA) at 30°C for 12 h. Fed-batch achieved by batch operation. Therefore, a fed-batch cul-
cultivation was carried out in a l-1 glass fermentor jar ture was carried out to obtain a high concentration of
(MDS-UlOO, B. E. Marubishi Co. Ltd., Tokyo) and the cells and P(3HB). To maintain the concentration of L-lac-
initial working volume was set at 400 ml. Various acidic tate in the culture liquid at low levels, a 20% (w/v) L-lac-
substrate solutions were fed into the fermentor to main- tic acid solution was fed by the pH-stat feeding method
tain the pH of the culture broth at 7.0 as monitored (Experiment 1). The result is shown in Fig. 1. L-Lactate
by a pH-controller (PHC-2201; Able Co. Ltd., Tokyo). concentration was kept below 5 g/l by feeding the L-lac-
The compositions of the acidic substrate solutions used tic acid solution but was not kept constant. After 6 h
in this study are shown in Table 1. The composition of of cultivation, ammonia water was added to the culture
the culture medium basically consisted of 3.Og of liquid to give an ammonium ion concentration of 1.Og/l
sodium L-lactate, 1.8 g of (NH&S04, 0.5 g of KH2P04, in order to stimulate cell growth. A maximum specific
1.O g of MgS04.7H20, 20 mg of CaS04. 2Hz0, and 0.5 growth rate of 0.45 h- * was observed between 4 and 8 h
ml of trace element solution in 1 1 of distilled water. of cultivation. At the end of this cultivation period
Lactic acid, used as the carbon source, was in the (29.5 h), the concentrations of the cells and P(3HB)
form of approximately 90% (w/v) L-lactic acid solution were 18.0 g/l and 11.1 g/l, respectively. Even though the
(Wako Pure Chemical Industry, Osaka) and 50% (w/v) nitrogen source (ammonium ions) was not limited for cell
sodium L-lactate solution (Showa-Kakou). The trace growth in this fermentation process, the content of
element solution contained 119 mg of CoC12, 16.2 mg P(3HB) in the ceils was relatively high [61.8% (w/w)],
FeC& . 6H20, 118 mg of NiC&. 6Hz0, 153 mg of CrClr . probably due to a shortage of nutrients such as phos-
6Hz0, 156 mg of CuS04.5H20, and 15.6g of citric acid
in 100 ml of 1.0 N HCl. The culture temperature was
controlled at 30°C. The partial pressure of dissolved oxy-
gen (DO), in the culture liquid was monitored by a mem-
brane-type DO electrode (Able) and maintained above
5 kPa by controlling the agitation speed and introducing
pure O2 gas into the fermentor.
Analytical methods Dry cell weight (DCW) was de-
termined by centrifuging 1 ml of culture broth at 4000 x
g for 10 min and drying the harvested cells at 105°C.
For determination of P(3HB) concentrations, cells from
0.2-l.Oml of the culture broth was centrifuged, the
resulting cell pellet was hydrolyzed with 0.5 ml of 2.0 N
NaOH at lOO”C, and the hydrolyzate was neutralized
with 0.5 ml of 2.0N HCl. The concentration of D-3-
30
hydroxybutyric acid in the hydrolyzed sample was deter-
mined by high-performance liquid chromatography as
20
described by Karr et af. (18). L-Lactate concentration
was determined by an enzymatic sensor (L-lactate ana-
10
lyzer YSI-23L; Yellow Springs Instruments, Yellow
Springs, OH, USA). The concentrations of ammonium 0
and phosphate in the culture were determined by a modi- 0 10 20 30
fied indophenol method (19) and Allen’s method (20), re-
spectively. Cultivation time (h)

FIG. 1. Time course of fed-batch culture with pH-stat feeding of

RESULTS 20% (w/v) L-lactic acid solution (Experiment 1). The arrows indicate
the addition of ammonia water into the culture liquid to obtain an
Feeding of L-lactic acid solution Since the cell ammonium concentration of 1.O g/l. Symbols: concentrations of L-
growth of A. eutrophus was inhibited when the lactate lactate (A), ammonium ions (A) and P(3HB) (0); DCW (0); and total
concentration in the culture liquid increased to 5 g/l amount of L-lactic acid fed to the fermentor ( 0 ).
406 TSUGE ET AL.

phate and magnesium.

Feeding of a mixture of L-lactic acid and ammonia
water To obtain a high concentration of P(3HB), it
is essential to increase the cell concentration before
P(3HB) starts to accumulate in the cells. Therefore, a
two-stage fed-batch culture, consist of a first culture for
cell growth and a subsequent culture for P(3HB) accumu-
lation, is often recommended. We first focused on how
to increase the cell concentration in the culture during
cell growth. In our previous study, a C/N molar ratio of
10 (mol carbon/mol nitrogen) in the substrate solution
was found to be the most effective for increasing the con-
centration of A. eutrophus cells, when acetic acid was
fed by the pH-stat feeding method (17). Therefore, a
mixture of L-lactic acid and ammonium solution, which
was prepared at a C/N molar ratio of 10, was fed as the
substrate solution into the culture system by the pH-stat
feeding method (Experiment 2). As shown in Fig. 2, a
specific growth rate of about 0.45 h -’ in the exponential
growth phase was observed and the cell concentration
was 26.5 g/l at the end of 15 h of cultivation. The con-
tent of P(3HB) in the cells at the end of 15 h of cultiva-
tion was 34.7% (w/w). As cultivation proceeded, the L-
lactate concentration in the culture liquid decreased to 25
almost zero (not completely zero), while the concentra-
tion of ammonium ions in the culture liquid increased.
The C/N molar ratio during cultivation was calculated 0
and is shown in Fig. 2. The value was almost 10 (mol 0 5 10 15 20
carbon/mol nitrogen) between 2.5 and 12.5 h of cultiva-
tion, but subsequently, increased markedly. This result Cultivation time (h)
suggests that a C/N molar ratio of 10 is suitable for cell
FIG. 2. Time course of fed-batch culture with pH-stat feeding of
growth in the exponential phase (between 2.5 and 12.5 h a substrate solution whose C/N molar ratio was 10 (Experiment 2).
of cultivation). On the other hand, the phosphate ions in Symbols: change in C/N molar ratio with substrate consumption by
the culture liquid were completely consumed after 12.5 h cells ( n ); concentrations of t-lactate (A), ammonium ions (A), phos-
of cultivation. We observed that the cell growth rate phate ions (0) and P(3HB) (0); DCW (0); and total amount of L-
decreased after 12.5 h of cultivation. This may be due to lactic acid fed to the fermentor ( 0 ).
the shortage of phosphate. For optimal cell growth, feed-
ing of carbon, nitrogen and phosphorus appeared to be changed, the concentration of acetate in the culture
important. We estimated the required amount of car- liquid also changed in accordance with the acid-base
bon, nitrogen and phosphorus for the production of 1 g balance of the culture liquid, in order to maintain a con-
of cells, based on the results of Experiment 2. The stant pH. Thus, the concentration of the inactive ion
results are summarized in Table 2. The required amounts works as a mediator controlling the acetic acid concentra-
of carbon, nitrogen and phosphorus were 5.22 X 10 2, tion in the culture liquid. We introduced this method
4.74 x 10~-3 and 2.89 x 10--4, respectively. Therefore, the into our culture system and used sodium ions as the inac-
C/N and phosphorus to nitrogen (P/N) molar ratios tive base. The substrate solution thus consisted of sodi-
were 1.11 x 10 and 6.10 x 10 m2, respectively. Based on um L-lactate with various sodium concentrations accord-
this analysis, we used a substrate solution with a P/N ing to the experimental aim (Experiment 3-l to 3-3). The
molar ratio of 6.10 x 10 2 for subsequent experiments. results are shown in Fig. 3. When a sodium-salt free-
Feeding of a substrate solution containing sodium ions substrate solution was fed (Experiment 3-l), the L-lactate
Ikeda et al. (21) were able to control the acetic acid con- concentration in the culture liquid gradually decreased
centration at levels lower than the inhibitory level in a with cultivation time. The concentrations of ammonium
pH-stat culture by feeding acetic acid as the sole carbon ions and phosphate ions in the culture liquid were main-
source. In their system, inactive acid or base for the tained at approximately 0.5 g/l and 0.3 g/l, respectively
microorganism was fed together with acetic acid. If the (data not shown). The cell concentration reached 22.4
pH of the culture liquid was kept constant, the acid-base g/l and the P(3HB) content in the cells was 39.5%
balance of the culture liquid did not change. When the (w/w) at the end of 15 h of cultivation. When a sub-
concentration of the inactive ion in the culture liquid strate solution containing 12.4% (w/v) sodium L-lactate

TABLE 2. The molar ratio of carbon or phosphorus to nitrogen consumed by the cells
Nutrient consumed by cells” Element consumed by cells” Molar ratio of C/N or P/N
Nutrient (mol/g-cells) Element (mol/g-cells) (C or P mol/N mol)
L-Lactic acid 1.74x10 2 c 5.22 x 10 m2 1.11 x 10
Ammonium ion 4.74 x IO-3 N 4.74x 10~3
Phosphate ion 2.89 x 10m4 P 2.89~ 10 4 6.10~ lo-*
a Data were obtained from the result of Fig. 2.
VOL. 88. 1999 P(3HB) PRODUCTION FROM LACTIC ACID 407

When a substrate solution containing 6.2% (w/v) sodium

L-lactate was fed (Experiment 3-2), feeding of the sub-
strate solution was activated and the L-lactate concen-
tration in the culture liquid was maintained at approxi-
mately 2 g/l until 15 h of cultivation. The concentrations
of ammonium ions and phosphate ions in the culture
liquid were also maintained at approximately 0.5 g/f and
0.3 g/l, respectively (data not shown). The cell concentra-
tion reached 27.4 g/l and the P(3HB) content in the cells
was 22.8% (w/w) at the end of 15 h of cultivation. Cell
growth was promoted by controlling the L-lactate concen-
tration at low and constant levels, which resulted in a
relatively low P(3HB) content in the cells.
Effect of C/N molar ratio on P(3HB) accumulation
We carried out a two-stage fed-batch culture to produce
P(3HB). In the first culture stage for the cell growth, the
substrate solution with a C/N molar ratio of 10, was fed
until the end of the exponential growth phase (12 h of
cultivation) as described above. In the subsequent cul-
ture stage for P(3HB) accumulation, substrate solutions
with C/N molar ratios of 23, 47 and 181 were fed to ex-
amine the effect of the C/N molar ratio on P(3HB)
accumulation (Experiment 4). The results are shown in
Fig. 4. In all cases, the concentrations of ammonium
ions in the culture liquid decreased to almost zero when
0 5 10 15 20 the C/N molar ratio of the substrate solution was
changed. When a substrate solution with a C/N molar
Cultivation time (h) ratio of 181 was used (Fig. 4, phase D), the cell concen-
FIG. 3. The changes in L-lactate concentration in culture liquid, tration reached 22.9 g/l and the P(3HB) content in the
DCW and P(3HB) content in the cells in the fed-batch culture with cells was 57.6% (w/w) at the end of 20 h of cultivation.
pH-stat feeding of substrate solutions containing various concentra- This was similar to that obtained when an ammonium-
tions of sodium L-lactate (Experiment 3-l to 3-3). Symbols: feeding of free substrate solution was fed in the P(3HB) accumula-
substrate solution containing 0% (w/v) ( q ), 6.2% (w/v) (0) and tion phase (data not shown). When a substrate solution
12.4% (w/v) (A) sodium L-lactate. with a C/N molar ratio of 47 was used (Fig. 4, phase
C), the cell concentration reached 45.3 g/l and the
was fed (Experiment 3-3), the L-lactate concentration in P(3HB) content in the cells was 33.5% (w/w) at the end
the culture liquid gradually increased and as a result of 32.5 h of cultivation. In this cultivation, the cell
poor cell growth was observed. The concentrations of growth rate slowed down when the C/N molar ratio of
ammonium ions and phosphate ions in the culture liquid the substrate solution was changed, and accumulation
also increased with cultivation time (data not shown). of P(3HB) in the cells was not promoted. When a C/N

I I
AIB A IC

25

0 10 20 30 40 50 600 10 20 30 40 50 600 10 20 30 40 SU bo

Cultivation time (h)

FIG. 4. Time course of fed-batch cultures with pH-stat feeding of substrate solutions, with various C/N molar ratios (Experiment 4). Until
12 h of cultivation, a solution with the C/N molar ratio of 10 [containing 6.2% (w/v) sodium L-lactate] was fed (indicated by phase A). Subse-
quently, the substrate solution with C/N molar ratios of 23 (indicated by phase B), 47 (C) or 181 (D) was fed to promote P(3HB) accumulation.
Symbols: concentrations of L-lactate (A), ammonium ions (A) and P(3HB) (0); DCW (0).
408 TSUGE ET AL.
molar ratio of 23 was used (Fig. 4, phase B), cell growth
associated with the formation of P(3HB) was observed.
The cell concentration increased to 103 g/l and the
P(3HB) content in the cells was 57.6% (w/w) after
51.5 h of cultivation. The overall productivity and yield
of P(3HB) were 1.15 g/l. h and 0.176 mol 3HB/mol
lactic acid, respectively. It appears that the formation of
P(3HB) was enhanced with a balance of activity of
P(3HB) biosynthesis and cell formation by feeding of
substrate solution with a C/N molar ratio of 23.
DISCUSSION
Lactic acid exhibits an inhibitory effect on cell growth,
therefore, it is necessary to maintain the concentration
of L-lactate in the culture liquid at low levels throughout
fermentation. When an organic acid is fed as the carbon
source, feedback control is probably more suitable than
other control methods such as constant rate feeding and
exponential rate feeding. The pH change in the culture
liquid can act as an indicator to control the feeding of
the substrate solution, and some studies have reported
the successful control of substrate concentrations by the
pH-stat feeding method. Choi et al. (22) reported that
high P(3HB) productivity [4.63 (g/l.h)] was obtained by
the pH-stat fed-batch culture of recombinant E. coli
strains harboring Alcaligenes latus PHA biosynthesis
genes, by feeding glucose at a concentration of 2Og/l.
Kim et al. (23) reported the production of poly(3-hydro-
xybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] by A.
eutrophus by feeding 2Og/l propionic acid solution as a
co-carbon source in a pH-stat culture, however, the cell
concentration and PHA yield were not high (PHA con-
centration was less than 2 g/Z). In a previous study (17),
we succeeded in the automatic control of the acetic acid
concentration at approximately 1 g/l, by pH-stat feeding
of a substrate solution with a C/N molar ratio of 10,
therefore the cell concentration reached 48.6 g/l at the
end of 21 h of cultivation. Similarly, we introduced the
pH-stat feeding method to the culture system for the
production of P(3HB) from L-lactic acid in this study.
However, the L-lactate concentration in the culture liq-
uid decreased with cultivation time and the pH-stat feed-
ing method did not work as successfully as it did when
acetic acid was used. We considered that the decrease in
L-lactate concentration in the culture liquid was a result
of factors such as, the buffer effect of CO* evolved from
the cells in the culture liquid and the oligomers of lactic
acid remaining in the culture liquid. Oligomers of lactic
acid are spontaneously formed by condensation, in a
high concentration lactic acid solution (24). As the sub-
strate solution used in our study contained 20-50% (w/v)
L-lactic acid, the formation of 2-5% (w/v) oligomers,
which are mainly dimers of lactic acid (lactyl lactic acid),
in substrate solution was estimated from Bezzi and
Watson’s data (24). Therefore, lactic acid and its oligo-
mers were fed into the fermentor according to the in-
crease in pH. The oligomers remained in the culture
liquid without being utilized by the cells. Consequently,
the concentration of oiigomers increased while the concen-
tration of L-lactic acid decreased with cultivation time.
We observed the presence of oligomers of lactic acid in
the culture supernatant by the following method. The
culture supernatant was subjected to hydrolysis with
2.ON NaOH at 100°C for 2 h, and the concentration
of L-lactic acid in the hydrolysate was measured using a
L-lactate analyzer. The concentration of L-lactic acid in the
hydrolysate was higher than that in the non-hydrolyzed
supernatant (the maximum concentration of oligomers
was approximately 2 g/l). In general, oligomers of lactic
acid dissociate into lactic acid at low concentrations of
lactic acid over a long period (24). Therefore, a portion
of the remaining oligomers gradually dissociate into
lactic acid in the culture liquid, and the cells consume
the dissociated L-lactic acid. However, it is necessary to
maintain the L-lactate concentration in the culture liquid
at an almost constant level for optimal cell growth. We
therefore introduced Ikeda’s method into our culture sys-
tem to maintain L-lactate concentration in culture liquid
at a constant level. Sodium ions were used to mediate
control the L-lactate concentration in the culture liquid.
When a substrate solution containing sodium I.-lactate
16.2% (w/v)], L-lactic acid, ammonia water and phos-
phate was fed, the L-lactate concentration in the culture
liquid could be maintained at approximately 2 g/l. which
is the optimal level for cell growth.
Most PHA-producing bacteria accumulate PHA in
their cells under nitrogen-limited conditions. However,
the activity of PHA biosynthesis is considerably damaged
under conditions of nitrogen deficiency (25). To obtain
higher yields of PHA, some researchers attempted the
addition of a small amount of a nitrogen source to the
culture system in the PHA accumulation phase. Suzuki
et al. (26) reported that a C/N molar ratio of 25 was the
most effective for the accumulation of P(3HB) in fed-
batch cultures of Protomonas extroquens sp. K using
methanol as the sole carbon source. In our results, a
C/N molar ratio of 23 was also effective for the accumu-
lation of P(3HB) from L-lactic acid. These findings indi-
cated that the activity of PHA biosynthesis is maintained
over a long time by the feeding of a substrate solution
with a C/N molar ratio of 23-25, using methanol or L-
lactic acid as the sole carbon source. On the other hand,
Rhee et al. (27) also reported that a C/N molar ratio of
139 (a molar ratio of glucose to ammonium ion of 23.1)
was the most effective for the production of P(3HB-
co-3HV) in fed-batch cultures of Alcaligenes sp. SH-69
from glucose as the sole carbon source. Therefore, it
seems that the most suitable C/N molar ratio for the
accumulation of P(3HB) differs with the strains and car-.
bon sources used. It is well known that an increase in
the C/N molar ratio of the substrate solution leads to an
increase in the P(3HB) content in the cell (26). In this
study, the C/N molar ratio of 47 (Experiment 4 phase
C) did not yield a higher P(3HB) content in the cells
than that obtained with a ratio of 23 (Experiment 4
phase B). We cannot however explain this result satisfac-
torily.
Linko et al. (28) carried out fed-batch cultures 0:‘ A.
eutrophus H16, by feeding a lactic acid solution periodi-
cally, and obtained cell and P(3HB) yields of 11.8 g/i
and 7.1 g/l, respectively. We investigated optimization of
the feeding of L-lactic acid in a two-stage fed-batch cul-
ture system and obtained high cell and P(3HB) yields of
103 g/l and 59 g/l, respectively. In this study, we estab-
lished a method for high-cell-density cultivation and
high production of P(3HB) using commercial-grade L-
lactic acid. We are now investigating the utilization of the
culture broth, obtained during lactic acid production by
L. lactis IO-1 from xylose, as the substrate solution for
the effective production of P(3HB) by A. eutrophus.
VOL. 88. 1999 P(3HB) PRODUCTION FROM LACTIC ACID 409

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