APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1997, p. 3703–3706 Vol. 63, No.

9
0099-2240/97/$04.0010
Copyright © 1997, American Society for Microbiology

Poly(3-Hydroxybutyrate) Production with High Productivity

and High Polymer Content by a Fed-Batch Culture
of Alcaligenes latus under Nitrogen Limitation
FULAI WANG AND SANG YUP LEE*
Department of Chemical Engineering and BioProcess Engineering Research Center, Korea Advanced
Institute of Science and Technology, 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, Korea
Received 4 April 1997/Accepted 18 June 1997

Alcaligenes latus has been known to produce poly(3-hydroxybutyrate) (PHB) in a growth-associated manner
even under nutrient-sufficient conditions. However, the PHB content obtained by fed-batch culture was always
low, at ca. 50%, which makes the recovery process inefficient. In this study, the effect of applying nitrogen
limitation on the production of PHB by A. latus was examined. In flask and batch cultures, the PHB synthesis
rate could be increased considerably by applying nitrogen limitation. The PHB content could be increased to
87% by applying nitrogen limitation in batch culture, which was considerably higher than that typically
obtainable (50%) under nitrogen-sufficient conditions. In fed-batch culture, cells were first cultured by the
DO-stat feeding strategy without applying nitrogen limitation. Nitrogen limitation was applied at a cell
concentration of 76 g (dry cell weight)/liter, and the sucrose concentration was maintained within 5 to 20 g/liter.
After 8 h of nitrogen limitation, the cell concentration, PHB concentration, and PHB content reached 111.7 g
(dry cell weight)/liter, 98.7 g/liter, and 88%, respectively, resulting in a productivity of 4.94 g of PHB/liter/h. The
highest PHB productivity, 5.13 g/liter/h, was obtained after 16 h.

Polyhydroxyalkanoates (PHAs) are intracellular energy and
carbon storage material synthesized by numerous microorgan-
isms (1, 9, 16). Even though PHAs are good candidates for
completely biodegradable polymers, the high production cost
of PHAs has hampered their commercial application (10).
Therefore, much effort has been devoted to reduce the pro-
duction cost of PHAs by developing more efficient processes
for the production and recovery of PHAs (10). Obviously, the
development of a fermentation strategy that allows high PHA
concentration and productivity is most important for reducing
the production cost (3, 10). High PHA content is also impor-
tant because the cost of polymer recovery increases signifi-
cantly with low PHA content (3).
Bacteria that are used for the production of PHAs can be
divided into two groups based on the culture conditions re-
quired for PHA synthesis (9, 10). The first group, which in-
cludes Alcaligenes eutrophus (7), methylotrophs (8), and pseu-
domonads (17), requires the limitation of an essential
nutritional element in the presence of an excess of carbon
source for the efficient synthesis of PHAs. The second group,
which includes Alcaligenes latus (2, 5, 6), Azotobacter vinelandii
(15), and recombinant Escherichia coli (11, 13, 14), does not
require nutrient limitation for PHA synthesis and can accumu-
late PHAs during growth. A. eutrophus has been employed
most often for the production of poly(3-hydroxybutyrate)
(PHB) since it accumulates a large amount of polymer in a
simple glucose-salts medium (7). A. latus has also been drawing
much attention as a candidate for PHA production since it
grows fast and accumulates PHA during growth without nutri-
ent limitation (2, 5, 6). Since it can utilize sucrose as a carbon
source, cheap substrates such as raw sugar, beets, or cane
molasses can also be used. These advantages created much
interest in developing a better process for the production of
PHAs by fed-batch culture of A. latus. It has been reported that
* Corresponding author. Fax: 042-869-8800. E-mail: leesy@sorak
.kaist.ac.kr.
a PHB concentration of 68.4 g/liter could be obtained in 18 h
with a pH-stat fed-batch culture of A. latus using a high inoc-
ulum concentration (13.7 g [dry cell weight]/liter), resulting in
a high PHB productivity of 3.97 g/liter/h (18). This productivity
would be close to 3 g/liter/h if a normal inoculum concentra-
tion were used. However, the PHB content obtained was
rather low, at 50% (18), and needs to be increased consider-
ably to make this process commercially attractive. Since A.
latus cells accumulate PHB during growth, it was reasoned that
PHB accumulation might be incomplete during the time frame
of fed-batch operation. In this study, we report that nitrogen
limitation can significantly enhance the PHB biosynthesis ca-
pability of A. latus.
Culture conditions. A. latus DSM1123 was used in this study.
Two-step flask cultures were created to examine the effect of
nitrogen limitation on PHB synthesis. Cells were first grown in
250-ml flasks containing 50 ml of nutrient broth for 24 h,
collected by centrifugation, washed once with AL1 medium,
and resuspended in AL1 medium. AL1 medium (pH 7.0) con-
tains (per liter): KH2PO4, 1.5 g; Na2HPO4 z 12H2O, 9 g;
MgSO4 z 7H2O, 0.2 g; CaCl2 z 2H2O, 0.01 g; citric acid, 0.1 g;
and trace element solution, 1 ml. Trace element solution con-
tains (per liter): FeSO4 z 7H2O, 20 g; H3BO4, 0.3 g; CoCl2 z
6H2O, 0.2 g; ZnSO4 z 7H2O, 0.03 g; MnCl2 z 4H2O, 0.03 g;
(NH4)6Mo7O24 z 4H2O, 0.03 g; NiSO4 z 7H2O, 0.03 g; and
CuSO4 z 5H2O, 0.01 g. Cells were then cultured under two dif-
ferent conditions: nitrogen-sufficient and nitrogen-limited condi-
tions. For nitrogen-sufficient cultures, sucrose and (NH4)2SO4
were supplemented to final concentrations of 20 and 3 g/liter,
respectively. For nitrogen-limited cultures, sucrose was supple-
mented to a concentration of 20 g/liter and no (NH4)2SO4 was
added. Cells were cultured in a shaking incubator at 30°C and
250 rpm.
Batch culture of A. latus was conducted in a 6.6-liter jar
fermentor (Bioflo3000; New Brunswick Scientific Co., Edison,
N.J.) containing 3 liters of AL2 medium (pH 6.8) supple-
mented with 30 g of sucrose per liter and 1 g of (NH4)2SO4 per

3703
3704 WANG AND LEE APPL. ENVIRON. MICROBIOL.

liter. AL2 medium contained (per liter): KH2PO4, 0.6 g;

Na2HPO4 z 12H2O, 3.6 g; MgSO4 z 7H2O, 1 g; CaCl2 z 2H2O,
0.1 g; citric acid, 0.1 g; and trace element solution, 3 ml.
Temperature and dissolved oxygen (DO) were controlled at
30°C and 40%, respectively. The pH was controlled at 6.8 with
NH4OH (28%, vol/vol), which also served as a nitrogen source.
Nitrogen limitation was applied by substituting 5 M NaOH
solution for NH4OH.
Fed-batch culture was carried out in a 6.6-liter jar fermentor
containing 1.6 liter of AL2 medium supplemented with 20 g of
sucrose per liter and 2 g of (NH4)2SO4 per liter. Seed culture
(400 ml) was prepared in AL2 medium. Temperature and pH
were controlled at 30°C and 6.8, respectively. DO was main-
tained at 40% of air saturation by automatic control of agita-
tion speed (up to 700 rpm) and the percentage of pure oxygen
supplied. The gas flow rate was fixed at 4 liters/min. Feeding
solution used before applying nitrogen limitation contained
(per liter): sucrose, 900 g; (NH4)2HPO4, 8 g; KH2PO4, 2 g;
NaH2PO4 z 2H2O, 10 g; MgSO4 z 7H2O, 4.5 g; citric acid, 0.2 g;
and trace element solution, 15 ml. The feeding solution used
during nitrogen limitation contained 900 g of sucrose per liter.
Two different feeding strategies were used for culturing cells
before and after the application of nitrogen limitation. During
the nitrogen-sufficient growth phase, the DO-stat feeding strat-
egy was used. When the DO exceeded its upper limit (50% of
air saturation), a calculated volume of feeding solution was
added to increase the sucrose concentration in the culture
broth to 20 g/liter. Nitrogen limitation was applied by substi-
tuting 5 M NaOH for NH4OH. There was no sharp DO in-
crease upon carbon depletion during the nitrogen-limited pe-
riod. The following continuous feeding model was therefore
developed:
FIG. 1. Time profiles of the cell concentration, PHB concentration, and
when Dt # 2, r 5 0.7 3 Cs (1) residual cell concentration (A) and the PHB synthesis rate and PHB content (B)
during the two-step flask culture of A. latus DSM1123. Cells were first cultured
when Dt . 2, r 5 ~0.7 3 Cs!/Dt (2) in nutrient broth and harvested and then reincubated in two different media:
nitrogen-sufficient (filled symbols) and nitrogen-limited (open symbols) defined
where Dt is the dimensionless time measured (in hours) after media.
the application of nitrogen limitation, r is the sucrose feeding
rate (grams of sucrose per hour), and Cs is the sucrose con-
sumption rate (grams of sucrose per hour) 1 h before nitrogen
depletion. residual cell concentration did not increase under nitrogen-
The concentrations of cells (grams [dry cell weight] per li- limited conditions. The final cell, and PHB concentrations
ter), PHB (grams per liter), residual cells (grams per liter), and were higher under nitrogen-sufficient conditions. However, the
sucrose (grams per liter) and the PHB content (percent) were PHB content and the PHB synthesis rate were higher under
determined as previously described (12, 14). The NH41 con- nitrogen-limited conditions. The PHB synthesis rate increased
centration (millimolar) was determined by nesslerization (4). significantly upon nitrogen limitation and then decreased to-
Flask and batch cultures. Since cell growth competes with wards the end of cultivation and was always higher than that
PHB synthesis under non-limiting nutritional conditions, a under nitrogen-sufficient conditions (Fig. 1B). The maximum
strategy of nutrient limitation was examined that allows PHB PHB synthesis rate under nitrogen limitation was 0.32 g of
production without further cell growth. After examining the PHB/g of residual cells/h. The PHB content also increased
effect of limiting nitrogen, phosphorus, magnesium, or sulfur in more rapidly under nitrogen-limited conditions. These results
flask cultures, nitrogen limitation was chosen as the best strat- suggest that in A. latus the PHB synthesis rate and PHB con-
egy since it allowed the greatest enhancement of PHB produc- tent can be increased by applying nitrogen limitation.
tion (data not shown). Two-step flask cultures were first cre- The effect of nitrogen limitation on cell growth and PHB
ated to examine the effect of nitrogen limitation on PHB synthesis was further examined by batch culture in a fermentor
synthesis. The reasons for applying two-step cultivation are as in which pH and DO level, in addition to temperature, can be
follows. When A. latus was grown in a defined medium con- perfectly controlled. Since the seed culture was prepared in the
taining sucrose as a carbon source, the PHB content obtained same medium used for the batch cultivation, cells had the
was about 50%. To examine the effect of nitrogen limitation on initially higher PHB content of ca. 50%. Nitrogen limitation
PHB synthesis, a low initial PHB content was desirable for was applied at 12 h. The PHB content increased very sharply,
better comparison of PHB synthesis rate and PHB content from 52% to 83%, 6.5 h after application of nitrogen limitation
under nitrogen-sufficient and nitrogen-limited conditions. By and then slightly increased afterwards (Fig. 2A). As before, the
first growing cells in nutrient broth, cells having a PHB content residual cell concentration stayed rather constant during the
of ca. 15% could be obtained. As shown in Fig. 1A, cell growth nitrogen-limited period, suggesting no further cell growth. The
under nitrogen-sufficient conditions was apparent from the NH41 concentration was 15.7 mM when nitrogen limitation
increase in residual cell concentration. On the other hand, the was applied and decreased steadily to zero in 4 h (Fig. 2B). The
VOL. 63, 1997
FIG. 2. Time profiles of the cell concentration, PHB concentration, and
residual cell concentration and the PHB content (A) and the concentrations of
sucrose (grams per liter) and NH41 (millimolar) and PHB synthesis rate (B)
during batch culture of A. latus DSM1123. NH4OH was replaced with NaOH at
12 h, and nitrogen was depleted at 16 h.
PHB synthesis rate increased very sharply when the NH41
concentration decreased to 1.1 mM. It can be seen from Fig.
2A and 2B that the increase in the PHB synthesis rate resulted
in the rapid increase of PHB concentration and PHB content.
The PHB synthesis rate reached a maximum value of 0.87 g of
PHB/g of residual cells/h 6.5 h after application of nitrogen
limitation and then decreased when the PHB content reached
a value of 83%. This seems to be due to the reduced metabolic
activity of cells containing a large amount of PHB, as was also
observed with A. eutrophus (7). This PHB synthesis rate is the
highest reported to date for any microorganism.
Fed-batch culture under nitrogen limitation. Fed-batch cul-
ture has been the most popular method to achieve high cell
density and high productivity of a desired product. At present,
Ceregen (a unit of Monsanto, St. Louis, Mo.) is producing poly
(3-hydroxybutyrate-co-3-hydroxyvalerate) by fed-batch culture
of A. eutrophus from glucose and propionic acid on a semi-
commercial scale. Therefore, it is important to demonstrate in
the fed-batch culture the beneficial effect of applying nitrogen
limitation found in flask and batch cultures. The DO-stat feed-
ing method was used since it was better than the pH-stat
method.
After the application of nitrogen limitation, the feeding
strategy was changed from the DO-stat method to the opti-
mally determined feeding profile (equations 1 and 2). This was
because there was no apparent DO increase upon carbon de-
pletion under nitrogen-limited conditions, which was expected
from no further significant cell growth under this condition.
PHB PRODUCTION BY ALCALIGENES LATUS 3705
FIG. 3. Time profiles of the cell concentration, PHB concentration, residual
cell concentration, and NH41 concentration (millimolar) and the PHB content
during fed-batch culture of A. latus DSM1123. NH4OH was replaced with NaOH
when the cell concentration reached 76 g/liter at 12 h. Nitrogen depletion oc-
curred at 12.5 h.
The feeding profile was determined experimentally by calcu-
lating the sucrose consumption rate during the nitrogen-lim-
ited period. It was found that, during the first two hours after
the application of nitrogen limitation, the sucrose consumption
rate was 70% of that one hour before nitrogen depletion. The
feeding profile shown in equation 2 was also developed based
on the sucrose consumption rate, which further decreased dur-
ing this period. The time profiles of the concentrations of cells,
residual cells, and PHB are shown in Fig. 3. A cell concentra-
tion of 76 g/liter was obtained in 12 h, resulting in the high cell
productivity of 6.33 g (dry cell weight)/liter/h. Nitrogen limita-
tion was applied at this point to trigger enhanced PHB synthe-
sis. The PHB concentration as well as the PHB content in-
creased rapidly upon nitrogen limitation. The residual cell
concentration did not increase but rather decreased under
nitrogen-limiting conditions, as is typically observed during the
fed-batch culture of other PHB producers (7, 14). At the end
of cultivation (20 h), the cell concentration, PHB concentra-
tion, and PHB content reached were as high as 111.7 g (dry cell
weight)/liter, 98.7 g/liter, and 88%, respectively, resulting in the
high productivity of 4.94 g of PHB/liter/h. The highest PHB
productivity was 5.13 g/liter/h, at 16 h. The maximum PHB
synthesis rate during the fed-batch culture was calculated to be
0.44 g/g of residual cells/h. This value is much higher than that
obtained with A. eutrophus (0.15 g of PHB/g of residual cells/h)
(7) or recombinant E. coli (0.18 g of PHB/g of residual cells/h)
(14). The yield of PHB on sucrose was 0.42 g/g of sucrose.
In this paper, we described a new strategy for the production
of PHB to a high concentration and high content with high
productivity and yield by fed-batch culture of A. latus from
sucrose. Increase in the PHB synthesis rate by applying nitro-
gen limitation together with the optimal sucrose feeding re-
sulted in unprecedentedly high PHB productivity with high
PHB content. This method should allow considerable reduc-
tion of PHB production cost and enhance the commercial
viability of PHB as a biodegradable polymer.
Fulai Wang was partially supported by the BioProcess Engineering
Research Center. This work was supported by the Ministry of Science
and Technology and by LG Chemicals, Ltd., Taejon, Korea.
REFERENCES
1. Anderson, A., and E. A. Dawes. 1990. Occurrence, metabolism, metabolic
role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Rev.
54:450–472.
3706 WANG AND LEE
2. Braunegg, G., and B. Bogensberger. 1985. Zur kinetik des wachstums und
der speicherung von poly-D(2)-3-hydroxybuttersaure bei Alcaligenes latus.
Acta Biotechnol. 5:339–345.
3. Choi, J. I., and S. Y. Lee. Process analysis and economic evaluation for
poly(3-hydroxybutyrate) production by fermentation. Bioprocess Eng., in
press.
4. Greenberg, A. E., L. S. Clesceri, and A. D. Eaton. (ed.). 1992. Standard
methods for the examination of water and wastewater. American Public
Health Association, Washington, D.C.
5. Hangii, U. J. 1990. Pilot scale production of PHB with Alcaligenes latus, p.
65–70. In E. A. Dawes (ed.), Novel biodegradable microbial polymers. Klu-
wer Academic Publishers, Dordrecht, The Netherlands.
6. Hrabak, O. 1992. Industrial production of poly-b-hydroxybutyrate. FEMS
Microbiol. Rev. 103:251–256.
7. Kim, B. S., S. C. Lee, S. Y. Lee, H. N. Chang, Y. K. Chang, and S. I. Woo.
1994. Production of poly(3-hydroxybutyric acid) by fed-batch culture of Al-
caligenes eutrophus with glucose concentration control. Biotechnol. Bioeng.
43:892–898.
8. Kim, S. W., P. Kim, H. S. Lee, and J. H. Kim. 1996. High production of
poly-b-hydroxybutyrate (PHB) from Methylobacterium organophilum under
potassium limitation. Biotechnol. Lett. 18:25–30.
9. Lee, S. Y. 1996. Bacterial polyhydroxyalkanoates. Biotechnol. Bioeng. 49:1–
14.
10. Lee, S. Y. 1996. Plastic bacteria? Progress and prospects for polyhydroxyal-
APPL. ENVIRON. MICROBIOL.
kanoate production in bacteria. Trends Biotechnol. 14:431–438.
11. Lee, S. Y. 1997. E. coli moves into plastic age. Nature Biotechnol. 15:17–18.
12. Lee, S. Y., and H. N. Chang. 1993. High cell density cultivation of Escherichia
coli by using sucrose as a carbon source. Biotechnol. Lett. 15:971–974.
13. Lee, S. Y., and H. N. Chang. 1995. Production of poly(3-hydroxybutyric acid)
by recombinant Escherichia coli strains: genetic and fermentation studies.
Can. J. Microbiol. 41(Suppl. 1):207–215.
14. Lee, S. Y., K. S. Yim, H. N. Chang, and Y. K. Chang. 1994. Construction of
plasmids, estimation of plasmid stability, and use of stable plasmids for the
production of poly(3-hydroxybutyric acid) in Escherichia coli. J. Biotechnol.
32:203–211.
15. Page, W. J., and A. Cornish. 1993. Growth of Azotobacter vinelandii UWD in
fish peptone medium and simplified extraction of poly-b-hydroxybutyrate.
Appl. Environ. Microbiol. 59:4236–4244.
16. Poirer, Y., C. Nawrath, and C. Somerville. 1995. Production of polyhydroxy-
alkanoates, a family of biodegradable plastics and elastomers, in bacteria and
plants. Bio/Technology 13:142–150.
17. Preusting, H., R. van Houten, A. Hoefs, E. K. van Langenberghe, O. Favre-
Bulle, and B. Witholt. 1993. High cell density cultivation of Pseudomonas
oleovorans: growth and production of poly(3-hydroxyalkanoates) in two-
liquid phase batch and fed-batch systems. Biotechnol. Bioeng. 41:550–556.
18. Yamane, T., M. Fukunaga, and Y. W. Lee. 1996. Increased PHB productivity
by high-cell-density fed-batch culture of Alcaligenes latus, a growth-associ-
ated PHB producer. Biotechnol. Bioeng. 50:197–202.