Journal of Applied Microbiology 2001, 90, 353±357

Biosynthesis of PHB tercopolymer by Bacillus cereus UW85

18 _
S. èabuzek and I. Radecka
Department of Biochemistry, Faculty of Biology and Environmental Protection, University of Silesia, Katowice,
Poland

417/6/00: received 19 June 2000, revised 13 October 2000 and accepted 7 November 2000

18 _ K A N D I . R A D E C K A . 2001.
S . è A B U ZE
Aims: The study was attempted to determine the ability of a Gram-positive Bacillus cereus
UW85 strain to biosynthesize poly (3-hydroxybutyrate) copolymers when e-caprolactone, or
e-caprolactone and glucose, were used as carbon sources.
Methods and Results: Bacillus cereus was grown for 24 h under nitrogen-limited conditions
in a mineral salts medium. Growth was monitored by measurement of turbidity. Glucose level
was determined by the colorimetric anthrone method. The e-caprolactone concentration was
determined by gas chromatography. The bacterial biopolymers were extracted with chloroform
in a Soxhlet extractor and then characterized by nuclear magnetic resonance and gel permeation
chromatography. When e-caprolactone was used as a carbon substrate, the bacterial strain
produced tercopolymer with 3-hydroxybutyrate, 3-hydroxyvalerate and 6-hydroxyhexanoate
units. However, when caprolactone and glucose were supplied together, only homopolymer of
poly (3-hydroxybutyrate) was produced.
Conclusions: All tercopolymers isolated from B. cereus UW85 cells were obtained with yields
up to 9% (w/w) and low number-average molecular weight compared with the homopolymer
PHB.
Signi®cance and Impact of the Study: Bacillus cereus UW85 produced tercopolymer with a
low molecular weight from one substrate (e-caprolactone) used as a carbon source. The results
are signi®cant for the potential future application of Bacillus biopolymers to bioplastics
1 production.

thermoplastic with an isotactic structure, a high degree of

INTRODUCTION
The microbial poly (hydroxyalkanoate) (PHA) family of
polyesters is synthesized by a wide variety of bacteria as
intracellular carbon and energy storage materials (Anderson
and Dawes 1990; Doi 1990). These polyesters are accumu-
lated as a result of limiting bacterial growth and supplying
an excess amount of a carbon source (Jackson and Dawes
2 1976; Bertrand et al. 1990; Dawes 1990; èabu_zek et al.
18 1994). The best known of the PHA family is the
homopolymer poly (3-hydroxybutyrate). Prokaryotic organ-
isms, including Gram-positive and Gram-negative bacteria,
are known to produce PHB amounting to as much as 80% of
their cellular dry weight (Doi 1990; Mans®eld et al. 1995).
The biopolymer is a biodegradable and biocompatible
Correspondence to: Dr Izabela Radecka, School of Applied Sciences, Universisty
Of Wolverhampton, Wulfruna street, Wolverhampton WV1 1SB, UK
(e-mail: cs1961@wlv.ac.uk).
crystallinity (approximately 80%), a high number-average
molecular weight (approximately 105±106) and a high
melting temperature (about 175 °C) (Dawes 1990; Scandola
1995; Madison and Huisman 1999).
However, there are drawbacks to the use of PHB as a
plastic material because of its tendency to be brittle. This
problem could be solved by the synthesis of copolymers of
3-hydroxybutyrate and other hydroxyalkanoates with a
relatively low molecular weight and melting point
3 (de Koning 1995; Scandola 1995; Fukui and Doi 1997).
Presently, more then 90 different monomeric units have
been found as constituents of PHA (Doi 1990; Seebach et al.
1995).
A wide variety of PHA copolymers have been isolated
from bacteria found in different environmental samples,
including marine sediments, marine and freshwater cyano-
bacteria, and sewage sludge. Varying the compositions of the
carbon substrates could control the copolymer compositions

ã 2001 The Society for Applied Microbiology

 354 _ K AND I. RADECKA
S . è A B U ZE

Table 1 Production of bacterial polyesters by Bacillus cereus UW85* grown in a mineral salts medium with glucose, glucose and
e-caprolactone, or e- caprolactone alone, as substrate

Substrate Cell dry PHA composition mol%

Initial concentration after 24 h weight PHA content Molecular
of substrate (g l)1) (g l)1) (g l)1) (weight %)  3HB 3HV 6HH weight (Mn)

10 g l)1 glucose 3á2 11á8 24á6 100 0 0 210 000

10 g l)1 glucose 2á5 0á9 2á32 100 0 0 97 000
1 g l)1 e-caprolactone 1
10 g l)1 glucose 2á4 1á3 3á28 100 0 0 81 000
3 g l)1 e-caprolactone 3
1 g l)1 e-caprolactone 0á07 0á43 5á9 75 21 4 21 500
3 g l)1 e-caprolactone 1á5 0á41 8á9 47 42 11 18 100

*24 h of incubation.
 Polyester content in dry cells.

(Doi 1990; Nakamura et al. 1991). Bacterial polyesters can
be divided into two groups, depending on the number of
carbon atoms in the monomeric units (Casini et al. 1997;
4 Matsusaki et al. 1998). The ®rst group consists of short-
chain-length PHA with C3 to C5 monomer units. For
example, copolymers of 3-hydroxybutyrate (3HB) and
3-hydroxyvaleriate (3HV) have been produced by Alcalig-
enes eutrophus from propionic acid or 1,5-pentanediol
(Nakamura et al. 1991) and copolymers of 3-hydroxybuty-
rate (3HB) and 4-hydroxybutyrate (4HB) when 4-hydroxy-
butyric acid or c-butyrolactone was used as the carbon
5 source (Doi 1990). Examples of the second group of
medium-chain-length copolyesters, produced by Pseudo-
monas oleovorans, contain 3-hydroxyalkanoate monomer
units of C6 to C12 from alkanes and alkanoic acids (Fukui
6 and Doi 1997).
The present study attempted to determine the biosyn-
thesis of PHA copolymers by Bacillus cereus UW85 strain,
when e-caprolactone was used as a carbon source. Bacterial
copolymers with e-caprolactone units might have value as
environmentally-degradable thermoplastics for a wide range
of agricultural, marine, and medical applications (Zhang and
Deng 1997).
M A T E R I A LS A N D M E T H O D S
Micro-organisms and culture conditions
Bacillus cereus UW85 was obtained from the Institute of
Immunology and Experimental Therapy, Polish Academy of
18 Science, Wroclaw, Poland. The shake-¯ask experiments
were performed in 500 ml conical ¯asks using 250 ml of
chemically-de®ned N-limited mineral salts medium at an
initial pH of 7á2 (Kojima et al. 1961). The medium
contained 10 g l)1 of glucose with 1 or 3 g l)1 e-caprolac-
tone, or 1 or 3 g l)1 of e-caprolactone alone, as potential
carbon source.
ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 353±357
Cultivation was carried out for 24 h at 30 °C with an
agitation rate of 125 rev min)1. To determine bacterial
growth, samples were taken at 0, 1, 2, 3, 4, 5, 6, 7, 8 and
24 h. Growth was monitored by measurement of turbidity
(k ˆ 550 nm) using a Beckman DU-50 spectrophotometer
(Beckman Coulter, Warsaw, Poland).
PHA extraction
After 24 h of cultivation, cells were harvested by centrifu-
gation, resuspended, washed twice with distilled water and
dried under vacuum at room temperature. The total cell dry
weight was measured. Dried cells were then suspended in
chloroform in a round-bottomed ¯ask. Biopolymers were
extracted with hot chloroform in a Soxhlet extractor (Sigma-
8 Aldrich, Poznan, Poland) running for 6±7 h. The resulting
solution was concentrated with a rotary vacuum evaporator.
Finally, the isolated biopolymers were precipitated with an
equal volume of hexane, separated by ®ltration and the total
PHA content measured gravimetrically. Samples of white
crystalline powder isolated from the bacterial mass were
dried at room temperature (20 °C) and stored for chemical
18 analysis (Doi 1990; Nakamura et al. 1991; èabu_zek et al.
1994).
Analytical procedures
A series of samples of the growing culture was taken to
determine glucose and e-caprolactone levels in the superna-
tant ¯uid after centrifugation. The e-caprolactone concen-
tration was determined by gas chromatography using a
9 Varian 3400 (Varian Inc, Warsaw, Poland) with a helium
¯ow rate of 5 ml min)1. Glucose level was determined by the
colorimetric anthrone method at k ˆ 620 nm, using Beck-
man DU-50 spectrophotometer (Klyszejko-Stefanowicz
1982).
 BIOSYNTHESIS OF TERCOPOLYMER 355

Fig. 1 1H-NMR spectrum of PHB isolated

from Bacillus cereus UW85, when glucose
(10 g l)1) and e-caprolactone (1 or 3 g l)1)
were used as a carbon source

The polymer isolated from the cells was identi®ed by
nuclear magnetic resonance. Spectra of 1H-NMR were run
in CDCl3 with tetramethylsilane (TMS) as internal standard
using a Varian-300 spectrometer (Varian Inc, Warsaw,
10 Poland). Proton 1H-NMR spectroscopy was also used to
determine copolymer composition. The molar fractions of
3HB, 3HV and 6HHx in the tercopolymers were calculated
from the peak of proton resonances of 3HB, 3HV and
6HHx.
The number-average molecular weight (Mn) was deter-
mined by gel permeation chromatography (GPC) calibrated
with polystyrene. The GPC experiments were carried out in
tetrahydrofuran (THF) with a ¯ow rate of 1 ml min)1, at
35 °C, using a Spectra Physics gel permeation chromato-
11 graph (Spectra Physics, Warsaw, Poland).
were used: 10 g l)1 glucose, and 1 or 3 g l)1 e-caprolactone.
It is known that the structure and monomeric composition
of the copolymer is generally dependent upon the concen-
tration of carbon substrate in the culture medium. A variety
of copolymers has been synthesized by co-feeding with
various substrates (Doi 1990; Madison and Huisman 1999).
When e-caprolactone (1 or 3 g l)1) was added to ¯asks
containing 10 g l)1 glucose, B. cereus UW85 synthesized a
biopolymer identi®ed by 1H-NMR (Fig. 1) as a homopoly-
mer of PHB (Table 1). This polymer was synthesized at
both concentrations of e-caprolactone. Poly (3-hydroxybu-
tyrate) accumulation was very low, and varied from 2á32%
(w/w) on glucose with 1 g l)1 e-caprolactone, to 2á85%

RESULTS AND DISCUSSION

The use of biological systems for the production of
biodegradable materials is becoming important as a solution
to the problems concerning non-biodegradable plastic waste
and the environment. The PHA family of polyesters offers a
range of thermoplastics varying in toughness and ¯exibility
(Dawes 1990; Doi 1990; Scandola 1995; Seebach et al. 1995;
Madison and Huisman 1999).
From previous work, it is known that organisms such as
18 Bacillus strains are able to produce poly hydroxybutyrate as
a carbon storage material (èabu_zek et al. 1994). For this
investigation, B. cereus UW85 strain was chosen as a
promising candidate from a number of bacteria known to
accumulate PHB and grow on glucose (1%) in nutrient
broth and mineral salts medium in shake ¯asks. Under such
conditions, B. cereus UW85 grew and produced PHB up to
25% (w/w) of dry biomass (Table 1).
To determine whether substrates such as glucose and Fig. 2 Growth of Bacillus cereus UW85 in shake ¯ask culture with
e-caprolactone, or e-caprolactone alone, could be used as glucose and e-caprolactone as substrates. (a) Glucose (10 g l)1) and
carbon sources for PHB copolymer production, B. cereus e-caprolactone (1 g l)1); (b) glucose (10 g l)1) and e-caprolactone
UW85 was grown on a nitrogen-limited medium in shake- (3 g l)1). Growth (s); glucose concentration (n); e-caprolactone
¯ask experiments. Two carbon substrate concentrations concentration (h)

ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 353±357
356 _ K AND I. RADECKA
S . è A B U ZE
(w/w) with 3 g l)1 e-caprolactone. It was found that when
B. cereus UW85 grew in the presence of both carbon
substrates together, it was only able to metabolize glucose
(Fig. 2), and accumulated PHB homopolymer with a higher
number-average molecular weight (up to 97 000) compared
with the tercopolymers synthesized (Table 1).
However, with e-caprolactone as the sole carbon source in
a mineral salts medium without glucose, copolymer with
short-and medium-chain-length monomer units was syn-
thesized by B. cereus UW85. Using the 1H-NMR method, it
was found that the copolymer was tercopolymer with
3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV) and
6-hydroxyhexanoate (6HHx) units (Fig. 3, Fig. 4). There
are only a few reports of bacteria able to synthesize both
ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 353±357
Fig. 3 1H-NMR spectrum of tercopolymer
isolated from Bacillus cereus UW85 with 3HB
(75 mol%), 3HV (21 mol%) and 6HH
(4 mol%) units, when 1 g l)1 e-caprolactone
was used as a carbon source
short- and medium-chain-length monomer units. For
example, Rhodospirillum rubrum or Rhodococcus ruber pro-
duce tercopolymers consisting of C4, C5 and C6 3-hydrox-
yalkanoate (3HA) units from hexanoate. Some pseudomonad
strains accumulate polyester with C4 to C12 3HA units
(Fukui and Doi 1997). Aeromonas caviae FA440, isolated
from soil, produced a random copolymer of 3-hydroxybu-
tyrate (3HB) and 3-hydroxyhexanoate (3HHx) (Matsusaki
et al. 1998) from alkanoic acid of even carbon number or
from plant oil. Yields were up to approximately 30% (w/w)
of the cellular dry weight, with a 3HHx fraction from 10 to
25 mol% (Fukui and Doi 1997; Matsusaki et al. 1998).
In the present experiments, all tercopolymers isolated
from B. cereus UW85 cells were obtained with yields of up to
Fig. 4 1H-NMR spectrum of tercopolymer
isolated from Bacillus cereus UW85 with 3HB
(47 mol%), 3HV (42 mol%) and 6HH
(11 mol%) units, when 1 g l)1 e-caprolactone
was used as a carbon source
 BIOSYNTHESIS OF TERCOPOLYMER 357

9% (w/w) and low number-average molecular weight De Koning, R. (1995) Physical properties of bacterial poly
compared with the homopolymer PHB. The yield increased 13,14 13,14 (R)-hydroxyalkanoates. Canadian Journals of Microbiology 41, 303±
from 5á9% (w/w) to 8á9% (w/w) when the concentration13,14 of 13,14 309.
)1 )1 15 Doi, Y. (1990) Microbial Polyester. New York: VCH.
e-caprolactone increased from 1 g l to 3 g l .
1 Fukui, T. and Doi, Y. (1997) Cloning and analysis of the poly (3-
H-NMR studies revealed that the ratio of 3HB units of
)1 hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of
tercopolymer isolated from bacteria when 3 g l e-capro-
Aeromonas caviae. Journal of Bacteriology 179, 4821±4830.
lactone was added to the mineral salts medium was lower Jackson, F. and Dawes, E.A. (1976) Regulation of the tricarboxylic acid
(47 mol%) compared with biopolymer isolated from medi- cycle and poly b-hydroxybutyrate metabolism in Azotobacter
um with 1 g l)1 (75 mol%). Nevertheless, the ratio of 3HB beijerinckii grown under nitrogen or oxygen limitation. Journal of
to 3HV and 6HHx units decreased (Table 1) as the General Microbiology 97, 303±312.
concentration of carbon substrate was increased from 1 to 18 Klyszejko-Stefanowicz, L. (ed.) (1982) Cwiczenia z biochemii, Warsz-
3 g l)1. As a result, the number-average molecular weight of 16 awa/Poznan: PWN.
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ACKNOWLEDGEMENTS Madison, L.L. and Huisman, G.W. (1999) Metabolic engineering of
poly (3-hydroalkanoates): form DNA to plastic. Microbiology and
The authors are grateful to friends from the Institute of Molecular Biology Reviews 63, 21±53.
Polymer Chemistry for technical assistance with NMR and Mans®eld, D.A., Anderson, A.J. and Naylor, L.A. (1995) Regulation of
GPC methods. Also the authors wish to thank to Dr David PHB metabolism in Alcaligenes eutrophus. Canadian Journal of
Necklen for English correction. Microbiology 41, 44±49.
Matsusaki, H., Manji, S., Taguchi, K., Kato, M., Fukui, T. and Doi,
Y. (1998) Cloning and molecular analysis of the poly (3-hydrox-
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ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 353±357