Poly(hydroxybutyric acid) (PHB) and other biodegradable
polyesters are promising candidates for the development of
environment-friendly, totally biodegradable plastics. The use
of cane molasses and corn steep liquor, two of the cheapest
substrates available in Egypt, may help to reduce the cost
of producing such biopolyesters. In this work, the effect of
different carbon sources was studied. Maximum production of
PHB was obtained with cane molasses and glucose as sole
carbon sources (40.8, 39.9 per mg cell dry matter, respective-
ly). The best growth was obtained with 3% molasses, while
maximum yield of PHB (46.2% per mg cell dry matter) was
obtained with 2% molasses. Corn steep liquor was the best
nitrogen source for PHB synthesis (32.7 mg per cell dry
matter), on the other hand, best growth was observed when
ammonium chloride, ammonium sulphate, ammonium oxa-
late or ammonium phosphate were used as nitrogen sources.
Key words: PHB – Bacillus megaterium – cane molasses –
corn steep liquor
Biodegradable polymers have recently attracted much
public and industrial interest as a consequence of exten-
sive discussions looking for better waste-management
strategies (Doi 1990; Schlegel 1992; Steinbüchel
1995). The use of biodegradable polymers allows com-
posting as an additional way for waste disposal.
Furthermore, the use of these polymers opens several
new applications in medicine, agriculture and industry.
Polyhydroxybutyrate (PHB) is an organic polymer
with commercial potential as a biodegradable thermo-
plastic and a biomaterial (Ramsay et al. 1990). PHB is
well known as a carbon and energy reserve produced by
a variety of microorganisms and its synthesis is favored
by environmental stresses such as nitrogen, phosphate
or oxygen limitation (Doi 1990; Steinbüchel 1991; Lee
1996). PHB and other PHAs are synthesized and depo-
sited intracellularly in the form of granules and might
amount up to 90% of the cellular dry weight (Schlegel
et al. 1961). Accumulation of intracellular storage poly-
mers has been considered a strategy used by bacteria
to increase survival in a changing environment. The
ability to store PHB is an example of this characteristic
and usually reflects a transient abundance of carbon
sources with respect to other nutrients such as nitrogen
and phosphorus (Pedrós-Alió et al. 1990; Steinbüchel
1991; Steinbüchel et al. 1992).
One of the limiting factors in the commercial success
of PHB and other PHAs production schemes is the cost
of the sugar substrate used for PHA formation. It has
been calculated that 3 tones of glucose must be used for
each tone of polymer produced (Collins 1987). PHB
can be produced from relatively cheaper substrates such
as methanol (Suzuki et al. 1986), beet molasses (Page
1992a, b) or ethanol (Alderet et al. 1993).
Molasses has been used extensively as a carbon sour-
ce for commercial production of baker’s yeast (White
1954). Since cane molasses contains vitamins and other
minor constituents (White 1954), it can be used as a
source of growth factors as can corn steep liquor
(Malathi and Chakraborty 1991).
In this study cane molasses and corn steep liquor
were used as cheaper carbon and energy sources for
0944-5013/01/156/03-201$15.00/0Microbiol. Res. 156 (2001) 3
Microbiol. Res. (2001) 156, 201–207
Production of PHB by a Bacillus megaterium strain
using sugarcane molasses and corn steep liquor as sole carbon
and nitrogen sources
Mona K. Gouda, Azza E. Swellam, Sanaa H. Omar
Botany Department, Faculty of Science, Alexandria University, Alexandria, Egypt
Accepted: January 21, 2001
Corresponding author: Mona K. Gouda
PHB production by a local isolate of Bacillus mega-
terium. The use of these substrates will also reduce
environmental pollution caused by these materials. A
trail to produce large amounts of PHB in a simple bio-
reactor was also investigated.
Materials and methods
Microorganism and culture medium. The strain used in
this study was isolated from the sewage treatment plant
of Alexandria and was identified by the German Culture
Collection of Microorganisms (DSMZ), Braunschweig,
Germany, as Bacillus megaterium.
The following medium was used for PHB production
(after DSMZ catalogue 1993) in g/l: fructose 20.0,
NH4Cl 0.5, KH2PO42.3, Na2HPO42.3, MgSO4 · 7H2O
0.5, NaHCO30.5, ferric citrate 0.05, CaCl20.01 and
5 ml trace element solution. Each liter of trace element
solution contained (g): ZnSO4 · 7H2O 0.01, MnCl2 ·
4H2O 0.003, H3BO40.003, CoCl2· 7H2O 0.02, CuCl2·
2H2O 0.001, NiCl2· 6H2O 0.002 and NaMo4· 2H2O
0.003. Carbon source, nitrogen source, magnesium
sulfate and ferric citrate were sterilized separately at
121°C for 20 min and added to the sterilized medium
at the desired concentration. pH of the medium was
adjusted to 7.0.
Sugarcane molasses was obtained from Starch and
Bakers’ yeast company, Alexandria, Egypt and was
used as the best carbon source.
Corn steep liquor was prepared from corn grains
according to the method of Liggette and Koffler (1948)
and was used as the best nitrogen source.
PHB production and analysis. Batch fermentations
were carried out in 250-ml Erlenmeyer flasks con-
taining 100 ml of culture medium. The flasks were
inoculated and maintained at 30°C and 130 rpm for the
requested time. For large-scale production, a 10-l bio-
reactor was used which contained 4 l of the culture
Cells were collected by centrifugation at 10,000 rpm
for 10 min in a centrifuge (Heraeus Sepatech Biofuge
28 Rs, Germany) at 20°C and lyophilized in a Lab.
Conco 4.5 lyophilizer.
PHA and PHB content were determined by metha-
nolysis of monomers according to the method of
Braunegg et al. (1978) and as modified by Lageveen
et al. (1988). The methylesters were then analyzed by
GLC using benzoic acid (2 mg/ml) as the internal
standard according to Brandl et al. (1988) and modified
by Füchtenbusch et al. (1996). A Perkin-Elmer gas
Chromatograph, Model 8420 was used. The gas
chromatograph was equipped with a Permaphase PEG
25 MX-capillary column (25 m by 0.32 mm), Perkin-
Elmer, Überlingen, Germany, and a flame ionization
detector. Helium (5cm/min) was used as the carrier gas.
The temperature of the injector and detector were
230°C and 275°C, respectively. Atemperature program
was used for efficient separation of the esters (120°C
for 5min, temperature ramp of 8°C per min, 180°C for
Gravimetrical determination of PHA. PHA was ex-
tracted from lyophilized cells with chloroform in a
Soxlet apparatus (Füchtenbusch et al. 1996) for 6 h.
Cell debris was removed by passing the suspension
through a folded Whatman filter paper. The diluted
extract in chloroform was then concentrated to a
viscous fluid by using a rotary evaporator (Rotavapor
BÜCHI R-114, Switzerland). This viscous solution
was then added drop by drop into a beaker containing
10 volumes of ethanol. A fluffy cottony precipitate
was seen to form at the interface of ethanol (98%).
This precipitate was then separated by centrifugation
(5000 rpm) at 4°C, washed twice with ethanol and then
dried in air for 16 h at room temperature to constant
Microscopic observation of PHB granules using trans-
mission electron microscopy (TEM). Bacterial samples
were harvested by centrifugation and prefixed at room
temperature for 30 min by adding equal volumes of
2% glutaraldehyde fixative buffer containing 0.1 so-
dium cacodylate (pH 7.2) according to the method of
Sun et al. (1995). After centrifugation, the pellets were
suspended and fixed in 2% glutaraldehyde in 0.1 M
sodium cacodylate for 2 h at room temperature.
Following fixation the samples were washed three
times with 0.1 M sodium cacodylate buffer (pH 7.2)
and then mixed with 1% OsO4plus 1% potassium
ferrocyanide in 0.1 M sodium cacodylate buffer (pH
7.2) for 2 h at room temperature. Specimens were then
washed twice with water, poststained with 2% uranyl
acetate for 1 h, dehydrated in graded concentrations
(first in 70 then 90 and then 95–100% v/v) of acetone
and embedded in Spurr’s epoxyresin (Spurr 1969) by
incubating at 70 ± 1°C over night. Thin sections of
70 nm were cut and stained in Reynold’s lead acetate
and photographed with a Jeol Transmission Electron
Microscope (JTM-Japan) in the Central laboratory,
Faculty of Science, Alexandria University.
Dry weight estimation. Dry weight was estimated
from 100 ml of culture broth. The cell suspension was
centrifuged at 10,000 rpm for 10 min (Heraeus
Sepatech Biofuge 28 Rs, Germany) at 20°C, washed
with warm distilled water several times, transferred to
preweighed vials and dried in an oven (Dental und
Laborbau GmbH, Germany) at 105°C till constant
Microbiol. Res. 156 (2001) 3
Results and discussion
The effect of different carbon sources on the production
of PHA and PHB by the test organism was studied, in
order to choose the best and most economical one.
Fructose, glucose, xylose, lactose, sucrose, maltose,
Na-gluconate and sugarcane molasses were added at
2% to the production medium, one at a time. The
amount of PHAand PHB and also the growth rate of the
organism after 48 h were determined.
As shown in Fig. 1 the best carbon source for growth
of cells was maltose (4.8 g/l). On the other hand, glu-
cose was the best carbon source for PHAs production
(45.6% per mg cell dry matter), this was followed by
sugarcane molasses (44.6% per mg cell dry matter).
Fructose was also a good carbon source for PHAs accu-
mulation while xylose was the poorest carbon source
(7.40% per mg cell dry matter).
Wilde (1962) reported that fructose is the only sugar
used by Alcaligenes eutrophus, whereas glucose and
disaccharides such as lactose, maltose or sucrose are
Linko et al. (1993) reported on the influence of some
carbon sources on the production of PHB by A. eutro-
phus. They reported that fructose was the best carbon
source followed by lactic acid. Glucose was also a
suitable source (Kim et al. 1994 a, b). PHB was accu-
mulated in a fair level 21% per mg cell dry matter using
lactose as sole carbon source in the production medium.
Lactose is found in high levels in whey, which is a
waste product of cheese production. As such it is a very
attractive and economical source of carbon. Janes et al.
(1990) pointed out the significance of lactose as a car-
bon source in PHB production.
The use of cane molasses as a growth factor maxi-
mized the production of PHB and showed some poten-
tial as a low-cost ingredient for industrial fermentation.
Since cane molasses contains trace elements and vit-
amins such as thiamine, riboflavin, pyridoxine and
niacinamide (White 1954; Crueger and Crueger 1984)
it can be used as a source of growth factors.
Different sugarcane molasses levels (1–5%) were
added to the production medium and the yield of
PHA and PHB as well as the growth of B. megaterium
were estimated after 48 h incubation. The results pre-
sented in Fig. 2 indicate that the amount of sugarcane
molasses affects the accumulation of the test products
and also the growth of the organism. The best growth
was obtained with 3% molasses, while the maximum
yield of PHA and PHB (46.5 and 46.3% per mg cell
dry matter) were obtained with 2% molasses, and
then decreased by increasing the level of molasses until
5% (18.3% for both PHA and PHB per mg cell dry
The effect of different concentrations of cane molas-
ses has been studied by Beaulieu et al. (1995) which
showed that the production of PHB was high between
0.1–0.3 g/l. However, the percentage of PHB on the
basis of cell dry weight was higher with a 0.1% con-
centration. Also Page (1992a) reported the production
of 59% PHB per cell dry matter when 5% cane molas-
ses were used with Azotobacter vinelandii UWD, and
32% PHB per cell dry matter when 3% glucose was
Microbiol. Res. 156 (2001) 3
Fig.1. Effect of different carbon sources (2%) on dry weight and accumulation of PHA and PHB by cells of B. megaterium.
A variety of different nitrogen sources were added
(on equal nitrogen basis using 0.05% ammonium chlo-
ride as control) to the medium with 2% sugarcane
molasses and the amount of PHA and PHB was deter-
mined after 48 h at 30°C and 130 rpm (Table 1). The
results indicate that ammonium chloride produced the
highest amount of PHA and PHB (40.1 and 38.4% per
mg cell dry matter, respectively) followed by corn steep
liquor and wheat bran. Ammonium nitrate and ammo-
nium acetate were also good nitrogen sources for the
accumulation of PHA and PHB. The use of some com-
plex nitrogen sources like yeast extract, peptone or beef
extract decreased the synthesis of PHA by about 70%,
although peptone and yeast extract support more
growth than wheat bran and CSL. Page (1992b) ob-
served that the addition of peptone to medium with
Microbiol. Res. 156 (2001) 3
Fig. 2. Effect of different sugar cane molasse levels on dry weight and accumulation of PHA and PHB by cells of B. mega-
Table1. Effect of different nitrogen sources (equivalent to 0.05% NH4Cl) on the dry weight and accumulation of PHA and
PHB in cells of B. megaterium, after 48 h incubation at 30°C.
Nitrogen SorcespH value Dry Weight (g/l) %/mg CDM
Corn steep liquor
CDM = Cell dry matter
impure sugar increased the yield of PHA, this obser-
vation is contradicting to the result obtained in this
work. On the other hand, the best growth was observed
with ammonium salts like chloride, sulphate, oxalate
and phosphate. These results prove that the yield of
PHAs is not related to the increase in growth. Beaulieu
et al. (1995) reported the production of PHB by
Alcaligenes eutrophus in a synthetic medium with 3%
glucose supplemented with several ammonium substra-
tes and found that the best growth and PHB production
were obtained with ammonium sulphate as nitrogen
Although pH values in this experiment fluctuated
between 5.12 and 6.9, it seems that it did not greatly
affect growth nor production of alkanoates by the orga-
nism under test. Beaulieu et al. (1995) reported that
(in the absence of pH control) at an ammonium con-
centration higher than 0.5 g/l, the growth of A. eutro-
phus rapidly stopped when the pH dropped below 5.4.
The optimum pH for growth and PHB production by
A. eutrophus was 6.9, and that of 5.4 inhibited its
growth (Repaske 1962).
In this study CSL was chosen as a cheap nitrogen
source for PHB production by B. megaterium in order
to reduce the cost of PHB production. Cho et al. (1997)
used swine waste liquor for PHB-co-PHV production
by A. vinelandii UWD.
It was found that the synthesis of PHAs is favored by
environmental stresses such as nitrogen limitation, so
PHAs formation was studied under nitrogen limiting
conditions. The production medium with 2% sugar-
cane molasses was supplemented with different levels
of CSL from 0.5–7%. Maximum yield of PHAs
(49.12% per cell dry matter) was obtained with 3%
CSL in the medium after 48 h incubation (Fig. 3). The
use of 7% CSL decreased the amount of PHAs by
about 62% in comparison with 3%. On the other hand,
maximum growth was obtained with 1% CSL and then
began to decrease. Lee and Chang (1994) studied the
effect of CSL concentration on PHB production by
recombinant E. coli from a concentration of 0.1–1%
and reported that the best concentration was 1% which
yielded 44.9% PHB. The effect of nitrogen limitation
was also studied by Ramsay et al. (1992).
In a trial to produce large quantities of biomass and,
therefore, considerable amounts of PHB, a simple bio-
reactor modified after Hoben and Somasegaran (1992)
was used. 4 l of the optimized production medium
[2% sugarcane molasses, 3% CSL, 0.1 g/l MgSO4
· 7H2O, 0.5 g/l Na2HPO4, 0.5 g/l KH2PO4, 0.5 g/l
NaHCO3, 0.05 g/l ferric citrate, 0.01 g/l CaCl2and 5 ml
trace element solution (as described under materials
and methods)] was used in a 10-l capacity glass fer-
mentor. The fermentor was incubated at room temp-
erature (30 ± 2°C) for 48 h on a magnetic stirrer
(supplemented with a simple pump to permit sterile air
entrance). The cells were centrifuged and lyophilized.
PHB was extracted in chloroform and precipitated in
ethanol. The white precipitate was then dried to con-
stant weight. The dry weight of the lyophilized cells
Microbiol. Res. 156 (2001) 3
Fig.3. Effect of different corn steep levels on dry weight and accumulation of PHA and PHB by cells of B. megaterium.
produced in the fermentor was 3.6 g/l, while the quan-
tity of PHB was 2.2 g, (59.4% on dry weight basis).
Transmission electron microscopy (TEM) of the cells
grown on the optimized medium (Fig. 4) illustrates the
formation of PHB granules which change the cell shape
from rod to oval and spherical to accommodate the size
of the growing granules within 24–48 h.
The authors wish sincerely to thank prof. Dr. A Steinbüchel
(Westfälische Wilhelms Universität, Münster, Germany) for
Alderet, J. E., Karl, D. W., Park, C. H. (1993): Production of
poly(hydroxybutyrate), homopolymer and copolymer from
ethanol and propanol in a fed-batch culture. Biotechnol.
Prog. 9, 520–525.
Beaulieu, M., Beaulieu, Y., Mélinard, J., Pandian, S., Goulet,
J. (1995): Influence of ammonium salts and cane molasses
on growth of Alcaligenes eutrophus and production of
polyhydroxybutyrate. Appl. Environ. Microbiol. 61,
Brandl, H., Gross, R. A., Lenz, R. W., Fuller, R. C. (1988):
Pseudomonas oleovorans as a source of poly (β-hydroxy-
alkanoates) for potential applications as biodegradable
polyesters. Appl. Environ. Microbiol. 54, 1977–1982.
Braunegg, G., Sonnleitner, B., Lafferty, R. M. (1978): Arapid
gas chromatographic method for the determination of poly-
β-hydroxybutyric acid in microbial biomass. Eur. J. Appl.
Microbiol. Biotechnol. 6, 29–37.
Cho, K.-S., Ryu, H. W., Park, C.-H., Goodrich, P. R. (1997):
Poly(hydroxybutyrate-co-hydroxyvalerate) from swine
waste liquor by Azotobacter vinelandii UWD. Biotechnol.
Lett. 19, 7–10.
Collins, S. (1987): Choice of substrate in polyhydroxybuty-
rate synthesis. Spec. Publ. Soc. General Microbiol. 21,
Crueger, W., Crueger, A. (1984): Biotechnology, A text book
of industrial microbiology. Science Tech. Inc., Madison,
Doi, Y. (1990): Microbial Polyesters. VCH Publisher,
Weinheim, Cambridge, NewYork.
DSM catalogue of strains (1993): Deutsche Stammsammlung
für Mikroorganismen und Zellkulturen GmbH (German
Culture Collection of Microorganisms and cell Cultures),
Füchtenbusch, B., Fabritius, D., Steinbüchel, A. (1996):
Incorporation of 2-methyl-3-hydroxybutyric acid into poly-
hydroxyalkanoic acids by axenic cultures in defined media.
FEMS Microbiol. Lett. 138, 153–160.
Hoben, H. J., Somasegaran, P. (1992): A small glass fermen-
tor for the production of Rhizobium inoculum. World J.
Microbiol. Biotechnol. 8, 333–334.
Microbiol. Res. 156 (2001) 3
Fig.4. TEM of B. megaterium cells cultivated in a bioreactor
under optimized culture conditions. (a) 24 h and (b & c) 48 h
incubation. (× 30,000)
Janes, B., Hollar, J., Dennis, D. (1990): Molecular character- Download full-text
ization of the poly-β-hydroxybutyrate biosynthetic path-
way of Alcaligenes eutrophus H16. In: Novel Biodegrad-
able Microbial Polymers. (Eds: Dawes, E. A.). Kluwer
Academic Publishers. Dordrecht, The Netherlands,
Kim, B. S., Lee, S. C., Lee, S. Y., Chang, H. N., Chang, Y. K.,
Woo, S. L. (1994a): Production of poly(3-hydroxybutyric
acid) by fed-batch culture of Alcaligenes eutrophus with
glucose concentration control. Biotechnol. Bioeng. 43,
Kim, B. S., Lee, S. C., Lee, S. Y., Chang, H. N., Chang, Y. K.,
Woo, S. L. (1994b). Production of poly(3-hydroxybutyric-
co-3-hydroxyvaleric acid) by fed-batch culture of
Alcaligenes eutrophus with substrate control using on-line
glucose analyzer. Enzyme Microbiol. Technol. 16,
Lageveen, R. G., Huisman, G. W., Preusting, H., Ketelaar, P.,
Eggink, G., Witholt, B. (1988): Formation of polyesters by
Pseudomonas oleovorans: Effect of substrates on forma-
tion and composition of poly-(R)-3-hydroxyalkanoates and
poly-(R)-3-hydroxyalkenoates. Appl. Environ. Microbiol.
Lee, S. Y., Chang, H. N. (1994): Effect of complex nitrogen
source on the synthesis and accumulation of poly(3-hydro-
xybutyric acid) by recombinant Escherichia coli in flask
and fed-batch cultures. J. Environ. Polymer Degrad. 2,
Lee, S. Y. (1996): Review: Bacterial polyhydroxyalkanoate.
Biotechnol. Bioeng. 49, 1–14.
Liggette, W. R., Koffler, H. (1948): Corn steep in microbiol-
ogy. Bacteriol. Rev. 12, 297–312.
Linko, S., Vahrte, H., Seepällä, J. (1993): Production of ploy-
β-hydroxybutyrates by Alcaligenes eutrophus on different
carbon sources. Appl. Microbiol. Biotechnol. 39, 11–15.
Malathi, S.,Chakraborty, R. (1991): Production of alkaline
protease by a new Aspergillus flavus isolate under solid-
substrate fermentation conditions for use as a depilation
agent. Appl. Environ. Microbiol. 57, 712–716.
Page, W. J. (1992a): Production of polyhydroxyalkanoates by
Azotobacter vinelandii UWD in beet molasses culture.
FEMS Microbiol. Rev. 103, 149–158.
Page, W. J. (1992b): Suitability of commercial beet molasses
fractions as substrates for polyhydroxyalkanoate produc-
tion by Azotobacter vinelandii UWD. Biotechnol. Lett. 14,
Pedrós-Alió, C., Mas, J.,Guerrero, R. (1990): Polyhydroxy-
alkanoate accumulation in planktonic and anaerobic en-
vironments. In: Novel Biodegradable Microbial Polymers
(Ed. Dawes, E. A.) Kluwer Academic Publisher. Dordrecht,
The Netherlands. 263–274.
Ramsay, B. A., Lomaliza, K., Chavarie, C., Dubé, B., Bataille,
P., Ramsay, J. A. (1990): Production of poly-(β-hydroxy-
butyric-Co-β-Hydroxyvaleric) acids. Appl. Environ.
Microbiol. 56, 2093–2098.
Ramsay, B. A., Saracovan, I., Ramsay, J. A. Marchessault, R.
H. (1992): Effect of nitrogen limitation on long-side-chain
poly-β-hydroxyalkanoate synthesis by Pseudomonas resi-
novorans. Appl. Environ. Microbiol. 58, 744–746.
Repaske, R. (1962): Nutritional requirements for Hydrogeno-
monas eutropha. J. Bacteriol. 83, 418–422.
Schlegel, H. G. (1992): Past and present cycle of carbon on
our planet. FEMS Microbiol. Rev. 103, 347–354.
Schlegel, H. G., Gottschalk, G., von Bartta, R. (1961):
Formation and utilization of poly-β-hydroxybutyric acid
by knallgas bacteria (Hydrogenomonas). Nature (London)
Spurr, A. R. (1969): A low viscosity epoxy resin embedding
medium for electron microscopy. J. Ultrastruct. Res. 26,
Steinbüchel, A. (1991): Polyhydroxyalkanoic acids. In:
Biomaterials, Novel materials from biological sources. (Ed.
Byrom, D.). MacMillan Publisher, Ltd, Basingstoke.
Steinbüchel, A. (1995): Use of biosynthetic, biodegradable
thermoplastics and elastomers from renewable resources,
The pros and cons. JMS Pure Appl. Chem. 32, 653–660.
Steinbüchel, A., Hustede, E., Liebergesell, M., Pieper, U.,
Timm, A., Valentin, H. (1992): Molecular basis for bio-
synthesis and accumulation of polyhydroxyalkanoic acids
in bacteria. FEMS Microbiol. Rev. 103, 217–230.
Sun, W., Teng, K., Meighen, E. (1995): Detection of poly(3-
hydroxybutyrate) granules by electron microscopy of
Vibrio harveyi stained with malachite green. Can. J.
Microbiol. 41, 131–137.
Suzuki, T., Yamane, T., Shimizu, S. (1986): Mass production
of poly-β-hydroxybutyric acid by fully automatic fed-batch
culture of methylotroph. Appl. Microbiol. Biotechnol. 23,
White, J. (1954): Yeast technology. Chapman & Hall, Ltd.,
Wilde, E. (1962): Untersuchungen über Wachstum und
Speicherstoffsynthese von Hydrogenomonas. Arch.
Mikrobiol. 43, 109–137.
Microbiol. Res. 156 (2001) 3