Biotechnol. J. 2012, 7, 293303 DOI 10.1002/biot.201100122 www.biotechnology-journal.

com

Research Article

Poly(3-hydroxybutyrate) production by Bacillus cereus SPV using

sugarcane molasses as the main carbon source

Everest Akaraonye1, Catalina Moreno1, Jonathan C. Knowles2,3, Tajalli Keshavarz1 and Ipsita Roy1
1 AppliedBiotechnology Research Group, Department of Molecular and Applied Biosciences, School of Life Sciences, University
of Westminster, London, UK
2 Department of Biomaterial and Tissue Engineering, Eastman Dental Institute, University College London, London, UK
3 WCU Research Centre of Nanobiomedical Science, Dankook University, Dongnam-gu, Cheonan-si, Chungnam, Republic of Korea

The main hindrance in the use of polyhydroxyalkanoates (PHAs) as a replacement for existing pe-
Received 12 April 2011
troleum-based plastics is their high production cost. The carbon source accounts for 50% of the Revised 6 June 2011
cost for PHA production. Thus, increasing the yield and productivity of PHAs on cheap substrates Accepted 25 August 2011
is an important challenge for biotechnologists to support the commercialization and further ap- Accepted
plications of these polymers. In this study, we have investigated the use of an agricultural raw ma- article online 30 August 2011
terial, sugarcane molasses, as the main carbon source for poly(3-hydroxybutyrate) (P(3HB)) pro-
duction by Bacillus cereus SPV. These studies were carried out in both shaken flasks and 2 L biore-
actors. Various conditions were evaluated for their effects on biomass and P(3HB) accumulation.
A high polymer yield was obtained, 61.07% dry cell weight (DCW) in a 1 L shaken flask study and
51.37% DCW in a 2 L fermenter study. These yields are 50% higher than previously observed with
Bacillus cereus SPV. Hence, the results are encouraging and show that sugarcane molasses are a
promising carbon source for an economical and commercially viable production of P(3HB).

Keywords: Biopolymers Biomaterials Polyhydroxyalkanoates PHA Sugar cane molasses

1 Introduction carbon sources primarily determine the efficiency

Recently, there has been increased scientific and
commercial interest in the development of fer-
mentation strategies to bio-convert renewable raw
materials into biodegradable polymers such as
polyhydroxyalkanoates (PHAs). This increase is
driven by environmental concerns, depleting pe-
troleum resources, and public awareness. Among
other factors such as the bacterial strain, fermen-
tation strategies and the purification processes,
Correspondence: Dr. Ipsita Roy, School of Life Sciences, University of
Westminster, 115 New Cavendish Street, London W1W 6UW, UK
E-mail: royi@wmin.ac.uk
Abbreviations: DCW, dry cell weight; DOT, dissolved oxygen tension; DSC,
differential scanning calorimetry; FT-IR, Fourier transform-infrared spec-
troscopy; PHA, polyhydroxyalkanoate; P(3HB), poly(3-hydroxybutyrate);
P(3HB-co-3HV), poly(3-hydroxybutyrate-co-3-hydroxyvalerate); RCM, resid-
ual cell mass
and economic production of PHAs [1]. The eco-
nomic evaluation of the cost of PHA production
suggested that the cost of carbon sources alone
accounts for 50% of the overall PHA production
cost [2, 3]. Hence, commercialization of PHAs
would be enhanced if the full potential of agricul-
tural raw materials and waste, as sources of carbon
and nitrogen for PHA production can be exploit-
ed. Agricultural raw materials including wheat
bran, corn steep liquor, rapeseed cake, starchy
wastewater, and molasses have been used as
biorefinery feedstock for the industrial production
of conventional chemicals and bioplastics [1]. Sug-
ar cane molasses have mostly been used in fer-
mentation processes because of its low price and
rich chemical composition [4]. Usually this in-
cludes the presence of sucrose, glucose, and fruc-
tose as the main carbohydrates [http://eprints.hec.
gov.pk/1489/1/1371.HTM].

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It is a well known fact that PHA produced by mi-
croorganisms can have different compositions and
structures which are dependent on the accumulat-
ing microorganisms and the carbon sources em-
ployed in the production. Also, the limiting factor(s)
employed to induce PHA accumulation can affect
the monomeric composition of the accumulated
polymer. For instance, Valappil et al., observed dur-
ing their work on PHA production by Bacillus
cereus SPV, that potassium limitation led to the ac-
cumulation of P(3HB-co-3HV) poly(3-hydroxybu-
tyrate-co-3-hydroxyvalerate) whilst, with sulphur,
phosphate, or nitrogen limitation, only P(3HB) was
accumulated by the organism [5, 6].
Different agricultural resources have been used
as substrates in order to obtain reasonable levels of
PHAs produced by different bacteria. This has
demonstrated the possibility of employing sustain-
able cheap materials as carbon sources to reduce
the production cost of PHAs. For instance, Acineto-
bacter venetianus has been shown to be able to uti-
lize soy molasses as the carbon substrate for PHA
production [7]. On the other hand Azotobacter
vinelandii has also been observed to accumulate
high amounts of PHAs by utilizing sugar cane
liquor as the main carbon source for growth and
PHA production. Other organisms known to have
the capacity of accumulating high amounts of
PHAs from agricultural resources include Pseudo-
monas fluorescens (sugarcane molasses); Azotobac-
ter chroococcum (beet molasses); Burkholderia
cepacia and B. sacchari (starch); and Pseudomonas
hydrogenovora (whey) [8]. Specifically in Bacillus
sp., the utilization of mahua flowers as a carbon
source produced high levels (5154%) of the PHA
copolymer, P(3HB-co-3HV) [9]. Reddy et al. have
also observed the accumulation of 60% DCW of
P(3HB-co-3HV) copolymer by Bacillus megaterium
strain OU303A, when glycerol, a cheap byproduct
of the biodiesel industry, was employed as the main
carbon source in the production medium [10].
These results confirm the potential of cheap agri-
cultural resources and waste material to reduce the
cost and hence enhance the commercialization of
PHAs.
Bacillus cereus SPV is a Gram positive, rod-
shaped aerobic, and chemo-organotrophic bacteri-
um. It is capable of accumulating between 4 and 6
hydrophobic PHA granules that range from 0.2 to
0.5 m in size. It accumulates both the homopoly-
mers of P(3HB) and copolymers, depending on the
carbon sources and the nutrient limiting factors
employed in the production medium [5, 6]. Fur-
thermore, the fermentation conditions used for
PHA production also affects the percentage of each
monomer present and the molecular weight of the
294
Biotechnol. J. 2012, 7, 293303
polymer produced. Finally, the extraction and pu-
rification techniques affect the physical properties
of the polymer such as the melting temperature,
crystallization temperature, and the glass transi-
tion temperatures.
Bacillus sp. are known to be spore forming bac-
teria that sporulate at pH above 7.4. Wu et al., have
reported sporulation of Bacillus sp. JMa5 in mo-
lasses rich production media containing high car-
bon to nitrogen ratio at low dissolved oxygen, lead-
ing to low PHA content and dry cell weight (DCW)
[11]. However, it was observed that in Bacillus
cereus T sporulation could be prevented by the ad-
dition of -picolinic acid or by maintaining low pH
[6, 12].
In this study, we have explored the use of the
agricultural waste material, sugarcane molasses, as
a low cost carbon source for the commercial pro-
duction of the biodegradable polymer, poly(3-hy-
droxybutyrate(P(3HB)). This process, if developed
well, would provide a sustainable means for agri-
cultural waste management as well as reduction in
the cost of carbon sources for P(3HB) production,
leading to enhanced commercial viability.
2 Materials and methods
Analytical grade chemicals and reagents obtained
from SigmaAldrich Company Ltd. (Dorset, Eng-
land) were used for media preparation and chemi-
cal analysis. Yeast extract was purchased from DIF-
CO (BD UK Ltd., Oxford, UK), chromatography
grade solvents were obtained from VWR (UK) for
P(3HB) extractions and quantifications. Standard
P(3HB) was purchased from Sigma-Aldrich.
2.1 Bacterial strain and maintenance
Bacillus cereus SPV used in this study was obtained
from the culture collection of University of West-
minster, London, UK. Stock cultures were grown at
30C in nutrient broth (containing in g L1): Lab-
Lemco powder 1; yeast extract, 2; peptone, 5; sodi-
um chloride, 5, and grown at 30C and stored at 4C
on solid nutrient agar (containing in g L1): Lab-
Lemco powder, 1; yeast extract, 2; peptone, 5; sodi-
um chloride, 5; agar, 15. Modified G-medium con-
taining the following nutrient composition in g L1
was used for the preparation of seed culture;
FeSO4 7H2O, 0.0005; CuSO4 5H2O, 0.005; ZnSO4
7H2O, 0.005; MnSO4 7H2O, 0.05; MgSO4, 0.2; CaCl2,
0.05; K2HPO4, 1.0; (NH4)2SO4, 1.0; (NH4)2PO4, 2, and
yeast extract, 4. The medium was further supple-
mented with sugarcane molasses. This medium
composition was developed by Akaraonye et al.,
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(unpublished data). For different evaluations, the
production medium was either supplemented or
substituted with the nutrient(s) under investiga-
tion. The culture was allowed to grow at 30C and
200 rpm for 10 h, after which the seed culture was
used at 10% volume to inoculate the production
medium in shaken flask or fermenter. The same
seed culture age and volume (10% of the total vol-
ume) was used throughout the study for different
investigations, unless otherwise stated. Also, based
on previous work carried out by Hanson et al. [12],
90 mM -picolinic acid was added to the production
medium after inoculation and the pH was allowed
to fall below 6.8 during the stationary growth to
prevent sporulation and degradation of the accu-
mulated polymer.
2.2 P(3HB) production in shaken flasks
Bacillus cereus SPV was grown at 30C in 60 mL
seed culture medium supplemented with 2 wt% of
sugarcane molasses in 250 mL flasks with rotary
shaking at 200 rpm (Stuart orbital incubator 51500,
UK). After about 10 h of growth, 15 mL of the cul-
ture broth was inoculated in 1 L Erlenmeyer flasks
containing 250 mL of the modified G medium con-
taining 2 wt% of sugarcane molasses. In all cases
the pH was adjusted to 6.8 prior to cultivation. Sam-
ples were collected at defined time intervals during
cultivation and analyzed for DCW, P(3HB) content,
and residual cell mass (RCM).
Continuous feeding of sugarcane molasses in
shaken flask was performed by addition of 1 g mL1
of sugarcane molasses at a feeding rate of
0.7 mL min1 after 12 h when the OD600 of the cul-
ture broth reached a value of 3. At this stage the col-
or of the culture broth changed from chocolate to
light brown indicating a reduction in the amount of
sugarcane molasses available to the growing or-
ganisms. Continuous feeding of sugarcane mo-
lasses was used to maintain excess carbon concen-
tration and thus enhance P(3HB) accumulation in
the organism. The small increase in volume was
compensated by samples taken for measurement
of various parameters, hence maintaining constant
volume in the culture.
2.3 P(3HB) production in a fermenter
For P(3HB) production in a fermenter, Bacillus
cereus SPV was first grown at 30C in 1 L shaken
flasks containing 200 mL of the production medium
(pH 6.8) containing sugarcane molasses (2 wt%),
with shaking at 250 rpm (New Brunswick Scientif-
ic Classic Series, UK), for 10 h. 150 mL of the seed
culture was then used to inoculate a 2 L fermenter
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vessel (Electro lab, UK) containing 1.35 L of pro-
duction medium. Two drops of 1 mM antifoam
(Down Corning, DB-110A) was added before inoc-
ulation of the fermenter with the seed culture and
pH was maintained at 6.8 using 0.5 M HCI/NaOH
where necessary. The air inflow rate and agitation
speed were initially adjusted to 0.5 L min1 and
250 rpm, respectively, during the fermentations.
When the dissolved oxygen tension (DOT) de-
creased below 20%, the air inflow was increased to
1 L min1 to avoid oxygen limitation. 10 mL samples
were collected at different time intervals for analy-
sis.
For P(3HB) production with continuous feeding
of sugarcane molasses in the fermenter, the DOT
was initially set at 100% at the beginning of the fer-
mentation before inoculation with the seed culture.
During fed-batch, 1 g mL1 sugarcane molasses was
constantly fed to the culture broth at a feeding rate
of 0.7 mL min1 after a reduction in the initial 100%
DOT to 20%. This was carried out in order to main-
tain excess carbon concentration and thus promote
P(3HB) accumulation in the organism. The small
increase in volume was compensated by samples
taken for measurement of various parameters,
hence maintaining constant volume in the fer-
menter.
2.4 P(3HB) isolation for different analysis
Bacillus cereus SPV cells, containing the biopoly-
ester, were harvested from 1 L of culture broth by
centrifugation at 10 000 g for 10 min. After centrifu-
gation, the supernatant was removed and the cells
were washed twice at each step, first with water fol-
lowed by acetone, ethanol, and diethyl ether. This is
necessary to remove media, extracellular protein
and lipids bound to the cells. The bacterial cells
were finally centrifuged for 30 min at 10 000 g.
P(3HB) was extracted by treating 1 g of washed wet
cells with a solution containing 50 mL of chloro-
form and 50 mL of a diluted (70%) sodium hypo-
chlorite solution in water in an orbital shaker (Stu-
art orbital incubator 51500, UK), at 100 rev min1.
The wet cells were treated at 37C for 3 h. The mix-
ture obtained was then centrifuged at 10 000 g for
10 min, which resulted in three separate phases.
The P(3HB) was recovered from the bottom phase
(i.e., that of chloroform), by precipitation using ten
volumes of ice-cold methanol.
2.5 Gas chromatography-mass spectrometry
(GC-MS)
For the identification of the PHA, a slight modifica-
tion of the gas chromatographic method of Hui-
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Journal
jberts et al. was employed [13]. 100 L of a
1 mg mL1 suspension of lyophilized cell containing
PHA and 100 L of 1 mg mL1 of methyl benzoate
in chloroform was added to a mixture of 2 mL 15%
sulfuric acid in methanol (ratio 1:1), at 100C, for 5 h
in a reflux apparatus. After the reaction, the tubes
were cooled on ice for 5 min, then 1.0 mL distilled
water was added and the tubes were vortexed for
1 min. After phase separation, the organic phase
was collected and dried over anhydrous sodium
sulphate. The analysis was performed using a Trace
GC-MS (Thermo, San Jose, CA, USA). The Trace
2000 gas chromatograph was equipped with a ZB-
5MS (Phenomenex, Torrance, CA, USA) column
(30 m length, 025 mm internal diameter, and
025 m film thickness). The sample (1 L), in chlo-
roform, was injected, with helium (1 mL min1) as
the carrier gas. The injector temperature was 220C
and the column temperature was increased from 40
to 320C at 20C min1 and held at the final tem-
perature for 6 min.
GC analysis was also used for the quantification
of the amount of the PHA produced at different
time points. Following the same protocol as above,
known amounts of purified P(3HB) was methano-
lyzed and injected for the generation of the stan-
dard curve. This was used to quantify the amount of
P(3HB) produced in the samples collected at dif-
ferent time points.
2.6 NMR-analysis
The 1H NMR spectrum was recorded on a Varian
300 MHz instrument with a Bruker ARX500 spec-
trometer at room temperature. Chemical shifts are
reported in ppm relative to deuterated chloroform
as the internal reference solvent. Spectra acquisi-
tions were recorded on 5% w/w polymer solutions
in CDCl3 using the following parameters: tempera-
ture 35C, pulse width 15 s, 8 k data points, 2.8 s re-
laxation delay, 48 k transients, and 5.0 line broad-
ening. The spectrum was evaluated using Bruker
UXNMR software.
2.7 Fourier transform-infrared spectroscopy
(FT-IR)
Characterization of PHA recovered from different
carbon sources by FT-IR was performed according
to the method described by Hong et al. [14]. Pre-
cipitated P(3HB) from B. cereus SPV was dissolved
in chloroform and used to prepare KBr discs (sam-
ple: KBr, 1:100). An FT-IR 1720 spectrometer
(Perkin Elmer) was used under the following con-
ditions: spectral range, 4000400 cm1; window ma-
terial, Csl; 16 scans; resolution 4 cm1; the detector
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Biotechnol. J. 2012, 7, 293303
was a temperature-stabilized, coated FR-DTGS de-
tector.
2.8 Differential scanning calorimetry (DSC)
DSC measurements were performed on the PHA
samples extracted from Bacillus cereus SPV grown
under different conditions. A differential scanning
calorimeter (Perkin-Elmer Pyris Diamond DSC
(Perkin-Elmer Instruments, USA) was used for the
measurements. The sample mass used for this
measurement, was in the range of 57 mg. To meas-
ure the thermal properties, each sample was en-
capsulated in standard aluminum pan and all tests
were performed under a nitrogen atmosphere.
Samples were heated/cooled/heated at a rate of
20C min1 between 50 and 200C. Measurements
were carried out in triplicates and the mean values
were calculated.
2.9 Quantitative analysis
DCW was determined by centrifuging 1 mL of the
culture sample at 12 000 g for 10 min; the pellet was
washed with distilled water and then dried at 75C
until constant weight was obtained.
P(3HB) content was quantified as described by
Law and Slepecky [15]. Pellets were hydrolyzed
with concentrated sulphuric acid heated for 1 h at
100C in a water bath to obtain crotonic acid which
was subsequently quantify by measuring ab-
sorbance at 235 nm.
RCM concentration was calculated as the differ-
ence between cell DCW concentration, i.e. of the
DCW, and P(3HB) concentration, while P(3HB)
content (% DCW) was obtained as the percentage of
the ratio of P(3HB) concentration to DCW accord-
ing to the definition by Lee et al. [16].
3 Results
3.1 P(3HB) production by Bacillus cereus SPV using
sugarcane molasses as the main carbon source
in shaken flasks
In order to evaluate the potential of sugarcane mo-
lasses as a replacement of the more expensive car-
bon sources in PHA production, B. cereus SPV was
grown on modified G-medium developed by
Akaraonye et al. (unpublished data), with 20 g L1
sugarcane molasses in 1 L shaken flasks. Figure 1A
shows the temporal profile of growth and polymer
accumulation. It was observed that polymer accu-
mulation occurred alongside growth until 30 h af-
ter which the rate of polymer accumulation became
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Figure 1. The temporal variation of DCW g L1 (d); % P(3HB) (); P(3HB) concentration g L1 (); and RCM g L1 () produced by Bacillus cereus SPV
in MGM medium (A) using sugarcane molasses as the main carbon source in shaken flask culture. (B) Using sugarcane molasses was as the main
carbon source in batch fermentation using 2 L bioreactors (C) using sugarcane molasses as the main carbon source in shaken flask culture with reduced
(NH4)2PO4 (D) using sugarcane molasses was as the main carbon source in batch fermentation using 2 L bioreactors with reduced (NH4)2PO4 (E) using a
continuous feeding mode of sugarcane molasses solution in 1 L shaken flask cultures (F) using a continuous feeding of sugarcane molasses solution in a
2 L fermenter. Experiments were performed in triplicates (for shaken flask culture)/duplicates (for 2 L fermenter study), while analyses of samples were
done in triplicates. Error bars show SD.

higher than the rate of biomass accumulation.
The maximum polymer yield (61.07% DCW) was
achieved at 55 h during the stationary phase of
growth. Initially, the RCM increased along with cell
growth and P(3HB) accumulation. However, after
27 h of growth, the RCM decreased and almost
reached a constant value of 2.48 g L1.
3.2 P(3HB) production by Bacillus cereus SPV using
sugarcane molasses as the main carbon source
in a 2 L fermenter
To further understand the potential of Bacillus
cereus SPV in utilizing sugarcane molasses as an
alternative carbon source for large scale PHA pro-
duction, studies were extended to a 2 L fermenter
level. The results (Fig. 1B), showed initial exponen-

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tial growth until 47 h, with the maximal DCW val-
ue of 6.93 g L1 observed at 55 h. Polymer accumu-
lation increased with cell growth until 47 h where
the maximum polymer yield of 51.37% DCW was
obtained, after which the yield decreased until the
end of the fermentation. The RCM increased along
with the cell growth until 36 h after which the val-
ue remained almost constant.
Comparison of the shaken flask and 2 L fer-
menter studies show that comparable maximal val-
ues of DCW (6.63 g L1) and P(3HB) concentration
(3.45 g L1) were observed in the shaken flask in-
vestigations, and the 2 L fermentation studies
which yielded maximal values of 6.93 and 4.05 g L1
of DCW and P(3HB) concentrations, respectively.
However, the maximum P(3HB) yield obtained was
61.07% DCW in shaken flask studies as compared
to a relatively lower value of 51.37% DCW obtained
in the 2 L fermenter studies.
3.3 Effect of reduced (NH4)2PO4 concentration on
biomass and P(3HB) accumulation by Bacillus
cereus SPV, using sugarcane molasses as the
main carbon source
In order to evaluate the effect of nutrients other
than the carbon source on cell growth and polymer
accumulation, the amount of (NH4)2PO4 present in
the production medium was reduced to half
(1 g L1) the amount used in the above two sections
(2 g L1). This investigation was carried out both at
the shaken flask level and in 2 L fermenter. Fig-
ure 1C shows the temporal variation of the para-
meters measured at shaken flask level. The expo-
nential growth phase was observed until 40 h with
simultaneous polymer accumulation. However, the
rate of polymer accumulation increased after 20 h
and the P(3HB) yield reached a maximum value of
52.27% DCW at 55 h, during which the cell had en-
tered the stationary phase. The P(3HB) concentra-
tion reached a maximum value of 3.64 g L1 at 45 h.
The RCM increased with the cell growth until 15 h,
after which the rate of polymer accumulation in-
creased with a simultaneous reduction in the rate
of increase of RCM. A similar pattern of cell growth
and polymer accumulation was also observed in
the study using the 2 L fermenter (Fig. 1D). How-
ever, the stationary growth phase was achieved
much earlier in the 2 L fermentation (21 h vs. 54 h
in shaken flask), thereby resulting in an overall
decrease in the fermentation time. Cell growth
decreased after 28 h while the highest polymer
yield (47.43% DCW) was achieved at 30 h of fer-
mentation and the maximum polymer concentra-
tion of 1.85 g L1 was observed at 28 h of fermenta-
tion.
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Biotechnol. J. 2012, 7, 293303
Thus, maximal values of 4.30 and 2.25 g L1 of
DCW and P(3HB) concentration, respectively, were
achieved in shaken flask studies as compared to
the maximal values of 4.30 and 1.85 g L1 of DCW
and P(3HB) concentrations, respectively, in 2 L fer-
menters. Also, the maximum P(3HB) yield of
52.27% DCW was achieved in shaken flask studies,
whereas a lower maximum value of 47.43% DCW
was achieved using 2 L fermenters.
3.4 Effect of continuous feeding of sugarcane
molasses on biomass and P(3HB)
accumulation by Bacillus cereus SPV
Since polymer accumulation occurs mostly in the
presence of excess carbon source, the effects of a
continuous supply of sugarcane molasses to the
growing cultures of Bacillus cereus SPV were in-
vestigated. In the shaken flask study, the rate of in-
crease of OD decreased at 12 h, indicating a de-
crease in cell growth; hence, continuous feeding of
sugarcane molasses solution (1 g mL1), at a feed-
ing rate of 0.7 mL min1 was initiated at this time
point. A DCW of 1.63 g L1 and 15.37% DCW PHA
yield was achieved at this time point. Both DCW
and PHA yield were found to increase to a maximal
value of 5.47 g L1 and 57.13% DCW, respectively, at
51 h, after the introduction of continuous feeding of
the sugarcane molasses solution (Fig. 1E).
In the 2 L fermenter study, continuous feeding
of the sugarcane molasses solution (1 g mL1) at a
feeding rate of 0.7 mL min1 was initiated after 23 h
of fermentation, when a decrease in the initial set
value of 100% DOT to 20% DOT was noticed. This
was carried out in order to enhance PHA accumu-
lation. Contrary to what was expected, the continu-
ous supply of the sugarcane molasses only in-
creased cell growth with a reverse effect on poly-
mer accumulation. Hence, the maximum polymer
content (28.10% DCW) achieved at 23 h, before the
continuous feeding of nutrient, reduced to 13.74%
DCW at 34 h when the maximum DCW of 6.83 g L1
was achieved (Fig. 1F).
3.5 Characterization of the isolated PHA
The PHA isolated from B. cereus SPV cells pro-
duced from each of the above mentioned condi-
tions were identified by NMR. The 1H NMR con-
firmed the polymers isolated from all the condi-
tions as the homopolymer of P(3HB) (Fig. 2). Fur-
thermore, the FT-IR analysis of the isolated poly-
mers revealed absorption bands at 1720 cm1
corresponding to the ester carbonyl group and
1282 cm1 corresponding to the CH group, charac-
teristic features of P(3HB) (Fig. 3).
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The DSC analysis performed on the isolated
P(3HB) produced showed that the melting temper-
ature, Tm (168.21C), crystallization temperature,
Tc (49.10C), and glass transition temperature, Tg
(3.61C) of the isolated P(3HB) was slightly lower
than the result obtained using the standard
commercial polymer obtained from Sigma, Tm
(173.11C), Tc (59.45C), and Tg (8.23C) (Fig. 4).
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Figure 2. 1H NMR spectrum of P(3HB)
isolated from Bacillus cereus SPV, when
sugarcane molasses was used as the
main carbon source.
Figure 3. FT-IR spectrum of P(3HB) produced using
B. cereus SPV grown in modified MGM media supple-
mented with sugarcane molasses () and Standard
P(3HB) from SigmaAldrich (- - -). The absorption bands
at 1282 and 1720 cm1 correspond to C=O and CO of
P(3HB), respectively. Analyses were carried out in dupli-
cate, a single spectrum for each sample is shown for
clarity.
4 Discussion
4.1 Comparison of DCW, P(3HB) yield, and P(3HB)
content accumulated at different conditions in
shaken flask expriments
Comparison of DCW achieved under different con-
ditions in the shaken flask studies shows that a
maximum value of 6.63 g L1 DCW was achieved in
the batch shaken flask investigations and the max-
imal value of DCW produced in both reduced
(NH4)2PO4 and the continuous feeding conditions,
in shaken flask, were lower values of 4.30 and
5.47 g L1, respectively (Fig. 5A). The highest
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B
Figure 4. Comparison of DSC thermograms of P(3HB) accumulated by
B. cereus SPV using sugar cane molasses (- - -) and commercial P(3HB)
purchased from Sigma-Aldrich (). Analyses were carried out in tripli-
cates, one thermogram is shown for clarity.

P(3HB) concentration was also obtained in the nor-

mal batch shaken flask fermentation where a max-
imum value of 4.05 g L1 was achieved as compared
to 2.25 and 3.09 g L1 achieved in the shaken flask,
with reduced (NH4)2PO4 concentration and the
fermentations with continuous feeding of sugar-
cane molasses, respectively. Finally, the maximum
P(3HB) yield (61.07% DCW) was also obtained in
the normal batch shaken flask fermentation as
compared to those achieved in the reduced
(NH4)2PO4 condition (52.27% DCW) and fermenta-
tions with continuous feeding of sugarcane mo-
lasses (57.13% DCW). Hence, the normal batch
shaken flask fermentation was the best P(3HB)
production condition in this study.
4.2 Comparison of DCW, P(3HB) yield, and P(3HB)
content accumulated at different conditions in
the 2 L fermenter experiments
In order to identify the best condition in the 2 L fer-
mentation study, i.e., that produces the highest
DCW in g L1, P(3HB) concentration in g L1, and
P(3HB) yield in % DCW, the values of these param-
eters were compared. As can be seen from the pro-
file in Fig. 5B, the maximum DCW (6.93 g L1) was
accumulated by Bacillus cereus SPV in the batch
fermentation and the fermentation with continu-
ous feeding of sugarcane molasses (6.83 g L1).
Much lower DCW (4.30 g L1) was achieved in the
condition with reduced (NH4)2PO4 concentration in
the medium. Comparison of the P(3HB) concentra-
tion accumulated by B. cereus SPV under the dif-
ferent conditions showed that the highest P(3HB)
Figure 5. (A) Comparison of DCW, P(3HB) g L1 and % P(3HB) DCW ac-
cumulated by Bacillus cereus SPV from different conditions at shaken flask
level (B) comparison of DCW, P(3HB) g L1 and % P(3HB) DCW accumu-
lated by Bacillus cereus SPV from different conditions at 2 L fermenter lev-
el. Experiments were carried out in duplicates and analyses were carried
out in triplicates. Error bars show the SD.
concentration was achieved in the batch fermenta-
tion (3.45 g L1). This was followed by the fermen-
tation with reduced (NH4)2PO4 concentration
(1.85 g L1) and the least amount of P(3HB) con-
centration was achieved when sugarcane molasses
was continuously fed to the culture (1.66 g L1). Fi-
nally, the highest P(3HB) yield was achieved in the
batch 2 L fermentation (51.37% DCW) as compared
to that achieved with reduced (NH4)2PO4 con-
centration (47.43% DCW) and the fermentation
with continuous feeding of sugarcane molasses
(16.03 g L1). Hence, as observed at the shaken flask
level, the batch fermentation was the best condition
where maximum DCW, P(3HB) concentration, and
P(3HB) yield was obtained.

300 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Biotechnol. J. 2012, 7, 293303
4.3 P(3HB) production by Bacillus cereus SPV using
sugarcane molasses as the main carbon source
in shaken flasks and a 2 L fermenter
The polymer accumulation observed alongside
growth during both shaken flask and 2 L fermenta-
tions (Fig. 1A and B) confirmed that B. cereus SPV
is capable of producing polymer under normal
growth conditions which was however enhanced
during the stationary phase where some essential
nutrients would be expected to have become limit-
ing. In the stationary phase the medium could no
longer support the growth of the organism, hence,
there was a decline in the growth rate and a simul-
taneous increase in the polymer accumulation by
the organism. At this stage, the amount of RCM de-
creased because of the increased polymer accumu-
lation without any simultaneous growth of the or-
ganism. A similar observation of growth-associated
P(3HB) production was also made with Alcaligenes
latus by Yamane et al. [17]. Alcaligenes latus is
known to produce P(3HB) alongside growth with
the maximal PHA production achieved at the sta-
tionary growth phase. Maekawa et al. [18] attrib-
uted such growth associated P(3HB) production to
a relatively lower kcat value of the 3-ketothiolase for
the cleavage reaction leading to the breakdown of
acetoacetyl-CoA to acetyl-CoA. The growth associ-
ated PHA production in this study is possibly due
to the same reason. In addition, trace elements play
an important role in the metabolic activity within a
bacterial cell. It is possible that the growth condi-
tions available to the organism at the start of fer-
mentation may be lacking in one of the trace ele-
ments required by Bacillus cereus SPV. This could
also result in early PHA accumulation. During the
stationary growth phase, there is no growth, how-
ever, the amount and possibly the size of the PHA
granules increases, occupying a large proportion of
the space in the cytoplasm. This leads to an in-
crease in PHA yield without a simultaneous in-
crease in the DCW during the stationary growth
phase.
4.4 Effects of reduced (NH4)2PO4 concentration on
biomass and P(3HB) accumulation by B. cereus
SPV using sugarcane molasses as the main
carbon source
Sugarcane molasses is a complex carbon source
containing sulfur, vitamins, calcium, magnesium,
potassium, iron, and minerals [1]. It is a well known
fact that the initial nitrogen and phosphate con-
centration in the production medium influences
both the biomass and PHA accumulation [16].
Ward et al. found an increase in PHA accumulation
2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotechnology-journal.com
by Pseudomonas putida CA-3 when the amount of
initial sodium ammonium phosphate concentra-
tion in the media was reduced from 134 to 67 mg L1
[19]. The results obtained in this study also demon-
strated that changes in the amount of initial phos-
phate concentration in the production medium re-
sulted in a significant effect on both DCW and
P(3HB) accumulation. However, in this study, both
DCW and P(3HB) content (% DCW) were reduced
from 6.63 g L1 and 61.07% DCW, respectively to
4.30 g L1 and 52.27% DCW when the amount of ini-
tial ammonium phosphate concentration was re-
duced from 2.0 to 1.0 g L1 in the shaken flask study.
Similarly, in 2 L fermentation studies, both DCW
and P(3HB) production reduced from 6.93 g L and
51.37% DCW respectively, to 4.17 g L1 and 47.43%
DCW when the amount of initial ammonium phos-
phate concentration was reduced from 2.0 to
1.0 g L1. The reason for the decrease in the amount
of accumulated DCW and P(3HB) content in this
study could possibly be due to the combined effect
of nitrogen and phosphate limitations. Since am-
monium phosphate is one of the sources of inor-
ganic nitrogen and the main source of phosphate in
the production medium, reduction to half the
amount available at the beginning of the study
could have had a direct effect on the growth of the
organism. In this study, the growth conditions, i.e.
medium composition used, perhaps also led to a si-
multaneous decrease in the polymer production
due to changes in the metabolic network in the or-
ganism leading to conditions unfavorable for poly-
mer production.
4.5 Effect of continuous feeding of sugarcane
molasses on the growth and P(3HB)
accumulation
The ability of the bacteria to accumulate sufficient
amounts of polymer is largely dependent on the
amount of carbon source present in the production
medium and its relative molar ratio with respect to
the limiting nutrient during the polymer accumula-
tion stage. This was investigated by supplying B.
cereus SPV with additional amounts (1 g mL1) of
sugarcane molasses, which contains a large per-
centage of carbon (80%), at a feed rate of 0.7 mL
min1, throughout the fermentation. It was found
that the organism utilized the additional sugarcane
molasses mainly for growth resulting in 5.47 g L1
DCW, achieved after 54 h in shaken flask and
6.83 g L1 DCW achieved in 2 L fermenter. Howev-
er, at the shaken flask level, a relative increase in
P(3HB) concentration and yield was observed as
compared to a drastic decrease in P(3HB) concen-
tration and yield obtained in the 2 L fermenter. In
301
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Journal
shaken flask conditions it is not possible to control
the dissolved oxygen level. Hence it is possible that
due to additional nutrients supplied by the feed,
there was an increase in the demand for available
oxygen, leading to almost anaerobic conditions of
growth, leading to enhanced P(3HB) production. In
contrast, in the 2 L fermenter, controlled air supply
would prevent the induction of anaerobic condi-
tions. In fact, the re-introduction of the limiting nu-
trient(s) by the feed, must have lead to a non-lim-
iting nutrient condition, thereby resulting in lower
PHA accumulation. Under the non-limiting condi-
tions, bacteria would begin to grow again, leading to
the breakdown of the previously accumulated
P(3HB), to be utilized as a carbon source, leading to
a reduction in the P(3HB) yield.
In general, it is expected that the polymer yields
obtained in fermenters should be greater than that
in shaken flask fermentation, since the physical
parameters can be well controlled in order to im-
prove the performance of the organism. Neverthe-
less, the results obtained in this study showed
maximum polymer yield in the shaken flask batch
fermentation. Hence, the conditions for polymer
production using Bacillus cereus SPV in the 2 L fer-
menter need further optimization for further en-
hanced production of the polymer. Overall, reason-
ably high level of polymer accumulation and cell
growth was achieved in this study; a maximal
P(3HB) content of 61.07% DCW and DCW of
6.63 g L1 were achieved from shaken flask investi-
gations while the 2 L fermenter study yielded
51.37% DCW and 6.60 g L1 of PHA content and
DCW respectively. These results are highly encour-
aging and confirm that sugarcane molasses are a
good sustainable carbon source for P(3HB) pro-
duction in Bacillus cereus SPV. These values are
much higher than the values of 30% DCW P(3HB)
yield and 3.5 g L1 DCW obtained by Albuquerque
et al. in their work on PHA production using sug-
arcane molasses as the sole carbon source in a se-
quencing batch reactor (SBR) [20]. Furthermore,
our results are comparable with that achieved by
Santimano et al. with another strain of Bacillus
(Bacillus sp. COLI/A6), where a maximum PHA
yield of 54.68% DCW and a DCW of 6.0 g L1 was ob-
tained using sugarcane molasses as the main car-
bon source contained in E2 mineral medium [21].
Studies are ongoing to further improve the current
substrate to P(3HB) transformation efficiency of
0.23 g g1 of sugarcane molasses achieved in this
work to a higher value. Improvement on substrate
transformation efficiency will help to further re-
duce cost of production as well as encourage sub-
strate utilization by the organism.
302
Biotechnol. J. 2012, 7, 293303
The chemical identity of the polymer produced
using sugarcane molasses was confirmed by 1H
NMR and FT-IR in comparison with a standard ref-
erence P(3HB) obtained from Sigma. The quality of
the NMR spectrum indicated a high level of purity
of the polymer due to the absence of any back-
ground signals. The DSC results showed a slight de-
crease in the Tm, Tc, and Tg values observed in the
P(3HB) isolated from B. cereus SPV as compared to
that obtained from Sigma. This indicates a slightly
lower crystallinity of the sample. Generally, PHAs,
including P(3HB), exist as amorphous granules
within the bacteria cells. However, when the poly-
mers are isolated from the bacteria, they form crys-
talline structures due to freeze-drying or the influ-
ence of the organic solvent treatment during re-
covery [22]. In this study, a unique wet cell extrac-
tion method was adopted in the isolation of the
polymer; leading to the recovery of the polymer
with relatively lower crystallinity. Also, the de-
creased values of the thermal properties might also
reflect lower molecular weight of the polymer iso-
lated in this work as compared to that obtained
from Sigma.
4.6 Conclusions
This study shows, for the first time, that the
biotechnological transformation of sugarcane mo-
lasses into the high-value green material, P(3HB),
is possible using Bacillus cereus SPV. It is a suitable
complex carbon source that can enhance both bio-
mass and P(3HB) accumulations in Bacillus cereus
SPV. The present work can form the basis of the de-
velopment of a biotechnological process for the
biotransformation of the renewable feedstock, sug-
arcane molasses, into P(3HB). This will decrease
the cost of P(3HB) production substantially, leading
to an enhancement in its widespread usage.
E. Akaraonye acknowledges the award of the Caven-
dish Scholarship by the University of Westminster,
London, UK. We are also grateful for the help provid-
ed by Dr. George Georgiou, Eastman Dental Institute,
UCL, for carrying out the DSC analysis. This work
was supported in part (JCK) by WCU Program
through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science
and Technology (No. R31-10069).
The authors declare no conflict of interest.
2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Biotechnol. J. 2012, 7, 293303 www.biotechnology-journal.com

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