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Production, optimization and characterization of

polyhydroxybutyrate, a biodegradable plastic by
Bacillus spp.
a a a a
Pabitra Bhagowati , Shreema Pradhan , Hirak R. Dash & Surajit Das
a
Laboratory of Environmental Microbiology and Ecology (LEnME), Department of Life
Science, National Institute of Technology, Rourkela, India
Published online: 22 Apr 2015.

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To cite this article: Pabitra Bhagowati, Shreema Pradhan, Hirak R. Dash & Surajit Das (2015): Production, optimization
and characterization of polyhydroxybutyrate, a biodegradable plastic by Bacillus spp., Bioscience, Biotechnology, and
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 Bioscience, Biotechnology, and Biochemistry, 2015
Production, optimization and characterization of polyhydroxybutyrate, a
biodegradable plastic by Bacillus spp.
Pabitra Bhagowatia, Shreema Pradhana, Hirak R. Dash and Surajit Das*
Laboratory of Environmental Microbiology and Ecology (LEnME), Department of Life Science, National Institute of
Technology, Rourkela, India
Received January 27, 2015; accepted March 14, 2015
http://dx.doi.org/10.1080/09168451.2015.1034651
Poly-β-hydroxybutyrate (PHB) is the intracellular
lipid reserve accumulated by many bacteria. The
most potent terrestrial bacterium Bacillus cereus
SE-1 showed more PHB accumulating cells (22.1
and 40% after 48 and 72 h) than that of the marine
Downloaded by [University of Alberta] at 10:44 25 April 2015
Bacillus sp. CS-605 (5 and 33% after 48 and 72 h).
Both the isolates harbored phbB gene and the
characteristics C=O peak was observed in the
extracted PHB by Fourier transformed infrared
spectroscopy analysis. Maltose was found to be the
most suitable carbon source for the accumulation of
PHB in B. cereus SE-1. The extracted PHB sample
from B. cereus SE-1 was blended with a thermoplas-
tic starch (TS) and an increased thermoplasticity
and decreased crystallinity were observed after
blending in comparison to the standard PHB. The
melting temperature (Tm), melting enthalpy (ΔHf),
and crystallinity (Xc) of the blended PHB sample
were found to be 109.4 °C, 64.58 J/g, and 44.23%,
respectively.
Key words: poly-β-hydroxybutyrate; Bacillus; fluo-
rescence activated cell sorter (FACS);
FTIR; melting enthalpy
Plastic materials have been regarded as an integral
part of our lifestyle due to their many desirable proper-
ties, i.e. durability and resistance to degradation. They
in turn accumulate in the environment at a rate of mil-
lion tonnes per year and recently, plastic pollution is
regarded as a major contributor to the environmental
pollution budget.1) The widespread use of plastic and
plastic products facilitates the continuous contact of
these materials with human body. This ultimately
increases the steady-state concentration of plastic
components in the body and disturbs the metabolism
and excretion of these compounds.2) Another problem
associated with traditional plastics is that it takes mil-
lions of years to be renewed as they are produced from
petrochemical sources.
*Corresponding author. Email: surajit@nitrkl.ac.in
a
These authors contributed equally to this work.
© 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
Thus, the recent issues concerning global environ-
ment and solid waste management have shifted the
interest for the development of biodegradable plastics
with desired physical and chemical properties of syn-
thetic plastics such as polypropylene (PP) and poly-
ethylene (PE).3) However, biologically degradable
polymers are still underdeveloped and need urgent
attention. In this context, biodegradable polymer, PHB,
has been found to be a promising material. PHB is a
polyhydroxyalkanoate (PHA) polymer which belongs
to the polyester class and is of huge interest due to
their biological origin and biodegradability.4)
In the case of PHB producing micro-organisms, syn-
thesis takes place when suitable carbon source is avail-
able in excess amount and cellular growth is limited
due to other nutrient limitation. More than 75 bacterial
genera have been reported so far for PHB accumulation
as intracellular granules. However, the most common
studied organisms for PHB accumulation include gen-
era of Alcaligenes, Azotobacter, Bacillus, Rhizobium,
Rhodospirillum, and Pseudomonas.5) It has also been
found that, micro-organisms rich in poly-beta-hydrox-
ybutyrate have a slower rate of autolysis than organ-
isms with poor PHB production capability, thus PHB
can act as a reserve carbon and energy source.6) PHB
production in bacteria involves the formation of inter-
mediates such as acetyl-CoA, acetoacetyl-CoA, and
(R)-3-hydroxybutyryl-CoA.4) 3-ketothiolase (PhbA),
acetoacetyl-CoA-reductase (PhbB), and PHB synthase
(PhbC) are the key enzymes during PHB synthesis and
phbB synthesized acetoacetyl-CoA reductase converts
acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA is the
prerequisite step of PHB synthesis.
PHB offers many advantages over the conventional
petrochemically derived plastics such as complete
biodegradability, use of renewable resources, better
physical properties than PP, and non-toxic.7) Mechani-
cal properties of PHB make them suitable to replace
polythenes, however, in contrast to these conventional
plastics; PHB can be degraded completely to form
carbon dioxide and water by natural microbial
 2 P. Bhagowati et al.
mineralization. Biomedical and biodegradable proper-
ties of PHB have led to the design of medical devices
and tissue engineering.8) However, PHB possesses
many deficiencies to be used as a practical polymer
material such as high crystallinity and thermal instabil-
ity near the melting point.9) Thus, physical blending
and chemical modifications have been carried out to
improve its properties such as strength, film formation,
mechanical strength, amphilicity, biodegradability, and
biocompatibility.10)
Though much advancement has taken place regard-
ing the microbial biosynthesis of PHB, industrial-scale
production and application are still at far away from
reality. In this regard, proper selection of the microbial
strains for PHB production holds the key and soil
microbes provides the better resources. Thus, the cur-
rent work aims to obtain a proper microbial system for
significant PHB production and their characterization to
increase the suitability of use of PHB with suitable
physical and chemical properties.
Downloaded by [University of Alberta] at 10:44 25 April 2015
Materials and methods
Sampling, isolation of bacteria, medium, and growth
conditions. Organic waste samples and marine sedi-
ment samples were collected from the garbage dumping
sites of Rourkela, Odisha (22.2492° N and 84.8828° E)
and Chilika Lake region of Bay of Bengal, Odisha
(19°44.582′ N and 85°12.768′ E), respectively. The
samples were processed immediately using standard
microbiological practice for the isolation of bacterial
culture using nutrient agar (peptone—5.0 g, NaCl—
5.0 g, beef extract—1.5 g, yeast extract—1.5 g, agar—
15 g, distilled water—1000 mL, and pH—7.4 ± 0.2) for
organic waste sample and sea water nutrient agar (pep-
tone—5.0 g, yeast extract—3.0 g, agar—15 g, aged sea
water—500 mL, deionized water—500 mL, and pH—
7.5 ± 0.1) for marine sediment sample. The plates were
incubated at 37 °C for 24 h. The colonies with distin-
guished colony morphology were selected and repeated
streaked on the respective plates to obtain the pure cul-
tures and were used for further study.
Screening of PHB accumulating isolates. In order
to screen the PHB positive isolates, Nile blue staining
technique was used.11) Briefly, the isolates were grown
in 10 mL of Minimal Davis broth (Hi-Media, India)
supplemented with 1% dextrose as sole carbon source.
The tubes were incubated at 37 °C for 72 h with shak-
ing at 180 rpm. Grown bacterial cultures were sub-
jected to centrifugation at 6000 rpm for 10 min at 4 °C.
The cell pellet was resuspended with 1 mL of sterile
distilled water. Smears of these cell suspensions were
heat fixed to glass slides and stained with 1% Nile blue
(Hi-Media, India) and observed under fluorescence
microscope (Olympus 1X71) with excitation wave-
length of 460 nm. Nile blue stains PHB granules in the
cells to orange.
The most potent PHB producers among the bacterial
isolates were selected by analyzing the intensity of Nile
blue staining and the number of positive cultures under
the microscope field by ImageJ analysis.12) The inte-
grated density of the PHB accumulating cells of the
isolates was carried out by analyzing the Nile blue-
stained images obtained from fluorescent microscopy
study by plugin of ImageJ program.
Characterization of the potent PHB-producing
isolates. Morphological and physiological character-
ization of the potent isolates was carried out by Gram
staining, Scanning electron microscopy (SEM),
biochemical tests, and antibiotic susceptibility testing.
Biochemical tests were performed manually13) for triple
sugar iron test, citrate utilization, mannitol utilization,
motility test, nitrate reduction, gelatin hydrolysis,
urease production, and oxidase test. Biochemical test
was carried out to study the comparative account of the
morphological as well as physiological differences
between the two most potent isolates. Similarly, antibi-
otic susceptibility test was conducted by disc diffusion
method14) using commercially available antibiotic discs
(Hi-Media, India) of ciprofloxacin (5 μg), amoxycillin
(30 μg), ampicillin (10 μg), chloramphenicol (30 μg),
and azithromycin (15 μg).
Molecular identification and GenBank submission.
Two most potent PHB producers one from organic
waste sample and another from marine sediment were
subjected to molecular identification by sequencing of
16S rRNA gene. Briefly, the 16S rRNA gene (~1.5 kb)
was amplified from the genomic DNA of the isolates
by PCR and the sequencing reactions were carried out
at Xcelris Genomics, India. Sequence data were com-
plied, and consensus sequence was obtained using
BioEdit (7.0.5.3) program and examined for sequence
homology with the archived 16S rDNA sequences from
GenBank employing BLASTN. The partial sequences
of 16S rRNA gene of both the isolates were submitted
at NCBI GenBank. The assigned accession number for
the isolates was KF241551 and KJ461699, respectively,
for Bacillus sp. CS-605 and B. cereus SE-1.
Amplification of phbB gene in the potent isolates.
phbB gene was amplified using bacterial lysate as tem-
plate. PCR was carried out using the primer set of
phbBF-5′-ATGAGCAATCAACGAATTGCA-3′ and
phbBR- 5′-TCATTGCATGTTCAGACCGC-3′.15) The
amplification reaction was performed in a total volume
of 25 μL using a thermal cycler (Bio-Rad, USA). The
PCR mixture contained 1 U/μL Taq polymerase
(Sigma-Aldrich), 1X enzyme buffer, 200 μM of each
dNTPs (Sigma-Aldrich), 1.25 mM mgCl2, and 0.5 μM
of each primer. The optimized amplification conditions
included a pre-denaturation step at 95 °C for 5 min fol-
lowed by 35 cycles of 95 °C for 2 min, 60 °C for 30 s,
72 °C for 2 min, and a final extension of 72 °C for
10 min and hold at 4 °C forever. The PCR product was
analyzed by 1% agarose gel electrophoresis and the
banding pattern was visualized in a Gel documentation
system (Bio-Rad, USA) under UV light.
 Characterization of biodegradable plastic
Comparative account of PHB production by flow
cytometry. A comparative account of PHB produc-
tion by B. cereus SE-1 and Bacillus sp. CS-605 was
studied for a period of 3 days at an interval of 24,
48, and 72 h. A Becton-Dickinson FACSCalibur flow
cytometer (Becton-Dickinson Immunocytometry Sys-
tem, San Jose, CA) with a 15-mW Ar laser with a
wavelength of 488 nm was used to analyze the single-
cell fluorescence intensity after staining. BODIPY
493/503 fluorescence was measured using a 530
± 30 nm band pass filter. Data were collected using
linear amplification. At least 30,000 cells were mea-
sured in each sample and the average channel number
of the height of each pulse was used to determine the
mean fluorescence of each sample. The cytometer
channels were standardized by setting the gains such
that signals from 8.1-μm fluorescence beads always
appear in the same channel. The isolate with higher
PHB accumulating capability was used for further
analyses.
Downloaded by [University of Alberta] at 10:44 25 April 2015
Optimization of carbon sources for PHB produc-
tion. The potent isolate was subjected to growth
under various carbon sources (dextrose, lactose,
sucrose, maltose, fructose, and galactose) for the opti-
mum production of PHB. Briefly, the isolate was
inoculated in Minimal Davis broth (Hi-Media, India)
supplemented with individual carbon sources (at a
final concentration of 1%) and incubated at 37 °C at
180 rpm for 72 h. After incubation, PHB was
extracted from the grown culture by sodium hypochlo-
rite-chloroform method.16) About 20 mL of grown cul-
ture was centrifuged at 6000 rpm for 20 min to collect
the cell pellet. The cell pellet was resuspended in
2.5 mL of 4% sodium hypochlorite for digestion fol-
lowed by addition of 2.5 mL of hot chloroform and
incubated at 37 °C for 1 h. The suspension was cen-
trifuged at 10,000 rpm for 10 min and the bottom
phase containing PHB and chloroform were collected.
Further extraction was carried out with hot chloroform
and precipitated with ethanol and acetone (1:1). The
precipitate was allowed to evaporate by incubating at
30 °C over-night to obtain PHB crystals. After
lyophilization, the dry weight of the PHB granules
was taken to determine the maximum production of
PHB under varying conditions of carbon sources. The
time course of PHB production at optimized condi-
tions was studied till 72 h with respect to the dried
cell mass.
Preparation of polymer blends. In order to
improve the thermal properties of extracted PHB sam-
ple, polymer blends were prepared with TS.17) TS
was obtained by mixing starch powder, water, and
glycerol in the ratio of 50:15:35 (w/v/v), respectively.
The contents were mixed for 15–30 min at room tem-
perature to obtain a paste. The paste was transformed
into TS by heating at 100 °C in water bath with con-
tinuous stirring for 15 min. This product so obtained
was mixed with PHB in the ratio of 58:52 (w/w) and
100:20 (w/w), and solvent cast films were obtained
from chloroform.
3
Characterization of polymer blend by Fourier trans-
formed infrared spectroscopy (FTIR). The presence
of the functional groups in the extracted PHB samples
was analyzed by FTIR spectroscopy. About 1 mg of
PHB sample was mixed with 10 mg of spectral pure
anhydrous potassium bromide crystals and the mixture
was compressed into translucent sample discs and fixed
in the FTIR spectrophotometer (Perkin-Elmer RX1).
The relative intensity of transmitted light energy was
measured against the wavelength of absorption on the
region 400–4000 cm−1 at ambient temperature.
Characterization of polymer blend by X-ray diffrac-
tion (XRD). XRD profile of the prepared samples
was collected on a PANalytical X-ray diffractometer
using nickel-filtered Cu Kα radiation and scanned from
10° to 60° at room temperature with a scan rate of
3° min−1.
Characterization of polymer blend by differential
scanning calorimetry (DSC). DSC thermograms were
recorded on a DSC instrument (DSC200 F3 Maia,
NETZSCH, Germany), and calibrated with indium (mp
—156.61 °C; ΔH = 28.54 J/g). The data were collected
by heat and cool method. Samples of cast films
weighed 5–10 mg were packed in aluminum pan and
then heated from 20 to 200 °C at a scanning rate of
10 °C per min under nitrogen atmosphere. The melting
temperature (Tm) and melting enthalpy (ΔHf) were
determined from DSC endothermal peaks. The crys-
tallinity (Xc) of PHB in blend was calculated as per the
equation given below.
X c ¼ DHf  100=DHo  w
where ΔHf is the melting enthalpy of the sample
(J/g), ΔHo is the melting enthalpy of the 100% crys-
talline PHB which is assumed to be 146 J/g, and w is
the weight fraction of PHB in the sample.
Results
Screening of potent PHB producers
A total of 32 bacterial isolates (12 from marine
sources and 20 from organic waste sample) were
screened for PHB accumulation. PHB accumulating
bacterial isolates flourish orange under fluorescence
microscope, whereas no such fluorescence was
observed for non-PHB producers. The acquired images
were processed for their average integral density
deducting the background noise, reflecting the level of
PHB production by ImageJ software. Based upon the
average integral density value, the most potent PHB
producers were selected for further study. Among the
bacterial isolates from organic waste sample, B. cereus
SE-1 (integral density 6513.558) was found to be the
most potent, whereas, among the marine isolates, Bacil-
lus sp. CS-605 (integral density 1876.942) was the
most potent PHB producer (Fig. 1). The raw integral
density data of all the isolates have been provided in
supplementary material (Table S1). The phylogenetic
relationship of these two potent isolates has been
 4 P. Bhagowati et al.

Fig. 1. Nile blue staining of the potent bacterial isolates as observed under fluorescence microscope.
Notes: The strains accumulating PHB produced bright orange color. (a) Bacillus sp. CS-605 and (b) Bacillus cereus SE-1.

revealed by constructing the phylogenetic tree and has
been provided in the supplementary material (Fig. S1).
Morphological and physiological characterization of
the isolates
Both the isolates Bacillus sp. CS-605 and B. cereus
Downloaded by [University of Alberta] at 10:44 25 April 2015
Bacillus sp. CS-605 was resistant to amoxycillin and
chloramphenicol and susceptible to ciprofloxacin,
ampicillin, and azithromycin. Both the isolates showed
significant similarities in their physiological charac-
teristics that have been provided in supplementary
material (Table S2).

SE-1 were found to be Gram positive rods under oil

immersion microscope. SEM reveals the rod-shaped
morphology of the isolates and a slight increase in the
size of the isolates was also marked after three days
incubation (Fig. 2). B. cereus SE-1 was found to be
susceptible to all the antibiotics tested, whereas
Amplification of phbB gene
Both the potential PHB producers B. cereus SE-1
and Bacillus sp. CS-605 were found to harbor phbB
gene in them. The clear distinct banding pattern of
744 bp confirms the presence of phbB gene in the two

Fig. 2. Revealing the morphology of bacteria during PHB synthesis by scanning electron microscopy.
Notes: (a) Bacillus sp. CS-605 before PHB accumulation, (b) Bacillus sp. CS-605 after PHB accumulation, (c) B. cereus SE-1 before PHB
accumulation, and (d) B. cereus SE-1 after PHB accumulation.
 Characterization of biodegradable plastic
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Fig. 3. Amplification of phbB gene in the potent PHB-producing
bacteria by polymerase chain reaction followed by agarose gel elec-
trophoresis.
Notes: 1. 100-bp DNA ladder, 2. Bacillus cereus SE-1, 3. Bacil-
lus sp. CS-605, and 4. negative control.
potent isolates (Fig. 3). None of the other screened iso-
lates showed the presence of phbB gene after PCR
amplification.
Comparative account of PHB production
PHB production ability was analyzed in both the iso-
lates B. cereus SE-1 and Bacillus sp. CS-605 at an
interval of 24, 48, and 72 h of incubation. The cellular
PHB distribution has been found as bivariate distribu-
tions (Fig. 4). The cytograms indicate that in B. cereus
SE-1, the PHB accumulation after 72 h is more than
that of Bacillus sp. CS-605 grown under same experi-
mental conditions. In the case of Bacillus sp. CS-605
5, 18.1 and 33% of the cells were found to accumulate
PHB at an interval of 24, 48, and 72 h, whereas 3.5,
22.1, and 40% cells accumulate PHB under same cir-
cumstances as accumulators. Thus, the higher PHB pro-
ducer B. cereus SE-1 was used further for extraction
and characterization of PHB.
Optimization of carbon sources for PHB production
Out of all the five carbon sources tested, in the pres-
ence of maltose, a higher level of PHB production
(5.63 g/L) was obtained, whereas a lower level of PHB
production was obtained with dextrose (0.4 g/L) alone
as the carbon source. Similarly, biomass of B. cereus
SE-1 was in the trend of maltose, fructose, galactose,
fructose, and dextrose (Fig. 5(a)). Time course of PHB
accumulation study revealed that with an increase in
time, the dry cell mass as well as PHB content also
increases (Fig. 5(b)).
Polymer blends of PHB and their characterization
Extracted PHB from B. cereus SE-1 using maltose as
sole carbon source was mixed with TS and the PHB,
5
blended PHB, and the PHB standard (Sigma-Aldrich)
were characterized by FTIR, XRD, and DSC. The
spectrum of FTIR reveals the presence of significant
peaks at wave numbers 3410 cm−1 corresponding to
the hydroxyl (–OH) stretching, C=O stretching at
1728 cm−1, and aliphatic stretching of carbonyl group
of RCO of that of the PHB. Other absorption bands
were found at 1288 cm−1 were that of the aliphatic –
CH stretching groups. The bands at 1320–1000 cm−1
were characteristic to that of carbon oxygen stretch of
the esters(C–O–C bond). The strong band at 1100 cm−1
was C–O–C stretching and 1650 cm−1 for CH2 stretch-
ing. PHB with different backbones and different chemi-
cal groups are previously studied (Fig. 6).
Non-isothermal DSC study reveals the blend PHB
and PHB standard having the characteristic endother-
mal peaks between 140 and 200 °C (Fig. S1, supple-
mentary file). The melting enthalpy (ΔHf) calculated
from the area of endothermal peaks. The crystallinity
degree (Xc) was calculated based on the melting
enthalpy of 146 J/g of 100% crystalline PHB. The
melting temperature of standard PHB and the TS blend
PHB were found to differ (Table 1) greatly. The
enthalpy of melting is 32.56 J/g for standard PHB,
whereas for the blend PHB, it was 64.58 J/g.
Diffraction patterns of PHB and blend PHB showed
2 θ values of 26.80°, 31.74°, 45.59°, and 56.22° at the
intensities 1800, 3100, 4700, and 700 a.u. (Fig. 7). The
increased intensity of peaks showed that the polymer
has more organized packed crystalline structure. The
X-ray diffraction pattern of standard PHB also followed
the same trend of the extracted PHB (data not shown
here).
Discussion
Global environment concerns and solid waste man-
agement problems have shifted the interest to the
development of biodegradable plastics that possess the
similar physical and chemical properties of the conven-
tional plastics. PHB accumulation as cytoplasmic inclu-
sions in certain bacteria during unbalanced growth
conditions has proved to be the best suitable alternative
to overcome this problem. More efforts to develop eco-
nomically feasible process for the synthesis of PHB
should take the center stage most preferably from
microbial origin. In this regard, many Bacillus strains
have been reported possessing tremendous potential of
PHB accumulation in their cytoplasm under nutrient
limiting conditions at a level of 6–97% of dry cell
weight.18–21) In a similar line with that, the two most
potent isolates of the current study were also identified
as Bacillus. Fluorescence microscopy is a useful tool
for the assessment of PHB accumulation by bacteria as
Nile blue selectively binds with the produced PHB and
is permeable to the bacterial cell. Screening of all the
isolates by Nile blue staining followed by fluorescent
microscope revealed the varied amount of PHB
production by the bacterial isolates. These results cor-
roborate with the previous results of many other work-
ers,22–24) but B. cereus SE-1 is a more superior strain
compared to the previous reports of PHB production
by other Bacillus species, i.e. Bacillus subtilis NG220
 6 P. Bhagowati et al.
Downloaded by [University of Alberta] at 10:44 25 April 2015

Fig. 4. Bivariate distribution of the FSC and PHB fluorescence of two isolates Bacillus cereus SE-1 and Bacillus sp. CS-605.
Notes: The bivariate distributions display data from (a) 5%, (b) 18.1%, (c) 33% for Bacillus sp. CS-605, and (d) 3.5%, (e) 22.1%, and (f)
40% for B. cereus SE-1 cells containing PHB after 24, 48, and 72 h, respectively. A gate was fitted by minimizing the difference and is indicated
as the black line. Cells with fluorescence above this line were considered to contain PHB and the cells present below do not produce PHB.

Fig. 5. Optimization of PHB production in potent isolate B. cereus SE-1.

(a) Optimization of carbon sources for the biomass and subsequently PHB production and (b) time-dependent PHB production in optimized culture
conditions.

(1.191 g/L)25) and Bacillus megaterium ATCC 6748
(0.056 g/L).26)
Two higher amount of PHB-producing bacterial iso-
lates, one from the terrestrial source and another from
the marine environment, were characterized in this
study. In this regard, there are many reports of the use
of terrestrial bacteria for bioplastic production.27–31)
However, the reports are scanty from the marine envi-
ronment sources.32) In comparison to terrestrial envi-
ronment, marine sources are the less explored
environmental conditions for the microbial diversity as
well as the screening of potential marine bacteria.33)
The study of comparative account of PHB production
by the two potent isolates revealed that the terrestrial
isolate B. cereus SE-1 produced marginally higher
amount of PHB than that of the marine isolate Bacillus
sp. CS-605 under same experimental setup. After 72 h
of incubation, the marine isolate was found to produce
33% of PHB, whereas 40% of cells produced PHB in
the case of B. cereus SE-1. In the same line with that,
 Characterization of biodegradable plastic
Fig. 6. Fourier transformed infrared spectroscopy (FTIR) analysis
of standard and extracted PHB.
Notes: FTIR analysis of extracted PHB from the B. cereus SE-1
reveals peaks for functional groups such as CH2, CH, and C=O, O–H.
Table 1. DSC result of the blend PHB sample in comparison to
the standard PHB.
Downloaded by [University of Alberta] at 10:44 25 April 2015
7
is a commonly occurring phenomenon in marine
environmental condition. This ultimately increases the
nutrient load in the marine environment. However, the
deep-sea environment contains fewer nutrients com-
pared to the estuary and coastal environment. In this
regard, the marine isolate Bacillus sp. CS-605 has been
isolated from the marine lake Chilika which is known
for its nutritional value due to continuous inflow of
river water and subsequent sedimentation. This in turn
increases the nutrient load of the lake and the isolated
bacterium might be exposed to a nutrient-rich environ-
ment. Thus, the marine bacterium no longer remains in
the nutrient-starved condition which is the prerequisite
for PHB accumulation. On the other hand, marine envi-
ronment is one of the most dynamic environmental
conditions, where the inhabiting bacteria are continu-
ously exposed to the ever-changing physicochemical
factors such as pH, salinity, sea surface temperature,
and others.29) However, limiting nutrient sources fairly
exist in marine environment leading to lower produc-
tion of PHB.

There exist three regulatory enzymes which are

Sample Tm (°C) ΔHf (J/g) X (%)
involved in the production and synthesis of PHB in
Standard PHB 174.5 32.56 22.30 bacteria. These enzymes include β-ketothiolase, ace-
Blend PHB 109.4 64.58 44.23 toacetyl-CoA reductase, and PHA synthase/polymerase
coded, respectively, by phbA, phbB, and phbC, respec-
tively.36) phbC is found to be located upstream from
0.9–45% of PHB-producing Saccharomyces cerevisiae phbA–phbB. The reductase when interacts with either
strain D603 cells has been reported by flow cytometric enoyl-CoA hydratase or epimerase or both leads to the
analysis.34) Two facts can be attributed to the higher formation of D(-)-3-hydroxybutyryl-CoA. Thus, the
production of PHB by the terrestrial isolate than that of presence of phbB gene in both the isolates revealed the
its marine counterpart. Marine environment is rich in phb gene cluster mediated PHB production in them.
organic nutrients (0.29 mg/mL of lipids) as well as Though there exist many different genetic mechanisms
other inorganic nutrients.35) Sedimentation is propor- of PHB production, most of the PHB production in
tional to the amount of new nutrients and sedimentation bacteria is mediated through phb gene cluster as

Fig. 7. Comparative account of XRD diffractogram of pristine PHB and blend PHB of B. cereus SE-1.
 8 P. Bhagowati et al.
37)
reported in Rhodobacter sphaeroides, Azotobacter
sp.,38) and Azospirillum brasilense.39) phbB mutants
have also been reported for poor growth and less PHB
accumulation in many bacteria, suggesting its useful-
ness during PHB synthesis process.40) Additionally,
many previous studies have amplified phbB gene in the
isolates to deduce the molecular nature of PHB produc-
tion in them.41,42) Thus, in order to confirm the role of
phbB gene of phb operon during pathway of PHB syn-
thesis, amplification of phbB gene was carried out in
the isolates. There are many factors affecting PHB pro-
duction by the environmental isolates that include the
nutrient conditions such as carbon, nitrogen, amino
acids, and metal ions.3) Out of which, carbon source is
the most limiting factor of PHB production. Thus, PHB
production by the potent isolate B. cereus SE-1 was
optimized in the presence of various carbon sources.
Out of all the carbon sources tested, the isolate
B. cereus SE-1 was found to produce PHB at a greater
amount in the presence of maltose as sole carbon
source. The isolate was found to produce a higher
Downloaded by [University of Alberta] at 10:44 25 April 2015
amount of biomass as well as PHB accumulation in the
presence of maltose which has also been reported for
many other bacterial species.43,44)
With the increase in time of incubation, the cell mass
as well as PHB accumulation increases at the optimized
culture conditions for B. cereus SE-1 using maltose as
sole carbon source till 72 h. This result also corrobo-
rates with the result obtained from the flow cytometry
analysis with maximum production of PHB after 72 h
of incubation. However, in natural environmental
conditions, PHB are the osmotic neutral reservoirs of
carbon and energy and are synthesized when more
carbon sources are available that can be consumed by
bacteria. These PHB are reutilized/mobilized during
times of starvation and greatly enhance the survival of
bacteria in the absence of a suitable exogenous carbon
source.45)
The presence of characteristic functional group of
the PHA granules is C=O group which has been con-
firmed by FTIR spectroscopy. Irrespective of the nature
and type of bioplastics, carbonyl group (C=O) is a
common feature in all structures which confirms the
extraction of PHB during the present study.46) It gives
further insights toward the chemical structure of the
polymer and its constituent monomeric units. During
this study, the functional group of PHA was confirmed
to be C=O, the similar result of which has also been
achieved by many previous workers 21) for 2983 cm−1
(CH, CH2, CH3); 2933 cm−1 (CH, CH2, CH3);
1720 cm−1 (ester C=O valence); 1639 cm−1 (thioester
C=O valence); 1380 cm−1; 1302 cm−1; 1260 cm−1
(CH2-S); and 1162 cm−1 (ester C–O). The IR spectrum
reflects the presence of both monomeric units as is
expected due to C=O valence vibration (1658 cm−1) of
a thioester group.47) Molecular weight of PHB accumu-
lated in Bacillus sp. under in vitro conditions has been
reported to range from 200–500 kDa,20,48,49) thus the
potent isolate B. cereus SE-1 is assumed to accumulate
PHB with the same range of molecular weight. How-
ever, the slight variation of molecular weight of PHB
may not impact negatively on the PHB extraction prac-
tices as well as in its further characterization.
Conclusion
PHB produced by the bacterial system are brittle and
break easily. Thus, blending of PHB with other poly-
mers in an economic way improves its mechanical
properties. The most commonly used blending materials
include thermostable starch, poly lactic acid, PE glycol,
and starch. During this study, TS was used to increase
the mechanical properties of the PHB sample. DSC and
XRD study reveals the increased thermoplasticity and
decreased crystallinity of the blend sample than that of
the PHB standard sample. The lower crystalline degree
and higher Tm of blend sample reveal that better plasti-
cizing effect depresses the crystallization of PHB which
further pushes the already formed microcrystals to pack
into more perfect structures in the blends. However, the
melting temperature of the blend sample decreased in
the blend samples to 109.4 °C from 174.5 °C as in the
case of the standard PHB sample which is in contrast to
the result obtained by Sindhu et al.17) Thus, the result
indicates that the blending of PHB with other polymers
will be advantageous for cost reduction as well as its
improved thermoplastic properties.
To deal with ever-increasing plastic pollution, PHB
from the bacterial sources is the better alternative. Both
the isolates B. cereus SE-1 and Bacillus sp. CS-605 are
capable of producing significant amount of PHB under
laboratory conditions. However, the terrestrial isolate
B. cereus SE-1 was found to be more capable of pro-
ducing PHB than that of its marine counterpart. Pres-
ence of C=O stretch in the extracted PHB sample
confirmed its integrity and purity. An increased thermo-
plasticity and decreased crystallinity of PHB blend
sample were found in comparison to the standard PHB.
Author contribution
P.B., S.P., H.R.D and S.D. designed the experiments.
P.B. carried out the isolation, characterization, and
flow-cytometric studies. S.P. and H.R.D. carried out
studies on the optimization, blending, and characteriza-
tion by FTIR, XRD, and DSC. H.R.D. and S.D. con-
tributed toward interpretation of results and manuscript
writing.
Supplemental material
The supplemental material for this paper is available
at http://dx.doi.org/10.1080/09168451.2015.1034651.
Acknowledgment
This paper is the Masters Project work of P.B. and
S.P. and we gratefully acknowledge the financial sup-
port and facilities provided by the authorities of
National Institute of Technology, Rourkela, Odisha.
 Characterization of biodegradable plastic
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