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ENHANCED BIOSYNTHESIS OF POLY(3-

HYDROXYBUTYRATE) FROM POTATO
STARCH BY Bacillus cereus STRAIN 64-INS
IN A LABORATORY-SCALE FERMENTER
a a
Iftikhar Ali & Nazia Jamil
a
Department of Microbiology and Molecular Genetics , University of
the Punjab, Quaid-i-Azam Campus , Lahore , Pakistan
Accepted author version posted online: 26 Nov 2013.Published
online: 11 Jul 2014.

To cite this article: Iftikhar Ali & Nazia Jamil (2014) ENHANCED BIOSYNTHESIS OF POLY(3-
HYDROXYBUTYRATE) FROM POTATO STARCH BY Bacillus cereus STRAIN 64-INS IN A LABORATORY-
SCALE FERMENTER, Preparative Biochemistry and Biotechnology, 44:8, 822-833, DOI:
10.1080/10826068.2013.867876

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 Preparative Biochemistry & Biotechnology, 44:822–833, 2014
Copyright # Taylor & Francis Group, LLC
ISSN: 1082-6068 print/1532-2297 online
DOI: 10.1080/10826068.2013.867876

ENHANCED BIOSYNTHESIS OF POLY(3-HYDROXYBUTYRATE)

FROM POTATO STARCH BY Bacillus cereus STRAIN 64-INS
IN A LABORATORY-SCALE FERMENTER
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Iftikhar Ali and Nazia Jamil

Department of Microbiology and Molecular Genetics, University of the Punjab,
Quaid-i-Azam Campus, Lahore, Pakistan

& To decrease the polyhydroxyalkanoate (PHA) production cost by supplying renewable carbon
sources has been an important aspect in terms of commercializing this biodegradable polymer.
The production of biodegradable poly(3-hydroxyalkanoates) (PHA) from raw potato starch by
the Bacillus cereus 64-INS strain isolated from domestic sludge has been studied in a lab-scale
fermenter. The bacterium was screened for the degradation of raw potato starch by a starch hydrolysis
method and for PHA production by Nile blue A and Sudan black B staining. Shake-flask cultures of
the bacterium with glucose [2% (w=v)] or raw potato starch [2% (w=v)] produced PHA of 64.35 %
and 34.68% of dry cell weight (DCW), respectively. PHA production was also carried out in a 5-L
fermenter under control conditions that produced 2.78 g=L of PHA and PHA content of 60.53%
after 21 hr of fermentation using potato starch as the sole carbon source. Gas chromatography–mass
spectroscopy (GC-MS) analyses confirmed that the extracted PHA contained poly(3-hydroxybutyrate)
(PHB) as its major constituent (>99.99%) irrespective of the carbon source used. The article
describes, for what we believe to be the first time, PHB production being carried out without any
enzymatic or chemical treatment of potato starch at higher levels by fermentation. More work
is required to optimize the PHB yield with respect to starch feeding strategies.

Keywords Bacillus cereus, biodegradable polymer, cheap carbon source, fermentation,

GC-MS, potato starch

INTRODUCTION
Polyhydroxyalkanoates (PHAs) are the aliphatic polyesters that are
accumulated intracellular in both Eubacteria and Achaea under the
unfavorable conditions. These PHAs not only bear physical and chemical
properties that are comparable to those of petroleum-based plastics but
Address correspondence to Iftikhar Ali, Department of Microbiology and Molecular Genetics,
University of the Punjab, Quaid-i-Azam Campus, Lahore-54590, Pakistan. E-mail: iftikhar_ali_iftikhar@
yahoo.com
Color versions of one or more of the figures in the article can be found online at www.tandfonline.
com/lpbb.
 Enhanced Biosythesis of Poly(3-hydrobutyrate) From Potato Starch 823

they are also biodegradable, which labels them as ‘‘environment-friendly

plastics’’ and recyclable.[1] These PHAs have already found a number of
applications in every field of life, such as drug targeting, protein purifi-
cation,[2] enzyme-linked assays, and bioimaging;[3] packaging containers
such as bottles; disposable items such as diapers;[4] as novel biofuels;[5]
and in medicine, food packaging, and industry.[6] A new wave of PHA
development with a focus on new applications is expected soon.[7]
To produce PHA at a price comparable to that of petroleum-based
plastics, one must seek ways that could possibly lower the final price of
theses PHAs at the consumer end. According to Gurieff and Lant,[8] the
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price of PHA is more than US$10=kg and it is almost 10 times higher than
that of petroleum-based plastics (London Metal Exchange, February 2011).
A number of factors are responsible for this price difference, such as PHA
productivity, method of recovery, cultivation strategy of microorganism,
choice of strain, and carbon substrate cost.[9]
To make polyhydroxyalkanoates production more economical,
researchers are focusing on several inexpensive carbon sources such
as plant oils,[10] cheese whey,[11] starch,[12] whey,[13] and municipal sewage
sludge[14] to produce PHAs, but still PHA remains underused mostly
because of its high cost.
Bacillus sp. have been well known for their ability to produce PHA, and
some of them have been well reported in this context, such as Bacillus
thrungiensis,[15] Bacillus spp.,[16,17] B. subtilis,[18] and B. megaterium strain
OU303A.[19] The production of PHA from potato starch has been
reported[20–23] to emphasize the need to utilize the second most abundant
starch source for the useful biopolymers production, but processing of
starch has been an issue. Here we used potato starch residue as sole carbon
source to grow locally isolated Bacillus cereus 64-INS. The aim of this
research was to isolate a microorganism capable of utilizing raw potato
starch as sole carbon source for its growth and for the subsequent
production of PHA in a chemically defined nitrogen-limited medium.

MATERIALS AND METHODS

Isolation, Purification, and Screening of PHA-Producing
Bacteria
Various bacterial colonies were isolated from a local domestic sludge sam-
ple on LB agar medium plates by a standard serial dilution method. The pur-
ified bacterial isolates were then screened for their ability to produce PHA by
Sudan black B staining.[9] For starch hydrolysis activity, bacteria were grown
on starch agar (0.2%); plates were incubated at 37 C for 24 hr, followed by
flooding with iodine solution to check the starch hydrolyzing ability.[16]
The PHA-producing bacterial isolate 64-INS used in this study was chosen
 824 I. Ali and N. Jamil

for its better starch-hydrolyzing ability and was maintained monthly on solid
LB agar medium containing (%, w=v): tryptone, 1.0; yeast extract, 0.5; NaCl,
1.0; and agar 1.5, pH 7.0. The bacterium was also characterized physiologi-
cally and biochemically for the colony morphology, growth requirements like
pH and temperature, and ability to utilize various sugars such as lactose, malt-
ose, sucrose, fructose, and so on[24]

Bacterial Growth on Various Carbon Sources

Bacillus cereus strain 64-INS was grown in 250-mL Erlenmeyer flasks
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containing 50 mL of nitrogen-limited mineral medium [(NH4)2SO4, 2 g=L;

KH2PO4, 13.3 g=L; MgSO4
7H2O, 1.2 g=L; citric acid, 1.7 g=L; and trace
elements solution 10 ml=L] with different carbon sources (fructose 1%;
lactose 1%; sodium gluconate 10 mM; molasses 0.1% and sodium octanoate
10 mM) at 30 C and 200 rpm. The cultures were centrifuged, washed
with 0.85% saline, and freeze-dried for dry cell weight (DCW) estimation,
subsequent PHA extraction, and analysis.

Pretreatment of Potato Waste

The solid waste of potatoes was obtained from a local market and
crushed to fine powder following its dissolution to distilled water. The
mixture was heated at 100 C for 10 min. After centrifugation the supernatant
was analyzed for its starch content against a standard curve. Supernatant
(0.5 ml) was added with 5 mL of 0.0007 N iodine and the optical density of
the mixture was measured at 660 nm to quantify the starch.[25]

Fermentative Production of PHA From Potato Starch

Bacillus cereus 64-INS was grown for 24 hr at 30 C and 150 rpm in 250-mL
Erlenmeyer flask containing 50 mL of rich medium containing tryptone
1.0 g, yeast extract 0.5 g, NaCl 1.0 g, and peptone 1.0 g=L. Bacterial cells were
harvested by centrifugation at 4000  g for 10 min, briefly dried, and 1%
(v=v) inoculum of cells was given to minimal medium containing
potato starch at final concentrations of 2% (w=v) in a 5-L fermenter
(Bioengineering, Wald, Switzerland) with a working capacity of 3 L. The
fermentation temperature was maintained at 30 C and pH was adjusted to
7.0 by the automatic addition of 2 N KOH solution. The dissolved oxygen
was set to 20% while airflow rate was 18 L=h with continuous agitation rate
of 600 rpm. A parallel set of experiments was conducted in 500-mL flasks
containing 100 mL of the already-described minimal medium supplemented
with either glucose or potato starch at 2% (w=v). The flasks were also
incubated in a reciprocal orbital shaker set at 200 rpm and 30 C.
 Enhanced Biosythesis of Poly(3-hydrobutyrate) From Potato Starch 825

Extraction and Purification of Polyhydroxyalkanoates

Samples were drawn at regular intervals from the PHA-producing cultures
and cells were harvested by centrifugation at 4000  g for 15 min at 8 C. The
cells were resuspended in deionized distilled water twice and were freeze-dried
in a lyophilizer for 24 hr. These cells were mixed by sodium hypochlorite
(13%) and chloroform solution (1:1) and agitated at 150 rpm for 100 min
at ambient temperatures to release the PHA granules from the cells. The mix-
ture was centrifuged and the lower dense organic phase containing the PHA
was precipitated by cold acetone (1:3) to precipitate the biopolymer. The PHA
polymer was obtained by centrifugation and dried at room temperature.[26]
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Analytical Procedure for PHA Composition

A small amount of purified PHA (8 mg) was methanolyzed by heating at
100 C for 140 min in a mixture of 1.0 mL chloroform, 0.85 mL methanol,
and 0.15 mL concentrated sulfuric acid.[27] A 1-mL sample of methanolyzed
PHA sample was injected in a gas chromatograph—mass spectrometer
(GCMS) QP2010 (Shimadzu) via split injection mode (70:30), and
nitrogen was used as the carrier gas at a flow rate of 3 mL=min. The oven
temperature was programmed as follows: 60 C for 2 min at the start and
then ramped at a rate of 5 C per min to 260 C, and held for 15 min.
The temperature of the injector was set at 260 C and column flow rate
was 0.57 cm=s. A DB-5 MS column (30 m, 0.25 mm, and 0.25 mm) was used.
The gas chromatography system was coupled with a mass spectrometry
system (GCMS QP2010 with D1), in which the temperature of the ion source
was set at 200 C, to analyze the monomers produced in this PHA sample.

16S rRNA and Phylogenetic Analysis of Bacillus cereus 64-INS

Genomic DNA of bacterium was isolated and 16S rRNA gene was
amplified using the 27F and 1492R primers following the gene sequencing
with 518F and 800R primers by Macrogen, Inc., Korea. The 16S rRNA
sequence obtained in this study was deposited in NCBI GenBank under
accession number JQ013099.1. A phylogenetic tree was constructed by
the MEGA software (version 5.0) using the neighbor-joining method and
a 1000-replicates bootstrap analysis.

RESULTS
Screening for PHA-Producing Bacteria and Biochemical
Characterization
The majority of bacterial colonies that were isolated from the sludge
sample were unable to produce PHA content in general. Only 11% of
 826 I. Ali and N. Jamil

bacteria were found to be positive for PHA production as depicted by the

screening methodology. Sludge environment has already been reported
for the presence of PHA-producing bacteria.[14] Bacterial isolates capable
of PHA production (positive for both Sudan black B and Nile blue A tests)
were characterized biochemically, and isolate 64-INS was selected due to its
best starch hydrolysis activity in terms of showing the biggest clearing zone
on starch-supplemented minimal medium agar (Supplemental Table S1).

Shake-Flask Studies With Glucose and Other Carbon Sources

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PHA was produced by the Bacillus cereus strain 64-INS at its maximum
levels (64.35% of DCW) when glucose was used as sole carbon source at
30 C after 24 hr of incubation. The bacterium also produced PHA from
molasses (16.59%) and sodium gluconate (9.84%) but in relatively lower
amounts as compared to glucose (Figure 1). PHA contents of 3.86% and
1.12% were obtained from fructose and sodium octanoate, respectively,
but no PHA was produced from lactose. The PHA quantity varied in case
of different concentrations of potato starch and it was at its maximum level
(34.68% of DCW) for 2% (w=v) solution of extracted starch (Table 1).

Fermentative Production of PHA by Bacillus cereus

Strain 64-INS
Growth of bacterium in a bioreactor showed a typical exponential
phase when grown on potato starch followed by biopolymer accumulation.
The quantity of PHA produced increased in the fermenter as the levels of
starch decreased, most likely due to bacterial amylase production. It can be
seen from the Figure 2 that cell dry weight increased steadily over the
passage of time. The production of PHA was related to the decreased
amount of starch present in the fermenter. The highest production of

FIGURE 1 Production of PHA by Bacillus cereus strain 64-INS on different carbon sources after 24 hr of
incubation at 30 C and 200 rpm in shake-flask experiments. Experiments were performed in duplicates
and standard deviation is represented here as error bars.
 Enhanced Biosythesis of Poly(3-hydrobutyrate) From Potato Starch 827

TABLE 1 PHA Production by Bacillus cereus 64-INS Grown on Starch and Glucose

Carbon Source (w=v) Growth DCW (g=L) PHA %a PHA (g=L) PHA Monomer

2% Glucose Shake flasks 2.52  0.31 64.35  0.78 1.62  0.23 3HB
2% Potato starch Shake flasks 1.72  0.23 34.68  0.27 0.59  0.12 3HB

a
PHA content in terms of bacterial dry cell weight (DCW).

PHA was at 21 hr when dry biomass was 4.59 g=L and PHA content was
60.53% of its DCW (Figure 2). Once the cells had reached considerable cell
density, the dissolved oxygen levels reached their minimum value and
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bacterial activity for the PHA accumulation was its peak. The yield of
PHA also reached its maximum value of 2.78 g=L when the starch in the
vessel had been consumed in considerable amounts (Figure 3).

FIGURE 2 Growth and production of PHB by Bacillus cereus strain 64-INS in the presence of potato
starch as sole carbon source in a bioreactor. Experiments were performed in duplicates, and standard
deviation is represented here as error bars.

FIGURE 3 PHB production and utilization of potato starch in the bioreactor by Bacillus cereus strain 64-INS.
Experiments were performed in duplicates, and standard deviation is represented here as error bars.
 828 I. Ali and N. Jamil

Characterization of Purified PHA

PHA isolated and purified from the shake-flask and fermentation
studies was analyzed by GC-MS. The chromatograms showed that the
methyl esters of poly(3-hydroxybutyric) acid were the dominant monomers
for the case of both glucose and starch as carbon sources.

Identification of the Bacterium

DNA sequence for the 16S rRNA gene of strain 64-INS was compared
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with the already deposited sequences in GenBank by NCBI BLAST, and it

was observed that this organism had 98% homology with the Bacillus cereus
strain CFR06 (Figure 4).

FIGURE 4 A phylogenetic tree of Bacillus cereus strain 64-INS constructed by neighbor-joining method
with 1000 bootstrap using MEGA5.0 software with Halolactibacillus alkaliphilus strain NBRC103919 as
an outgroup.
 Enhanced Biosythesis of Poly(3-hydrobutyrate) From Potato Starch 829

DISCUSSION
With the diminution of crude oil source to produce conventional
plastics, there is a possibility of producing biodegradable plastics from
renewable sources like cellulose, starch, and sugars.[28] Since 50% of the
cost of PHA production is due to its carbon source,[29] various inexpensive
carbon sources have been employed by researchers to combat the high
production costs of PHA. The present work reports the production
and characterization of bioplastic by using potato starch as a cheap carbon
source from a bacterium isolated from a sludge sample. Due to its
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abundance, renewability, and low cost, starch has been used in different
forms, such as granular starch, plasticized starch, modified starch, and
starch blends.[30] So far, Bacillus sp. has been in use for the utilization of
starch due to its a-amylase production.[31–33] Moreover, Bacillus cereus has
been exploited to produce PHA from different unusual carbon sources like
pea-shells slurry, in addition to H2 production.[34,35]
The bacterium isolated in this study was identified as a Bacillus cereus
strain according to 16 s rRNA gene similarity to already reported species
at the NCBI GenBank. Bacillus cereus 64-INS was able to utilize the molasses
and sodium gluconate as well and produced DCW of 0.53 and 0.75 g=L,
respectively, but the amount of PHA produced was very low. It produced
PHA content of 16.59 and 9.84%, which was very low as compared to
64.35% when grown on glucose, while other carbon sources (especially
lactose) could sustain neither healthy biomass for bacterium nor the
PHA (Figure 1). The bacterium produced very little amount of PHA from
fructose and sodium octanoate, but it seems to prefer more simplified
monomers (as present in molasses) for its growth and PHA production.
There have been a number of ways reported to utilize starch in either its
raw form or its processed form to produce PHA. The processing of starch
involves liquification and saccharification processes that are costly due to
their enzyme consumption. Hence, the idea has been to use a bacterium
that has both starch hydrolysis and PHA biosynthetic capabilities so that
we may save the extra cost of starch hydrolysis. In shake-flask studies,
strain 64-INS showed good bacterial growth on potato starch as well, that
is, 1.72 g=L as compared to 2.52 g=L when grown on glucose, while PHA
content was almost double in the case of glucose (Table 1).
Strain 64-INS continuously used the starch up to 21 hr of fermentation,
resulting in >60% poly(3-hydroxybutyrate) (PHB) of its DCW, although
some starch content was still there that was never used. In fact, the bacterial
metabolism was shifted at this point and resulted in consumption of PHB as
well. This was the point where we obtained maximum yield of PHB (2.78 g=L),
as indicated in Figure 3. Bacillus cereus 64-INS in this study produced
a considerable amount of 60% PHA in a relatively short period of time
(21 hr), as compared to other studies where nonprocessed starch has been
 Downloaded by [Memorial University of Newfoundland] at 00:52 18 July 2014

TABLE 2 Production of PHA Using Various Forms of Starch by Different Bacteria Reported in Literature

Organism Carbon Source Cultivation PHA (%)a PHA (g=L) PHA type Reference

Bacillus megaterium Hydrolyzed cassava starch Shake flask 29.70 1.48 PHB [33]
Ralstonia eutropha NCIMB 11599 Saccharified waste potato starch Fed-batch 52.51b 94.00 PHB [39]
Halomonas boliviensis LC1 Starch hydrolysate Shake flask 56.00 NA PHB [40]
Batch fermenter 35.00 NA PHB
Haloferax mediterranei Extruded cornstarch Repeated fed-batch 38.70 24.20 NA [41]
Haloferax mediterranei Enzymatic extruded starch Fed-batch 50.80 NA PHBV [12]
Alcaligenes eutrophus DSM 545 Enzyme treated potato waste Batch fermenter 77.00 5.00 PHB [21]
Pseudomonas aeruginosa NCIB 950 Cassava starch hydrolysate Batch fermenter 57.70 NA PHB [38]
Saccharophagus degradans Raw starch Fed-batch 17.46 2.71 PHB [36]
ATCC 43961 Batch fermenter 7.12 0.53 PHB

830
Pseudomonas fluorescens Cassava starch hydrolysate Batch fermenter 71.66 1.25 PHB [42]
Bacillus cereus CFR06 Soluble starch Shake flask 48.00 0.48 PHB [31]
Bacillus sp. CFR 67 Corn starch Shake flask 2.93b 0.09 NA [37]
Potato starch Shake flask 17.14b 0.24 NA
Topioca starch Shake flask 30.00b 0.39 NA
Soluble starch Shake flask 18.5b 0.37 NA
Alcaligenes eutrophus Acidogenic starchy wastewater Batch fermenter 34.10 1.20 PHB [43]
Azotobacter chroococcum 23 Soluble starch Shake flask 74.00 NA PHB [44]
Soluble starch Batch fermenter 44.00 NA PHB
Soluble starch Fed-batch 46.00 NA PHB
Bacillus cereus 64-INS Potato starch Batch fermenter 60.53 2.78 PHB This study

a
Percentage amount of PHA in its DCW.
b
Calculated on basis of given data in respective articles.
 Enhanced Biosythesis of Poly(3-hydrobutyrate) From Potato Starch 831

used as carbon source[31,36,37]; hence, it is suggested as a potential

organism to produce PHB from raw potato starch as compared to other
bacteria (Table 2). Alcaligenes eutrophus DSM 545 has produced PHB
content up to 77% in batch fermentation as well, but it was fed with enzy-
matically treated potato starch.[21] Pseudomonas aeruginosa NCIB 950 could
also produce PHB in batch fermentation but in the presence of cassava
starch hydrolysate.[38] The raw starch has been used in fed-batch fermen-
tation by Gonzalez-Garcia et al.[36] using Saccharophagus degradans ATCC
43961, but it could only produce a PHA content of about 18% of its
DCW. Other processed starch feeding has also been reported, but the more
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simplified and cost-effective form of starch may be the most economical way
of converting this cheap carbon source into biotechnologically important
polymers like PHB. As we know that the presence of 3-hydroxybutyrate
(3HB) monomers contributes to the brittle nature of PHA polymer, this
requires a sufficient amount of other monomers (e.g., 3-hydroxyvalerate
[3HV] and 3-hydroxyhexanoate [3HHx]) to form copolymers that may
be used as a thermoplastic polymer. Hence, it is necessary to find the ways
and develop strategic fermentation technologies for the conversion of
abundant quantities of starch present in the environment into more
thermoplastic PHA polymer.

CONCLUSION
The newly isolated bacterium in this study, Bacillus cereus 64-INS, has
shown the ability to produce biodegradable polymer PHB (poly-3-hydroxy-
butyrate) in considerable amounts by using potato starch, especially in
batch fermentation. The novelty in this work is found in both the strain
of biocatalyst used and the fact that this is the first account of a PHA-
producing bacterium that can utilize untreated starch as a carbon source,
suggesting the presence of some highly active amylase enzymes. The use
of potato starch for this purpose would certainly reduce the costs of carbon
sources at industrial level to produce PHA. This is for the first time, to the
best of our knowledge, that PHB production has been carried out without
any enzymatic or chemical treatment of potato starch, and it gave high PHA
content as well; however, further studies are still necessary to optimize the
PHB yield by varying the starch feeding strategies.

FUNDING
The authors are thankful to the Higher Education Commission (HEC),
Pakistan, and the University of the Punjab, Lahore, Pakistan, for providing
support and funds to complete this research work.
 832 I. Ali and N. Jamil

SUPPLEMENTAL DATA
Supplemental data for this article can be accessed on the publisher’s
website.

REFERENCES
1. Ariffin, H.; Nishida, H.; Hassan, M.A.; Shirai, Y. Chemical Recycling of Polyhydroxyalkanoates
as a Method Towards Sustainable Development. Biotechnol. J. 2010, 5(5), 484–492.
2. Williams, S.F.; Martin, D.P. Applications of PHAs in Medicine and Pharmacy. Biopolymers 2002, 4, 91–97.
3. Grage, K.; Jahns, A.C.; Parlane, N.; Palanisamy, R.; Rasiah, I.A.; Atwood, J.A.; Rehm, B.H. Bacterial
Downloaded by [Memorial University of Newfoundland] at 00:52 18 July 2014

Polyhydroxyalkanoate Granules: Biogenesis, Structure, and Potential Use as Nano-=Micro-Beads

in Biotechnological and Biomedical Applications. Biomacromolecules 2009, 10(4), 660–669.
4. Snell, K.D.; Peoples, O.P. PHA Bioplastic: A Value-Added Coproduct for Biomass Biorefineries.
Biofuel Bioprod. Biorg. 2009, 3(4), 456–467.
5. Zhang, X.J.; Luo, R.C.; Wang, Z.; Deng, Y.; Chen, G.Q. Application of (R)-3-Hydroxyalkanoate
Methyl Esters Derived From Microbial Polyhydroxyalkanoates as Novel Biofuels. Biomacromolecules
2009, 10(4), 707–711.
6. Ikada, Y.; Tsuji, H. Biodegradable Polyesters for Medical and Ecological Applications. Macromol.
Rapid. Commun. 2000, 21(3), 117–122.
7. Chen, G.Q. A Microbial Polyhydroxyalkanoates (PHA) Based Bio- and Materials Industry. Chem. Soc.
Rev. 2009, 38(8), 2434–2446.
8. Gurieff, N.; Lant, P. Comparative Life Cycle Assessment and Financial Analysis of Mixed Culture
Polyhydroxyalkanoate Production. Bioresource Technol. 2007, 98(17), 3393–3403.
9. Lee, S.; Choi, J. Polyhydroxyalkanoates: Biodegradable Polymer. In Manual of Industrial Microbiology
and Biotechnology, 2nd ed.; Demain, A.L., Davies, J.E., Atlas, R.M., Eds.; ASM Press: Washington, DC,
1999; pp. 616–627.
10. Lee, W.H.; Loo, C.Y.; Nomura, C.T.; Sudesh, K. Biosynthesis of Polyhydroxyalkanoate Copolymers From
Mixtures of Plant Oils and 3-Hydroxyvalerate Precursors. Bioresource Technol. 2008, 99(15), 6844–6851.
11. Obruca, S.; Marova, I.; Melusova, S.; Mravcova, L. Production of Polyhydroxyalkanoates From
Cheese Whey Employing Bacillus megaterium CCM 2037. Ann. Microbiol. 2011, 61(4), 947–953.
12. Chen, C.W.; Don, T.M.; Yen, H.F. Enzymatic Extruded Starch as a Carbon Source for the Production
of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Haloferax mediterranei. Process Biochem. 2006,
41(11), 2289–2296.
13. Koller, M.; Bona, R.; Chiellini, E.; Fernandes, E.G.; Horvat, P.; Kutschera, C.; Hesse, P.; Braunegg, G.
Polyhydroxyalkanoate Production From Whey by Pseudomonas hydrogenovora. Bioresource Technol.
2008, 99(11), 4854–4863.
14. Reddy, S.V.; Thirumala, M.; Reddy, T.V.K.; Mahmood, S.K. Isolation of Bacteria Producing
Polyhydroxyalkanoates (PHA) From Municipal Sewage Sludge. World J. Microbiol. Biotechnol. 2008,
24(12), 2949–2955.
15. Singh, M.; Kumar, P.; Patel, S.K.; Kalia, V.C. Production of Polyhydroxyalkanoate Co-Polymer by
Bacillus thuringiensis. Ind. J. Microbiol. 2013, 53(1), 77–83.
16. Porwal, S.; Kumar, T.; Lal, S.; Rani, A.; Kumar, S.; Cheema, S.; Purohit, H.J.; Sharma, R.; Patel, S.K.S.;
Kalia, V.C. Hydrogen and Polyhydroxybutyrate Producing Abilities of Microbes From Diverse
Habitats by Dark Fermentative Process. Bioresource Technol. 2008, 99(13), 5444–5451.
17. Kumar, T.; Singh, M.; Purohit, H.J.; Kalia, V.C. Potential of Bacillus sp. to Produce Polyhydroxybutyrate
From Biowaste. J. Appl. Microbiol. 2009, 106(6), 2017–2023.
18. Singh, M.; Patel, S.K.S.; Kalia, V.C. Bacillus subtilis as Potential Producer for Polyhydroxyalkanoates.
Microb. Cell. Fact. 2009, 8, 38.
19. Reddy, S.V.; Thirumala, M.; Mahmood, S. Production of PHB and P (3HB-co-3HV) Biopolymers by
Bacillus megaterium Strain OU303A Isolated From Municipal Sewage Sludge. World J. Microbiol.
Biotechnol. 2009, 25(3), 391–397.
20. Ramadas, N.V.; Singh, S.K.; Soccol, C.R.; Pandey, A. Polyhydroxybutyrate Production Using
Agro-Industrial Residue as Substrate by Bacillus sphaericus NCIM 5149. Braz. Arch. Biol. Technol.
2009, 52(1), 17–23.
 Enhanced Biosythesis of Poly(3-hydrobutyrate) From Potato Starch 833

21. Rusendi, D.; Sheppard, J.D. Hydrolysis of Potato Processing Waste for the Production of
Poly-beta-hydroxybutyrate. Bioresource Technol. 1995, 54(2), 191–196.
22. Sheu, D.S.; Chen, W.M.; Yang, J.Y.; Chang, R.C. Thermophilic Bacterium Caldimonas taiwanensis
Produces Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) From Starch and Valerate as Carbon Sources.
Enzyme Microb. Technol. 2009, 44(5), 289–294.
23. Van-Thuoc, D.; Quillaguaman, J.; Mamo, G.; Mattiasson, B. Utilization of Agricultural Residues for
Poly(3-hydroxybutyrate) Production by Halomonas boliviensis LC1. J. App. Microbiol. 2008, 104(2), 420–428.
24. Reddy, C.; Beveridge, T.J.; Breznak, J.; Marzluf, G.; Schmidt, T.; Snyder, L. Methods for General and
Molecular Microbiology. ASM Press: Washington, DC, 2007.
25. Lillo, J.G.; Rodriguez-Valera, F. Effects of Culture Conditions on Poly(beta-hydroxybutyric acid)
Production by Haloferax mediterranei. Appl. Environ. Microbiol. 1990, 56(8), 2517–2521.
26. Hahn, S.K.; Chang, Y.K.; Kim, B.S.; Chang, H.N. Optimization of Microbial Poly(3-hydroxybutyrate)
Recovery Using Dispersions of Sodium-Hypochlorite Solution and Chloroform. Biotechnol. Bioeng.
Downloaded by [Memorial University of Newfoundland] at 00:52 18 July 2014

1994, 44(2), 256–261.

27. Sin, M.C.; Gan, S.N.; Annuar, M.S.M.; Tan, I.K.P. Thermodegradation of Medium-Chain-Length
Poly(3-hydroxyalkanoates) Produced by Pseudomonas putida From Oleic Acid. Polym. Degrad. Stabil.
2010, 95(12), 2334–2342.
28. Du, C.; Sabirova, J.; Soetaert, W.; Ki, C.; Lin, S. Polyhydroxyalkanoates Production From Low-Cost
Sustainable Raw Materials. Curr. Chem. Biol. 2012, 6(1), 14–15.
29. Kim, B.S. Production of Poly(3-hydroxybutyrate) From Inexpensive Substrates. Enzyme Microbiol.
Technol. 2000, 27(10), 774–777.
30. Fabunmi, O.O.; Tabil, L.; Chang, P.; Panigrahi, S. Developing Biodegradable Plastics From Starch.
American Society of Agricultural and Biological Engineers: St. Joseph, MI, 2007.
31. Halami, P.M. Production of Polyhydroxyalkanoate From Starch by the Native Isolate Bacillus cereus
CFR06. World J. Microbiol. Biotechnol. 2008, 24(6), 805–812.
32. Hayashida, S.; Teramoto, Y.; Inoue, T. Production and Characteristics of Raw-Potato-Starch-
Digesting a-Amylase From Bacillus subtilis 65. Appl. Environ. Microbiol. 1988, 54(6), 1516–1522.
33. Krueger, C.L.; Radetski, C.M.; Bendia, A.G.; Oliveira, I.M.; Castro-Silva, M.A.; Rambo, C.R.; Antonio,
R.V.; Lima, A.O.S. Bioconversion of Cassava Starch By-Product Into Bacillus and Related Bacteria
Polyhydroxyalkanoates. Electron. J. Biotechnol. 2012, 15(3), http://dx.doi.org/10.2225/vol2215-
issue2223-fulltext-2226
34. Patel, S.K.S.; Singh, M.; Kalia, V.C. Hydrogen and Polyhydroxybutyrate Producing Abilities of
Bacillus spp. From Glucose in Two Stage System. Ind. J. Microbiol. 2011, 51(4), 418–423.
35. Patel, S.K.S.; Singh, M.; Kumar, P.; Purohit, H.J.; Kalia, V.C. Exploitation of Defined Bacterial
Cultures for Production of Hydrogen and Polyhydroxybutyrate From Pea-Shells. Biomass. Bioenerg.
2012, 36, 218–225.
36. Gonzalez-Garcia, Y.; Rosales, M.A.; Gonzalez-Reynoso, O.; Sanjuan-Duenas, R.; Cordova, J.
Polyhydroxybutyrate Production by Saccharophagus degradans Using Raw Starch as Carbon Source.
Eng. Life Sci. 2011, 11(1), 59–64.
37. Sreekanth, M.; Vijayendra, S.V.N.; Joshi, G.; Shamala, T. Effect of Carbon and Nitrogen Sources
on Simultaneous Production of a-Amylase and Green Food Packaging Polymer by Bacillus sp.
CFR 67. J. Food Sci. Technol. Mysore 2013, 50(2), 404–408.
38. Aremu, M.; Layokun, S.; Solomon, B. Production of Poly (3-Hydroxybutyrate) From Cassava
Starch Hydrolysate by Pseudomonas aeruginosa NCIB 950. Am. J. Sci. Ind. Res. 2010, 1(3), 421–426.
39. Haas, R.; Jin, B.; Zepf, F.T. Production of Poly (3-Hydroxybutyrate) From Waste Potato Starch. Biosci.
Biotechnol. Biochem. 2008, 72(1), 253–256.
40. Quillaguaman, J.; Hashim, S.; Bento, F.; Mattiasson, B.; Hatti-Kaul, R. Poly(b-Hydroxybutyrate)
Production by a Moderate Halophile, Halomonas boliviensis LC1 Using Starch Hydrolysate as Substrate.
J. Appl Microbiol. 2005, 99(1), 151–157.
41. Huang, T.Y.; Duan, K.J.; Huang, S.Y.; Chen, C.W. Production of Polyhydroxyalkanoates From
Inexpensive Extruded Rice Bran and Starch by Haloferax mediterranei. J. Ind. Microbiol. Biotechnol.
2006, 33(8), 701–706.
42. Aremu, M.; Olu-Arotiowa, O.; Layokun, S.; Solomon, B. Growth of Pseudomonas fluorescens on Cassava
Starch Hydrolysate for Polyhydroxybutyrate Production. J. Appl. Sci. Environ. Manage. 2011, 14(4), 61–66.
43. Yu, J. Production of PHA From Starchy Wastewater Via Organic Acids. J. Biotechnol. 2001, 86(2), 105–112.
44. Kim, B.S.; Chang, H.N. Production of Poly (3-Hydroxybutyrate) From Starch by Azotobacter
chroococcum. Biotechnol. Lett. 1998, 20(2), 109–112.