Purification and characterization of two polyhydroxyalcanoates from Bacillus cereus

International Journal of Biological Macromolecules 61 (2013) 82–88

Contents lists available at SciVerse ScienceDirect

International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Purification and characterization of two polyhydroxyalcanoates from Bacillus cereus Emna Zribi-Maaloul, Imen Trabelsi, Lobna Elleuch, Hichem Chouayekh, Riadh Ben Salah ∗ Laboratory of Microorganisms and Biomolecules (LMB), Centre of Biotechnology of Sfax, Road of Sidi Mansour Km 6, P.O. Box 1177, Sfax 3018, Tunisia

a r t i c l e

i n f o

Article history: Received 17 April 2013 Received in revised form 21 June 2013 Accepted 26 June 2013 Available online xxx Keywords: Bacillus Bioplastic Polyhydroxyalkanoates GC–MS FT-IR

a b s t r a c t This work aimed to study the potential of 155 strains of Bacillus sp., isolated from a collection of Tunisian microorganisms, for polyhydroxyalcanoates production. The strains were submitted to a battery of standard tests commonly used for determining bioplastic properties. The findings revealed that two of the isolates, namely Bacillus US 163 and US 177, provided red excitations at a wavelength of approximately 543 nm. The polyhydroxyalcanoates produced by the two strains were purified. Gas chromatography–mass spectroscopy (GC–MS), Fourier transformed infrared spectroscopy (FTIR), and gel permeation chromatography (GPC) were used to characterize the two biopolymers. Bacillus US 163 was noted to produce a poly methyl-3-hydroxy tetradecanoic acid (P-3HTD) with an average molecular weight of 455 kDa, a completely amorphous homopolymer without crystallinity. The US 177 strain produced a homopolymer of methyl-3-hydroxy octadecanoic acid (P3-HOD) with an average molecular weight of 555 kDa. Exhibiting the highest performance, US 163 and US 177 were submitted to 16S rRNA gene sequencing, and the results revealed that they belonged to the Bacillus cereus species. Overall, the findings indicated that the Bacilli from petroleum soil have a number of promising properties that make them promising candidates for bioplastic production. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Bioplastics are a special type of biopolymer defined by the American Society for Testing Materials as “degradable plastic in which the degradation results from the action of naturally occurring microorganisms such as bacteria, fungi and algae” [1]. They are polyesters, produced by a range of microoganisms, such as Bacillus, Pseudomonas, Aeromonas [2], Aeromonascaviae, Burkholderia sp. [3], Comamonas sp. EB172 [4], and fungi, such as Rhizopus oryzae [5], cultured under different nutrient and environmental conditions. These polymers, which are usually lipid in nature, are accumulated as storage materials (in the form of mobile, amorphous, and liquid granules), allowing microbial survival under stress conditions [6,7]. The number and size of these granules, monomer composition, macromolecular structure, and physico-chemical properties vary, depending on the producer microorganisms [8,9]. They can be observed as intracellular light-refracting granules or as electronlucent bodies that, in overproducing mutants, cause a striking alteration of the bacterial shape.

∗ Corresponding author at: Laboratoire de Microorganismes et de Biomolécules (LMB), Centre de Biotechnologie de Sfax, Route de Sidi Mansour Km 6, BP “1177”, 3018 Sfax, Tunisia. Tel.: +216 74 87 04 51; fax: +216 74 87 04 51. E-mail addresses: riadh [email protected], [email protected] (R. Ben Salah). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.06.043

Polyhydroxyalkanoates (PHAs) are a class of biopolymers formed as naturally occurring storage polyesters by a wide diversity of microorganisms [10]. They are deposited as spherical intracellular inclusions with an amorphous hydrophobic PHA core which is mainly surrounded by proteins involved in PHA metabolism [11,12]. The weight of the polymer can range from 200 to 3000 kDa, depending on the organism and conditions under which it was produced [13]. PHAs can vary substantially in composition, as there are over 150 known constituents, resulting in a wide diversity of material properties. These bio-polymers also exhibit a crystallinity index ranging from 30% to 70% and a melting temperature of 50 ◦ C to 180 ◦ C, two thermoplastic material properties that make them valuable alternatives to oil-based plastics [10]. PHAs can be classified by chain length, with medium-chain-length PHAs (which have constituent C6 C14 chains) being produced mainly by Pseudomonas [14] and short-chain-length PHAs (which have constituent C3 C5 chains) being produced by a wide range of bacteria and archaea [14,15]. PHAs have a wide range of industrial applications particularly due to their desired properties, including biocompatibility, biodegradability, and low cytotoxicity to cells. They have, for instance, often been considered as efficient substitutes to petrochemically-based polymers in various fields and processes involving packaging and coating materials. Their compounding and blending properties have also broadened the scope of their

E. Zribi-Maaloul et al. / International Journal of Biological Macromolecules 61 (2013) 82–88

performances as potential end-use applications [16]. These biopolymers have also been applied in the production of packaging materials, such as films, boxes, coating, and fibers, as well as of foam materials, biofuels, medical implants, and drug delivery carriers. Due to their valuable properties, cost-effectiveness and ecofriendliness, PHAs have been extensively employed in large-scale applications involving biodegradable packaging materials [17]. They have been manufactured for non-woven materials, polymer films, sutures, and pharmaceutical products used in surgery, transplantology, tissue engineering, pharmacology [18], urological stents, neural- and cardiovascular-tissue engineering, fracture fixation, treatment of narcolepsy and alcohol addiction, drug-delivery vehicles, cell microencapsulation, support of hypophyseal cells, or as precursors of molecules with anti-rheumatic, analgesic, radiopotentiator, chemopreventive, antihelmintic or anti-tumuoral properties (those containing aromatic monomers or those linked to nucleosides) [7,19–24]. In the medical field, PHAs offer a distinct advantage over silicon, a conventional polymer often associated with malign effects, including the induction of cancer cell growth [25]. In order for PHAs to gain access as biomaterial substitutes to silicon, they need to fulfill five key criteria required for application in tissue engineering, namely biocompatibility; support of cell growth and adhesion; guidance and organization of cells; promotion of cell growth, passage of nutrients, and waste products; and biodegradability without the production of harmful compounds [26]. Considering the promising properties and attributes of Bacillus strains, the present study was undertaken to investigate and evaluate 155 Bacillus strains, designated US 100 to US 255, isolated from Tunisian petroleum soils, in terms of their viability and potential for the production of polyhydroyalcanoates. The strains were screened for a number of traits, namely red excitation in agar plates and culture media, at wavelengths between 280 nm and 543 nm. The bioplastics produced were purified and characterized by GC–MS, GPC, and FTIR. Two stains, namely US 163 and US 177, which exhibited high levels of bioplastic production were submitted to further 16S ribosomal RNA (rRNA) gene sequencing, and were identified as belonging to the Bacillus cereus species. 2. Materials and methods 2.1. Strains Bacillus strains were purchased from the Tunisian Collection of Microorganisms of Centre of Biotechnology of Sfax. 2.2. Screening of Bacillus strains in agar plates for PHA productions A staining solution of Nile blue A was prepared by dissolving 0.05 g of Nile blue A in l00 m1 of ethanol. Colonies on the agar plate were stained with 5 ml of the staining solution. After 20 min, the staining solution was removed from the agar plate, and the plate was left for a period of time sufficient to dry the surface [27]. The Nile blue A stained colonies were irradiated with a short wave ultraviolet light at 520, 320, 360, and 280 nm from a Mineralight UV lamp [27]. 2.3. Screening of Bacillus strains in culture medium for PHA productions Heat-fixed smears of bacterial cells were stained with the Nile blue A solution (1%; w:v) at 55 ◦ C for 10 min in a coplin staining jar. The slides were then washed with tap water and 8% aqueous acetic acid for 1 min to remove excess of stain. After that, the stained smear was washed and blotted dry with bibulous paper,

83

remoistened with tap water, and covered with a glass cover slip. The preparation was examined using a confocal microscope with an episcopic fluorescence attachment. A red excitation method that provided an excitation wavelength of approximately 543 nm was used [28]. 2.4. Growth kinetics of Bacillus strains Bacillus inoculum preparation was performed by transferring the microorganism from the stock solution to Luria Bertani (LB) agar plates and subsequent incubation for 24 h at 37 ◦ C. A loopful of cells was then transferred from the LB agar plates to 100-ml conical flasks containing 50 ml of sterile LB media and incubated for 24 h at 37 ◦ C and 250 rpm. This culture was used as the inoculum. Fermentation was carried out in 250-ml Erlenmeyer flasks containing 50 ml of the sterile production medium. The latter was inoculated with 5% (v/v) of 24-h old Bacillus culture. A sample was taken every 2 h, and OD was measured by a spectrophotometer at 600 nm. The medium used for growth and maintenance (LB-agar) contained (g/l): peptone, 10; yeast extract, 5; NaCl, 10; and agar, 17 (pH 7). Bacterial cells in the agar slants were incubated for 24 h at 37 ◦ C. 2.5. Product characterization 2.5.1. Gas chromatography Both lyophilized cells and the extracted pure PHA were submitted to methanolysis [29]. Benzoic acid was used as an internal standard. After fermentation, the culture broth was concentrated by centrifugation at 4000 rpm for 20 min. The residues were filtered and freeze-dried. The PHAs were extracted from the dried cell through esterification, which consisted of the following reagents: 0.29 g of benzoic acid, 3 ml of concentrated 98% H2SO4, and 97 ml of methanol. During extraction, 1 ml of the esterification solution and 1 ml of chloroform were added to the tubes containing 10–14 mg of the samples. The mixed samples were heated for 4 h at 100 ◦ C. Afterward, 1 ml of distilled water was added to the cooled solution, and the mixture was vortexed for 1 min. The mixture was then left overnight to separate into two layers. The bottom layer, which contained dissolved PHA, was used for subsequent analysis [29]. PHA content and composition were determined by Agilent 19091S-433 gas chromatography equipped with a fused HP-5MS 5% Phenyl Methyl Siloxane column (length 30 m; diameter 250 ␮m; and film thickness 0.25 ␮m) (Agilent, USA). The resulting 0.4 ml of methyl esters were injected into the gas chromatography column. This was initially performed at 100 ◦ C for 3 min, and the temperature was then increased at a rate of 8 ◦ C/min to reach 220 ◦ C, which was maintained for 5 min before the end of analysis. A PHA standard mixture containing various long-chain-length monomers (kindly provided by Dr. Lobna Jlaiel was used for PHA monomer band identification. 2.5.2. FT-IR spectroscopy FT-IR spectra were recorded using a JASCOFT/IR430 spectrometer (JASCO Corp., Japan) over the 400–4000 cm−1 range at a spectral resolution of 4 cm−1 . The PHA was directly extracted using chloroform. Initially, the bacterial cultures were harvested by centrifugation at 5000 rpm for 10 min. The lipids were then removed from the cell pellet-using methanol (10 times the volume of cell pellets), and the cells were incubated for 1 h at 95 ◦ C. The suspension was then filtered to fully remove methanol, and the sediment granules were incubated in an oven at 65 ◦ C till becoming dry. Chloroform was added to the dried granules and incubated at for 10 min 95 ◦ C. After cooling, the solution was gently mixed overnight and then filtered to get the debris. Finally, the PHA was precipitated

84

E. Zribi-Maaloul et al. / International Journal of Biological Macromolecules 61 (2013) 82–88

from the debris using 7:3 (v/v) mixtures of methanol and water. The precipitated PHA was washed with acetone and dried [30]. 2.6. Study of PHA molecular weight using GPC Average molecular weights were estimated by GPC (Waters 1525, USA) with a combination of four styragel column series (Styragel HR, 5 mm) at 40 ◦ C using a 2414 differential refractive index detector and a UV detector, respectively. Chloroform was used as an eluent at a flow rate of 1.0 ml/min. The sample was prepared at a concentration of 1 mg/ml and injected at a volume of 50 ␮L. A calibration curve was generated using polystyrene standards of low polydispersity.

matches for the sequences was performed using the Basic Local Alignment Search Tool (BLAST) program available at the National Center for Biotechnology Information (NCBI; Bethesda, MD, USA) [33]. 2.8. Phylogenetic analysis of Bacillus strains The nucleotide sequences of the whole 16S rRNA gene (1555 bp) of the US 163 and US 177 strains have been deposited in GenBank (EMBL) under accession numbers ID1600075 and ID1600108, respectively. The sequences were aligned using clustalW [34], and a phylogenetic analysis was performed using the PHYLIP software package [35]. The phylogenetic tree was constructed using the neighbor joining (NJ) method [36].

2.7. Sequencing of the 16S rRNA gene of the Bacillus isolates 2.9. Statistical analysis The chromosomal DNA from the Bacillus strains was extracted as previously described elsewhere [31]. The isolate was identified by sequencing the total sequence of the 16S rRNA gene amplified with primers S73 (5 -AGAGTTTGATCCTGGCTCAG-3 ) and S74 (5 -AAGGAGGTGATCCAAGCC-3 ) [32]. The amplification program consisted of 1 cycle of 95 ◦ C/5 min and 30 cycles of 94 ◦ C/40 s, 52 ◦ C/1 min, and 72 ◦ C/1 min 30 s, with a final extension at 72 ◦ C/10 min. The PCR products were purified using the Wizard SV Gel and PCR Clean-Up system (Promega). Sequencing was performed with an ABI 3100 Capillary DNA Sequencer (Applied Biosystems Inc., Foster City, CA, USA). A search for the closest 16S

The data presented in the current study are the means of three replications and are expressed as the mean ± standard deviation (X¯ ± SD). 3. Results and discussion 3.1. Screening of Bacillus strains in LB agar plates A collection of 155 strains of Bacillus sp. isolated from petroleum soil samples was investigated for their abilities to produce PHA.

Fig. 1. Fluorescence of PHA granules using Nile Blue A staining at 280 nm: (A) US 100, US 106, US 177, US 500 strains; (B) US 183, US 531, US 554, US 516 strains, (C): US 195, US 400, US 113, US 163 strains, and (D) Beta 5, Beta 4 strains. The white colonies are the bioplastic strains produced. The dark colonies are the bioplastic non-producing strains.

E. Zribi-Maaloul et al. / International Journal of Biological Macromolecules 61 (2013) 82–88

85

Fig. 2. PHA granules within Bacillus strains stained with Nile blue A observed under fluorescent light. The preparations were from exponential phase cells. The Nile blue A stained cells were scanned with ZEISS LSM 510 confocal laser scanning microscopy at 543 nm and 64 magnifications: (A) US 195 (negative control), (B) US 163; and (C) US 177.

The bacteria were initially screened for PHA production in Luria Bertani agar plates and using Nile Blue A staining to investigate their abilities to synthesize PHA granules. The formation of low-molecular weight fluorescent compounds was observed at an emission wavelength of 280 nm. Based on the intensity of the fluorescence observed, 8 strains were identified as potential PHA producers: US 100; US 106; US 163; US 177; US 183; US 531; US 554 and Beta 5) (Fig. 1). The PHA non-producing strains were noted to yield a dark smear (Fig. 1). (Insert Fig. 1)

3.2. Screening of Bacillus strains in LB liquid medium Confocal laser scanning fluorescence microscopy is a powerful technique for the detection of PHA granule formation at a wavelength of 543 nm. A ZEISS LSM 510 confocal laser scanning microscopy was, therefore, employed to investigate the PHA production abilities of the 8 Bacillus strains selected. The results

revealed that 2 among the 8 strains under investigation, namely US163 and US177, displayed reddish orange fluorescence (Fig. 2). The PHA non-producing strains were, on the other hand, noted to exhibit black smear or red spots (Fig. 2). (Insert Fig. 2)

3.3. Growth kinetic of PHA produced strains The growth kinetics of the two strains were investigated in LB media at 37 ◦ C and 250 rpm to determine the growth phase corresponding to the production of PHA. Growth kinetics were monitored by measuring optical density at 600 nm (OD600 nm ) every 2 h for 24 h (data not shown). Based on the intensity of the fluorescence observed for the lag, exponential, stationary and decline phases during the staining procedure, the phase corresponding to PHA production was identified to occur during the exponential phase (data not shown).

Fig. 3. FT-IR spectrum of PHA produced by the new US 163 strain.

86

E. Zribi-Maaloul et al. / International Journal of Biological Macromolecules 61 (2013) 82–88

Fig. 4. FT-IR spectrum of PHA produced by the new US 177 strain.

3.4. FTIR analysis of the novel PHA The FTIR spectra of the PHA produced by the two strains were determined. An intensity band was observed at around 1220 cm−1 , which was assigned to the stretching vibration of the C O C group. The region of 1675–1735 cm−1 was associated with the C O stretching of the ester carbonyl bond. The bond at 1530 cm−1 was characteristic of the stretching and deformation vibration of the C H group, and those at 2930 cm−1 and 3272 cm−1 were

characteristic of the stretching and deformation vibrations of the O H groups (Figs. 3–4). The FTIR peaks were identical to those displayed by commercial PHA (Fig. 5). The functional groups of the polymer PHA were confirmed as C O groups by FT-IR spectroscopy. The results described above are congruent with the findings previously reported by several studies in the literature [37], particularly 2933 cm−1 (CH, CH2, CH3), 1720 cm−1 (ester C O valence), and 1639 cm−1 (thioester C O valence). Phukon et al. [38] also sunbmitted the PHA produced by Bacillus circulans (MTCC 8167) to

Fig. 5. FT-IR spectrum of the contol (commercial PHA).

E. Zribi-Maaloul et al. / International Journal of Biological Macromolecules 61 (2013) 82–88

87

Table 1 Structure of the two polyhydroxyalacanoates produced by Bacillus cereus US 163 and Bacillus cereus US 177. Strains

Retention time (min)

Monomers of produced PHA

Bacillus cereus US 163

16.548

Tetradecanoic acid, 3-hydroxy, methylester

Bacillus cereus US 177

18.560

Octadecanoic acid, 3-hydroxy, methylester

FT-IR characterization. The FTIR spectra showed high absorbance at 3360, 2922, 1735, and 1206 cm−1 , which resulted in the vibration functions of O H, C H, C O, and C O C, respectively. Likewise, Phukon et al. [39] analyzed the P(3HB-co-3HV) bioplastic produced by Bacillus circulans MTCC 8167 strain by FT-IR. They indicated that the biopolymer obtained after solvent extraction exhibited C H and carbonyl stretching bands similar to those of standard PHA. The presence of intense absorption bands at 1735 cm−1 and 1206 cm−1 were reported to be characteristic of the C O and C O stretching groups. Arcos-Hernandez et al. [40] also characterized the functions of the P(3-HB-co-3HV) produced by a mixed culture of bacterial systems by FT-IR. The FT-IR spectra showed high absorbance at 1753 and 1200–900 cm−1 , which resulted in the vibration functions of C O and C O C, respectively. (Insert Figs. 3–5)

Lee and Choi [41] have previously analyzed the PHA product after hydrolysis with GC–MS. They reported on the presence of the 4-hydroxy butanoic, 4-hydroxy valeric and 4-hydroxy hexanoic acids among the hydrolysis products. Phukon et al. [39] have also submitted the bioplastic produced by Bacillus circulans MTCC 8167 to GC–MS analysis and showed that the strain produced the P(3HB-co-3HV). Likewise, He et al. [42] submitted Pseudomonas stutzeri 1317 to GC–MS analysis and reported on the production of a novel polyhydroxyalkanoates from glucose and soybean oil. These included the methyl esters of 3-hydroxy-hexanoic acid, 3hydroxy-octanoic acid, 3-hydroxy decanoic acid 3HD, 3-hydroxy 5-dodecenoic acid, 3-hydroxy dodecanoic acid, and 3-hydroxy tetradecanoic acid. 3.6. GPC analysis of the novel PHA

3.5. GC–MS analysis of the novel PHA The bioplastics produced by the US 163 and US 177 strains were analyzed by GC–MS. The molecules that were obtained are shown in Table 1. Compared to the database molecules, the US 163 strain was noted to produce the methyl-3-hydroxy tetradecanoic acid, and the US 177 strain produced the methyl-3-hydroxy octadecanoic acid belonging to the long-chain-length PHA class. (Insert Table 1)

GPC analysis showed that the PHA produced by strains US163 and US177 were a poly tetradecanoic acid (PTD) and poly octadecanoic acid (PTO) homopolymers with average molecular weights of 435 and 555 kDa, respectively. In fact, Oliveira et al. [43] reported the molecular weight of the extracted PHB to be about 5.2 × 105 Da. Phukon et al. [39] have previously submitted the PHBV polymer produced by Bacillus circulans (MTCC 8167) to GPC analysis and reported that the polymer had a molecular mass of 5.1 × 104 Da.

Fig. 6. Phylogenetic tree derived from 16S rDNA sequences; the positions of US 163 and US 177 strains were indicated. All the sequences used were from the Bacillus type strains.

88

E. Zribi-Maaloul et al. / International Journal of Biological Macromolecules 61 (2013) 82–88

3.7. Identification and phylogenetic analysis of Bacillus US163 and US177 The total nucleotide sequences of 1.472 pb and 1.496 pb were determined from the whole 16S rRNA genes of strains US163 and US177. The alignment of these sequences with previous 16S rRNA gene sequences available at the Gene bank database showed high similarity (99%) with the Bacillus 16S rRNA reference genes. The closest similarity recorded for the new isolates US 163 and US 177 strains were with Bacillus cereus GQ28038.1 and Bacillus cereus FJ982658.1, respectively (Fig. 6). Based on the nucleotide sequence of the 16S rRNA gene of the two strains and the findings from subsequent phylogenetic analyses, the new isolates were identified as Bacillus cereus US163 and Bacillus cereus US 177, respectively. (Insert Fig. 6)

[17]

[18] [19] [20] [21]

[22]

[23]

Acknowledgements The authors would like to express their sincere gratitude to Dr. Lobna Jlaiel for kindly providing the long-chain-length monomers used in this study. Thanks are also due to Mr Anouar Smaoui from the English Language Unit at the Sfax Faculty of Science for his constructive proofreading and language polishing services.

[24] [25]

[26]

[27]

References [1] B.P. Mooney, The second green revolution; production of plant-based biodegradable plastics, Biochemical Journal 418 (2009) 219–232. [2] L.L. Madison, G.W. Huisman, Metabolic engineering of poly(3hydroxyalkanoates): from DNA to plastic, Microbiology and Molecular Biology Reviews 63 (1999) 21–53. [3] J.Y. Chee, Y. Tan, M.R. Samian, K. Sudesh, Isolation, Characterization of a Burkholderia sp. USM (JCM15050) capable of producing polyhydroxyalkanoate (PHA) from triglycerides, fatty acids and glycerols, Journal of Polymers and the Environment 18 (2010) 584–592. [4] M.R. Zakaria, M. Tabatabaei, F.M. Ghazali, S. Abd-Aziz, Y. Shirai, M.A. Hassan, Polyhydroxyalkanoate production from anaerobically treated palm oil mill effluent by new bacterial strain Comamonas sp. EB172, World Journal of Microbiology and Biotechnology 26 (2010) 767–774. [5] C. Accinelli, M.L. Saccà, M. Mencarelli, A. Vicari, Deterioration of bioplastic carrier bags in the environment and assessment of a new recycling alternative, Chemosphere 89 (2012) 136–143. [6] G.N. Barnard, J.K. Sanders, The polyhydroxybutyrate granule in vivo. A new insight based on NMR spectroscopy of whole cells, Journal of Biological Chemistry 264 (1989) 3286–3291. [7] K. Sudesh, H. Abe, Y. Doi, Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters, synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters, Progress in Polymer Science 25 (2000) 1503–1555. [8] A.J. Anderson, E.A. Dawes, Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates, Microbiology Reviews 54 (1990) 450–472. [9] R.G.E. Murray, R.D. Doetsch, C.F. Robinow, Determinative and cytological light microscopy, in: Manual of Methods for General Bacteriology, American Society for Microbiology, Washington, 1994, pp. 21–41. [10] B.H.A. Rehm, Bacterial polymers: biosynthesis, modifications and applications applied and industrial microbiology, Nature Reviews Microbiology 8 (2010) 578–592. [11] K. Grage, A.C. Jahns, N. Parlane, R. Palanisamy, I.A. Rasiah, J.A. Atwood, B.H.A. Rehm, Bacterial polyhydroxyalkanoate granules: biogenesis, structure, and potential use as nano-micro-beads in biotechnological and biomedical applications, Biomacromolecules 10 (2009) 660–669. [12] D. Jendrossek, Polyhydroxyalkanoate granules are complex subcellular organelles (carbonosomes), Journal of Bacteriology 191 (2009) 3195–3202. [13] S. Khanna, A.K. Srivastava, Recent advances in microbial polyhydroxyalkanoates, Process Biochemistry 40 (2005) 607–619. [14] A.J. Anderson, G.W. Haywood, E.A. Dawes, Biosynthesis and composition of bacterial polyhydroxyalkanoates, International Journal of Biological Macromolecules 12 (1990) 102–105. [15] J. Reemmer, Advances in the synthesis and extraction of biodegradable polyhydroxyalkanoates in plant systems: a review, MMG 445 Basic Biotechnology 5 (2009) 44–49. [16] J.Y. Chee, S.S. Yoga, N.S. Lau, S.C. Ling, R.M.M. Abed, K. Sudesh, Bacterially produced polyhydroxyalkanoate (PHA): converting renewable resources into

[28]

[29]

[30]

[31]

[32]

[33] [34]

[35] [36] [37]

[38]

[39]

[40]

[41]

[42]

[43]

bioplastics, Current Research, Technology and Education Topics in Applied Microbial Biotechnology (2010) 1395–1404. G.-Q. Chen, Q. Wu, Y.K. Jung, S.Y. Lee, Comprehensive Biotechnology: Industrial Biotechnology and Commodity Products, Academic Press, Canada, 2011, pp. 217–227. I. Noda, Process for recovering polyhydroxyalkanoates using air classification, U.S. Patent 5 (1998) 849–854. A. Steinbuchel, B. Fuchtenbusch, Bacterial and other biological systems for polyester production, Trends in Biotechnology 16 (1998) 419–427. N. Angelova, D. Hunkeler, Rationalizing the design of polymeric biomaterials, Trends in Biotechnology 17 (1999) 409–421. B. Witholt, B. Kessler, Perspectives of medium chain length poly(hydroxyalkanotes), a versatile set of bacterial bioplastics, Current Opinion in Biotechnology 10 (1999) 279–285. G.A. Abraham, A. Gallardo, J. San Roman, E.R. Olivera, R. Jodra, B. Garcia, B. Minambres, J.L. Garcia, J.M. Luengo, Microbial synthesis of poly(bhydroxyalkanoates) bearing phenyl groups from Pseudomonas putida: chemical structure and characterization, Biomacromolecules 2 (2001) 562–567. E.R. Olivera, D. Carnicero, R. Jodra, B. Minambres, B. Garcia, G.A. Abraham, A. Gallardo, J. San Roman, J.L. Garcia, G. Naharro, Genetically engineered Pseudomonas: a factory of new bioplastics with broad applications, Environmental Microbiology 3 (2001) 612–618. D. Samid, Methods for therapy of cancer, U.S. Patent 6037376 (2000). M. Zinn, B. Witholt, T. Egli, Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate, Advanced Drug Delivery Reviews 53 (2001) 5–21. S. Cammas, M.M. Bear, L. Moine, R. Escalup, G. Ponchel, K. Kataoka, P. Guerin, Polymers of malic acid and 3-alkylmalic acid as synthetic PHAs in the design of biocompatible hydrolyzable devices, International Journal of Biological Macromolecules 25 (1999) 273–282. S. Kitamura, Y. Doi, Staining method of poly(3-hydroxyalkanoic acids) producing bacteria by Nile blue, Biotechnology Technology 8 (1994) 345–350. A.G. Ostle, J.G. Holt, Nile Blue A as a fluorescent stain for poly3-hydroxybutyrate, Applied and Environmental Microbiology 44 (1982) 238–241. K.W. Lo, H. Chua, H. Lawford, W.H. Lo, P.H.F. Yu, Effects of fatty acids on growth and poly-3-hydroxybutyrate production in bacteria, Applied Biochemistry and Biotechnology 121–124 (2005) 575–580. A. Santhanam, S. Sasidharan, Microbial production of polyhydroxyalkanotes (PHA) from Alcaligens spp.and Pseudomonas oleovorans using different carbon sources, African Journal of Biotechnology 9 (2010) 3144–3150. E. Malinen, R. Laitinen, A. Palva, Genetic labeling of Lactobacilli in a food grade manner for strain-specific detection of industrial starters and probiotic strains, Food Microbiology 18 (2001) 309–317. J. Doré, A. Sghir, G. Hannequart-Gramet, G. Corthier, P. Pochart, Design and evaluation of a 16S rRNA-targeted oligonucleotide probe for specific detection and quantitation of human faecal bacteroides populations, Systematic and Applied Microbiology 21 (1998) 65–71. S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman, Basic local alignment search tool, Journal of Molecular Biology 215 (1990) 403–410. J.D. Thompson, D.G. Higgins, T.J. Gibson, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice, Nucleic Acids Research 22 (1994) 4673–4680. J. Felsenstein, PHYLIP-phylogeny inference package, version 3.2, Cladistics 5 (1989) 164–166. N. Saitou, M. Nei, Center for demographic and population genetics, Molecular Biology and Evolution 4 (1987) 406–425. K.R. Shah, FTIR analysis of polyhydroxyalkanoates by novel Bacillus sp. AS 3-2 from soil of Kadi region, North Gujarat, India, Journal of Biochemical Technology 3 (2012) 0974–2328. P. Phukon, J.P. Saikia, B.K. Konwar, Enhancing the stability of colloidal silver nanoparticles using polyhydroxyalkanoates (PHA) from Bacillus circulans (MTCC 8167) isolated from crude oil contaminated soil, Colloids and Surfaces B 86 (2011) 314–318. P. Phukon, J.P. Saikia, B.K. Konwar, Bio-plastic (P-3HB-co-3HV) from Bacillus circulans (MTCC 8167) and its biodegradation, Colloids and Surfaces B 92 (2012) 30–34. M.V. Arcos-Hernandez, N. Gurieff, S. Pratt, P. Magnusson, A. Werker, A. Vargas, P. Lant, Rapid quantification of intracellular PHA using infrared spectroscopy: an application in mixed cultures, Journal of Biotechnology 150 (2010) 372–379. E.Y. Lee, C.Y. Choi, Structural identification of polyhydroxyalkanoic acid (PHA) containing 4-hydroxyalkanoic acids by gas chromatography–mass spectrometry (GC–MS) and its application to bacteria screening, Biotechnology Techniques 11 (1997) 167–171. W. He, W. Tian, G. Zhang, G.Q. Chen, Z. Zhang, Production of novel polyhydroxyalkanoates by Pseudomonas stutzeri 1317 from glucose and soybean oil, FEMS Microbiology Letters 169 (1998) 45–49. F.C. Oliveira, M.L. Dias, L.R. Castilho, D.M.G. Freire, Characterization of poly(3hydroxybutyrate) produced by Cupriavidus necator in solid state fermentation, Bioresource Technology 98 (2007) 633–638.