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

Contents lists available at SciVerse ScienceDirect

International Journal of Biological Macromolecules

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urification and characterization of two polyhydroxyalcanoates fromacillus cereus

mna Zribi-Maaloul, Imen Trabelsi, Lobna Elleuch, Hichem Chouayekh, Riadh Ben Salah ∗

aboratory 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

rticle history:eceived 17 April 2013eceived in revised form 21 June 2013ccepted 26 June 2013vailable online xxx

eywords:acillusioplastic

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 ofTunisian microorganisms, for polyhydroxyalcanoates production. The strains were submitted to a bat-tery of standard tests commonly used for determining bioplastic properties. The findings revealed thattwo of the isolates, namely Bacillus US 163 and US 177, provided red excitations at a wavelength ofapproximately 543 nm. The polyhydroxyalcanoates produced by the two strains were purified. Gaschromatography–mass spectroscopy (GC–MS), Fourier transformed infrared spectroscopy (FTIR), andgel permeation chromatography (GPC) were used to characterize the two biopolymers. Bacillus US 163was noted to produce a poly methyl-3-hydroxy tetradecanoic acid (P-3HTD) with an average molecu-

olyhydroxyalkanoatesC–MST-IR

lar weight of 455 kDa, a completely amorphous homopolymer without crystallinity. The US 177 strainproduced a homopolymer of methyl-3-hydroxy octadecanoic acid (P3-HOD) with an average molecularweight of 555 kDa. Exhibiting the highest performance, US 163 and US 177 were submitted to 16S rRNAgene sequencing, and the results revealed that they belonged to the Bacillus cereus species. Overall, thefindings indicated that the Bacilli from petroleum soil have a number of promising properties that makethem promising candidates for bioplastic production.

© 2013 Elsevier B.V. All rights reserved.

. Introduction

Bioplastics are a special type of biopolymer defined by themerican Society for Testing Materials as “degradable plastic inhich the degradation results from the action of naturally occur-

ing microorganisms such as bacteria, fungi and algae” [1]. They areolyesters, produced by a range of microoganisms, such as Bacillus,seudomonas, Aeromonas [2], Aeromonascaviae, Burkholderia sp. [3],omamonas sp. EB172 [4], and fungi, such as Rhizopus oryzae [5],ultured under different nutrient and environmental conditions.hese polymers, which are usually lipid in nature, are accumulateds storage materials (in the form of mobile, amorphous, and liq-id granules), allowing microbial survival under stress conditions6,7]. The number and size of these granules, monomer composi-ion, macromolecular structure, and physico-chemical propertiesary, depending on the producer microorganisms [8,9]. They can

e observed as intracellular light-refracting granules or as elec-ronlucent bodies that, in overproducing mutants, cause a strikinglteration of the bacterial shape.

∗ Corresponding author at: Laboratoire de Microorganismes et de BiomoléculesLMB), Centre de Biotechnologie de Sfax, Route de Sidi Mansour Km 6, BP “1177”,018 Sfax, Tunisia. Tel.: +216 74 87 04 51; fax: +216 74 87 04 51.

E-mail addresses: riadh fss@yahoo.fr, riadh.bensalah@cbs.rnrt.tn (R. Ben Salah).

141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijbiomac.2013.06.043

Polyhydroxyalkanoates (PHAs) are a class of biopolymersformed as naturally occurring storage polyesters by a wide diversityof microorganisms [10]. They are deposited as spherical intracel-lular inclusions with an amorphous hydrophobic PHA core whichis 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 pro-duced [13]. PHAs can vary substantially in composition, as thereare over 150 known constituents, resulting in a wide diversity ofmaterial properties. These bio-polymers also exhibit a crystallinityindex ranging from 30% to 70% and a melting temperature of50 ◦C to 180 ◦C, two thermoplastic material properties that makethem valuable alternatives to oil-based plastics [10]. PHAs canbe classified by chain length, with medium-chain-length PHAs(which have constituent C6 C14 chains) being produced mainly byPseudomonas [14] and short-chain-length PHAs (which have con-stituent C3 C5 chains) being produced by a wide range of bacteriaand archaea [14,15].

PHAs have a wide range of industrial applications particularlydue to their desired properties, including biocompatibility,biodegradability, and low cytotoxicity to cells. They have,

for instance, often been considered as efficient substitutes topetrochemically-based polymers in various fields and processesinvolving packaging and coating materials. Their compoundingand blending properties have also broadened the scope of their

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erformances as potential end-use applications [16]. These bio-olymers have also been applied in the production of packagingaterials, such as films, boxes, coating, and fibers, as well as of foamaterials, biofuels, medical implants, and drug delivery carriers.Due to their valuable properties, cost-effectiveness and eco-

riendliness, PHAs have been extensively employed in large-scalepplications involving biodegradable packaging materials [17].hey have been manufactured for non-woven materials, poly-er films, sutures, and pharmaceutical products used in surgery,

ransplantology, tissue engineering, pharmacology [18], urologicaltents, neural- and cardiovascular-tissue engineering, fracture fix-tion, treatment of narcolepsy and alcohol addiction, drug-deliveryehicles, cell microencapsulation, support of hypophyseal cells, ors precursors of molecules with anti-rheumatic, analgesic, radiopo-entiator, chemopreventive, antihelmintic or anti-tumuoral prop-rties (those containing aromatic monomers or those linked toucleosides) [7,19–24].

In the medical field, PHAs offer a distinct advantage over sili-on, a conventional polymer often associated with malign effects,ncluding the induction of cancer cell growth [25]. In order forHAs to gain access as biomaterial substitutes to silicon, theyeed to fulfill five key criteria required for application in tissuengineering, namely biocompatibility; support of cell growth anddhesion; guidance and organization of cells; promotion of cellrowth, passage of nutrients, and waste products; and biodegrad-bility without the production of harmful compounds [26].

Considering the promising properties and attributes of Bacillustrains, the present study was undertaken to investigate and evalu-te 155 Bacillus strains, designated US 100 to US 255, isolated fromunisian petroleum soils, in terms of their viability and potential forhe production of polyhydroyalcanoates. The strains were screenedor a number of traits, namely red excitation in agar plates andulture media, at wavelengths between 280 nm and 543 nm. Theioplastics produced were purified and characterized by GC–MS,PC, and FTIR. Two stains, namely US 163 and US 177, which exhib-

ted high levels of bioplastic production were submitted to further6S ribosomal RNA (rRNA) gene sequencing, and were identified aselonging to the Bacillus cereus species.

. Materials and methods

.1. Strains

Bacillus strains were purchased from the Tunisian Collection oficroorganisms of Centre of Biotechnology of Sfax.

.2. Screening of Bacillus strains in agar plates for PHAroductions

A staining solution of Nile blue A was prepared by dissolving.05 g of Nile blue A in l00 m1 of ethanol. Colonies on the agar plateere stained with 5 ml of the staining solution. After 20 min, the

taining solution was removed from the agar plate, and the plateas left for a period of time sufficient to dry the surface [27]. Theile blue A stained colonies were irradiated with a short wave ultra-iolet light at 520, 320, 360, and 280 nm from a Mineralight UV lamp27].

.3. Screening of Bacillus strains in culture medium for PHAroductions

Heat-fixed smears of bacterial cells were stained with the Nile

lue A solution (1%; w:v) at 55 ◦C for 10 min in a coplin staining

ar. The slides were then washed with tap water and 8% aque-us acetic acid for 1 min to remove excess of stain. After that, thetained smear was washed and blotted dry with bibulous paper,

iological Macromolecules 61 (2013) 82– 88 83

remoistened with tap water, and covered with a glass cover slip.The preparation was examined using a confocal microscope withan episcopic fluorescence attachment. A red excitation method thatprovided an excitation wavelength of approximately 543 nm wasused [28].

2.4. Growth kinetics of Bacillus strains

Bacillus inoculum preparation was performed by transferringthe microorganism from the stock solution to Luria Bertani (LB)agar plates and subsequent incubation for 24 h at 37 ◦C. A loop-ful of cells was then transferred from the LB agar plates to 100-mlconical flasks containing 50 ml of sterile LB media and incubatedfor 24 h at 37 ◦C and 250 rpm. This culture was used as the inocu-lum. Fermentation was carried out in 250-ml Erlenmeyer flaskscontaining 50 ml of the sterile production medium. The latter wasinoculated with 5% (v/v) of 24-h old Bacillus culture. A sample wastaken every 2 h, and OD was measured by a spectrophotometer at600 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 at37 ◦C.

2.5. Product characterization

2.5.1. Gas chromatographyBoth lyophilized cells and the extracted pure PHA were sub-

mitted to methanolysis [29]. Benzoic acid was used as an internalstandard. After fermentation, the culture broth was concentratedby centrifugation at 4000 rpm for 20 min. The residues were fil-tered and freeze-dried. The PHAs were extracted from the dried cellthrough esterification, which consisted of the following reagents:0.29 g of benzoic acid, 3 ml of concentrated 98% H2SO4, and 97 ml ofmethanol. During extraction, 1 ml of the esterification solution and1 ml of chloroform were added to the tubes containing 10–14 mgof the samples. The mixed samples were heated for 4 h at 100 ◦C.Afterward, 1 ml of distilled water was added to the cooled solu-tion, and the mixture was vortexed for 1 min. The mixture was thenleft overnight to separate into two layers. The bottom layer, whichcontained dissolved PHA, was used for subsequent analysis [29].

PHA content and composition were determined by Agilent19091S-433 gas chromatography equipped with a fused HP-5MS5% Phenyl Methyl Siloxane column (length 30 m; diameter 250 �m;and film thickness 0.25 �m) (Agilent, USA). The resulting 0.4 ml ofmethyl esters were injected into the gas chromatography column.This was initially performed at 100 ◦C for 3 min, and the temper-ature 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 PHAstandard mixture containing various long-chain-length monomers(kindly provided by Dr. Lobna Jlaiel was used for PHA monomerband identification.

2.5.2. FT-IR spectroscopyFT-IR spectra were recorded using a JASCOFT/IR430 spectrom-

eter (JASCO Corp., Japan) over the 400–4000 cm−1 range at aspectral resolution of 4 cm−1. The PHA was directly extracted usingchloroform. Initially, the bacterial cultures were harvested by cen-trifugation at 5000 rpm for 10 min. The lipids were then removedfrom the cell pellet-using methanol (10 times the volume of cellpellets), and the cells were incubated for 1 h at 95 ◦C. The suspen-sion was then filtered to fully remove methanol, and the sediment

granules were incubated in an oven at 65 ◦C till becoming dry. Chlo-roform was added to the dried granules and incubated at for 10 min95 ◦C. After cooling, the solution was gently mixed overnight andthen filtered to get the debris. Finally, the PHA was precipitated

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rom the debris using 7:3 (v/v) mixtures of methanol and water.he precipitated PHA was washed with acetone and dried [30].

.6. Study of PHA molecular weight using GPC

Average molecular weights were estimated by GPC (Waters525, USA) with a combination of four styragel column seriesStyragel HR, 5 mm) at 40 ◦C using a 2414 differential refractivendex detector and a UV detector, respectively. Chloroform wassed as an eluent at a flow rate of 1.0 ml/min. The sample was pre-ared at a concentration of 1 mg/ml and injected at a volume of0 �L. A calibration curve was generated using polystyrene stan-ards of low polydispersity.

.7. Sequencing of the 16S rRNA gene of the Bacillus isolates

The chromosomal DNA from the Bacillus strains was extracteds previously described elsewhere [31]. The isolate was iden-ified by sequencing the total sequence of the 16S rRNA genemplified with primers S73 (5′-AGAGTTTGATCCTGGCTCAG-3′)nd S74 (5′-AAGGAGGTGATCCAAGCC-3′) [32]. The amplificationrogram consisted of 1 cycle of 95 ◦C/5 min and 30 cycles of4 ◦C/40 s, 52 ◦C/1 min, and 72 ◦C/1 min 30 s, with a final extension

t 72 ◦C/10 min. The PCR products were purified using the Wiz-rd SV Gel and PCR Clean-Up system (Promega). Sequencing waserformed with an ABI 3100 Capillary DNA Sequencer (Appliediosystems Inc., Foster City, CA, USA). A search for the closest 16S

ig. 1. Fluorescence of PHA granules using Nile Blue A staining at 280 nm: (A) US 100, U95, US 400, US 113, US 163 strains, and (D) Beta 5, Beta 4 strains. The white colonies are

trains.

iological Macromolecules 61 (2013) 82– 88

matches for the sequences was performed using the Basic LocalAlignment Search Tool (BLAST) program available at the NationalCenter 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 Gen-Bank (EMBL) under accession numbers ID1600075 and ID1600108,respectively. The sequences were aligned using clustalW [34], anda phylogenetic analysis was performed using the PHYLIP softwarepackage [35]. The phylogenetic tree was constructed using theneighbor joining (NJ) method [36].

2.9. Statistical analysis

The data presented in the current study are the means of threereplications and are expressed as the mean ± standard deviation(X̄ ± 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 petroleumsoil samples was investigated for their abilities to produce PHA.

S 106, US 177, US 500 strains; (B) US 183, US 531, US 554, US 516 strains, (C): USthe bioplastic strains produced. The dark colonies are the bioplastic non-producing

E. Zribi-Maaloul et al. / International Journal of Biological Macromolecules 61 (2013) 82– 88 85

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ig. 2. PHA granules within Bacillus strains stained with Nile blue A observed undetained cells were scanned with ZEISS LSM 510 confocal laser scanning microscopy77.

he bacteria were initially screened for PHA production in Luriaertani agar plates and using Nile Blue A staining to investi-ate their abilities to synthesize PHA granules. The formation ofow-molecular weight fluorescent compounds was observed at anmission wavelength of 280 nm. Based on the intensity of the flu-rescence observed, 8 strains were identified as potential PHAroducers: US 100; US 106; US 163; US 177; US 183; US 531; US54 and Beta 5) (Fig. 1). The PHA non-producing strains were notedo yield a dark smear (Fig. 1). (Insert Fig. 1)

.2. Screening of Bacillus strains in LB liquid medium

Confocal laser scanning fluorescence microscopy is a power-

ul technique for the detection of PHA granule formation at aavelength of 543 nm. A ZEISS LSM 510 confocal laser scanningicroscopy was, therefore, employed to investigate the PHA pro-

uction abilities of the 8 Bacillus strains selected. The results

Fig. 3. FT-IR spectrum of PHA produ

rescent light. The preparations were from exponential phase cells. The Nile blue A3 nm and 64 magnifications: (A) US 195 (negative control), (B) US 163; and (C) US

revealed that 2 among the 8 strains under investigation, namelyUS163 and US177, displayed reddish orange fluorescence (Fig. 2).The PHA non-producing strains were, on the other hand, noted toexhibit 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 inLB media at 37 ◦C and 250 rpm to determine the growth phasecorresponding to the production of PHA. Growth kinetics weremonitored by measuring optical density at 600 nm (OD600 nm) every2 h for 24 h (data not shown). Based on the intensity of the fluo-

rescence observed for the lag, exponential, stationary and declinephases during the staining procedure, the phase corresponding toPHA production was identified to occur during the exponentialphase (data not shown).

ced by the new US 163 strain.

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

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.4. FTIR analysis of the novel PHA

The FTIR spectra of the PHA produced by the two strains wereetermined. An intensity band was observed at around 1220 cm−1,hich was assigned to the stretching vibration of the C O C

roup. The region of 1675–1735 cm−1 was associated with the

O stretching of the ester carbonyl bond. The bond at 1530 cm−1

as characteristic of the stretching and deformation vibrationf the C H group, and those at 2930 cm−1 and 3272 cm−1 were

Fig. 5. FT-IR spectrum of the c

ced by the new US 177 strain.

characteristic of the stretching and deformation vibrations of theO H groups (Figs. 3–4). The FTIR peaks were identical to those dis-played by commercial PHA (Fig. 5). The functional groups of thepolymer PHA were confirmed as C O groups by FT-IR spectroscopy.

The results described above are congruent with the findingspreviously reported by several studies in the literature [37], partic-

ularly 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] alsosunbmitted the PHA produced by Bacillus circulans (MTCC 8167) to

ontol (commercial PHA).

E. Zribi-Maaloul et al. / International Journal of Biological Macromolecules 61 (2013) 82– 88 87

Table 1Structure 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

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T-IR characterization. The FTIR spectra showed high absorbance at360, 2922, 1735, and 1206 cm−1, which resulted in the vibrationunctions of O H, C H, C O, and C O C, respectively. Likewise,hukon et al. [39] analyzed the P(3HB-co-3HV) bioplastic producedy Bacillus circulans MTCC 8167 strain by FT-IR. They indicated thathe biopolymer obtained after solvent extraction exhibited C Hnd carbonyl stretching bands similar to those of standard PHA. Theresence of intense absorption bands at 1735 cm−1 and 1206 cm−1

ere reported to be characteristic of the C O and C O stretchingroups. Arcos-Hernandez et al. [40] also characterized the functionsf the P(3-HB-co-3HV) produced by a mixed culture of bacterial sys-ems by FT-IR. The FT-IR spectra showed high absorbance at 1753nd 1200–900 cm−1, which resulted in the vibration functions of

O and C O C, respectively. (Insert Figs. 3–5)

.5. GC–MS analysis of the novel PHA

The bioplastics produced by the US 163 and US 177 strains werenalyzed by GC–MS. The molecules that were obtained are shown in

able 1. Compared to the database molecules, the US 163 strain wasoted to produce the methyl-3-hydroxy tetradecanoic acid, and theS 177 strain produced the methyl-3-hydroxy octadecanoic acidelonging to the long-chain-length PHA class. (Insert Table 1)

ig. 6. Phylogenetic tree derived from 16S rDNA sequences; the positions of US 163 andtrains.

Lee and Choi [41] have previously analyzed the PHA productafter hydrolysis with GC–MS. They reported on the presence ofthe 4-hydroxy butanoic, 4-hydroxy valeric and 4-hydroxy hex-anoic acids among the hydrolysis products. Phukon et al. [39] havealso submitted the bioplastic produced by Bacillus circulans MTCC8167 to GC–MS analysis and showed that the strain produced theP(3HB-co-3HV). Likewise, He et al. [42] submitted Pseudomonasstutzeri 1317 to GC–MS analysis and reported on the productionof a novel polyhydroxyalkanoates from glucose and soybean oil.These included the methyl esters of 3-hydroxy-hexanoic acid, 3-hydroxy-octanoic acid, 3-hydroxy decanoic acid 3HD, 3-hydroxy5-dodecenoic acid, 3-hydroxy dodecanoic acid, and 3-hydroxytetradecanoic acid.

3.6. GPC analysis of the novel PHA

GPC analysis showed that the PHA produced by strains US163and US177 were a poly tetradecanoic acid (PTD) and poly octade-canoic acid (PTO) homopolymers with average molecular weightsof 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 polymerproduced by Bacillus circulans (MTCC 8167) to GPC analysis andreported that the polymer had a molecular mass of 5.1 × 104 Da.

US 177 strains were indicated. All the sequences used were from the Bacillus type

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.7. Identification and phylogenetic analysis of Bacillus US163nd US177

The total nucleotide sequences of 1.472 pb and 1.496 pb wereetermined from the whole 16S rRNA genes of strains US163 andS177. The alignment of these sequences with previous 16S rRNAene sequences available at the Gene bank database showed highimilarity (99%) with the Bacillus 16S rRNA reference genes. Thelosest similarity recorded for the new isolates US 163 and US77 strains were with Bacillus cereus GQ28038.1 and Bacillus cereusJ982658.1, respectively (Fig. 6). Based on the nucleotide sequencef the 16S rRNA gene of the two strains and the findings from sub-equent phylogenetic analyses, the new isolates were identifieds Bacillus cereus US163 and Bacillus cereus US 177, respectively.Insert Fig. 6)

cknowledgements

The authors would like to express their sincere gratitude to Dr.obna Jlaiel for kindly providing the long-chain-length monomerssed in this study. Thanks are also due to Mr Anouar Smaoui fromhe English Language Unit at the Sfax Faculty of Science for hisonstructive proofreading and language polishing services.

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