Dielectric relaxation analysis of biopolymer poly(3-hydroxybutyrate)

Dielectric Relaxation Analysis of BiopolymerPoly(3-hydroxybutyrate)

Taha A. Hanafy,1 Khaled Elbanna,2 Somyia El-Sayed,1 Arafa Hassen1

1Department of Physics, Faculty of Science, Fayoum University, Fayoum 63514, Egypt2Department of Microbiology, Faculty of Agriculture, Fayoum University, Fayoum 63514, Egypt

Received 13 July 2010; accepted 7 December 2010DOI 10.1002/app.33950Published online 11 April 2011 in Wiley Online Library (wileyonlinelibrary.com).

ABSTRACT: Poly(3-hydroxybutyrate), PHB, is a widelydistributed carbon storage polymer among prokaryotesincluding Rhizobium. Capacities of Rhizobium etli R13 toproduce the bioplastic during growth on media with dif-ferent carbon sources appeared to be specific carbon-source. In fed batch fermentation, R. etli R13 resulted incell dry weight 6.2 g/L and PHB 51.4%. Gas chromatogra-phy-mass spectrometry and gel permeation chromatogra-phy analysis revealed that PHB produced from R. etli R13was solely composed of 3-hydroxybutyric acid and themolecular mass of the purified PHB was 3.4 � 105 Dawith polydispersity 1.47. Dielectric relaxation of PHB hasbeen studied in the temperature and frequency ranges300–440 K and 10 kHz–4 MHz, respectively. A clear dielec-tric a and q-relaxation processes are observed in these

studied ranges of temperature and frequency. The firstprocess is due to the dipole relaxation in the crystallinephase of PHB. The second one is due to the space-chargeformation or Maxwell-Wagner-polarization. The a-relaxa-tion process has been investigated by semiempirical Havri-liak-Negami relaxation function. The activation energy (Ea)and the relaxation time (s0) are calculated using the Arrhe-nius equation. The dielectric relaxation strength (De) isstrongly temperature dependent. The calculated values ofEa for ac conductivity, ln(r), of PHB provide informationabout the presence of electronic conduction. VC 2011 WileyPeriodicals, Inc. J Appl Polym Sci 121: 3306–3313, 2011

Key words: poly(3-hydroxybutyrate); Rhizobium etli R13;relaxation processes; conduction mechanism

INTRODUCTION

Polyhydroxyalkanoates (PHAs) are a sort of biologi-cal polyester, which function as carbon and energyreserves in prokaryotic cells; many different bacteriasynthesize PHA when a carbon source is providedin excess and one essential growth nutrient is lim-ited.1–5 These bacterial polyesters have attractedindustrial attentions as environmentally nontoxic,biodegradable, and biocompatible thermoplastics tobe used for a wide range of industrial, agricultural,and medical applications.6–8 They have a highdegree of polymerization, are highly crystalline,optically active and isotactic, piezoelectric and insol-uble in water. These features make them highlycompetitive with polypropylene, polyethylene andthe petrochemical-derived plastics.9 Poly(3-hydroxy-butyrate) (PHB) is a homopolymer of 3-hydroxybu-teric acid. X-ray diffraction reveals that the purifiedpolymer has crystallinity ratio of 67% with ortho-rhombic crystal shape.10 Differential scanning calo-rimetry (DSC) showed a melting enthalpy (DHm) of62.31 J/g and melting temperature (Tm) of 448 K.

The glass rubber transition temperature (Tg) is� 285–291 K, while thermal degradation occursaround 523 K.Relaxation properties are very important in poly-

mer processing. Many processes are characterized asbeing elastic, while others as viscous. Relaxationproperties can be studied by dynamic mechanicalspectroscopy (DMS), dielectric relaxation spectros-copy (DRS), or nuclear magnetic resonance (NMR)spectroscopy. Relaxation processes play a dominantrole and in a complex pattern of temperature andfrequency-dependent properties.11 DRS is a usefulmethod to investigate structure property relation-ships of polymers. This method is sensitive to molec-ular fluctuation of dipoles within the system. Thesefluctuations are related to the molecular mobility ofgroups, segments or wholly polymer chains whichshow up as different relaxation processes. The relax-ation time (s0) of these processes was found todepend on the molecular shape and the molecularfriction forces encountered by the rotating dipoles.Moreover, the dipole motions within the amorphousand crystalline phases have a big effect on the semi-crystalline polymer.12,13 Structural transitions inpolymers are generally accompanied by changes inthe relaxation properties. When an oscillatory elec-tric field is applied to a polymeric material, severaltypes of polarization are operative: electronic, ionic,

Correspondence to: T. A. Hanafy ([email protected]).

Journal of Applied Polymer Science, Vol. 121, 3306–3313 (2011)VC 2011 Wiley Periodicals, Inc.

orientational, or space charge. A dielectric spectrumis a complicated manifestation of the combination ofthese operative polarization sources. Orientationaland space charge polarization are particularly im-portant when structural transitions are concerned.For polymers, many relaxation modes are involvedin orientational polarization. They can be groupedinto local processes, cooperative processes in longerchain sequences, chain diffusion, and specific proc-esses in semicrystalline states.14

Reported DRS studies on PHB and its blends arescarce. DRS was used to investigate the influence ofsemicrystalline morphology on the molecular mobil-ity of the crystallized PHBs15,16 and its blend withpolyvinyl acetate PVAc.17,18 The presence of thecrystallinity had a significant effect on the a-relaxa-tion characteristics of the various cold-crystallizedPHBs when compared with the wholly amorphousmaterial. The dielectric spectrum of the previousblends was resolved into three processes a, a0-relaxa-tion processes and ionic conductivity based on Hav-riliak-Negami and ionic conductivity equations. TheMaxwell-Wagner-Sillars (MWS) interfacial polariza-tion effect (i.e., q-relaxation process) can also be seenin the dielectric permittivity curve of semicrystallinepolymer such as polyvinyl alcohol PVA.19,20 Thiswork is aimed to study the production of PHB byRhizobium etli R13 to explore the dielectric relaxationproperties of PHB over a wide range of temperatureand frequency. Comparison to previous results andsimilar materials will be discussed.

SAMPLE PREPARATION ANDEXPERIMENTAL DETAILS

Microorganism and production of biomasscontaining PHB

The microorganism used in the present study wasRhizobium etli R13 (accession number FJ263092).21 Toevaluate the production of PHB from different car-bon sources by R. etli R13, batch fermentations werecarried out in 250 mL Erlenmeyer flasks containing100 mL of sterile YEM broth.22 To optimize produc-tion of PHB from R. etli R13, fed batch fermentation(80 h) were performed using YEM broth containing1% mannitol. The flasks were inoculated with 4% offreshly grown preculture and incubated at 30�C on arotary shaker at 150 rpm and 2% glucose was addedafter 48 h. At regular intervals, the samples weretaken (as whole flasks in duplicate) and certificatedat 5000 rpm for 30 min at 4�C and washed twicewith saline solution, then lyophilized for furtheranalysis. For polymer isolation, lyophilized cellswere resuspended in 250 mL of chloroform for3 days, and then filtered through filter paper. PHBextract was concentrated and precipitated with

diethyl ether. The precipitate was redissolved inchloroform and the process was repeated twice toobtain pure PHB.

Polymer samples preparation for GC andGC/MS analysis

Lyophilized cells (5–10 mg) were suspended in1.0 mL chloroform and subjected to methanolysisin 1 mL methanol in the presence of 15% (v/v)sulfuric acid. The methanolysis was performed for3–5 h at 100�C in an oil bath, and then 1 mL waterwas added to the cooled mixture and mixed thor-oughly for 30 s. After phase separation, the resultingmethylesters of the corresponding fatty acids constit-uent were assayed by gas chromatography (GC). Gelpermeation chromatography (GPC) was used to esti-mate the molecular weight of the purified PHB.Therefore, GPC system (model 410, Waters Corp,Milford) was used. The purified polymer was dis-solved in chloroform (5–10 mg/mL) and subjected toGPC system; applying four sequentially arrangedStyragel HR3-6 columns for separation and a model410 differential refractometer for detection. Polysty-rene standards dissolved in chloroform (0.1% w/v)were employed to construct the calibration curve.

Preparation of PHB films and dielectricspectroscopy measurements

Poly(3-hydroxybutyrate) film was obtained by dis-solving the polymer in chloroform at temperature30�C with continuous stirring. The aqueous solu-tion was cast into a Petridish, placed on a leveledplate at room temperature for 5 days until the sol-vent was completely evaporated. The obtained PHBfilm, 0.1 mm thickness, was cut into square piecesand coated with silver paste to achieve ohmic con-tacts. Dielectric spectroscopy measurements wereaccomplished using a Hioki (Ueda, Nagano, Japan)model 3532 High Tester LCR, with the accuracy oforder 6 0.08%. The dielectric constant (e0), anddielectric loss (e’’) were recorded at frequency andtemperature ranging from 10 kHz to 4 MHz andfrom 300 to 440 K, respectively. Both dielectric con-stant, e0, and dielectric losses, e00, were calculated asfollows:

e0 ¼ Cd

eoAand e00 ¼ e0 tan d (1)

where C is the capacitance of the sample filled ca-pacitor, d is the sample thickness, eo is the vacuumpermittivity, and A is the electrode area. The tem-perature was measured with a T-type thermocouplewith its junction just in contact with the sample withaccuracy better than 6 1 K.

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RESULTS AND DISCUSSION

Poly(3-hydroxybutyrate) is a widely distributed car-bon storage polymer among prokaryotes includingRhizobium.23 It was found that capacities of R. etliR13 to produce the bioplastic during growth onmedia with different carbon sources appeared to bespecific carbon-source. Five different carbon sourcessuch mannitol, glucose, sucrose, fructose and lactosewere evaluated for production of high cell densityand PHB accumulation. Among these substratesmannitol appears to be the most suitable for biomassproduction (5 g/L) followed by glucose (4 g/L), su-crose (3.2 g/L), fructose (3.0 g/L) and lactose (2.7 g/L), Whereas, glucose was the most favorable sub-strate for PHB accumulation (46%) followed by su-crose (42.2%), fructose (34.6%) and lactose (33.5%).To optimize bacterial growth and production ofPHB, fed batch fermentation was performed. Thecell dry mass and PHB percentage of R. etli R13during the time course of 80 h were presented in Fig-ure 1. As seen, the maximum biomass was 6.2 g/Land the maximum PHB accumulation was 51.4% percell dry weight.

Many nitrogen-fixing microorganisms synthesizePHB. According to Tombolini and Nuti,24 the con-tent of this polymer in rhizobia ranges from 30 to55% of dry cell weight. It was found that PHB syn-thesis can be selectively induced either in active orless active Rhizobium strains by sources of carbonand nitrogen25. Because chemical structure and mo-lecular mass are important factors to determine thephysical properties of polymers, the purified poly-mer was analyzed using gas chromatography-massspectrometry (GC/MS) and GPC. Figure 2 representsGC/MS spectrum of methyl ester 3HB. The pro-duced PHB by Rhizobium etli R28 was solely com-posed of 3-hydroxybutyric acid. In addition, GPC

analysis showed that the molecular mass of the puri-fied PHB was 3.4 � 105 Da with polydispersity 1.47.The temperature dependence of e’’ of PHB at

selected frequencies is shown in Figure 3. A broadrelaxation peak in e’’ was observed at 370 K. Thispeak becomes narrow and moves towards the lowtemperatures as the frequency increases. Suchbehavior can be attributed to the space charge polar-ization (i.e., the q-relaxation process). This effectoccurs at temperature higher than Tg (285–291 K)10

and at a low field frequency. The nature of q-relaxa-tion process seems to vary depending on the poly-meric material. For semicrystalline polymer, chaintrapping at interfaces inside the sample or Maxwell-Wagner polarization (MWS) phenomena is expected.

Figure 1 PHB yield and cell dry weight of R. etli duringthe fed batch fermentation. R. etli grown on YEM brothsupplemented with 1% mannitol at the beginning of fer-mentation. After 48 h, glucose was added with 0.5% (w/v)until the final concentration reached 2% (w/v).

Figure 2 GC/MS spectrum of 3HB-methyl ester indicat-ing that the PHB produced by Rhizobium etli R28 wassolely composed of 3-hydroxybutyric acid.

Figure 3 The temperature dependence of e00 for PHB atsome selected frequencies.

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For an amorphous materials, q-relaxation processmay be attributed to cooperative parameters such asimpurities, injected space charges and electrodeeffects.20

The isothermal plots for e0 and e’’ versus fre-quency at selective fixed temperatures are presentedin Figure 4(a,b). The dielectric constant, e0, increasessmoothly with increasing temperature and fre-quency. This can be attributed to the increase of thethermal energy, which is absorbed by the dipoles ofPHB. Consequently, the dipoles have sufficientenergy to orientate themselves easily in the directionof the applied field. On the other hand, the increasein e0 with temperature is assigned to the disentangle-ment of the molecular chains which becomes easierdue to molecular vibrations.26 The dependence of e0

on temperature reflects the orientational distributionof the polymer chains in the crystalline as well as inthe amorphous regions inside the sample.16,27 Inother words, the number of the polar C¼¼O andmethyl, CH3, groups become free to rotate withincreasing applied field and temperature. From Fig-ure 4(b), one can observe that e" of PHB undergoes asingle relaxation peak at 316 kHz in the studied fre-quency range due to a-relaxation process. The peakposition of a-process shifts to higher frequencieswith increasing temperatures and it can be charac-terized as a dipolar relaxation.

The origin of the a-transition has been attributedto several mechanisms, such as a rotation of crystal-line sequence followed by a translation along the

chain axis, torsional twisting in crystalline sequence,fold movements, or even point defect mechanisms.28

The crystallinity ratio of PHB has a significant effecton the a-relaxation characteristics of various cold-crys-tallized samples when compared with the whollyamorphous materials. The constraining influences ofthe crystallites produce a progressive relaxationbroadening and a positive offset in relaxation temper-ature.16 Also, all cold-crystallized PHB/PVAc blendsexhibit two glass-rubber (a and a’) relaxations accord-ing to the coexisting of mixed amorphous interlamel-lar phase, and a pure PVAc phase residing in interfi-billar regions.18 The a’- relaxation process is related tothe rigid amorphous phase located between adjacentlamellar inside the lamellar stacks.29–33 The a’-relaxa-tion process was observed in the PHB blends with 30wt % of PVAc.17 So, a-relaxation process of PHB canbe assigned to the segmental motion in the crystallinephases within the sample. By other words this processis probably due to the dipole relaxation inside the la-mellar stacks.The dielectric dispersion of the aliphatic polyester

as a function of frequency in the vicinity of Tg relax-ation can be described by Havriliak-Negami (HN)phenomenological equation.34–37 Then, the complexdielectric permittivity e* is given by:

e� ¼ e1 þ De

1þ ixsoð Þbh ic (2)

where De ¼ es � e1, is the dielectric strength, es ande1 are the relaxed and unrelaxed dielectric con-stants, respectively. s0 is the relaxation time, b and care the shape parameters that describe the symmet-ric and asymmetric broadening of s0, respectively.The study of the dielectric behavior of PHB by

Cole-Cole plot (e’’ vs. e’) provides valuable informa-tion about the dielectric relaxation process. Cole-Cole equation is given by38:

e� ¼ e1 þ De

1þ ðixsoÞ1�c (3)

where s0 is the mean relaxation time and c is thedistribution parameter that ranges from 0 to 1. So,Figure 5 shows the Cole-Cole representation at dif-ferent fixed temperatures, where data points (e’’ vs.e’) from an arc when a dielectric relaxation occurs inthe examined frequency interval. The values so andc have been evaluated using the equation39:

U

V¼ ðxsoÞ1�c (4)

where U is the distance from a particular data pointin the Cole-Cole plot from point e1 and V is the dis-tance of the same data point from point es.

Figure 4 Frequency dependence of: (a) e0 and (b) e00 ofPHB at some selected temperatures.

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The calculated values of c and s0, are tabulatedin Table I. The Arrhenius law for a-relaxationprocess is:

soðTÞ ¼ A expEa

kT

� �(5)

where Ea is the activation energy, k is Boltzmannconstant, and A is a pre-exponential factor. Figure6(a) depicts the relaxation time (s0) as a function ofthe inverse of the temperature as calculated from theCole-Cole curves. The calculated value of Ea was

found to be of �0.3 eV. Figure 4(b) shows that themaximum frequency, fmax, of the e’’ peak as a func-tion of the inverse temperature. fmax was describedby the equation:

fmax ¼ fo exp�Ea

kT

� �(6)

where fo is constant. The calculated value of Ea,according to eqs. (5) and (6), of a-relaxation processis 0.3 eV. This value is lower than that of the earlier

Figure 5 Argand plots at some selected temperatures for PHB.

Figure 6 (a) Arrhenius plot of the logarithm of fmax ver-sus 1000/T for PHB. (b) Arrhenius plot of ln(s) against1000/T for PHB.

TABLE IThe Calculated Values of the Distribution Parameter (c)

and the Average Relaxation Time (so) According toCole-Cole Plots of PHB

T (K) c so (s)

308 0.877 7.70 � 10�4

323 0.766 3.22 � 10�4

338 0.866 3.01 � 10�4

353 0.855 1.69 � 10�5

368 0.833 1.06 � 10�4

383 0.833 6.77 � 10�5

398 0.888 6.61 � 10�5

413 0.833 6.19 � 10�5

428 0.900 6.63 � 10�5

443 0.877 4.56 � 10�5

3310 HANAFY ET AL.

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report (1.0 eV).40 The low value of Ea of a-relaxationprocess could be explained by the lower bulkiness ofthe CH3 and C¼¼O groups within the amorphousand the crystalline phases of PHB. A similar behav-ior has been reported for polyethylene terephatha-late (PET).11 Consequently, the low value of Ea ofPET compared with those obtained for poly ethylenenaphthalate (PEN) was assigned to the lower bulki-ness of the terephthalate ring compared to the morebulky naphthalate group in PEN.11

The temperature dependence of dielectric strength,De, of PHB obtained from the above analysis is dis-played in Figure 7. It is clear that the values of Deincrease with the increase of the temperature. Thiscan be attributed to the cooperation exists betweenthermal energy and the electric field effects of thedipole alignments. Increasing the thermal energy ofCH3 and C¼¼O groups will tend to enhance the align-ment of themselves with the direction of the appliedelectric field and therefore De increases. It has beenreported for the amorphous PHB that De is decreasedwith increasing temperature due to the decrease ofthe volume fraction of the mobile amorphous regionin the PHB/PVAc blends.11 However, the increase ofDe with temperature for the crystalline PHB andPHB/PVAc blends were interpreted to the existenceof a rigid amorphous phase which relaxes graduallyabove the Tg of the mobile amorphous material10.When the crystalline ratio of pure PHB increases upto 67%, an increase of De is expected.

To suppress the electrode effect, the complex elec-tric modulus M* is used to explore and analyze thedielectric spectra. The electric modulus M* has thefollowing form40,41:

M� ¼ 1

e�¼ M0 þ iM00 (7)

M0ðxÞ ¼ e0ðxÞe0ðxÞ2 þ e00ðxÞ2 (8)

M00ðxÞ ¼ e00ðxÞe0ðxÞ2 þ e00ðxÞ2 (9)

Figure 8 shows M00(T) spectra obtained by trans-forming the data of Figure 3(b) to M* formalism [eq.(8)]. It is observed that PHB undergoes q-relaxationprocess, which shifts to lower temperature withincreasing frequencies. Also, the peak values of M00

were found to be lower than the values of e’’ thatobtained from Figure 3(b). This indicates the re-moval of the electrode polarization. It is also inter-esting to note that q-relaxation process becomes evi-dent only when the temperatures are higher than Tg

(285–291 K). At temperature higher than 370 K, agrowth in M00 with decreasing field frequency isobserved. Dc conduction alone will not affect thebehavior of M00. Another relaxation process musthave something to do with the mobilization of spacecharges. It is assigned to MWS interfacial polariza-tion process which originates from the createdcharges by contact of different phases of differentcharge conductivity. It can be suggested that theintrinsic relaxation spectra of PHB samples are char-acterized by a combination of an MWS process anddc conductivity effect. In addition, the charge car-riers which are trapped by the surrounding crystalli-tes, accumulate at the interface and move throughthe amorphous phase under the influence of anapplied field. The mobility of these charge carriersshould also increase with the increase of the temper-ature so that the relaxation time becomes shorter.The bulk conductivity in amorphous samples isclosely related to ion mobility. The behavior of thetrapped charge carriers should resemble those of theglobal charge carriers which are responsible for bulkconductivity.

Figure 7 The temperature dependence of the dielectricrelaxation strength (De) for PHB.

Figure 8 The variation of the dielectric modulus M00 as afunction of temperature at some fixed frequencies forPHB.

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The frequency dependence of M00 is represented inFigure 9. At f ¼ 316 kHz, a-relaxation process ofPHB is observed and shifted to higher frequencieswith increasing temperature. This peak contributesto the conductivity as seen in Figure 4(b). The shiftsof the maximum M00 with temperature correspondsto the conductivity current relaxation. At each tem-perature, the region to the left of the conductivitycurrent relaxation peak (low frequency side) extendsto a long distance since the charge carriers are mo-bile. On the other hand, the conductivity currentrelaxation peak (high frequency side) does not moveto contribute to the conduction process because thecharge carriers are spatially confined to their poten-tial wells.

The temperature dependence of ac conductivityln(r) of PHB is shown in Figure 10. Within the stud-ied range of temperature, ln(r) exhibits two straightregions; I at (303–357 K) and II at (400–454 K). The

behavior of ln(r) within these two regions can bedescribed according to the Arrhenius equation:

r ¼ ro exp � E

kT

� �(10)

where ro is constant, and E is the activation energy.The E values were calculated for PHB sample at dif-ferent frequencies and listed in Table II. It is clearthat the values of lnr in both regions (I and II) werefound to increase with the increase of the field fre-quency. This may be due to the increase of theabsorbed energy which leads to increase the numberof the charge carriers that contribute to the conduc-tion process. Moreover, this reveals that the conduc-tion mechanism could be a hopping one.42,43 Also,the variation of the conductance with temperature isdue to a combined effect of a change in the conduct-ance with temperature and the nature of the trapdistribution inside the matrix of PHB. It might indi-cate that the conductance takes place via hopping ofcarriers between randomly distributed trapping cen-ters in amorphous and crystalline phases. From Ta-ble II, one can recognize that the values of E varyfrom 0.22 to 0.45 eV. Therefore, the conductionmechanism is mainly electronic and partially ionicas a result of bulk conduction with the differentphases that exist in PHB.

CONCLUSIONS

GC/MS and GPC analysis revealed that PHB pro-duced from R. etli R13 was solely composed of 3-hy-droxybutyric acid and PHB has a high molecularweight to be used in different industrial applica-tions. The dielectric behavior of PHB undergoes tworelaxation processes namely a and q. The first one isattributed to the dipole relaxation inside the crystal-line regions. The second one is the space-chargetransition which is due to the chain trapping at theinterfaces or MWS polarization within PHB. Thedielectric strength De of PHB was strongly

Figure 9 Variation of the dielectric modulus M00 as afunction of frequency at some fixed temperatures for PHB.

Figure 10 The temperature dependence of the ac conduc-tivity, ln(r), for PHB at some selected frequencies.

TABLE IIThe Calculated Values of the Activation Energy (E)

Based on the ac Conductivity of PHB

f (kHz) E (I) (eV) E (II) (eV)

1 0.76 0.3010 0.20 0.30

100 0.23 0.86300 0.45 0.45500 0.45 0.45700 0.45 0.14900 0.45 0.23

2000 0.27 0.223000 0.31 0.204000 0.27 0.20

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temperature dependent. The Cole-Cole exponent cshows that there is a distribution in the relaxationtimes for PHB. The mean relaxation time s0 and fmax

of a-relaxation process verify Arrhenius type de-pendence. The behavior of ac conductivity indicatesthat, the conduction mechanism of PHB could behopping one.

The authors would like to sincerely thank Prof. Dr. A. Stein-buchel (Westfalische Wilhelms Universitat, Munster, Ger-many) for GC/MS analysis.

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